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Clinical Focus
- Allergy and Immunology
- Adult and Pediatric Asthma, Allergy, and Immunology
Academic Appointments
Associate Professor - Med Center Line, Pediatrics - Immunology and Allergy
Associate Professor - Med Center Line (By courtesy), Otolaryngology (Head and Neck Surgery)
Member, Child Health Research Institute
Administrative Appointments
Director, Sean N. Parker Center for Allergy Research at Stanford University (2014 - Present)
Associate Professor, Affiliate appointment in Otolaryngology (2011 - Present)
Associate Professor, Pediatrics (2011 - Present)
Faculty Member, Multidisciplinary Program in Immunology (2007 - Present)
Faculty Member, Stanford Institute of Immunity, Transplantation and Infectious Disease (2007 - Present)
Assistant Professor, Pediatrics (2007 - 2011)
Assistant Professor, Affiliate appointment in Otolaryngology (2007 - 2011)
Medical Director, Allergy & Immunology Clinics (2007 - 2011)
Assistant Program Director, Allergy & Immunology Fellowship Program (2007 - 2011)
Instructor, ENT (2007 - 2008)
Instructor, Allergy and Immunology (2006 - 2007)
Volunteer Clinical Instructor, General Pediatrics (2003 - 2005)
Honors & Awards
Junior Faculty Award, Clinical Immunological Society (2010)
National Junior Faculty Award, American Lung Association (2009)
Fellow, American Academy of Asthma, Allergy and Immunology (2008-present)
Speaker Award, La Entrada Inspirational Speaker Series (2007)
National Junior Faculty Award, American Academy of Asthma, Allergy and Immunology (2007)
Stanford Free Clinics Teaching Award, Stanford Medical School (June 2007)
Mary Hewitt Loveless Award, Loveless Foundation (July 2006-July 2009)
Parker B Francis Award, Francis Foundation (July 2006-July 2009)
Pediatric Research Award, CHRP (July 2007-July 2008)
Professional Education
Board Certification: Pediatrics, American Board of Pediatrics (2006)
Fellowship:Stanford University Medical Center (2006) CA
Residency:Children's Hospital Boston (1997) MA
Residency:Stanford University Medical Center (2004) CA
Internship:Children's Hospital Boston (1996) MA
Medical Education:Harvard Medical School (1995) MA
Board Certification: Allergy and Immunology, American Board of Allergy and Immunology (2007)
PhD, Harvard Medical School, Biochemistry and Immunology (1995)
B.S., Haverford College, Biology (1988)
Community and International Work
Volunteer Clinical Faculty, Menlo Park VA
Clinic for underserved
Partnering Organization(s)
Stanford/Menlo Park VA
Populations Served
Ongoing Project
Opportunities for Student Involvement
Current Research and Scholarly Interests
The Nadeau Laboratory focuses on the mechanisms of immune dysfunction in primary immune disease (PID), allergy, and asthma. In recent years, allergic disorders have reached epidemic proportions in children and adults. Many studies have determined that the immune system of patients with allergies (also called atopy), such as asthma, atopic dermatitis, food allergies, allergic rhinitis, allergic conjunctivitis and other atopic disorders, is overactive and skewed toward a certain subtype of immune cell called the Th2 cell. So far, there is little understanding of how cells turn off this abnormal proliferation and activation of the Th2 cell.
The Nadeau Laboratory has found that a type of cell, called the natural regulatory T cells (nTreg), can decrease Th2 cell overactivation in allergies, thus leading to improvement or even reversal of the allergic condition. By understanding how these Treg cells work, we hope to discover new diagnostic and therapeutic ways to treat or prevent allergic conditions.
The Nadeau Laboratory maintains a database and sample/tissue bank of healthy controls and patients treated at LPCH/Stanford Medical Center with allergic disorders. The main focus of our laboratory is five-fold:
1. To determine the role of STAT5a and STAT5b in Treg development and function;
2. Since Tregs may respond to pollution, we are studying the effect of ambient air pollution on Tregs;
3. Since most allergic conditions start in childhood, we are examining the role of Th2 and Treg in different age groups with and without allergies;
4. Since the activity of Th2 and Treg is determined by their interactions with other cell types, such as epithelial cells and dendritic cells, we are studying their effects on Th2/Treg interactions; and
5. Since improvement of Treg function is associated with improvement of allergic conditions, we are designing new allergy treatments (for example, sublingual immunotherapy, small molecule chemokines) that enhance Treg function.
2014-15 Courses
- Translating Science to Disease Treatment
PEDS 50N (Win)
Independent Studies (11)
- Directed Reading in Immunology
IMMUNOL 299 (Aut, Win, Spr, Sum)
- Directed Reading in Pediatrics
PEDS 299 (Aut, Win, Spr, Sum)
- Early Clinical Experience
PEDS 280 (Aut, Win, Spr, Sum)
- Early Clinical Experience in Immunology
IMMUNOL 280 (Aut, Win, Spr, Sum)
- Graduate Research
IMMUNOL 399 (Aut, Win, Spr, Sum)
- Graduate Research
PEDS 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
PEDS 370 (Aut, Win, Spr, Sum)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Sum)
- Teaching in Immunology
IMMUNOL 290 (Aut, Win, Spr, Sum)
- Undergraduate Directed Reading/Research
PEDS 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
IMMUNOL 199 (Aut, Win, Spr, Sum)
- Directed Reading in Immunology
Prior Year Courses
2013-14 Courses
- Translating Science to Disease Treatment
PEDS 50N (Win)
- Translating Science to Disease Treatment
Postdoctoral Advisees
Graduate and Fellowship Programs
Journal Articles
Whole-exome sequencing identifies tetratricopeptide repeat domain 7A (TTC7A) mutations for combined immunodeficiency with intestinal atresias.
journal of allergy and clinical immunology
2013; 132 (3): 656-664 e17
Combined immunodeficiency with multiple intestinal atresias (CID-MIA) is a rare hereditary disease characterized by intestinal obstructions and profound immune defects.We sought to determine the underlying genetic causes of CID-MIA by analyzing the exomic sequences of 5 patients and their healthy direct relatives from 5 unrelated families.We performed whole-exome sequencing on 5 patients with CID-MIA and 10 healthy direct family members belonging to 5 unrelated families with CID-MIA. We also performed targeted Sanger sequencing for the candidate gene tetratricopeptide repeat domain 7A (TTC7A) on 3 additional patients with CID-MIA.Through analysis and comparison of the exomic sequence of the subjects from these 5 families, we identified biallelic damaging mutations in the TTC7A gene, for a total of 7 distinct mutations. Targeted TTC7A gene sequencing in 3 additional unrelated patients with CID-MIA revealed biallelic deleterious mutations in 2 of them, as well as an aberrant splice product in the third patient. Staining of normal thymus showed that the TTC7A protein is expressed in thymic epithelial cells, as well as in thymocytes. Moreover, severe lymphoid depletion was observed in the thymus and peripheral lymphoid tissues from 2 patients with CID-MIA.We identified deleterious mutations of the TTC7A gene in 8 unrelated patients with CID-MIA and demonstrated that the TTC7A protein is expressed in the thymus. Our results strongly suggest that TTC7A gene defects cause CID-MIA.
View details for DOI 10.1016/j.jaci.2013.06.013
View details for PubMedID 23830146
Differentiating the roles of STAT5B and STAT5A in human CD4(+) T cells
2013; 148 (2): 227-236
STAT5A and STAT5B are highly homologous proteins whose distinctive roles in human immunity remain unclear. However, STAT5A sufficiency cannot compensate for STAT5B defects, and human STAT5B deficiency, a rare autosomal recessive primary immunodeficiency, is characterized by chronic lung disease, growth failure and autoimmunity associated with regulatory T cell (Treg) reduction. We therefore hypothesized that STAT5A and STAT5B play unique roles in CD4(+) T cells. Upon knocking down STAT5A or STAT5B in human primary T cells, we found differentially regulated expression of FOXP3 and IL-2R in STAT5B knockdown T cells and down-regulated Bcl-X only in STAT5A knockdown T cells. Functional ex vivo studies in homozygous STAT5B-deficient patients showed reduced FOXP3 expression with impaired regulatory function of STAT5B-null Treg cells, also of increased memory phenotype. These results indicate that STAT5B and STAT5A act partly as non-redundant transcription factors and that STAT5B is more critical for Treg maintenance and function in humans.
View details for DOI 10.1016/j.clim.2013.04.014
View details for Web of Science ID 000322101300009
Markers of Antigen Presentation and Activation on Eosinophils and T Cells in the Esophageal Tissue of Patients With Eosinophilic Esophagitis
2013; 56 (3): 257-262
Evidence suggests eosinophils may be acting as antigen-presenting cells (APCs) by presenting antigen to T cells. We investigated the surface proteins of eosinophils and T cells in the esophageal biopsies of patients with eosinophilic esophagitis (EoE), patients with gastroesophageal reflux disease (GERD), and healthy controls (HCs).: Subjects were categorized as EoE, GERD, or HC. In esophageal tissue, EG2+ eosinophils were stained for the APC markers, CD40 or CD80, via immunohistochemistry. CD3+ T cells were stained for costimulatory markers, CD40L or CD28, and for activation markers, CD69 or CD134, via immunofluorescence or immunohistochemistry.Eosinophils stained with CD40 and CD80. The number of EG2+CD40+ cells was increased in EoE (mean 19.1±14.8 cells/high-power field [HPF], n=11), compared with GERD (mean 0.13±0.19 cells/HPF, n=5, P<0.01) and HC (mean 0.3±0.7 cells/HPF, n=5, P<0.01). There was an elevation in EG2+CD80+ cells in EoE (mean 18.1±16.2 cells/HPF, n=10), GERD (mean 1.7±2.8 cells/HPF, n=6, P<0.01), or HC (mean 0.8±1.3 cells/HPF, n=6, P<0.01). CD3+ T cells stained with CD40L (not quantified). CD3+ T cells stained with CD28 at elevated levels in EoE (mean 14±8.7 cells/HPF, n=9) versus GERD (mean 3.3±1.2 cells/HPF, n=6, P<0.05) or HC (mean 3.0±3.2 cells/HPF, n=7, P<0.01). The number of CD3+CD69+ cells was highest in EoE (mean 14.8±7.5 cells/HPF, n=6) versus GERD (mean 0.8±0.9 cells/HPF, n=6, P<0.001) or HC (mean 2.7±2.5 cells/HPF, n=6, P<0.001).We show that esophageal eosinophils express CD40 and CD80, and T cells with CD40L, CD28, and CD69. The number of double-stained cells was higher in EoE in comparison to control groups. These data support the hypothesis that eosinophils in EoE may act as APCs, activating T cells.
View details for DOI 10.1097/MPG.0b013e3182758d49
View details for Web of Science ID 000315461400010
View details for PubMedID 23059644
Immunomodulatory Effect of Vancomycin on Treg in Pediatric Inflammatory Bowel Disease and Primary Sclerosing Cholangitis
2013; 33 (2): 397-406
Vancomycin has been shown to affect tumor necrosis factor-alpha (TNF-α) pathways as an immunomodulator; this is thought to be separate from its function as an antibiotic [1]. Previous studies have shown that oral vancomycin (OV) is an effective treatment for concomitant primary sclerosing cholangitis (PSC) and inflammatory bowel disease (IBD) in children [2, 3]. Since both diseases are associated with immune dysfunction, we hypothesized that vancomycin's therapeutic effect in IBD and PSC occurs through immunomodulation. Therefore, we examined the in vivo immunological changes that occur during OV treatment of 14 children with PSC and IBD. Within 3 months of OV administration, peripheral gamma-glutamyl transpeptidase (GGT) and alanine aminotransferase (ALT) concentrations, white blood cell (WBC) counts, and neutrophil counts normalized from elevated levels before treatment. Patients also demonstrated improved biliary imaging studies, liver biopsies and IBD symptoms and biopsies. Additionally, plasma transforming growth factor beta (TGF-β) levels were increased without concurrent shifts in Th1-or Th2-associated cytokine production. Peripheral levels of CD4 + CD25hiCD127lo and CD4 + FoxP3+ regulatory T (Treg) cells also increased in OV-treated PSC + IBD patients compared to pretreatment levels. A unique case study shows that the therapeutic effects of OV in the treatment of PSC + IBD do not always endure after OV discontinuation, with relapse of PSC associated with a decrease in blood Treg levels; subsequent OV retreatment was then associated with a rise in blood Treg levels and normalization of liver function tests (LFTs). Taken together, these studies support immune-related pathophysiology of PSC with IBD, which is responsive to OV.
View details for DOI 10.1007/s10875-012-9801-1
View details for Web of Science ID 000323122000016
View details for PubMedID 23054338
Asthma Discordance in Twins Is Linked to Epigenetic Modifications of T Cells
2012; 7 (11)
T cells mediate the inflammatory responses observed in asthma among genetically susceptible individuals and have been suspected to be prone to epigenetic regulation. However, these relationships are not well established from past clinical studies that have had limited capacity to control for the effects of variable genetic predisposition and early environmental exposures. Relying on a cohort of monozygotic twins discordant for asthma we sought to determine if epigenetic modifications in T cells were associated with current asthma and explored whether such modifications were associated with second hand smoke exposures. Our study was conducted in a monozygotic twin cohort of adult twin pairs (n?=?21) all discordant for asthma. Regulatory T cell (Treg) and effector T cell (Teff) subsets were assessed for levels of cellular function, protein expression, gene expression and CpG methylation within Forkhead box P3 (FOXP3) and interferon gamma-? (IFN?) loci. Comparisons by asthma and current report of exposure to second hand smoke were made. Treg from asthmatic discordant twins demonstrated decreased FOXP3 protein expression and impaired Treg function that was associated with increased levels of CpG methylation within the FOXP3 locus when compared to their non-asthmatic twin partner. In parallel, Teff from discordant asthmatic twins demonstrated increased methylation of the IFN? locus, decreased IFN? expression and reduced Teff function when compared to Teff from the non-asthmatic twin. Finally, report of current exposure to second hand smoke was associated with modifications in both Treg and Teff at the transcriptional level among asthmatics. The results of the current study provide evidence for differential function of T cell subsets in monozygotic twins discordant for asthma that are regulated by changes in DNA methylation. Our preliminary data suggest exposure to second hand smoke may augment the modified T cell responses associated with asthma.
View details for DOI 10.1371/journal.pone.0048796
View details for Web of Science ID 000312376100014
View details for PubMedID 23226205
Epigenetic modifications and improved regulatory T-cell function in subjects undergoing dual sublingual immunotherapy
2012; 130 (1): 215-?
Allergen-specific immunotherapy is the only mode of therapy that has been demonstrated to offer a cure in patients with IgE-mediated respiratory allergies.We sought to demonstrate the safety and efficacy of timothy grass (TG) and dust mite (DM) dual sublingual immunotherapy (SLIT) and to begin to investigate the immune mechanisms involved in successful immunotherapy with multiple allergens.The safety and efficacy of dual SLIT with TG and DM in children and adults with demonstrated allergies to TG and DM were investigated in a single-center, randomized, double-blind, controlled phase I study. Thirty subjects received either TG and DM dual SLIT (n= 20) or placebo (n = 10). Immune parameters were evaluated for differentiation of desensitized subjects from control subjects.Subjects treated with dual SLIT had decreased rhinoconjunctivitis scores (P < .001) and medication use scores (P < .001) and reduced responses to TG and DM allergen based on results of skin prick tests or nasal disk challenges (P < .01 and P < .001, respectively) compared with placebo-treated control subjects. An increase in TG- and DM-specific IgG(4) levels, reduced allergen-specific IgE levels, and subsequent basophil activation were observed in the active treatment group. Dual SLIT promoted allergen-specific suppressive CD4(+)CD25(high)CD127(low)CD45RO(+) forkhead box protein 3 (Foxp3)(+) memory regulatory T cells with reduced DNA methylation of CpG sites within the Foxp3 locus.The results of this pilot study suggest that dual SLIT could be an effective means to treat subjects with sensitivities to a variety of allergens and that long-term tolerance might be induced by epigenetic modifications of Foxp3 in memory regulatory T cells.
View details for DOI 10.1016/j.jaci.2012.04.021
View details for Web of Science ID 000306644800030
View details for PubMedID 22677046
Modulation of mTOR Effector Phosphoproteins in Blood Basophils from Allergic Patients
2012; 32 (3): 565-573
The mammalian target of rapamycin (mTOR) pathway contributes to various immunoinflammatory processes. Yet, its potential involvement in basophil responses in allergy remains unclear. In this pilot study, we quantified two key mTOR effector phosphoproteins, the eukaryotic initiation factor 4E (peIF4E) and S6 ribosomal protein (pS6rp), in blood basophils from nut allergy patients (NA, N?=?16) and healthy controls (HC, N?=?13). Without stimulation in vitro, basophil peIF4E levels were higher in NA than HC subjects (P?=?0.014). Stimulation with nut (offending) but not chicken / rice (non-offending) extract increased basophil peIF4E and pS6rp levels (+32%, P?=?0.018, and +98%, P?=?0.0026, respectively) in NA but not HC subjects, concomitant with increased surface levels of CD203c and CD63, both known to reflect basophil activation. Pre-treatment with the mTOR inhibitor rapamycin decreased pS6rp and CD203c responses in nut extract-stimulated basophils in NA subjects. Thus, basophil responses to offending allergens are associated with modulation of mTOR effector phosphoproteins.
View details for DOI 10.1007/s10875-012-9651-x
View details for Web of Science ID 000305982100019
View details for PubMedID 22350221
Personal Omics Profiling Reveals Dynamic Molecular and Medical Phenotypes
2012; 148 (6): 1293-1307
Personalized medicine is expected to benefit from combining genomic information with regular monitoring of physiological states by multiple high-throughput methods. Here, we present an integrative personal omics profile (iPOP), an analysis that combines genomic, transcriptomic, proteomic, metabolomic, and autoantibody profiles from a single individual over a 14 month period. Our iPOP analysis revealed various medical risks, including type 2 diabetes. It also uncovered extensive, dynamic changes in diverse molecular components and biological pathways across healthy and diseased conditions. Extremely high-coverage genomic and transcriptomic data, which provide the basis of our iPOP, revealed extensive heteroallelic changes during healthy and diseased states and an unexpected RNA editing mechanism. This study demonstrates that longitudinal iPOP can be used to interpret healthy and diseased states by connecting genomic information with additional dynamic omics activity.
View details for DOI 10.1016/j.cell.2012.02.009
View details for Web of Science ID 000301889500023
View details for PubMedID 22424236
Multiplex meta-analysis of RNA expression to identify genes with variants associated with immune dysfunction
2012; 19 (2): 284-288
We demonstrate a genome-wide method for the integration of many studies of gene expression of phenotypically similar disease processes, a method of multiplex meta-analysis. We use immune dysfunction as an example disease process.We use a heterogeneous collection of datasets across human and mice samples from a range of tissues and different forms of immunodeficiency. We developed a method integrating Tibshirani's modified t-test (SAM) is used to interrogate differential expression within a study and Fisher's method for omnibus meta-analysis to identify differentially expressed genes across studies. The ability of this overall gene expression profile to prioritize disease associated genes is evaluated by comparing against the results of a recent genome wide association study for common variable immunodeficiency (CVID).Our approach is able to prioritize genes associated with immunodeficiency in general (area under the ROC curve = 0.713) and CVID in particular (area under the ROC curve = 0.643).This approach may be used to investigate a larger range of failures of the immune system. Our method may be extended to other disease processes, using RNA levels to prioritize genes likely to contain disease associated DNA variants.
View details for DOI 10.1136/amiajnl-2011-000657
View details for Web of Science ID 000300768100023
View details for PubMedID 22319178
Oral Immunotherapy and Anti-IgE Antibody-Adjunctive Treatment for Food Allergy
2012; 32 (1): 111-?
One of the most promising therapies for food allergy is oral immunotherapy (OIT), in which small amounts of allergen are administered in increasing amounts, with the immediate goal of desensitization and the long-term goal of tolerance. However, safety and standardization concerns prevent its widespread use, and a subgroup of patients may experience severe allergic reactions. These concerns might be addressed by another promising therapy involving anti-IgE monoclonal antibodies (mAb), which can reduce allergic reactions associated with food administration. A recent pilot study combining anti-IgE mAb with OIT suggests that anti-IgE mAb might improve the safety, rapidity, and efficacy of OIT.
View details for DOI 10.1016/j.iac.2011.11.004
View details for Web of Science ID 000300467200010
View details for PubMedID 22244236
The STAT5b Pathway Defect and Autoimmunity.
Frontiers in immunology
2012; 3: 234-?
The signal transducer and activator of transcription (STAT) 5b is a universal transcription factor that plays key biological roles in allergic diseases, immunodeficiencies, autoimmunities, cancers, hematological diseases, growth disorders, and lung diseases. The identification of distinct pathological manifestations of STAT5b deficiency in humans has highlighted the critical role of the STAT5b pathway. Proper gene transcription at IL-2R ?, FOXP3, Bcl-2, and growth hormone (GH) associated loci are thought to be associated with normal STAT5b transcriptional activity. These genes are thought to play important roles in allergy/autoimmunity, immunodeficiency, cancer/anemia, and growth, respectively. The STAT5A and STAT5B genes are collocated on 17q11. Although these two monomeric proteins exhibit peptide sequence similarities of >90%, it is known through observations of STAT5b deficient subjects that STAT5a and STAT5b are not fully redundant in humans. Patients with STAT5b deficiency have decreased numbers of regulatory CD4(+)CD25(high) T cell (Treg) despite their STAT5a levels being normal. Prior studies on STAT5b deficient subjects have revealed immunological aberrations associated with the following disease phenotype: modest lymphopenia and decreased populations of Treg, ?-? T cells, and natural killer (NK) cells. Most subjects with STAT5b deficiency show severe eczema, and autoimmune disease (juvenile idiopathic arthritis, autoimmune thyroiditis, idiopathic thrombocytic purpura) which are thought to be associated with Treg dysfunction. We will review the likely pathophysiological mechanisms associated with STAT5b deficiency.
View details for DOI 10.3389/fimmu.2012.00234
View details for PubMedID 22912632
The Safety of Peanut Oral Immunotherapy in Peanut-Allergic Subjects in a Single-Center Trial
2012; 159 (2): 179-182
Peanut allergy is the leading cause of food-related anaphylaxis, and accidental exposures are common. Oral immunotherapy (OIT) has been posited as a potential treatment.Patients aged 3-65 years with peanut-specific IgE ?7 kU/l and/or a positive skin prick test with a history of an allergic reaction to peanut were recruited to undergo an OIT protocol. All adverse reactions were recorded by research staff or patients in real time.Twenty-four patients received 6,662 doses. Symptoms were mostly mild (84%), and only 3 severe gastrointestinal reactions required the administration of epinephrine. Abdominal pain was the most common reaction, followed by oropharyngeal and lip pruritus. Respiratory symptoms were rare.In this trial of OIT in adults and children, most reactions were mild.
View details for DOI 10.1159/000336391
View details for Web of Science ID 000305801300011
View details for PubMedID 22678151
Genotype, phenotype, and outcomes of nine patients with T-B plus NK plus SCID
2011; 15 (7): 733-741
There are few reports of clinical presentation, genotype, and HCT outcomes for patients with T-B+NK+ SCID. Between 1981 and 2007, eight of 84 patients with SCID who received and/or were followed after HCT at UCSF had the T-B+NK+ phenotype. One additional patient with T-B+NK+ SCID was identified as the sibling of a patient treated at UCSF. Chart reviews were performed. Molecular analyses of IL7R, IL2RG, JAK3, and the genes encoding the CD3 T-cell receptor components ? (CD3D), ? (CD3E), and ? (CD3Z) were carried out. IL7R mutations were documented in four patients and CD3D mutations in two others. Three patients had no defects found. Only two of nine patients had an HLA-matched related HCT donor. Both survived, and neither developed GVHD. Five of seven recipients of haploidentical grafts survived. Although the majority of reported cases of T-B+NK+ SCID are caused by defects in IL7R, CD3 complex defects were also found in this series and should be considered when evaluating patients with T-B+NK+ SCID. Additional genes, mutations in which account for T-B+NK+ SCID, remain to be found. Better approaches to early diagnosis and HCT treatment are needed for patients lacking an HLA-matched related donor.
View details for DOI 10.1111/j.1399-3046.2011.01563.x
View details for Web of Science ID 000296049000020
View details for PubMedID 21883749
T(H)1, T(H)2, and T(H)17 cells instruct monocytes to differentiate into specialized dendritic cell subsets
2011; 118 (12): 3311-3320
Monocytes and T helper (T(H)) cells rapidly infiltrate inflamed tissues where monocytes differentiate into inflammatory dendritic cells (DCs) through undefined mechanisms. Our studies indicate that T(H) cells frequently interact with monocytes in inflamed skin and elicit the differentiation of specialized DC subsets characteristic of these lesions. In psoriasis lesions, T(H)1 and T(H)17 cells interact with monocytes and instruct these cells to differentiate into T(H)1- and T(H)17-promoting DCs, respectively. Correspondingly, in acute atopic dermatitis, T(H)2 cells interact with monocytes and elicit the formation of T(H)2-promoting DCs. DC formation requires GM-CSF and cell contact, whereas T(H) subset specific cytokines dictate DC function and the expression of DC subset specific surface molecules. Moreover, the phenotypes of T cell-induced DC subsets are maintained after subsequent stimulation with a panel of TLR agonists, suggesting that T(H)-derived signals outweigh downstream TLR signals in their influence on DC function. These findings indicate that T(H) cells govern the formation and function of specialized DC subsets.
View details for DOI 10.1182/blood-2011-03-341065
View details for Web of Science ID 000295120900018
View details for PubMedID 21813450
Immunophenotyping of Peripheral Eosinophils Demonstrates Activation in Eosinophilic Esophagitis
2011; 53 (1): 40-47
Eosinophilic esophagitis (EoE) is a chronic inflammatory disorder characterized by upper gastrointestinal symptoms and the presence of high numbers of eosinophils in the esophagus. Although eosinophils in the esophagus have been found to be activated in subjects with EoE, detailed studies of intracellular signaling pathways involved in the mechanism of activation of eosinophils in EoE have heretofore been limited. The aim of the study was to assess whether any surface molecules or transcription factors are activated in peripheral eosinophils in subjects with EoE.Eosinophils and CD3+ lymphocytes were identified directly from 50 ?L of whole blood of EoE and control subjects. Using Hi-FACS, levels of surface activation markers, including CD66b, and intracellular phosphoepitopes, including phosphorylated forms of signal transducer and activator of transcription (phospho-STAT) 1 and 6, were measured within each cell subset.Levels of surface CD66b as well as levels of intracellular phospho-STAT1 and phospho-STAT6 in peripheral blood eosinophils were significantly higher for untreated subjects with EoE vs healthy controls (P < 0.05). Levels of phospho-STAT1 and phospho-STAT6 in peripheral blood eosinophils were lower in subjects with EoE on therapy versus untreated subjects with EoE (P < 0.05).Levels of phospho-STAT1 and phospho-STAT6, transcription factors involved in inflammatory processes, were both significantly higher in peripheral eosinophils from untreated (ie, newly diagnosed) subjects with EoE versus subjects with EoE on therapy, healthy controls. Blood-based measurements of CD66b and phospho-STAT levels in peripheral eosinophils may be beneficial for identifying EoE.
View details for DOI 10.1097/MPG.0b013e318212647a
View details for Web of Science ID 000291925500006
View details for PubMedID 21694534
Rapid oral desensitization in combination with omalizumab therapy in patients with cow's milk allergy
2011; 127 (6): 1622-1624
View details for DOI 10.1016/j.jaci.2011.04.009
View details for Web of Science ID 000291048500045
View details for PubMedID 21546071
Basophil CD203c Levels Are Increased at Baseline and Can Be Used to Monitor Omalizumab Treatment in Subjects with Nut Allergy
2011; 154 (4): 318-327
Basophils contribute to anaphylaxis and allergies. We examined the utility of assessing basophil-associated surface antigens (CD11b/CD63/CD123/CD203c/CD294) in characterizing and monitoring subjects with nut allergy.We used flow cytometry to analyze basophils at baseline (without any activation) and after ex vivo stimulation of whole blood by addition of nut or other allergens for 2, 10, and 30 min. We also evaluated whether basophil expression of CD11b/CD63/CD123/CD203c/CD294 was altered in subjects treated with anti-IgE monoclonal antibody (omalizumab) to reduce plasma levels of IgE.We demonstrate that basophil CD203c levels are increased at baseline in subjects with nut allergy compared to healthy controls (13 subjects in each group, p < 0.0001). Furthermore, we confirm that significantly increased expression of CD203c occurs on subject basophils when stimulated with the allergen to which the subject is sensitive and can be detected rapidly (10 min of stimulation, n = 11, p < 0.0008). In 5 subjects with severe peanut allergy, basophil CD203c expression following stimulation with peanut allergen was significantly decreased (p < 0.05) after 4 and 8 weeks of omalizumab treatment but returned toward pretreatment levels after treatment cessation.Subjects with nut allergy show an increase of basophil CD203c levels at baseline and following rapid ex vivo stimulation with nut allergen. Both can be reduced by omalizumab therapy. These results highlight the potential of using basophil CD203c levels for baseline diagnosis and therapeutic monitoring in subjects with nut allergy.
View details for DOI 10.1159/000321824
View details for Web of Science ID 000288529200007
View details for PubMedID 20975283
- STAT5b Deficiency: An Unsuspected Cause of Growth Failure, Immunodeficiency, and Severe Pulmonary Disease J Pediatr 2011; 158 (5): 701-8
Migration of regulatory T cells toward airway epithelial cells is impaired in chronic rhinosinusitis with nasal polyposis
2010; 137 (1): 111-121
The pathogenesis of chronic rhinosinusitis with nasal polyposis (CRSwNP) is still unclear. To evaluate the role of regulatory T cells (Treg) in the pathogenesis of nasal polyposis, we tested migration potential of Treg purified from subjects with CRSwNP, CRS without NP and controls. The nasal tissue expressions of FOXP3 were analyzed by means of RT-PCR and double immunohistochemistry. Chemotaxis assays were used to evaluate the migration potential of Treg onto bronchial epithelial cells and primary nasal epithelial cells, and toward chemokines. FOXP3(+)CD3(+) cells frequency and FOXP3 transcript expression in nasal tissue, and migration potentials of Treg toward airway epithelial cells and CCL1 were significantly lower in CRSwNP compared with other groups (P<0.05). These results indicate that migration potential of Treg is decreased in CRSwNP subjects, and this may be one of the reasons why tissue infiltration of Treg was decreased as seen in the immunohistochemistry of nasal polyps from CRSwNP subjects.
View details for DOI 10.1016/j.clim.2010.05.013
View details for Web of Science ID 000282204900013
View details for PubMedID 20598643
Individual Variation in the Germline Ig Gene Repertoire Inferred from Variable Region Gene Rearrangements
2010; 184 (12): 6986-6992
Individual variation in the Ig germline gene repertoire leads to individual differences in the combinatorial diversity of the Ab repertoire, but the study of such variation has been problematic. The application of high-throughput DNA sequencing to the study of rearranged Ig genes now makes this possible. The sequencing of thousands of VDJ rearrangements from an individual, either from genomic DNA or expressed mRNA, should allow their germline IGHV, IGHD, and IGHJ repertoires to be inferred. In addition, where previously mere glimpses of diversity could be gained from sequencing studies, new large data sets should allow the rearrangement frequency of different genes and alleles to be seen with clarity. We analyzed the DNA of 108,210 human IgH chain rearrangements from 12 individuals and determined their individual IGH genotypes. The number of reportedly functional IGHV genes and allelic variants ranged from 45 to 60, principally because of variable levels of gene heterozygosity, and included 14 previously unreported IGHV polymorphisms. New polymorphisms of the IGHD3-16 and IGHJ6 genes were also seen. At heterozygous loci, remarkably different rearrangement frequencies were seen for the various IGHV alleles, and these frequencies were consistent between individuals. The specific alleles that make up an individual's Ig genotype may therefore be critical in shaping the combinatorial repertoire. The extent of genotypic variation between individuals is highlighted by an individual with aplastic anemia who appears to lack six contiguous IGHD genes on both chromosomes. These deletions significantly alter the potential expressed IGH repertoire, and possibly immune function, in this individual.
View details for DOI 10.4049/jimmunol.1000445
View details for Web of Science ID 000278516700047
View details for PubMedID 20495067
Eotaxin and FGF enhance signaling through an extracellular signal-related kinase (ERK)-dependent pathway in the pathogenesis of Eosinophilic esophagitis.
Allergy, asthma, and clinical immunology : official journal of the Canadian Society of Allergy and Clinical Immunology
2010; 6 (1): 25-?
Eosinophilic esophagitis (EoE) is characterized by the inflammation of the esophagus and the infiltration of eosinophils into the esophagus, leading to symptoms such as dysphagia and stricture formation. Systemic immune indicators like eotaxin and fibroblast growth factor were evaluated for possible synergistic pathological effects. Moreover, blood cells, local tissue, and plasma from EoE and control subjects were studied to determine if the localized disease was associated with a systemic effect that correlated with presence of EoE disease.Real-time polymerase chain reaction from peripheral blood mononuclear cells (PBMC), immunohistochemistry from local esophageal biopsies, fluid assays on plasma, and fluorescence-activated cell sorting on peripheral blood cells from subjects were used to study the systemic immune indicators in newly diagnosed EoE (n = 35), treated EoE (n = 9), Gastroesophageal reflux disease (GERD) (n = 8), ulcerative colitis (n = 5), Crohn's disease (n = 5), and healthy controls (n = 8).Of the transcripts tested for possible immune indicators, we found extracellular signal-regulated kinase (ERK), Bcl-2, bFGF (basic fibroblast growth factor), and eotaxin levels were highly upregulated in PBMC and associated with disease presence of EoE. Increased FGF detected by immunohistochemistry in esophageal tissues and in PBMC was correlated with low levels of pro-apoptotic factors (Fas, Caspase 8) in PBMC from EoE subjects. Plasma-derived bFGF was shown to be the most elevated and most specific in EoE subjects in comparison to healthy controls and disease control subjects.We describe for the first time a possible mechanism by which increased FGF is associated with inhibiting apoptosis in local esophageal tissues of EoE subjects as compared to controls. Eotaxin and FGF signaling pathways share activation through the ERK pathway; together, they could act to increase eosinophil activation and prolong the half-life of eosinophils in local tissues of the esophagus in EoE subjects.
View details for DOI 10.1186/1710-1492-6-25
View details for PubMedID 20815913
- Ambient Air Pollution Impairs Regulatory T-Cell Function in Asthma. J Allergy Clin Immunol 2010; 126 (4): 845-852
- Increased Number of Regulatory T Cells in Children with Eosinophilic Esophagitis. J Pediatr Gastroenterol Nutri 2010; 51 (3): 283-9
- Epoprostenol-Associated Pneumonitis: Diagnostic Use of a T Cell Proliferation Assay J Heart Lung Transplant 2010; 29 (9): 1071-5
- Increased HLA-DR Expression on Tissue Eosinophils in Eosinophilic Esophagitis. J Pediatr Gastroenterol Nutr 2010; 51 (3): 290-4
TSLP directly impairs pulmonary Treg function: association with aberrant tolerogenic immunity in asthmatic airway.
Allergy, asthma, and clinical immunology : official journal of the Canadian Society of Allergy and Clinical Immunology
2010; 6 (1): 4-?
Even though thymic stromal lymphopoietin (TSLP) has been implicated in the development of allergic inflammation, its influence on immune tolerance mediated by regulatory T cells (Treg) have not been explored. We aimed to dissect the influence of TSLP on immunosuppressive activities of Treg and its potential consequences in human allergic asthma.In vitro culture system was utilized to study the effects of TSLP on human Treg. The functional competency of pulmonary Treg from a cohort of 15 allergic asthmatic, 15 healthy control, and 15 non-allergic asthmatic subjects was also evaluated by suppression assays and flow cytometric analysis.Activated pulmonary Treg expressed TSLP-R and responded to TSLP-mediated activation of STAT5. TSLP directly and selectively impaired IL-10 production of Treg and inhibited their suppressive activity. In human allergic asthma, pulmonary Treg exhibited a significant decrease in suppressive activity and IL-10 production compared to healthy control and non-allergic asthmatic counterparts. These functional alterations were associated with elevated TSLP expression in bronchoalveolar lavage fluid (BAL) of allergic asthmatic subjects. Furthermore, allergic asthmatic BAL could suppress IL-10 production by healthy control pulmonary Treg in a TSLP-dependent manner.These results provide the first evidences for a direct role of TSLP in the regulation of suppressive activities of Treg. TSLP mediated inhibition of Treg function might present a novel pathologic mechanism to dampen tolerogenic immune responses in inflamed asthmatic airway.
View details for DOI 10.1186/1710-1492-6-4
View details for PubMedID 20230634
- Bradykinin Inhibitors in Hereditary Angioedema J Angioedema 2010; 1 (1): 27-30
Measurement and Clinical Monitoring of Human Lymphocyte Clonality by Massively Parallel V-D-J Pyrosequencing
2009; 1 (12)
The complex repertoire of immune receptors generated by B and T cells enables recognition of diverse threats to the host organism. In this work, we show that massively parallel DNA sequencing of rearranged immune receptor loci can provide direct detection and tracking of immune diversity and expanded clonal lymphocyte populations in physiological and pathological contexts. DNA was isolated from blood and tissue samples, a series of redundant primers was used to amplify diverse DNA rearrangements, and the resulting mixtures of barcoded amplicons were sequenced using long-read ultra deep sequencing. Individual DNA molecules were then characterized on the basis of DNA segments that had been joined to make a functional (or nonfunctional) immune effector. Current experimental designs can accommodate up to 150 samples in a single sequence run, with the depth of sequencing sufficient to identify stable and dynamic aspects of the immune repertoire in both normal and diseased circumstances. These data provide a high-resolution picture of immune spectra in normal individuals and in patients with hematological malignancies, illuminating, in the latter case, both the initial behavior of clonal tumor populations and the later suppression or re-emergence of such populations after treatment.
View details for DOI 10.1126/scitranslmed.3000540
View details for Web of Science ID 000277263200001
View details for PubMedID 20161664
Impaired IL-10-dependent Induction of Tolerogenic Dendritic Cells by CD4(+)CD25(hi)CD127(lo/-) Natural Regulatory T Cells in Human Allergic Asthma
2009; 180 (9): 823-833
Tolerogenic dendritic cells and natural regulatory T cells have been implicated in the process of infectious tolerance in human allergic asthma. However, the significance of the influence of natural regulatory T cells on tolerogenic dendritic cells in the disease has not been investigated.We aimed to characterize the mechanism of induction of the tolerogenic phenotype in circulating blood dendritic cells by allergic asthmatic natural regulatory T cells.The study was performed in a cohort of 21 subjects with allergic asthma, 21 healthy control subjects, and 21 subjects with nonallergic asthma. We cultured blood dendritic cells with natural regulatory T cells to study the induction of tolerogenic dendritic cells. Flow cytometry and proliferation assays were employed to analyze phenotype and function of dendritic cells as well as IL-10 production from natural regulatory T cells.Dendritic cells cultured with natural regulatory T cells up-regulated IL-10, down-regulated costimulatory molecules, and stimulated the proliferation of CD4(+)CD25(-) effector T cells less potently. Allergic asthmatic natural regulatory T cells were significantly less efficient in inducing this tolerogenic phenotype of dendritic cells compared with healthy control and nonallergic asthmatic counterparts. Furthermore, this defective function of natural regulatory T cells was associated with their decreased IL-10 expression, disease severity, and could be reversed by oral corticosteroid therapy.These results provided the first evidences of impaired induction of tolerogenic dendritic cells mediated by natural regulatory T cells in human allergic asthma.
View details for DOI 10.1164/rccm.200905-0761OC
View details for Web of Science ID 000271215500006
View details for PubMedID 19679691
Neonatal Alloimmune Thrombocytopenia and Neutropenia Associated With Maternal Human Leukocyte Antigen Antibodies
2009; 53 (1): 97-99
Neonatal thrombocytopenia or neutropenia may result from passive transfusion of maternally derived antibodies. Antibodies against platelet antigens are commonly associated with neonatal alloimmune thrombocytopenia (NAIT), and anti-neutrophil antibodies are frequently identified in alloimmune neonatal neutropenia (ANN). Combined alloimmune cytopenias in the newborn are rarely reported; even fewer reports document human leukocyte antigen (HLA) antibodies as a potential cause of neonatal thrombocytopenia or neutropenia. We describe neutropenia and thrombocytopenia in a newborn associated with markedly elevated maternal HLA antibodies in the absence of anti-neutrophil or anti-platelet antibodies to highlight consideration of HLA antibodies in the pathogenesis of ANN and NAIT.
View details for DOI 10.1002/pbc.21979
View details for Web of Science ID 000266186200021
View details for PubMedID 19229975
Regulatory T cell dysfunction in subjects with common variable immunodeficiency complicated by autoimmune disease
2009; 131 (2): 240-253
Approximately 25% of subjects with common variable immunodeficiency (CVID) develop autoimmune disease. We analyzed T cell subsets, specifically regulatory T cells along with B cell subsets to determine whether there were changes in regulatory T cells which would correlate with the autoimmune disease clinical phenotype in CVID subjects. We hypothesized that regulatory T cell (CD4+CD25hiCD127lo) suppressive function would be impaired in CVID subjects with autoimmune disease. Using purified, sorted Treg from CVID subjects (n=14) and from healthy controls (HC, n=5) in standard suppression assays, we found the suppressive function of Treg from CVID subjects with autoimmune disease (CVID w/ AI, n=8) to be significantly attenuated compared to CVID subjects with no autoimmune disease (CVID w/o AI, n=6) and to HC (n=5). A number of proteins associated with Treg function were decreased in expression as detected through immunofluorescent antibody via flow cytometry (mean fluorescence intensity (MFI) of FoxP3, Granzyme A, XCL1, pSTAT5, and GITR in Treg was significantly lower (by up to 3 fold) in CVID w/ AI compared to CVID w/o AI and HC. Furthermore, a statistically significant correlation was found between intracellular MFI of FoxP3, Granzyme A, and pSTAT5 in Treg and the degree of Treg dysfunction. These results suggest that attenuation of Treg function is associated with autoimmune disease in CVID subjects and may contribute to autoimmune pathogenesis.
View details for DOI 10.1016/j.clim.2008.12.006
View details for Web of Science ID 000265420000007
View details for PubMedID 19162554
Selective deregulation in chemokine signaling pathways of CD4(+)CD25(hi)CD127(lo/-) regulatory T cells in human allergic asthma
2009; 123 (4): 933-939
CD4+CD25(hi)CD127(lo)/(-) regulatory T cells have been suggested to be critical regulators of inflammatory processes in allergic asthma. Recent studies reported a selective decrease in the frequency of regulatory T cells in the bronchoalveolar lavage fluid of allergic asthmatic (AA) subjects, prompting the possibility of defective recruitment of these cells to the airway in response to chemokines produced during asthmatic inflammation.This study aimed to characterize the chemotactic profile of circulating regulatory T cells in AA subjects in response to chemokines abundantly produced in airway inflammation, such as CCL1, CCL17, and CCL22.The study was performed in a cohort of 26 AA, 16 healthy control, and 16 non-AA subjects. We used chemotaxis assays to evaluate cell migration, flow cytometry to examine chemokine receptor expression, and phospho-ELISA to study consequent signaling pathways in regulatory T cells.Regulatory T cells, but not CD4+CD25(-)T cells, from AA subjects showed decreased chemotactic responses, specifically to CCL1, in comparison with their healthy control and non-AA counterparts. Decreased CCL1-mediated chemotaxis in AA regulatory T cells was associated with decreased phosphorylation of protein kinase B (AKT), a protein involved in chemokine intracellular signaling. Furthermore, the decreased chemotactic response to CCL1 in AA regulatory T cells significantly correlated with asthma severity and decreased pulmonary function in AA subjects.These results provide the first evidence of dysfunction in the chemokine signaling pathway in AA regulatory T cells.
View details for DOI 10.1016/j.jaci.2008.11.037
View details for Web of Science ID 000265058600023
View details for PubMedID 19152963
- T Lymphocytes and Its Subsets in Transplanted Small Bowel Treated with Alemtuzumab (Campath-1H) Proc XI Int Small Bowel Transplant Sym 2009; Dec: 37-42
Idiopathic neutropenia of childhood is associated with Fas/FasL expression
2008; 129 (3): 438-447
Idiopathic neutropenia (IN) in children is characterized by decreased neutrophil counts (<1500/microl), can be acute or chronic (greater than 6 months duration). The pathophysiology is not well understood; therefore, potential mechanisms of pediatric IN were investigated. An increase in Fas transcripts in neutrophils of IN patients compared to age-matched healthy control (HC) neutrophils was observed (p<0.005). Increased expression of Fas protein was found in IN neutrophils, while Fas surface expression on other immune cells was similar. Plasma from acute IN patients had higher protein levels of soluble FasL than chronic IN patients. When HC neutrophils were incubated in plasma from IN patients, greater rates of apoptosis were observed. Biochemical studies suggest the apoptotic factor(s) in plasma is heat-sensitive, non-IgG, and 12-50 kD protein. Addition of anti-sFasL blocking antibodies to patient plasma caused a statistically significant decrease in neutrophil apoptosis. These studies show that the Fas/FasL pathway could be associated with neutrophil apoptosis in childhood IN.
View details for DOI 10.1016/j.clim.2008.08.006
View details for Web of Science ID 000261011100007
View details for PubMedID 18819843
XCL1 enhances regulatory activities of CD4(+)CD25(high)CD127(low/-) T cells in human allergic asthma
2008; 181 (8): 5386-5395
Chemokine-mediated recruitment of regulatory cell subsets to the airway during inflammation and enhancement of their activities are potential strategies for therapeutic development in allergic asthma (AA). In this study, we aim to explore the role of XCL1, a chemokine associated with immune suppression and allergy, on CD4(+)CD25(high)CD127(low/-) regulatory T cell (Treg) function in AA. Flow cytometry and PCR analysis showed a reduction in XCL1 and XCR1 expression in AA Treg compared with healthy control and nonallergic asthmatic counterparts. This reduction in XCL1 expression was associated with the suboptimal regulatory function of Treg in AA. Interestingly, incubation with recombinant human XCL1 significantly increased Treg-mediated suppression and cytotoxicity by up-regulating expression of XCL1 and chief effector molecules of Treg function. Altogether, these results suggest an association between dysregulated XCL1 expression and reduced Treg activities in AA, as well as a potential role of XCL1 in reversing defective Treg function in the disease.
View details for Web of Science ID 000260025300030
View details for PubMedID 18832695
Increased cytotoxicity of CD4(+) invariant NKT cells against CD4(+)CD25(hi)CD127(lo/-) regulatory T cells in allergic asthma
2008; 38 (7): 2034-2045
CD4+CD25(hi)CD127(lo/-) regulatory T cells (Treg) have been implicated in the resolution of asthma-associated inflammation while the opposite role of CD4+ invariant NKT (iNKT) cells has been the subject of recent investigations. Studies here focused on mechanisms of interaction between CD4+ iNKT cells and Treg to further explore their roles in allergic asthma (AA). Flow cytometry analysis revealed a significant increase in the expression of the natural cytotoxicity receptors NKp30 and NKp46 by CD4+ iNKT cells in AA subjects compared to healthy controls (HC) and non-allergic asthmatics (NA). Subsequent intracellular staining showed that CD4+ iNKT cells also expressed higher levels of granzyme B and perforin in AA than HC. In in vitro killing assays, AA CD4+ iNKT cells selectively killed autologous Treg, but not CD4+CD25- T cells, more potently than HC and NA counterparts. This increased cytotoxicity positively correlated with asthma severity and granzyme B/perforin expression of CD4+ iNKT cells. Furthermore, it could be abrogated by either inhibition of the granzyme B-/perforin-dependent cell death pathway or oral corticosteroid administration. Altogether, these findings suggest that increased cytotoxicity of CD4+ iNKT cells against Treg might contribute to dysfunctional cellular interactions in AA.
View details for DOI 10.1002/eji.200738082
View details for Web of Science ID 000257826300027
View details for PubMedID 18581330
A novel mutation associated with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy
2008; 100 (2): 169-169
View details for Web of Science ID 000253136500012
View details for PubMedID 18320920
Altered phosphorylated signal transducer and activator of transcription profile of CD4(+)CD161(+) T cells in asthma: Modulation by allergic status and oral corticosteroids
2007; 120 (6): 1441-1448
Asthma is a complex immunologic disorder linked to altered cytokine signaling.We tested whether asthmatic patients showed any change in cytokine-dependent signal transducer and activator of transcription (STAT) levels, focusing on the central/effector-memory CD4(+)CD161(+) subset, which represents 15% to 25% of circulating T cells.We quantified intracellular levels of active phosphorylated STAT (phospho-STAT) 1, 3, 5, and 6 by means of flow cytometry, without any activation or expansion.Baseline phospho-STAT1 and phospho-STAT6 levels were increased in CD4(+)CD161(+) T cells from asthmatic patients compared with those from healthy control subjects (by 10- and 8-fold, respectively). This asthma-associated alteration was both subset specific because no change was seen in CD4(+)CD161(-)CD25(+) (regulatory T cells) and CD4(+)CD161(-)CD25(-) subsets and isoform specific because phospho-STAT5 and phospho-STAT3 levels were unchanged. Among asthmatic patients, phospho-STAT1 and phospho-STAT6 levels correlated negatively with each other, suggesting antagonistic regulation. Oral corticosteroid (OCS) treatment significantly decreased phospho-STAT6 and IL-4 levels but not phospho-STAT1 levels. Disease parameters showing significant correlations with phospho-STAT1, phospho-STAT6, or both included age at onset, plasma IgE levels, and levels of the T(H)2 cytokines IL-4 and IL-10 and the T(H)1 cytokine IL-2. Overall, combined phospho-STAT1 and phospho-STAT6 measurements showed excellent predictive value for identifying (1) asthmatic patients versus healthy control subjects, (2) allergic versus nonallergic asthmatic patients, and (3) asthmatic patients taking versus those not taking OCSs.Baseline changes in phospho-STAT1 and phospho-STAT6 levels in blood CD4(+)CD161(+) T cells identify asthmatic patients and mirror their allergic status and response to OCSs.These results confirm the pathologic importance of activated STAT1 and STAT6 in asthma and suggest their potential use as clinical biomarkers.
View details for DOI 10.1016/j.jaci.2007.08.012
View details for Web of Science ID 000251653800029
View details for PubMedID 17919711
Cutting edge: Decreased accumulation and regulatory function of CD4(+)CD25(high) T cells in human STAT 5b deficiency
2006; 177 (5): 2770-2774
We show that STAT5b is important for the in vivo accumulation of CD4+ CD25(high) T cells with regulatory cell function. A patient homozygous for a missense A630P STAT5b mutation displayed immune dysregulation and decreased numbers of CD4+ CD25(high) T cells. STAT5b(A630P/A630P) CD4+ CD25(high) T cells had low expression of forkhead box P3 and an impaired ability to suppress the proliferation of or to kill CD4+ CD25- T cells. Expression of CD25, a component of the high-affinity IL-2R, was also reduced in response to IL-2 or after in vitro propagation. The impact of the STAT5b mutation was selective in that IL-2-mediated up-regulation of the common gamma-chain cytokine receptor and perforin, and activation-induced expressions of CD154 and IFN-gamma were normal. These results indicate that STAT5b propagates an important IL-2-mediated signal for the in vivo accumulation of functional regulatory T cells.
View details for Web of Science ID 000240002800007
View details for PubMedID 16920911
Lgl1 is suppressed in oxygen toxicity animal models of bronchopulmonary dysplasia and normalizes during recovery in air
2006; 59 (3): 389-395
Bronchopulmonary dysplasia (BPD), a major cause of morbidity in premature infants, is characterized by arrest of lung growth and inhibited alveologenesis. We had earlier cloned late-gestation lung 1 (LGL1), a glucocorticoid (GC)-induced, developmentally regulated gene in lung mesenchyme, and showed that reduced levels of late-gestation lung 1 protein (lgl1) inhibit lung branching. Maximal fetal expression of LGL1 is concordant with the onset of alveolar septation, suggesting an additional role for lgl1 in alveologenesis. At postnatal d 7, during the period of maximal septation in postnatal rat lung, lgl1 concentrates at the tips of budding secondary alveolar septa. We studied two models of impaired postnatal alveologenesis generated by exposure of newborn rats to 60% O2 for 2 wk or 95% O2 for 1 wk. A profound decrease of lgl1 expression with oxygen exposure was observed in both animal models. Animals exposed to 95% O2 for 1 wk recovered in air over a 3-wk period, associated with normalization of lgl1 levels. Changes in lung levels of alpha-actin (a marker of myofibroblast differentiation associated with alveologenesis) and the mesenchymal marker vimentin were significant but less marked. Our findings support a role for lgl1 in postnatal lung development. We speculate that deficiency of lgl1 contributes to the arrested alveolar partitioning observed in BPD and that recovery is associated with normalization of lgl1 levels.
View details for DOI 10.1203/01.pdr.0000198819.81785.f1
View details for Web of Science ID 000235626700009
View details for PubMedID 16492977
Inhibition of CD40 ligand (CD154) in the treatment of factor VIII inhibitors.
2000; 85 (10): 35-39
The development of persistent, high titer inhibitors represents a serious complication of the treatment of patients with severe hemophilia A. Elimination of these inhibitory antibodies is usually attempted through repeated administration of high doses of factor VIII. Such regimens are costly, time-consuming and often fail when the inhibitor is of very high titer or of longstanding duration. A potential alternative approach to inhibit the production of antifactor VIII antibodies is blockade of the T-cell/B-cell collaboration that is required to generate humoral responses. One cognate receptor pair that is required for T-cell-dependent B-cell activation consists of CD40, which is expressed on B-lymphocytes and other antigen presenting cells, and CD40 ligand (CD40L, CD154), which is transiently expressed on activated T-cells. To determine whether blockade of the CD40-CD40L pathway can inhibit the production of anti-factor VIII antibodies, a clinical study has been designed in which patients with hemophilia A and a high titer inhibitor (> 10 BU) receive monthly exposures to factor VIII in the presence of a humanized mouse monoclonal antibody to human CD40L (hu5c8*). Subjects must be between the ages of 5 and 60 years old and be HIV seronegative. To date, three subjects have received at least three doses of hu5c8 at the initial protocol dose of 10 mg/kg. Preliminary results suggest that anti-CD40L inhibition may be effective in blocking anamnestic responses to factor VIII in some patients. It remains to be determined whether this effect will persist and whether patients may eventually become tolerant to factor VIII in the absence of hu5c8 co-administration.
View details for PubMedID 11187868
Renal allograft protection with losartan in Fisher -> Lewis rats: Hemodynamics, macrophages, and cytokines
2000; 57 (6): 2618-2625
We sought to assess the effects of angiotensin receptor blockade on glomerular hypertension, macrophage recruitment, and cytokine expression, all of which contribute to the development of chronic graft injury in this model.The effects of treatment with the specific angiotensin II type 1 (AT1) receptor antagonist, losartan, were assessed over 24 weeks in F344-->LEW rats (LOS, N = 9) versus vehicle-treated F344-->LEW controls (CON, N = 9).UprotV rose progressively in CON (from 7.0 +/- 2.9 to 41 +/- 17 mg/day at 24 wk) but remained at baseline in LOS (4.2 +/- 0.6 to 9.4 +/- 1.3 mg/day, P < 0.05 vs. CON). Glomerular capillary pressure (PGC) was increased in CON (71 +/- 1 mm Hg at week 20), but remained within the normal range in LOS rats (54 +/- 2 mm Hg, P < 0.05). Glomerulosclerosis averaged 0.3 +/- 0.2% in LOS versus 4 +/- 2% in CON rats (P < 0.05). Tubulointerstitial injury was minimal in both LOS and CON rats (+). The overexpression of renal cortical cytokine mRNA levels for the monocyte chemoattractants, monocyte chemoattractant protein-1 (MCP-1) and RANTES, as well as interleukin-1, inducible nitric oxide synthase, and transforming growth factor-beta, assessed by competitive reverse transcription-polymerase chain reaction, was suppressed in LOS versus CON rats at 20 weeks. Macrophage and T-cell numbers were decreased, and MCP-1, RANTES, and intercellular adhesion molecule-1 staining in the graft, identified by immunohistochemistry, were attenuated in LOS versus CON rats.The renoprotective effects of losartan in F344-->LEW rats were associated with lowered PGC, inhibition of macrophage chemoattractants and recruitment, and suppression of macrophage-associated cytokines at 20 weeks. These findings suggest that chronic allograft injury in F344-->LEW rats is, to a large extent, mediated by angiotensin II-dependent mechanisms and that these involve glomerular hemodynamics, macrophages, and macrophage-associated cytokines.
View details for Web of Science ID 000087346100044
View details for PubMedID 10844632
Cloning and characterization of the two enzymes responsible for trypanothione biosynthesis in Crithidia fasciculata
1998; 273 (31): 19383-19390
Protozoa of the order Kinetoplastida differ from other organisms in their ability to conjugate glutathione (gamma-Glu-Cys-Gly) and spermidine to form trypanothione (N1,N8-bis(glutathionyl)spermidine), which is involved in maintaining intracellular thiol redox and in defense against oxidants. In this study, the genes from Crithidia fasciculata, Cf-GSS and Cf-TRS, which encode, respectively, glutathionylspermidine synthetase (EC 18.104.22.168) and trypanothione synthetase (EC 22.214.171.124) have been cloned and expressed. The deduced amino acid sequence of both Cf-GSS and Cf-TRS share 50% sequence similarity with the Escherichia coli glutathionylspermidine synthetase/amidase. Both genes are present as single copies in the C. fasciculata genome. When expressed in E. coli and Saccharomyces cerevisiae, neither protein was present in an active soluble form. However, thiol analysis of S. cerevisiae demonstrated that cells transformed with the Cf-GSS gene contained substantial amounts of glutathionylspermidine, whereas cells expressing both the Cf-GSS and Cf-TRS genes contained glutathionylspermidine and trypanothione, confirming that these genes encode the functional glutathionylspermidine and trypanothione synthetases from C. fasciculata. The translation products of Cf-GSS and Cf-TRS show significant homology to the amidase domain present in E. coli glutathionylspermidine synthetase, which can catalyze both synthesis and degradation of glutathionylspermidine. Glutathionylspermidine synthetase isolated from C. fasciculata was found to possess a similar amidase activity.
View details for Web of Science ID 000075125200007
View details for PubMedID 9677355
Effects of explosive brain death on cytokine activation of peripheral organs in the rat
1998; 65 (12): 1533-1542
The success rate of transplanted organs from brain-dead cadaver donors is consistently inferior to that of living sources. As cadaver and living unrelated donors are equally genetically disparate with a given recipient, the difference must lie within the donor himself and/or the effects of organ preservation and storage. We have hypothesized that irreversible central nervous system injury may up-regulate proinflammatory mediators and cell surface molecules in peripheral organs to be engrafted, making them more prone to host inflammatory and immunological responses.Rats undergoing surgically induced acutely increased intracranial pressure (explosive brain death) were followed for 6 hr. Their peripheral tissues were examined by reverse transcriptase polymerase chain reaction and immunohistology, serum factors were assessed by enzyme-linked immunosorbent assay, and the influence of inflammatory molecules in the blood stream was determined by cross-circulation experiments with normal animals.mRNA expression of both lymphocyte- and macrophage-associated products increased dramatically in all tissues. Similar factors in serum were coincidentally increased; these were shown to be active in vivo by cross-circulation with normal animals. The organs of all control groups, including animals with important ischemic injury and with hemorrhagic shock, were negative. Up-regulation of MHC class I and II antigens and the co-stimulatory molecule B7 suggests increased immunogenicity of the peripheral organs. These changes could be inhibited by: (i) administration of a recombinant soluble P-selectin glycoprotein ligand-Ig, a P- and E-selectin antagonist; and (ii) a fusion protein, cytotoxic T lymphocyte antigen 4-Ig, which blocks B7-mediated T-cell co-stimulation.Activation of peripheral organs following explosive brain death may be caused by various interrelated events, including the effects of massive acute central injury, hypotension, and circulating factors. Almost complete suppression of these changes could be produced by biological agents. Such interventions, if reproducible in humans, could improve the quality of organs from "marginal" donors, broadening the criteria for donor acceptance.
View details for Web of Science ID 000074557600001
View details for PubMedID 9665067
Late blockade of T cell costimulation interrupts progression of experimental chronic allograft rejection
1998; 101 (11): 2309-2318
Early blockade of T cell-costimulatory activation pathways prevents development of experimental chronic allograft rejection. Ongoing T cell recognition of alloantigen and activation may also play an important role in progression of chronic rejection, but definitive evidence is lacking. We used the fusion protein CTLA4Ig to block CD28-B7 T cell costimulation late after the onset of initial graft injury. Using the F334 into LEW rat model of chronic renal allograft rejection, transplant recipients were treated with a 10-d course of cyclosporine, and a subgroup received a single injection of CTLA4Ig at 8 wk after transplant. Functionally, CTLA4Ig administration prevented development of progressive proteinuria (14.3+/-4.1 mg/24 h versus 41.0+/-12.0 mg/24 h at 24 wk after transplant, P < 0.05). Histologically, graft mononuclear cell infiltration, glomerular hypertrophy, focal and segmental glomerulosclerosis, and intimal vascular hyperplasia were all attenuated in CTLA4Ig-treated animals. Lastly, reverse transcriptase-PCR and immunohistologic studies showed a significant reduction in the intragraft expression of key products of T cell and macrophage activation, and upregulation of what have recently been termed as "protective" genes, including the bcl family members, Bcl-2 and Bcl-xL, and hemoxygenase. Our data are the first to demonstrate that blocking T cell-costimulatory activation late after transplantation, after initial graft injury, prevents progression of chronic allograft rejection supporting the hypothesis that ongoing T cell recognition of alloantigen and activation are key mediators of ongoing chronic allograft rejection.
View details for Web of Science ID 000074165900003
View details for PubMedID 9616202
Influence of initial antigen-independent events on acute allograft rejection: Inhibition by a soluble P-selectin ligand and low-dose cyclosporine in combination
1998; 30 (4): 1027-1028
View details for Web of Science ID 000074150800039
View details for PubMedID 9636414
Prevention of late renal changes after initial ischemia/reperfusion injury by blocking early selectin binding
1997; 64 (11): 1520-1525
Increasing clinical evidence suggests that delayed initial function secondary to ischemia/reperfusion injury alone, and particularly in combination with early episodes of acute rejection, reduces kidney allograft survival over time.We investigated changes developing over the long term following a standardized ischemia/reperfusion insult in a Lewis rat model. The left kidney was isolated in a uninephrectomized host and cooled, and the pedicle was clamped for 45 min. Animals were followed for 48 weeks after initial renal injury. Organs were removed serially (4, 8, 16, 24, 32, 40, and 48 weeks) for immunohistology and reverse transcriptase polymerase chain reaction.Progressive proteinuria developed after 8 weeks. By immunohistology, CD4+ leukocytes and ED-1+ macrophages infiltrated the ischemic organs in parallel with up-regulation of major histocompatibility complex class II antigen expression. Because macrophages have been shown to be critical in chronic changes in other models, they were examined primarily in these studies. By reverse transcriptase polymerase chain reaction, macrophage-derived, fibrosis-inducing factors (transforming growth factor-beta, interleukin 6, and tumor necrosis factor-alpha) remained highly and constantly expressed throughout the follow-up period. The long-term influence of initial treatment with the soluble form of P-selectin glycoprotein ligand-1, a soluble ligand for P- and E-selectin, was then examined. All functional and structural changes remained at relative baseline, similar to uninephrectomized controls.These data suggest that blocking the initial selectin-mediated step after ischemia/reperfusion injury, which triggers significant early cellular and molecular events, also reduces later renal dysfunction and tissue damage over time. In part, the findings may be explained by the sparing of functioning nephron units, which if destroyed or compromised by the original insult, may contribute to long-term graft failure. This approach may be important clinically in the transplantation of kidneys from non-heart-beating or marginal donors or organs experiencing prolonged ischemic times.
View details for Web of Science ID 000071034100003
View details for PubMedID 9415550
Infection-associated macrophage activation accelerates chronic renal allograft rejection in rats
1997; 64 (11): 1602-1605
The influence of infection-associated cellular activation on chronic rejection of kidney grafts was assessed in an established rat model by administration of lipopolysaccharide (LPS), an endotoxin and a potent stimulator of various cell populations including mononuclear cells and renal epithelial cells.Lewis recipients of F344 kidneys were treated with low-dose cyclosporine (1.5 mg/kg/day x 10 days). Animals with well-functioning grafts received a single dose of LPS (2 mg in 1 ml of NaCl, intraperitoneally) at 4 or 8 weeks after engraftment. Untreated control rats, which later experienced chronic rejection, were given 1 ml of NaCl.Administration of LPS during the early quiescent phase of chronic rejection accelerated the chronic process, functionally (proteinuria), morphologically, immunohistologically, and by reverse-transcriptase polymerase chain reaction as compared with untreated controls. Infiltration of macrophages and their associated factors was especially affected.As the later events of chronic rejection seem to be mediated primarily by macrophages and their products, administration of LPS accelerated the tempo and activity of these cells in the development of chronic rejection. These findings may explain the clinical observation that infection may be an important risk factor for chronic allograft rejection.
View details for Web of Science ID 000071034100018
View details for PubMedID 9415565
CD28-B7 blockade in organ dysfunction secondary to cold ischemia/reperfusion injury - Rapid Communication
1997; 52 (6): 1678-1684
Ischemic injury to cadaver organs is a major risk factor for development of chronic organ dysfunction. We have recently shown that the B7 costimulatory pathway plays a critical role in early organ dysfunction developing after renal cold ischemia/reperfusion injury. We extended these observations to investigate the role of this pathway in the development and progression of chronic organ dysfunction following such injury. Uninephrectomized rats which underwent cold ischemia/reperfusion injury developed progressive proteinuria as compared to uninephrectomized controls. Animals treated with CTLA4Ig, which blocks B7 costimulation, starting on the day of injury had significantly better long-term survival and developed significantly less proteinuria than control animals treated with control Ig. RT-PCR analysis of kidney tissue showed significant reduction in expression of activation and inflammatory cytokines, chemoattractants, and growth factors, as compared to controls. Delaying administration of CTLA4Ig for one week, but not four weeks, after injury was still effective in ameliorating development of progressive proteinuria. Interestingly, selective blockade of B7-1 by a mutant form of CTLA4Ig had no effect on early or chronic organ dysfunction. These findings indicate the long-term functional and molecular consequences of experimental cold ischemia/reperfusion injury, and suggest that B7-2 is critical in the development of organ dysfunction following ischemic injury, even in the absence of alloantigen.
View details for Web of Science ID A1997YH57300028
View details for PubMedID 9407517
The role of the B7 costimulatory pathway in experimental cold ischemia/reperfusion injury
1997; 100 (5): 1199-1203
Ischemia/reperfusion injury associated with organ retrieval and storage influences the development of chronic graft dysfunction, the major clinical problem in solid organ transplantation. The potential role of mononuclear cells (T cells and monocyte/macrophages) in this type of injury is unknown. Inbred male Lewis rats were uninephrectomized and the left kidney perfused in situ with 10 ml of iced University of Wisconsin solution. Immunohistological studies showed mononuclear cell infiltration of the ischemic organs associated with the upregulation of MHC class II antigen expression. Reverse transcriptase-PCR indicated that T cell associated cytokines and monocyte/macrophage activation markers/products are upregulated early after the ischemic insult. B7 expression occurred within 24 h and peaked at 3 d. Plasma creatinine levels rose transiently with complete recovery of renal function by 5 d. Animals began to develop progressive proteinuria after 8-12 wk, indicative of the long-term functional consequences of early ischemia/reperfusion injury. Blockade of T cell CD28-B7 costimulation with CTLA4Ig resulted in significant inhibition of T cell and macrophage infiltration and activation in situ. Treated animals did not exhibit transient renal dysfunction, nor developed proteinuria over time. This is the first demonstration that blocking T cell costimulatory activation in the absence of alloantigen can prevent the early and late consequences of ischemia/reperfusion injury.
View details for Web of Science ID A1997XV75300029
View details for PubMedID 9276737
Cellular and molecular predictors of chronic renal dysfunction after initial ischemia/reperfusion injury of a single kidney
1997; 64 (2): 190-197
Initial ischemia/reperfusion injury occurring secondary to organ retrieval, storage, and transplantation has been associated with late renal allograft deterioration and failure. In addition, there is an apparent synergy, reported in several clinical series, between the initial injuries of ischemia/reperfusion and acute rejection; the long-term results of graft survival are significantly deceased after both events in combination as compared with either alone or if no such episodes occur.In the present study, we examined patterns of proteinuria, cellular infiltration, cytokine expression, and glomerulosclerosis over time in Lewis and Fischer 344 rats after 45 min of warm ischemia of a single kidney and with or without contralateral nephrectomy. Both early (4 hr to 7 days) and late (2-52 weeks) events were studied serially in the affected kidneys morphologically, by immunohistology and by reverse transcriptase polymerase chain reaction.Intercellular adhesion molecule 1, endothelin, and major histocompatibility complex class II expression were up-regulated within 2 to 5 days after injury; T cells and macrophages increased transiently. Proteinuria developed after approximately 8 weeks only in animals bearing a single injured kidney, and not in those with a retained native organ. Progressive morphological changes occurred after 16 weeks, including glomerulosclerosis, arterial obliteration, and interstitial fibrosis. After a period of relative quiescence, expression of intercellular adhesion molecule 1 again increased in relation to progressive macrophage infiltration and their associated products, particularly, interleukin 1, tumor necrosis factor-alpha, transforming growth factor-beta, and inducible nitric oxide synthase. Monocyte chemotactic protein 1 was intensely up-regulated by 24 weeks, coincident with a dramatic rise in this infiltrating population. These changes remained virtually at baseline in animals with a retained native kidney.These data imply that chronic injury after significant initial ischemia and reperfusion occurs when there is already a 50% renal mass reduction, but not when two kidneys remain in place. Permanent nephron loss resulting from such an insult could account for this phenomenon. Early ischemia and reperfusion, if severe enough in a single kidney, may be an important antigen-independent risk factor for later renal deterioration and failure. In the context of a renal allograft, it may contribute to chronic rejection.
View details for Web of Science ID A1997XN86900002
View details for PubMedID 9256172
The cytokine-adhesion molecule cascade in ischemia/reperfusion injury of the rat kidney - Inhibition by a soluble P-selectin ligand
1997; 99 (11): 2682-2690
Ischemia/reperfusion (I/R) injury associated with renal transplantation may influence both early graft function and late changes. The initial (= 7 d) events of warm and in situ perfused cold ischemia of native kidneys in uninephrectomized rats were examined. mRNA expression of the early adhesion molecule, E-selectin, peaked within 6 h; PMNs infiltrated in parallel. T cells and macrophages entered the injured kidney by 2-5 d; the associated upregulation of MHC class II antigen expression suggested increased immunogenicity of the organ. Th1 products (IL-2, TNFalpha, IFNgamma) and macrophage-associated products (IL-1, IL-6, TGFbeta) remained highly expressed after 2 d. To examine directly the effects of selectins in I/R injury, a soluble P-selectin glycoprotein ligand (sPSGL) was used. Ischemic kidneys were perfused in situ with 5 microg of sPSGL in UW solution; 50 microg was administered intravenously 3 h after reperfusion. E-selectin mRNA remained at baseline, leukocytes did not infiltrate the injured organs throughout the 7-d period, and their associated products were markedly inhibited. Class II expression did not increase. No renal dysfunction secondary to I/R occurred. The early changes of I/R injury may be prevented by treatment with soluble P- and E-selectin ligand. This may reduce subsequent host inflammatory responses after transplantation.
View details for Web of Science ID A1997XD35700018
View details for PubMedID 9169498
Sequential cellular and molecular kinetics in acutely rejecting renal allografts in rats
1997; 63 (8): 1101-1108
The initial (0-24 hr), early (3-5 days), and late (7-14 days) events occurring in LBNF1 renal allografts transplanted into Lew recipients were examined to define precisely the sequential cellular and molecular kinetics during acute rejection. Grafts and spleens were harvested at 3, 6, 12, and 24 hr, and at 3, 5, 7, and 14 days and processed for morphology, immunohistology, and reverse transcriptase-polymerase chain reaction. Various factors (mRNA) were up-regulated sequentially in the allografts over time. In the initial phase, E-selectin and complement (C1 and C3) expression was noted within 6 hr, peaking by 24 hr. RANTES (regulated upon activation, normal T cell expressed and secreted) increased within 6 hr, and then again between 3 and 6 days. By immunohistology, MHC class II was up-regulated consistently after day 1. Intercellular adhesion molecule-1 expression increased after day 3; lymphocyte function-associated antigen-1+ infiltrating leukocytes peaked at day 5. Infiltrating CD8+ T lymphocytes increased strikingly between days 1 and 3, peaking at day 5; CD4+ cells infiltrated more slowly until day 5. The kinetics of ED1+ macrophages were similar to those of lymphocyte function-associated antigen-1+ cells. The CD4+ T cell-derived product, interleukin (IL)-2, peaked at 7 days. Interferon-gamma increased progressively up to 14 days. By 3 days, the macrophage-associated factor, transforming growth factor-beta, peaked; this was followed by increased IL-6 expression by day 5. IL-1, tumor necrosis factor-alpha, and inducible nitric oxide synthase increased slowly until day 7, declining thereafter. Endothelin increased progressively over the 14-day follow-up period. Cytokine dynamics occurring in host spleen were similar to those noted in the allografts. Although acute rejection is primarily T cell mediated, adhesion molecules, macrophages, and their associated products may influence initial and later changes. The brisk expression of complement, E-selectin, and RANTES within the first few hours after engraftment may occur secondary to ischemic injury and trigger subsequent immunological events. Macrophages and their products may play a larger role in the process than hitherto appreciated.
View details for Web of Science ID A1997WW66000009
View details for PubMedID 9133471
Unaccompanied children in detention in the US
1997; 99 (4): 653-653
View details for Web of Science ID A1997WQ80200052
View details for PubMedID 9093327
Immigrating unaccompanied miners - A neglected minority?
1997; 166 (3): 221-221
View details for Web of Science ID A1997WW66700014
View details for PubMedID 9143205
Nephron mass modulates the hemodynamic, cellular, and molecular response of the rat renal allograft
1997; 63 (4): 519-528
Functioning nephron mass has recently been implicated as a risk factor for development of chronic "rejection" of kidney allografts. Reductions in nephron number below 50% may induce glomerular hypertension and hyperfiltration in surviving units, which in turn lead to graft injury. In the present study, which extends and amplifies our previous investigations, cellular and molecular characteristics of single allografts from F344 donors in bilaterally nephrectomized LEW recipients, our standard experimental model of chronic renal allograft dysfunction, were compared with allografts from recipients where total renal mass was reduced (by ligating branches of the graft renal artery) or restored to normal levels by transplanting or retaining a second kidney. Our findings in this study confirm that progressive proteinuria and structural injury in recipients of single allografts were accentuated in grafts with reduced mass but virtually absent in rats with increased kidney mass. A striking observation was that patterns of cell surface molecule expression, cellular infiltration, and expression of all T cell- and macrophage-associated products studied were all markedly modulated by changes in renal mass. Moreover, several molecules that are up-regulated before evidence of graft injury are down-regulated by providing increased renal mass. These data show that the quantity of functioning renal mass is not only an important independent determinant of the tempo and intensity of chronic renal allograft failure, but also a potent modulator of fundamental cellular and molecular components of a complex process. This phenomenon involves antigen-dependent and antigen-independent elements that ultimately result in chronic allograft failure.
View details for Web of Science ID A1997WL85500006
View details for PubMedID 9047144
Sequential cytokine expression in renal allografts in rats immunosuppressed with maintenance cyclosporine or mycophenolate mofetil
1996; 62 (9): 1363-1366
Although the immunosuppressive agents used clinically modulate acute rejection of organ allografts, their ability to prevent chronic rejection has been less clear. To ascertain the effects of prolonged maintenance treatment with cyclosporine (CsA) and mycophenolate mofetil, we examined sequential patterns of cytokine regulation by reverse transcriptase polymerase chain reaction in long-surviving renal allografts in treated recipients. In renal allografts in animals on long-term CsA therapy, there is important up-regulation of transforming growth factor-beta, Hsp70, and endothelin as compared with control animals. Conversely, interleukin-2 receptor, interferon-gamma, and tumor necrosis factor-alpha in kidney grafts in this group were expressed at lower levels compared with those noted in chronically rejecting grafts in control animals that had received only CsA for 10 days after transplantation. Morphologically, the long-term CsA-treated kidneys had more extensive arterial obliterative changes and glomerulosclerosis after 24 weeks than control organs; these changes can presumably be attributed to the nephrotoxic effects of this drug combined with the progressive changes of chronic rejection. In contrast, mycophenolate mofetil inhibited the production of all lymphocyte and macrophage-derived cytokines throughout the entire follow-up period. Allograft kidneys in these latter recipients showed no late morphological abnormalities. This agent may be important clinically in preventing chronic rejection.
View details for Web of Science ID A1996VU11700034
View details for PubMedID 8932288
Blockade of T-cell costimulation prevents development of experimental chronic renal allograft rejection
1996; 93 (22): 12439-12444
Blocking CD28-B7 T-cell costimulation by systemic administration of CTLA4Ig, a fusion protein which binds B7 molecules on the surface of antigen-presenting cells, prevents rejection and induces tolerance in experimental acute allograft rejection models. We tested the effect of CTLA4Ig therapy on the process of chronic renal allograft rejection using an established experimental transplantation model. F344 kidneys were transplanted orthotopically into bilaterally nephrectomized LEW recipients. Control animals received low dose cyclosporine for 10 days posttransplantation. Administration of a single injection of CTLA4Ig on day 2 posttransplant alone or in addition to the low dose cyclosporine protocol resulted in improvement of long-term graft survival as compared with controls. More importantly, control recipients which received cyclosporine only developed progressive proteinuria by 8-12 weeks, and morphological evidence of chronic rejection by 16-24 weeks, including widespread transplant arteriosclerosis and focal and segmental glomerulosclerosis, while animals treated with CTLA4Ig alone or in addition to cyclosporine did not. Competitive reverse transcriptase-PCR and immunohistological analysis of allografts at 8, 16, and 24 weeks showed attenuation of lymphocyte and macrophage infiltration and activation in the CTLA4Ig-treated animals, as compared with cyclosporine-alone treated controls. These data confirm that early blockade of the CD28-B7 T-cell costimulatory pathway prevents later development and evolution of chronic renal allograft rejection. Our results indicate that T-cell recognition of alloantigen is a central event in initiating the process of chronic rejection, and that strategies targeted at blocking T-cell costimulation may prove to be a valuable clinical approach to preventing development of the process.
View details for Web of Science ID A1996VP93700071
View details for PubMedID 8901600
Inflammatory mediators, cells and their products in acute host alloresponsiveness.
Annals of transplantation
1996; 1 (3): 5-13
View details for PubMedID 9869913
1995; 92 (19): 8729-8733
Chronic rejection, the most important cause of long-term graft failure, is thought to result from both alloantigen-dependent and -independent factors. To examine these influences, cytokine dynamics were assessed by semiquantitative competitive reverse transcriptase-PCR and by immunohistology in an established rat model of chronic rejection lf renal allografts. Isograft controls develop morphologic and immunohistologic changes that are similar to renal allograft changes, although quantitatively less intense and at a delayed speed; these are thought to occur secondary to antigen-independent events. Sequential cytokine expression was determined throughout the process. During an early reversible allograft rejection episode, both T-cell associated [interleukin (IL) 2, IL-2 receptor, IL-4, and interferon gamma] and macrophage (IL-1 alpha, tumor necrosis factor alpha, and IL-6) products were up-regulated despite transient immunosuppression. RANTES (regulated upon activation, normal T-cell expressed and secreted) peaked at 2 weeks; intercellular adhesion molecule (ICAM-1) was maximally expressed at 6 weeks. Macrophage products such as monocyte chemoattractant protein (MCP-1) increased dramatically (to 10 times), presaging intense peak macrophage infiltration at 16 weeks. In contrast, in isografts, ICAM-1 peaked at 24 weeks. MCP-1 was maximally expressed at 52 weeks, commensurate with a progressive increase in infiltrating macrophages. Cytokine expression in the spleen of allograft and isograft recipients was insignificant. We conclude that chronic rejection of kidney allografts in rats is predominantly a local macrophage-dependent event with intense up-regulation of macrophage products such as MCP-1, IL-6, and inducible nitric oxide synthase. The cytokine expression in isografts emphasizes the contribution of antigen-independent events. The dynamics of RANTES expression between early and late phases of chronic rejection suggest a key role in mediating the events of the chronic process.
View details for Web of Science ID A1995RU75900040
View details for PubMedID 7568006
1994; 33 (9): 2561-2567
Deoxyspergualin (DSG), a spermidinyl, alpha-hydroxyglycyl, 7-guanidinoheptanoyl peptidomimetic, shows immunosuppressive activity. In confirmation of a recent report that immobilized methoxyDSG selectively retains the heat shock protein Hsc70, we report here quantitative binding of DSG and analogs to both Hsc70 and the 90-kDa heat shock protein Hsp90. We have utilized affinity capillary electrophoresis to obtain Kd values for DSG and analogs, and stimulation of the ATPase activity of Hsc70 to obtain Km values for DSG, that are comparable and corroborative. Kd values are 4 microM for DSG binding to Hsc70 and 5 microM for DSG binding to Hsp90. Two active analogs, methoxy- and glycylDSG, bind with similar affinities. Glyoxylylspermidine and des(aminopropyl)DSG, two inactive metabolites, have much reduced affinity for Hsc70 and Hsp90. These data validate binding of these novel immunosuppressant agents to these molecular chaperones, at concentrations in the range of pharmacologically active doses, and indicate that further characterization of Hsc70 and/or Hsp90 as potential targets for DSG is warranted.
View details for Web of Science ID A1994MZ69700027
View details for PubMedID 8117717
1994; 269 (5): 3868-3874
An analysis of the genetic organization, regulated expression and biochemical properties of the cytoplasmic/nuclear (hsp70) and mitochondrial (mtp70) 70-kDa heat shock proteins of Trypanosoma cruzi is presented. The two proteins are encoded by tandemly arranged gene families that are located on different chromosomes. Both are mildly heat-inducible but have different optimal temperatures for expression. During the switch from proliferation to differentiation that occurs during the growth of T. cruzi in culture, the hsp70 level decreases dramatically while the mtp70 level falls only slightly. The subcellular locations of the two proteins differ during heat shock. While mtp70 remains associated with the kinetoplast at all temperatures, hsp70 becomes more concentrated in the nucleus at higher temperatures. Biochemical analysis of hsp70 and mtp70 revealed both to be potent ATPases. Each protein binds ATP with a Km of about 70 microM and hydrolyzes ATP with a kcat of about 100 min-1, 100 times greater than the kcat of human hsp70. The high ATPase activities of hsp70 and mtp70 are further stimulated by incubation with peptides, suggesting that these trypanosome heat shock proteins have protein chaperone activity. Finally, mtp70, but not hsp70, was found to possess autophosphorylation activity in vitro, a property that it shares with prokaryotic hsp70. These findings demonstrate unique cellular and biochemical characteristics of T. cruzi mtp70 and hsp70 that suggest that they play distinct physiologic roles in the biology of the cell.
View details for Web of Science ID A1994MV63100107
View details for PubMedID 8106432
1993; 268 (2): 1479-1487
Heat shock proteins of the 82-90 kDa class (hsp82 and hsp90) are abundant, conserved, and ubiquitous from prokaryotes to eukaryotes. Although proposed to be chaperones, they had not been reported to possess enzymatic activity until our recent observation that pure trypanosomatid hsp83 had potent ATPase activity (Nadeau, K., Sullivan, M., Engman, D., and Walsh, C. T. (1992) Protein Sci. 1, 970-979). We have now purified the hsp90 homolog from Escherichia coli (HtpG) and from Saccharomyces cerevisiae (hsp82) to homogeneity and observe ATPase activity with kcat values of 3 min-1 and 140 min-1. In addition, examinations of purified rat hsp90 and human hsp90 detect ATPase activity with a kcat of 0.6 min-1 and 10 min-1. Each of these hsp90s undergoes autophosphorylation on serine or threonine residues. In prokaryotes and eukaryotes, the induction of hsps during heat shock is controlled, respectively, by the binding of an alternate sigma 32 or a transcriptional activator (heat shock factor or HSF) at heat shock promoter elements. Here we show that E. coli HtpG immobilized to Affi-Gel beads selectively retains sigma 32 while the yeast hsp90 and rat hsp90 retain HSF. The peptidyl prolyl isomerase hsp59 of the FK506 binding class is known to bind to hsp90. We also detect binding of the other family of PPIases, the cyclophilins, to immobilized hsp90, consistent with a functional convergence of protein foldases.
View details for Web of Science ID A1993KG07700106
View details for PubMedID 8419347
1992; 1 (8): 970-979
A Crithidia fasciculata 83-kDa protein purified during a separate study of C. fasciculata trypanothione synthetase was shown to have ATPase activity and to belong to the hsp90 family of stress proteins. Because no ATPase activity has previously been reported for the hsp90 class, ATP utilization by C. fasciculata hsp83 was characterized: this hsp83 has an ATPase kcat of 150 min-1 and a Km of 60 microM, whereas the homologous mammalian hsp90 binds ATP but has no ATPase activity. Crithidia fasciculata hsp83 undergoes autophosphorylation on serine and threonine at a rate constant of 3.3 x 10(-3) min-1. Similar analysis was performed on recombinant Trypanosoma cruzi hsp83, and comparable ATPase parameters were obtained (kcat = 100 min-1, Km = 80 microM, kautophosphorylation = 6.3 x 10(-3) min-1). The phosphoenzyme is neither on the ATPase hydrolytic pathway nor does it affect ATPase catalytic efficiency. Both C. fasciculata and T. cruzi hsp83 show up to fivefold stimulation of ATPase activity by peptides of 6-24 amino acids.
View details for Web of Science ID A1992JM67800002
View details for PubMedID 1304385
1992; 31 (28): 6414-6420
African trypanosomes are devoid of glutathione reductase activity, and instead contain a unique flavoprotein variant, trypanothione reductase, which acts on a cyclic derivative of glutathione, trypanothione. The high degree of sequence similarity between trypanothione reductase and glutathione reductase, as well as the obvious similarity in the reactions catalyzed, led us to investigate the pH dependence of the kinetic parameters, and the isotopic behavior of trypanothione reductase. The pH dependence of the kinetic parameters V, V/K for NADH, and V/K for oxidized trypanothione has been determined for trypanothione reductase from Trypanosoma congolense. Both V/K for NADH and the maximum velocity decrease as single groups exhibiting pK values of 8.87 +/- 0.09 and 9.45 +/- 0.07, respectively, are deprotonated. V/K for oxidized trypanothione, T(S)2, decreases as two groups exhibiting experimentally indistinguishable pK values of 8.74 +/- 0.03 are deprotonated. Variable magnitudes of the primary deuterium kinetic isotope effects on pyridine nucleotide oxidation are observed on V and V/K when different pyridine nucleotide substrates are used, and the magnitude of DV and D(V/K) is independent of the oxidized trypanothione concentration at pH 7.25. Solvent kinetic isotope effects, obtained with 2',3'-cNADPH as the variable substrate, were observed on V only, and plots of V versus mole fraction of D2O (i.e., proton inventory) were linear, and yielded values of 1.3-1.6 for D2OV. Solvent kinetic isotope effects obtained with alternate pyridine nucleotides as substrates were also observed on V, and the magnitude of D2OV decreases for each pyridine nucleotide as its maximal velocity relative to that of NADPH oxidation decreases.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1992JE53600008
View details for PubMedID 1633154
1992; 1 (7): 874-883
Two enzymes involved in the biosynthesis of the trypanosomatid-specific dithiol trypanothione-glutathionylspermidine (Gsp) synthetase and trypanothione (TSH) synthetase--have been identified and purified individually from Crithidia fasciculata. The Gsp synthetase has been purified 93-fold and the TSH synthetase 52-fold to apparent homogeneity from a single DEAE fraction that contained both activities. This constitutes the first indication that the enzymatic conversion of two glutathione molecules and one spermidine to the N1,N8-bis(glutathionyl)spermidine (TSH) occurs in two discrete enzymatic steps. Gsp synthetase, which has a kcat of 600/min, shows no detectable TSH synthetase activity, whereas TSH synthetase does not make any detectable Gsp and has a kcat of 75/min. The 90-kDa Gsp synthetase and 82-kDa TSH synthetase are separable on phenyl Superose and remain separated on gel filtration columns in high salt (0.8 M NaCl). Active complexes can be formed under low to moderate salt conditions (0.0-0.15 M NaCl), consistent with a functional complex in vivo.
View details for Web of Science ID A1992JF88300005
View details for PubMedID 1304372
1992; 33: 409-417
View details for Web of Science ID A1992MD69600023
View details for PubMedID 1354149
1991; 16 (8): 305-309
Trypanosoma and Leishmania are parasitic protozoa that cause a variety of diseases, which include African sleeping sickness and oriental sore. Attempts to determine pharmaceutically exploitable differences between host and parasite biochemistry have identified the unique trypanothione pathway as a possible target. This pathway includes the enzyme trypanothione reductase, the parasite analogue of glutathione reductase.
View details for Web of Science ID A1991GA13000013
View details for PubMedID 1957352
1991; 38 (3): 224-228
We tested if genetic exchange was observable between two strains of Leishmania major (Trypanosomatidae) during mixed infection of the sand fly Phlebotomus papatasi. Previous studies suggested that genetic exchange may occur in natural populations of Leishmania at a low frequency, but experimental crosses examining small numbers of progeny (less than 60) did not reveal hybrid parasites. Accordingly, a strategy was devised to increase the number of progeny that could be screened by 100-fold. Clonal derivatives from two strains that were infective to flies and contained numerous restriction fragment length polymorphisms were characterized and selected for resistance to methotrexate or tunicamycin by gene amplification. A successfully mixed infection of P. papatasi was obtained, and a method was developed for directly plating promastigotes from the gut contents of infected flies onto selective media. Twenty-five hundred independent progeny were scored for the presence of both drug resistance markers. No hybrid parasites were observed, indicating that the frequency of genetic exchange in this cross must be less than 4 x 10(-4). The lines and methods established in this work may prove useful in future studies of the mechanism and frequency of gene exchange in Leishmania.
View details for Web of Science ID A1991FQ06400010
View details for PubMedID 1880760
1991; 88 (1): 48-52
A 203-base-pair sequence 5' of the latency-associated transcripts (LATs) of herpes simplex virus type 1 contains a 7-base consensus sequence TGCGTCA that is identical to the cAMP-response element of the proenkephalin gene. This consensus sequence is at -38 relative to the putative 5' end of the LATs with a TATA box at the -24 position. In transient chloramphenicol acetyltransferase assays in rat pheochromocytoma (PC12) cells, this enhancer region stimulated gene expression up to 3-fold in the presence of dibutyryl cAMP, forskolin, nerve growth factor, or phorbol 12-myristate 13-acetate. Mutation of the cAMP-response element to TGCG-CAA resulted in a 4-fold reduction of basal activity and a complete loss of inducible stimulation. In DNA gel retardation assays, purified cAMP-response element-binding protein and a nuclear protein from PC12 cells were shown to bind specifically to this element. Furthermore, it was demonstrated that the reactivation of wild-type herpes simplex virus type 1 from dissociated latently infected murine trigeminal ganglia was significantly accelerated (P less than 0.005) by the addition of cAMP analogs or adenylate cyclase activators. However, these reagents did not accelerate reactivation of a deletion mutant that lacks the putative cAMP-response element-containing promoter region, transcriptional start site, and 1015 base pairs of the LATs. These studies demonstrate that the promoter region of the LATs contains a functional cAMP-response element and that expression of the LATs is likely controlled by second messenger signal transduction and imply a role for cAMP in triggering viral reactivation.
View details for Web of Science ID A1991EQ54400011
View details for PubMedID 1846042
Conference Proceedings
Successful conversion from conventional immunosuppression to anti-CD154 monoclonal antibody costimulatory molecule blockade in rhesus renal allograft recipients
Several conventional forms of immunosuppression have been shown to antagonize the efficacy of anti-CD154 monoclonal antibody- (mAb) based costimulatory molecule blockade immunotherapy. Our objective was to determine if allograft recipients treated with a conventional immunosuppressive regimen could be sequentially converted to anti-CD154 mAb monotherapy without compromising graft survival.Outbred juvenile rhesus monkeys underwent renal allotransplantation from MHC-disparate donors. After a 60-day course of triple therapy immunosuppression with steroids, cyclosporine, and mycophenolate mofetil, monkeys were treated with: (1) cessation of all immunosuppression (control); (2) seven monthly doses of 20 mg/kg hu5C8 (maintenance), or; (3) 20 mg/kg hu5C8 on posttransplant days 60, 61, 64, 71, 79, and 88 followed by five monthly doses (induction+maintenance). Graft rejection was defined by elevation in serum creatinine>1.5 mg/dl combined with histologic evidence of rejection.Graft survival for the three groups were as follows: group 1 (control): 70, 75, >279 days; group 2 (maintenance): 83, 349, >293 days, and; group 3 (induction+maintenance): 355, >377, >314 days. Acute rejection developing in two of four monkeys after treatment with conventional immunosuppression was successfully reversed with intensive hu5C8 monotherapy.Renal allograft recipients can be successfully converted to CD154 blockade monotherapy after 60 days of conventional immunosuppression. An induction phase of anti-CD154 mAb appears to be necessary for optimal conversion. Therefore, although concurrent administration of conventional immunosuppressive agents including steroids and calcineurin inhibitors has been shown to inhibit the efficacy of CD154 blockade, sequential conversion from these agents to CD154 blockade appears to be effective.
View details for Web of Science ID 000170968400006
View details for PubMedID 11544416
Inhibition of CD28-B7 T-cell costimulatory activation pathway affects intragraft cytokine and growth factor expression in chronic renal allograft rejection
View details for Web of Science ID A1997WM12700453
View details for PubMedID 9123187
Initial ischemia/reperfusion injury influences late functional and structural changes in the kidney
View details for Web of Science ID A1997WM12700678
View details for PubMedID 9123412
Early cellular and molecular changes in ischemia/reperfusion injury: Inhibition by a selectin antagonist, P-selectin glycoprotein ligand-1
View details for Web of Science ID A1997WM12700591
View details for PubMedID 9123325
Prevention of functional, structural, and molecular changes of chronic rejection of rat renal allografts by a specific macrophage inhibitor
Chronic rejection is the primary cause of long-term allograft loss. Macrophages and their products have been shown to be critical in the development of this process in an established kidney allograft rat model. A new synthetic agent, Gamma lactone, is a specific inhibitor of macrophages and monocytes that inhibits the generation of these populations in vitro and their activities in the effector phase of host alloresponsiveness. We tested its effects on the development of chronic changes in the model. Untreated control allograft recipients developed increasing proteinuria after 12 weeks; progressive glomerulosclerosis, interstitial fibrosis, and arterial obliteration developed thereafter. Infiltrating ED1+ macrophages as noted by immunohistology increased dramatically between 12 and 16 weeks, localizing preferentially in glomeruli and perivascular areas. The presence of these cells was associated with dense expression of their products. Reverse transcription polymerase chain reaction confirmed and expanded the immunohistological findings, showing significant gene expression of macrophage-derived mediators. In contrast, recipients treated with G-Lac daily for 32 weeks never developed proteinuria; macrophage infiltration was dramatically reduced, and expression of their products was virtually absent. At 32 weeks, most glomeruli and arteries remained histologically normal. In another group in which treatment was stopped at 24 weeks, however, proteinuria began to develop by 32 weeks; macrophages infiltrated the organs and expression of their products became manifest. These results confirm the importance of macrophages and macrophage-derived factors in chronic rejection and suggest that a specific inhibitor of macrophage activation may be useful in the prevention of the process over the long term.
View details for Web of Science ID A1995TN23000034
View details for PubMedID 8545893
View details for Web of Science ID A1993BZ60M00047
View details for PubMedID 8109853