Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 24
Filter
1.
Mol Ther Methods Clin Dev ; 31: 101155, 2023 Dec 14.
Article in English | MEDLINE | ID: mdl-38074412

ABSTRACT

Ex vivo autologous hematopoietic stem cell lentiviral-based gene therapy with betibeglogene autotemcel has been studied in patients with transfusion-dependent ß-thalassemia in Phase III clinical trials (HGB-207 and HGB-212), with 90% of patients reaching transfusion independence (TI). Here, we explore manufacturing parameters, drug product quality attributes, and limited patient characteristics that had an impact on clinical efficacy in HGB-207 and HGB-212. Retrospective analysis revealed that the peripheral blood vector copy number (VCN) was related to TI, with a strong correlation between peripheral blood VCN at 6 months and gene therapy-derived therapeutic protein (HbAT87Q) expression at 6 months (correlation coefficient, 0.8681; p < 0.0001; R2 = 0.7536). A peripheral blood VCN threshold of ≥0.75 copies per diploid genome at 6 months post betibeglogene autotemcel infusion provided a stringent surrogate biomarker for TI and was used as the outcome variable for multivariate analysis using a random forest classifier. The top predictive feature of clinical efficacy was found to be the percentage of lentiviral vector-positive cells in the drug product. This retrospective analysis is critical to understanding the key product quality attributes that can predict clinical efficacy in lentiviral vector gene therapy within this clinical trial population.

3.
N Engl J Med ; 386(2): 138-147, 2022 01 13.
Article in English | MEDLINE | ID: mdl-34898140

ABSTRACT

Gene therapy with LentiGlobin for sickle cell disease (bb1111, lovotibeglogene autotemcel) consists of autologous transplantation of a patient's hematopoietic stem cells transduced with the BB305 lentiviral vector that encodes the ßA-T87Q-globin gene. Acute myeloid leukemia developed in a woman approximately 5.5 years after she had received LentiGlobin for sickle cell disease as part of the initial cohort (Group A) of the HGB-206 study. An analysis of peripheral-blood samples revealed that blast cells contained a BB305 lentiviral vector insertion site. The results of an investigation of causality indicated that the leukemia was unlikely to be related to vector insertion, given the location of the insertion site, the very low transgene expression in blast cells, and the lack of an effect on expression of surrounding genes. Several somatic mutations predisposing to acute myeloid leukemia were present after diagnosis, which suggests that patients with sickle cell disease are at increased risk for hematologic malignant conditions after transplantation, most likely because of a combination of risks associated with underlying sickle cell disease, transplantation procedure, and inadequate disease control after treatment. (Funded by Bluebird Bio.).


Subject(s)
Anemia, Sickle Cell/therapy , Gene Expression , Genetic Therapy/adverse effects , Hematopoietic Stem Cell Transplantation , Leukemia, Myeloid, Acute/etiology , beta-Globins/genetics , Adult , Anemia, Sickle Cell/complications , Anemia, Sickle Cell/genetics , Carcinogenesis , Female , Genetic Vectors , Humans , Lentivirus , Risk Factors , Sequence Analysis, RNA , Transgenes , Transplantation, Autologous
4.
N Engl J Med ; 386(5): 415-427, 2022 02 03.
Article in English | MEDLINE | ID: mdl-34891223

ABSTRACT

BACKGROUND: Betibeglogene autotemcel (beti-cel) gene therapy for transfusion-dependent ß-thalassemia contains autologous CD34+ hematopoietic stem cells and progenitor cells transduced with the BB305 lentiviral vector encoding the ß-globin (ßA-T87Q) gene. METHODS: In this open-label, phase 3 study, we evaluated the efficacy and safety of beti-cel in adult and pediatric patients with transfusion-dependent ß-thalassemia and a non-ß0/ß0 genotype. Patients underwent myeloablation with busulfan (with doses adjusted on the basis of pharmacokinetic analysis) and received beti-cel intravenously. The primary end point was transfusion independence (i.e., a weighted average hemoglobin level of ≥9 g per deciliter without red-cell transfusions for ≥12 months). RESULTS: A total of 23 patients were enrolled and received treatment, with a median follow-up of 29.5 months (range, 13.0 to 48.2). Transfusion independence occurred in 20 of 22 patients who could be evaluated (91%), including 6 of 7 patients (86%) who were younger than 12 years of age. The average hemoglobin level during transfusion independence was 11.7 g per deciliter (range, 9.5 to 12.8). Twelve months after beti-cel infusion, the median level of gene therapy-derived adult hemoglobin (HbA) with a T87Q amino acid substitution (HbAT87Q) was 8.7 g per deciliter (range, 5.2 to 10.6) in patients who had transfusion independence. The safety profile of beti-cel was consistent with that of busulfan-based myeloablation. Four patients had at least one adverse event that was considered by the investigators to be related or possibly related to beti-cel; all events were nonserious except for thrombocytopenia (in 1 patient). No cases of cancer were observed. CONCLUSIONS: Treatment with beti-cel resulted in a sustained HbAT87Q level and a total hemoglobin level that was high enough to enable transfusion independence in most patients with a non-ß0/ß0 genotype, including those younger than 12 years of age. (Funded by Bluebird Bio; HGB-207 ClinicalTrials.gov number, NCT02906202.).


Subject(s)
Biological Products/therapeutic use , Genetic Therapy/methods , beta-Globins/genetics , beta-Thalassemia/therapy , Adolescent , Adult , Biological Products/adverse effects , Busulfan/therapeutic use , Child , Erythrocyte Transfusion/adverse effects , Erythropoiesis , Female , Genetic Vectors , Genotype , Hemoglobins/analysis , Humans , Iron Overload/prevention & control , Lentivirus/genetics , Male , Middle Aged , Myeloablative Agonists/therapeutic use , beta-Thalassemia/blood , beta-Thalassemia/genetics
5.
J Drug Assess ; 7(1): 66-74, 2018.
Article in English | MEDLINE | ID: mdl-30370176

ABSTRACT

Objective: Infection with hepatitis C virus is the leading indication for liver transplantation and most common cause of infectious disease-related mortality in the United States. BZF961 is a novel inhibitor of the hepatitis C virus NS3-4A protease. Methods: This sequential, three part exploratory first-in-human study investigated the safety and pharmacokinetics of single and multiple ascending oral doses of BZF961 in healthy subjects. The first two parts were randomized, double-blind, placebo-controlled, time-lagged, single and multiple ascending oral dose segments. The third part analyzed the effect of ritonavir on BZF961 pharmacokinetics. Results: BZF961 was generally safe and well-tolerated in single and multiple oral doses in healthy subjects. There were no deaths and no serious adverse events. The most common adverse events were nausea and other gastrointestinal symptoms. Co-administration of ritonavir with BZF961 was well tolerated and increased BZF961 exposure by up to 60-fold, as well as reduced the overall exposure variability. Conclusions: BZF961 was generally safe and well-tolerated and its exposure was boosted by the co-administration of ritonavir.

6.
Clin Ther ; 40(9): 1567-1581.e4, 2018 09.
Article in English | MEDLINE | ID: mdl-30185394

ABSTRACT

PURPOSE: Infection with hepatitis C virus is the leading cause of infectious disease mortality in the United States. BZF961 is a novel small molecule inhibitor of the hepatitis C virus NS3-4A protease. Here we present the results of a randomized, double-blinded, placebo-controlled, multicentered study in treatment-naïve patients with chronic hepatitis C virus genotype-1 infection. METHODS: Patients were enrolled sequentially in 2 parts and treated for 3days. BZF961 was administered as monotherapy (500mg BID for 3 days) or in combination with the cytochrome P450 3A4 inhibitor ritonavir to boost its exposure (BZF961 10, 20, or 50mg QD or BID). FINDINGS: BZF961 was safe and well tolerated in the patients studied with no serious adverse events. There were no appreciable differences in adverse events among patients who received BZF961, BZF961 with ritonavir, or placebo. There was a significant, clinically meaningful reduction in viral load from baseline in patients treated either with BZF961 500mg every 12hours alone or BZF961 50mg every 12hours in combination with ritonavir. Activity against the hepatitis C virus of the lower-dose regimens was apparent but more modest. There were no relevant changes from baseline viral loads in placebo-treated patients. IMPLICATIONS: Coadministration of ritonavir with BZF961 boosted BZF961 exposure (including Cmin, which is the clinically relevant parameter associated with antiviral activity) in a therapeutic range with less variability compared with BZF961 alone. For strategic reasons, BZF961 is no longer under development.


Subject(s)
Antiviral Agents/therapeutic use , Hepatitis C, Chronic/drug therapy , Organic Chemicals/therapeutic use , Ritonavir/therapeutic use , Adult , Antiviral Agents/administration & dosage , Antiviral Agents/adverse effects , Double-Blind Method , Drug Therapy, Combination , Female , Genotype , Hepacivirus/genetics , Humans , Male , Middle Aged , Organic Chemicals/administration & dosage , Organic Chemicals/adverse effects , United States , Viral Load/drug effects , Viral Nonstructural Proteins/antagonists & inhibitors
7.
ACS Infect Dis ; 3(2): 144-151, 2017 02 10.
Article in English | MEDLINE | ID: mdl-27788579

ABSTRACT

Hepatitis C virus (HCV) infection is a significant cause of liver disease affecting 80-150 million people globally. Diacylglycerol transferase 1 (DGAT-1), a triglyceride synthesis enzyme, is important for the HCV life cycle in vitro. Pradigastat, a potent DGAT-1 inhibitor found to lower triglycerides and HgbA1c in patients, was investigated for safety and efficacy in patients with HCV. This was a two-part study. In the in vitro study, the effect of pradigastat on virus production was evaluated in infected cells in culture. In the clinical study ( https://clinicaltrials.gov/ct2/show/NCT01387958 ), 32 patients with HCV infection were randomized to receive pradigastat or placebo (26:6) once daily for 14 days. Primary efficacy outcomes were serum viral RNA and alanine aminotransferase levels. In vitro, pradigastat significantly reduced virus production, consistent with inhibition of viral assembly and release. However, the clinical study was prematurely terminated for lack of efficacy. There was no significant change in serum viral RNA levels after dosing with pradigastat or placebo for 14 days. Pradigastat was safe and well-tolerated in this population. Most treatment-emergent adverse events were gastrointestinal; there were no hepatic adverse events. Although pradigastat had a potent antiviral effect in vitro, no significant antiviral effect was observed in patients at predicted efficacious exposures.


Subject(s)
Acetates/administration & dosage , Aminopyridines/administration & dosage , Antiviral Agents/administration & dosage , Diacylglycerol O-Acyltransferase/antagonists & inhibitors , Hepatitis C, Chronic/drug therapy , Acetates/pharmacology , Adult , Alanine Transaminase/blood , Aminopyridines/pharmacology , Antiviral Agents/pharmacology , Female , Hepacivirus/drug effects , Hepacivirus/genetics , Humans , Male , Middle Aged , RNA, Viral/blood , Treatment Outcome , Virus Replication/drug effects , Young Adult
8.
J Gastroenterol Hepatol ; 30(1): 184-91, 2015 Jan.
Article in English | MEDLINE | ID: mdl-24995515

ABSTRACT

BACKGROUND AND AIMS: Albinterferon is a fusion of albumin and interferon-α2b developed to improve the pharmacokinetics, convenience, and potential efficacy of interferon-α for the treatment of chronic hepatitis infections. METHODS: This open-label, randomized, active-controlled, multicenter study investigated the safety and efficacy of albinterferon in patients with chronic hepatitis B virus (HBV) infection who were e-antigen (HBeAg) positive. One hundred and forty-one patients received one of four albinterferon doses/regimens or pegylated-interferon-α2a. Primary efficacy outcomes were changes in serum HBeAg and antibody, HBV-DNA, and alanine aminotransferase. Principal safety outcomes were changes in laboratory values, pulmonary function, and adverse events. RESULTS: The study was prematurely terminated as phase III trials in hepatitis C infection indicated noninferior efficacy but inferior safety compared with pegylated-interferon-α2a. Here, all treatment groups had a significant reduction in HBV-DNA from baseline. Reductions in HBV-DNA were not significantly different, except the 1200 µg every 4 weeks albinterferon dose which was inferior compared with pegylated-interferon-α2a. The serum alanine aminotransferase levels decreased in all arms. The per-patient incidence of adverse events was not significantly different for albinterferon (96.4-100%) and pegylated-interferon-α2a (93.1%). Total adverse events, however, were higher for albinterferon and correlated to dose. Decreased lung function was found in all arms (∼93% of patients), and was more common in some albinterferon groups. CONCLUSIONS: Albinterferon doses with similar anti-HBV efficacy to pegylated-interferon-α2a had higher rates of certain adverse events, particularly changes in lung diffusion capacity (http://www.clinicaltrials.gov number NCT00964665).


Subject(s)
Albumins/administration & dosage , Antiviral Agents/administration & dosage , Hepatitis B, Chronic/drug therapy , Interferon-alpha/administration & dosage , Adult , Alanine Transaminase/blood , Albumins/adverse effects , Antiviral Agents/adverse effects , Biomarkers/blood , DNA, Viral/blood , Female , Hepatitis B e Antigens/blood , Hepatitis B virus/genetics , Hepatitis B virus/immunology , Hepatitis B, Chronic/diagnosis , Hepatitis B, Chronic/virology , Humans , Interferon-alpha/adverse effects , Male , Polyethylene Glycols/administration & dosage , Polyethylene Glycols/adverse effects , Recombinant Proteins/administration & dosage , Recombinant Proteins/adverse effects , Treatment Outcome , Young Adult
9.
Nat Immunol ; 12(2): 167-77, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21217759

ABSTRACT

Mouse CCL8 is a CC chemokine of the monocyte chemoattractant protein (MCP) family whose biological activity and receptor usage have remained elusive. Here we show that CCL8 is highly expressed in the skin, where it serves as an agonist for the chemokine receptor CCR8 but not for CCR2. This distinguishes CCL8 from all other MCP chemokines. CCL8 responsiveness defined a population of highly differentiated, CCR8-expressing inflammatory T helper type 2 (T(H)2) cells enriched for interleukin (IL)-5. Ccr8- and Ccl8-deficient mice had markedly less eosinophilic inflammation than wild-type or Ccr4-deficient mice in a model of chronic atopic dermatitis. Adoptive transfer studies established CCR8 as a key regulator of T(H)2 cell recruitment into allergen-inflamed skin. In humans, CCR8 expression also defined an IL-5-enriched T(H)2 cell subset. The CCL8-CCR8 chemokine axis is therefore a crucial regulator of T(H)2 cell homing that drives IL-5-mediated chronic allergic inflammation.


Subject(s)
Chemokine CCL1/metabolism , Chemokine CCL8/metabolism , Dermatitis, Atopic/immunology , Skin/pathology , Th2 Cells/metabolism , Adoptive Transfer , Animals , Calcium Signaling/immunology , Cells, Cultured , Chemokine CCL1/genetics , Chemokine CCL1/immunology , Chemokine CCL8/genetics , Chemokine CCL8/immunology , Chemotaxis/genetics , Chemotaxis/immunology , Cloning, Molecular , Disease Models, Animal , Humans , Interleukin-5/immunology , Interleukin-5/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Lymphocyte Homing/immunology , Th2 Cells/immunology , Th2 Cells/pathology
10.
Cell Adh Migr ; 5(1): 56-8, 2011.
Article in English | MEDLINE | ID: mdl-20798599

ABSTRACT

Leukocyte chemotaxis plays an essential role in generating and delivering immune responses and is a critical component of inflammation. In order to identify novel genes and pathways important for regulating chemotaxis, we performed an RNAi-based screen and identified several genes involved with vesicle movement and fusion as mediators of chemotaxis. Our recently published data show that during chemotaxis vesicle trafficking proteins are required for lysosome fusion, uropod release and efficient directed cell migration.


Subject(s)
Cell Movement/physiology , Chemotaxis, Leukocyte/physiology , RNA Interference , Synaptotagmins/genetics , Synaptotagmins/metabolism , Biological Transport , Cell Line, Tumor , Gene Library , Humans , Lysosomal-Associated Membrane Protein 1/metabolism , Membrane Fusion , Receptors, CXCR/metabolism , rab GTP-Binding Proteins/metabolism
11.
PLoS One ; 5(9): e12700, 2010 Sep 13.
Article in English | MEDLINE | ID: mdl-20856926

ABSTRACT

CXCL10 (or Interferon-inducible protein of 10 kDa, IP-10) is an interferon-inducible chemokine with potent chemotactic activity on activated effector T cells and other leukocytes expressing its high affinity G protein-coupled receptor CXCR3. CXCL10 is also active on other cell types, including endothelial cells and fibroblasts. The mechanisms through which CXCL10 mediates its effects on non-leukocytes is not fully understood. In this study, we focus on the anti-proliferative effect of CXCL10 on endothelial cells, and demonstrate that CXCL10 can inhibit endothelial cell proliferation in vitro independently of CXCR3. Four main findings support this conclusion. First, primary mouse endothelial cells isolated from CXCR3-deficient mice were inhibited by CXCL10 as efficiently as wildtype endothelial cells. We also note that the proposed alternative splice form CXCR3-B, which is thought to mediate CXCL10's angiostatic activity, does not exist in mice based on published mouse CXCR3 genomic sequences as an in-frame stop codon would terminate the proposed CXCR3-B splice variant in mice. Second, we demonstrate that human umbilical vein endothelial cells and human lung microvascular endothelial cells that were inhibited by CXL10 did not express CXCR3 by FACS analysis. Third, two different neutralizing CXCR3 antibodies did not inhibit the anti-proliferative effect of CXCL10. Finally, fourth, utilizing a panel of CXCL10 mutants, we show that the ability to inhibit endothelial cell proliferation correlates with CXCL10's glycosaminoglycan binding affinity and not with its CXCR3 binding and signaling. Thus, using a very defined system, we show that CXCL10 can inhibit endothelial cell proliferation through a CXCR3-independent mechanism.


Subject(s)
Cell Proliferation , Chemokine CXCL10/metabolism , Down-Regulation , Endothelial Cells/cytology , Receptors, CXCR3/metabolism , Animals , Base Sequence , Cells, Cultured , Chemokine CXCL10/genetics , Endothelial Cells/metabolism , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , RNA Splicing , Receptors, CXCR3/genetics
12.
Clin Infect Dis ; 51(7): 813-22, 2010 Oct 01.
Article in English | MEDLINE | ID: mdl-20738185

ABSTRACT

In this review, we collate 25 clinically useful human immunodeficiency virus (HIV)-related Web sites to facilitate efficient access to online resources according to themes of clinical inquiry: (1) comprehensive clinical information, (2) opportunistic infections, (3) antiretroviral drug interactions, (4) care of HIV-infected women and children, and (5) continuing medical education. We evaluated these Web sites for clinical content and quality using criteria including the currency of information, inclusion of references, sponsors, whether the site is useful in resource-limited settings, ease of navigation, and content specific for each theme. Using the specified criteria, we provided overall ratings for each Web site. We conclude that the Web sites listed in this review can help extend knowledge about best practices and provide real-time patient care support to clinicians.


Subject(s)
Education, Medical, Continuing/methods , HIV Infections/diagnosis , HIV Infections/drug therapy , Infectious Disease Medicine/education , Internet , Medical Informatics/methods , HIV Infections/epidemiology , Humans , Physicians
13.
Nat Immunol ; 11(6): 495-502, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20473299

ABSTRACT

Chemokines and other chemoattractants direct leukocyte migration and are essential for the development and delivery of immune and inflammatory responses. To probe the molecular mechanisms that underlie chemoattractant-guided migration, we did an RNA-mediated interference screen that identified several members of the synaptotagmin family of calcium-sensing vesicle-fusion proteins as mediators of cell migration: SYT7 and SYTL5 were positive regulators of chemotaxis, whereas SYT2 was a negative regulator of chemotaxis. SYT7-deficient leukocytes showed less migration in vitro and in a gout model in vivo. Chemoattractant-induced calcium-dependent lysosomal fusion was impaired in SYT7-deficient neutrophils. In a chemokine gradient, SYT7-deficient lymphocytes accumulated lysosomes in their uropods and had impaired uropod release. Our data identify a molecular pathway required for chemotaxis that links chemoattractant-induced calcium flux to exocytosis and uropod release.


Subject(s)
Cell Movement/physiology , Synaptotagmins/metabolism , Animals , Chemokine CXCL12/metabolism , Chemotaxis , Immunoblotting , Mice , Mice, Inbred C57BL , Mice, Knockout , Polymerase Chain Reaction , Receptors, CXCR4/metabolism , Synaptotagmin II/metabolism , Synaptotagmins/genetics , T-Lymphocytes/immunology
14.
J Biol Chem ; 284(29): 19189-95, 2009 Jul 17.
Article in English | MEDLINE | ID: mdl-19473985

ABSTRACT

Recent studies have demonstrated that kynurenic acid (KYNA), a compound produced endogenously by the interferon-gamma-induced degradation of tryptophan by indoleamine 2,3-dioxygenase, activates the previously orphaned G protein-coupled receptor, GPR35. This receptor is expressed in immune tissues, although its potential function in immunomodulation remains to be explored. We determined that GPR35 was most highly expressed on human peripheral monocytes. In an in vitro vascular flow model, KYNA triggered the firm arrest of monocytes to both fibronectin and ICAM-1, via beta(1) integrin- and beta(2) integrin-mediated mechanisms, respectively. Incubation of monocytes with pertussis toxin prior to use in flow experiments significantly reduced the KYNA-induced monocyte adhesion, suggesting that adhesion is triggered by a G(i)-mediated process. Furthermore, KYNA-triggered adhesion of monocytic cells was reduced by short hairpin RNA-mediated silencing of GPR35. Although GPR35 is expressed at slightly lower levels on neutrophils, KYNA induced firm adhesion of these cells to an ICAM-1-expressing monolayer as well. KYNA also elicited neutrophil shedding of surface L-selectin, another indicator of leukocyte activation. Taken together, these data suggest that KYNA could be an important early mediator of leukocyte recruitment.


Subject(s)
Endothelial Cells/drug effects , Kynurenic Acid/pharmacology , Leukocytes, Mononuclear/drug effects , CD18 Antigens/metabolism , Cell Adhesion/drug effects , Cell Line , Dose-Response Relationship, Drug , Endothelial Cells/cytology , Endothelial Cells/metabolism , Excitatory Amino Acid Antagonists/pharmacology , Fibronectins/metabolism , Gene Expression/drug effects , Humans , Integrin beta1/metabolism , Intercellular Adhesion Molecule-1/metabolism , Kynurenic Acid/chemistry , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/metabolism , Molecular Structure , RNA Interference , Receptors, G-Protein-Coupled/genetics , Reverse Transcriptase Polymerase Chain Reaction , Rheology
15.
J Exp Med ; 206(3): 637-53, 2009 Mar 16.
Article in English | MEDLINE | ID: mdl-19237602

ABSTRACT

Receptors involved in innate immunity to fungal pathogens have not been fully elucidated. We show that the Caenorhabditis elegans receptors CED-1 and C03F11.3, and their mammalian orthologues, the scavenger receptors SCARF1 and CD36, mediate host defense against two prototypic fungal pathogens, Cryptococcus neoformans and Candida albicans. CED-1 and C03F11.1 mediated antimicrobial peptide production and were necessary for nematode survival after C. neoformans infection. SCARF1 and CD36 mediated cytokine production and were required for macrophage binding to C. neoformans, and control of the infection in mice. Binding of these pathogens to SCARF1 and CD36 was beta-glucan dependent. Thus, CED-1/SCARF1 and C03F11.3/CD36 are beta-glucan binding receptors and define an evolutionarily conserved pathway for the innate sensing of fungal pathogens.


Subject(s)
Caenorhabditis elegans/microbiology , Candida albicans/immunology , Conserved Sequence , Cryptococcus neoformans/immunology , Evolution, Molecular , Immunity, Innate , Receptors, Scavenger/immunology , Animals , CD36 Antigens/deficiency , CD36 Antigens/immunology , Caenorhabditis elegans/immunology , Caenorhabditis elegans Proteins/immunology , Candida albicans/cytology , Candidiasis/immunology , Candidiasis/microbiology , Cell Adhesion , Cryptococcosis/immunology , Cryptococcosis/microbiology , Cryptococcus neoformans/cytology , Cytokines/biosynthesis , Macrophage Activation , Macrophages/cytology , Macrophages/immunology , Macrophages/microbiology , Membrane Proteins/immunology , Mice , RNA, Small Interfering/metabolism , Survival Analysis , Toll-Like Receptor 2/metabolism
16.
Proc Natl Acad Sci U S A ; 105(12): 4814-9, 2008 Mar 25.
Article in English | MEDLINE | ID: mdl-18347328

ABSTRACT

Cerebral malaria is a significant cause of global mortality, causing an estimated two million deaths per year, mainly in children. The pathogenesis of this disease remains incompletely understood. Chemokines have been implicated in the development of cerebral malaria, and the IFN-inducible CXCR3 chemokine ligand IP-10 (CXCL10) was recently found to be the only serum biomarker that predicted cerebral malaria mortality in Ghanaian children. We show that the CXCR3 chemokine ligands IP-10 and Mig (CXCL9) were highly induced in the brains of mice with murine cerebral malaria caused by Plasmodium berghei ANKA. Mice deficient in CXCR3 were markedly protected against cerebral malaria and had far fewer T cells in the brain compared with wild-type mice. In competitive transfer experiments, CXCR3-deficient CD8(+) T cells were 7-fold less efficient at migrating into the infected brains than wild-type CD8(+) T cells. Adoptive transfer of wild-type CD8(+) effector T cells restored susceptibility of CXCR3-deficient mice to cerebral malaria and also restored brain proinflammatory cytokine and chemokine production and recruitment of T cells, independent of CXCR3. Mice deficient in IP-10 or Mig were both partially protected against cerebral malaria mortality when infected with P. berghei ANKA. Brain immunohistochemistry revealed Mig staining of endothelial cells, whereas IP-10 staining was mainly found in neurons. These data demonstrate that CXCR3 on CD8(+) T cells is required for T cell recruitment into the brain and the development of murine cerebral malaria and suggest that the CXCR3 ligands Mig and IP-10 play distinct, nonredundant roles in the pathogenesis of this disease.


Subject(s)
Chemokine CXCL10/immunology , Chemokine CXCL9/immunology , Malaria, Cerebral/immunology , Malaria, Cerebral/pathology , Receptors, CXCR3/immunology , Animals , Brain/parasitology , Brain/pathology , CD3 Complex/immunology , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/parasitology , Cell Movement , Chemokine CXCL10/genetics , Chemokine CXCL9/genetics , Ligands , Malaria, Cerebral/parasitology , Malaria, Cerebral/prevention & control , Mice , Mice, Inbred C57BL , Mice, Knockout , Plasmodium berghei/immunology , Receptors, Cytokine/deficiency , Spleen/metabolism , Spleen/pathology , Survival Rate , Up-Regulation/genetics
17.
J Immunol Methods ; 331(1-2): 127-39, 2008 Feb 29.
Article in English | MEDLINE | ID: mdl-18206159

ABSTRACT

Trafficking of leukocytes to sites of inflammation is an important step in the establishment of an immune response. Chemokines are critical regulators of leukocyte trafficking and are widely studied molecules for their important role in disease and for their potential as new therapeutic targets. The ability of chemokines to induce leukocyte recruitment has been mainly measured by in vitro chemotaxis assays, which lack many components of the complex biological process of leukocyte migration and therefore provide incomplete information about chemokine function in vivo. In vivo assays to study the activity of chemokines to induce leukocyte recruitment have been difficult to establish. We describe here the development of a robust in vivo recruitment assay for CD8(+) and CD4(+) T lymphocytes induced by the CXCR3 ligands IP-10 (CXCL10) and I-TAC (CXCL11). For this assay, in vitro activated T lymphocytes were adoptively transferred into the peritoneum of naïve mice. Homing of these transferred T lymphocytes into the airways was measured following intratracheal instillation of chemokines. High recruitment indices were achieved that were dependent on chemokine concentration and CXCR3 expression on the transferred lymphocytes. Recruitment was also inhibited by antibodies to the chemokine. The assay models the natural condition of chemokine-mediated lymphocyte migration into the airways as chemokines are expressed in the airways during inflammation. The nature of this model allows flexibility to study wildtype and mutant chemokines and chemokine receptors and the ability to evaluate chemokine antagonists and antibodies in vivo. This assay will therefore help elucidate a deeper understanding of the chemokine system in vivo.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Chemokines, CXC/metabolism , Chemotaxis, Leukocyte , Receptors, Chemokine/metabolism , Animals , CD4-Positive T-Lymphocytes/physiology , CD8-Positive T-Lymphocytes/physiology , Chemokines, CXC/immunology , Inflammation/immunology , Inflammation/metabolism , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Receptors, Chemokine/immunology
18.
Circ Res ; 102(2): 218-25, 2008 Feb 01.
Article in English | MEDLINE | ID: mdl-17991878

ABSTRACT

Obese individuals often have low plasma adiponectin and concomitant chronic inflammation with a predisposition to metabolic and cardiovascular diseases. The present study reports a novel antiinflammatory action of adiponectin in human monocyte-derived macrophages (MPhi) suppressing T-lymphocyte accumulation in atherogenesis. RNA profiling of lipopolysaccharide-stimulated human MPhi identified CXC chemokine ligands (CXCLs), such as IP-10 (interferon [IFN]-inducible protein 10) (CXCL10), I-TAC (IFN-inducible T-cell alpha chemoattractant) (CXCL11), and Mig (monokine induced by IFN-gamma) (CXCL9), T-lymphocyte chemoattractants associated with atherogenesis, among the top 14 transcripts suppressed by adiponectin. Real-time quantitative RT-PCR and ELISA verified that adiponectin inhibited expression of these chemokines at both the mRNA and protein levels in a concentration-dependent manner. Adiponectin reduced the release by lipopolysaccharide-stimulated MPhi of chemoattractant activity for CXC chemokine receptor 3-transfected (receptor for IP-10, Mig, and I-TAC) lymphocytes. Adiponectin decreased lipopolysaccharide-inducible IP-10 promoter activity in promoter-transfected THP-1 MPhi but did not change IP-10 mRNA stability. In lipopolysaccharide-stimulated MPhi, reduction of IFN-beta by adiponectin preceded inhibition of IP-10 mRNA expression. Immunoblot and chromatin immunoprecipitation analyses demonstrated that adiponectin attenuated activation of the transcription factor IFN regulatory factor 3, involved in the MyD88-independent pathway of Toll-like receptor 4 signaling, and subsequent IFN regulatory factor 3 binding to IFN-beta promoter. In vivo studies further demonstrated that apolipoprotein E/adiponectin double-deficient (apoE-/-APN-/-) mice had increased plasma IP-10 levels, accelerated T-lymphocyte accumulation in atheromata, and augmented atherogenesis compared with apoE single-deficient (apoE-/-APN+/+) mice. This study establishes that low levels of adiponectin associated with obesity, the metabolic syndrome, and diabetes favor T-lymphocyte recruitment and contribute to adaptive immune response during atherogenesis.


Subject(s)
Adiponectin/pharmacology , Arteriosclerosis/immunology , Chemokines, CXC/antagonists & inhibitors , Chemotaxis, Leukocyte/drug effects , Receptors, CXCR3/metabolism , T-Lymphocytes/drug effects , Cells, Cultured , Diabetes Mellitus , Dose-Response Relationship, Drug , Humans , Immunity/drug effects , Macrophages , Metabolic Syndrome , Obesity , Receptors, CXCR3/genetics , Transfection
19.
J Biol Chem ; 282(13): 9547-9555, 2007 Mar 30.
Article in English | MEDLINE | ID: mdl-17289675

ABSTRACT

G protein-coupled receptors (GPCRs) convey extracellular stimulation into dynamic intracellular action, leading to the regulation of cell migration and differentiation. T lymphocytes express G alpha(i2) and G alpha(i3), two members of the G alpha(i/o) protein family, but whether these two G alpha(i) proteins have distinguishable roles guiding T cell migration remains largely unknown because of a lack of member-specific inhibitors. This study details distinct G alpha(i2) and G alpha(i3) effects on chemokine receptor CXCR3-mediated signaling. Our data showed that G alpha(i2) was indispensable for T cell responses to three CXCR3 ligands, CXCL9, CXCL10, and CXCL11, as the lack of G alpha(i2) abolished CXCR3-stimulated migration and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) incorporation. In sharp contrast, T cells isolated from G alpha(i3) knock-out mice displayed a significant increase in both GTPgammaS incorporation and migration as compared with wild type T cells when stimulated with CXCR3 agonists. The increased GTPgammaS incorporation was blocked by G alpha(i3) protein in a dose-dependent manner. G alpha(i3)-mediated blockade of G alpha(i2) activation did not result from G alpha(i3) activation, but instead resulted from competition or steric hindrance of G alpha(i2) interaction with the CXCR3 receptor via the N terminus of the second intracellular loop. A mutation in this domain abrogated not only G alpha(i2) activation induced by a CXCR3 agonist but also the interaction of G alpha(i3) to the CXCR3 receptor. These findings reveal for the first time an interplay of G alpha(i) proteins in transmitting G protein-coupled receptor signals. This interplay has heretofore been masked by the use of pertussis toxin, a broad inhibitor of the G alpha(i/o) protein family.


Subject(s)
GTP-Binding Protein alpha Subunit, Gi2/antagonists & inhibitors , GTP-Binding Protein alpha Subunit, Gi2/physiology , GTP-Binding Protein alpha Subunits, Gi-Go/physiology , Receptors, Chemokine/physiology , Signal Transduction/physiology , Animals , Cells, Cultured , Chemokine CXCL10 , Chemokine CXCL11 , Chemokine CXCL9 , Chemokines, CXC/metabolism , Female , GTP-Binding Protein alpha Subunit, Gi2/deficiency , GTP-Binding Protein alpha Subunit, Gi2/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/deficiency , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, CXCR3 , Receptors, Chemokine/metabolism , Signal Transduction/genetics , T-Lymphocytes/immunology , T-Lymphocytes/metabolism
20.
J Immunol ; 177(10): 6991-8, 2006 Nov 15.
Article in English | MEDLINE | ID: mdl-17082614

ABSTRACT

The chemokine IFN-gamma-inducible protein of 10 kDa (IP-10; CXCL10) plays an important role in the recruitment of activated T lymphocytes into sites of inflammation by interacting with the G protein-coupled receptor CXCR3. IP-10, like other chemokines, forms oligomers, the role of which has not yet been explored. In this study, we used a monomeric IP-10 mutant to elucidate the functional significance of oligomerization. Although monomeric IP-10 had reduced binding affinity for CXCR3 and heparin, it was able to induce in vitro chemotaxis of activated T cells with the same efficacy as wild-type IP-10. However, monomeric IP-10 was unable to induce recruitment of activated CD8+ T cells into the airways of mice after intratracheal instillation. Use of a different IP-10 mutant demonstrated that this inability was due to lack of oligomerization rather than reduced CXCR3 or heparin binding. Molecular imaging demonstrated that both wild-type and monomeric IP-10 were retained in the lung after intratracheal instillation. However, in vitro binding assays indicated that wild-type, but not monomeric, IP-10 was retained on endothelial cells and could induce transendothelial chemotaxis of activated T cells. We therefore propose that oligomerization of IP-10 is required for presentation on endothelial cells and subsequent transendothelial migration, an essential step for lymphocyte recruitment in vivo.


Subject(s)
Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Chemokines, CXC/chemistry , Chemokines, CXC/physiology , Endothelium, Vascular/immunology , Endothelium, Vascular/metabolism , Animals , Antigen Presentation/genetics , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/transplantation , Cell Adhesion/immunology , Cell Line , Cell Line, Transformed , Cell Line, Tumor , Chemokine CXCL10 , Chemokines, CXC/administration & dosage , Chemokines, CXC/genetics , Chemotaxis, Leukocyte/genetics , Chemotaxis, Leukocyte/immunology , Egg Proteins/immunology , Egg Proteins/metabolism , Endothelium, Vascular/cytology , Humans , Intubation, Intratracheal , Mice , Mice, Inbred C57BL , Mice, Transgenic , Ovalbumin/immunology , Ovalbumin/metabolism , Peptide Fragments
SELECTION OF CITATIONS
SEARCH DETAIL
...