Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 27
Filter
1.
Nat Immunol ; 16(1): 75-84, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25347465

ABSTRACT

In lymph nodes, fibroblastic reticular cells (FRCs) form a collagen-based reticular network that supports migratory dendritic cells (DCs) and T cells and transports lymph. A hallmark of FRCs is their propensity to contract collagen, yet this function is poorly understood. Here we demonstrate that podoplanin (PDPN) regulates actomyosin contractility in FRCs. Under resting conditions, when FRCs are unlikely to encounter mature DCs expressing the PDPN receptor CLEC-2, PDPN endowed FRCs with contractile function and exerted tension within the reticulum. Upon inflammation, CLEC-2 on mature DCs potently attenuated PDPN-mediated contractility, which resulted in FRC relaxation and reduced tissue stiffness. Disrupting PDPN function altered the homeostasis and spacing of FRCs and T cells, which resulted in an expanded reticular network and enhanced immunity.


Subject(s)
Collagen/metabolism , Fibroblasts/cytology , Lectins, C-Type/metabolism , Lymph Nodes/cytology , Membrane Glycoproteins/metabolism , Amides/pharmacology , Animals , Cell Survival/immunology , Collagen/immunology , Cytoskeleton/immunology , Cytoskeleton/ultrastructure , Enzyme Inhibitors/pharmacology , Female , Fibroblasts/immunology , Fibroblasts/ultrastructure , Lectins, C-Type/immunology , Lymph Nodes/immunology , Lymph Nodes/ultrastructure , Male , Membrane Glycoproteins/immunology , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Confocal , Phosphorylation , Pyridines/pharmacology , Specific Pathogen-Free Organisms
2.
J Neurosci ; 44(29)2024 Jul 17.
Article in English | MEDLINE | ID: mdl-38830764

ABSTRACT

Human genetics and preclinical studies have identified key contributions of TREM2 to several neurodegenerative conditions, inspiring efforts to modulate TREM2 therapeutically. Here, we characterize the activities of three TREM2 agonist antibodies in multiple mixed-sex mouse models of Alzheimer's disease (AD) pathology and remyelination. Receptor activation and downstream signaling are explored in vitro, and active dose ranges are determined in vivo based on pharmacodynamic responses from microglia. For mice bearing amyloid-ß (Aß) pathology (PS2APP) or combined Aß and tau pathology (TauPS2APP), chronic TREM2 agonist antibody treatment had limited impact on microglia engagement with pathology, overall pathology burden, or downstream neuronal damage. For mice with demyelinating injuries triggered acutely with lysolecithin, TREM2 agonist antibodies unexpectedly disrupted injury resolution. Likewise, TREM2 agonist antibodies limited myelin recovery for mice experiencing chronic demyelination from cuprizone. We highlight the contributions of dose timing and frequency across models. These results introduce important considerations for future TREM2-targeting approaches.


Subject(s)
Alzheimer Disease , Membrane Glycoproteins , Microglia , Multiple Sclerosis , Receptors, Immunologic , Animals , Receptors, Immunologic/agonists , Receptors, Immunologic/metabolism , Receptors, Immunologic/genetics , Membrane Glycoproteins/agonists , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Mice , Multiple Sclerosis/drug therapy , Multiple Sclerosis/immunology , Female , Male , Microglia/drug effects , Microglia/metabolism , Disease Models, Animal , Mice, Inbred C57BL , Mice, Transgenic , Antibodies/pharmacology , Humans , Amyloid beta-Peptides/metabolism , tau Proteins/metabolism
3.
Proc Natl Acad Sci U S A ; 116(33): 16420-16429, 2019 08 13.
Article in English | MEDLINE | ID: mdl-31371506

ABSTRACT

Multiple myeloma (MM) arises from malignant immunoglobulin (Ig)-secreting plasma cells and remains an incurable, often lethal disease despite therapeutic advances. The unfolded-protein response sensor IRE1α supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may co-opt the IRE1α-XBP1s pathway; however, the validity of IRE1α as a potential MM therapeutic target is controversial. Genetic disruption of IRE1α or XBP1s, or pharmacologic IRE1α kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice and augmented efficacy of two established frontline antimyeloma agents, bortezomib and lenalidomide. Mechanistically, IRE1α perturbation inhibited expression of key components of the endoplasmic reticulum-associated degradation machinery, as well as secretion of Ig light chains and of cytokines and chemokines known to promote MM growth. Selective IRE1α kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138- cells derived from bone marrows of newly diagnosed or posttreatment-relapsed MM patients, in both US- and European Union-based cohorts. Effective IRE1α inhibition preserved glucose-induced insulin secretion by pancreatic microislets and viability of primary hepatocytes in vitro, as well as normal tissue homeostasis in mice. These results establish a strong rationale for developing kinase-directed inhibitors of IRE1α for MM therapy.


Subject(s)
Endoribonucleases/genetics , Multiple Myeloma/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/genetics , Aged , Animals , Bortezomib/pharmacology , Endoplasmic Reticulum Stress/genetics , Endoribonucleases/antagonists & inhibitors , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lenalidomide/pharmacology , Male , Mice , Middle Aged , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Signal Transduction/drug effects , Unfolded Protein Response/genetics , X-Box Binding Protein 1/genetics , Xenograft Model Antitumor Assays
4.
J Neurosci ; 40(9): 1956-1974, 2020 02 26.
Article in English | MEDLINE | ID: mdl-31980586

ABSTRACT

TREM2 is an Alzheimer's disease (AD) risk gene expressed in microglia. To study the role of Trem2 in a mouse model of ß-amyloidosis, we compared PS2APP transgenic mice versus PS2APP mice lacking Trem2 (PS2APP;Trem2ko) at ages ranging from 4 to 22 months. Microgliosis was impaired in PS2APP;Trem2ko mice, with Trem2-deficient microglia showing compromised expression of proliferation/Wnt-related genes and marked accumulation of ApoE. Plaque abundance was elevated in PS2APP;Trem2ko females at 6-7 months; but by 12 or 19-22 months of age, it was notably diminished in female and male PS2APP;Trem2ko mice, respectively. Across all ages, plaque morphology was more diffuse in PS2APP;Trem2ko brains, and the Aß42:Aß40 ratio was elevated. The amount of soluble, fibrillar Aß oligomers also increased in PS2APP;Trem2ko hippocampi. Associated with these changes, axonal dystrophy was exacerbated from 6 to 7 months onward in PS2APP;Trem2ko mice, notwithstanding the reduced plaque load at later ages. PS2APP;Trem2ko mice also exhibited more dendritic spine loss around plaque and more neurofilament light chain in CSF. Thus, aggravated neuritic dystrophy is a more consistent outcome of Trem2 deficiency than amyloid plaque load, suggesting that the microglial packing of Aß into dense plaque is an important neuroprotective activity.SIGNIFICANCE STATEMENT Genetic studies indicate that TREM2 gene mutations confer increased Alzheimer's disease (AD) risk. We studied the effects of Trem2 deletion in the PS2APP mouse AD model, in which overproduction of Aß peptide leads to amyloid plaque formation and associated neuritic dystrophy. Interestingly, neuritic dystrophies were intensified in the brains of Trem2-deficient mice, despite these mice displaying reduced plaque accumulation at later ages (12-22 months). Microglial clustering around plaques was impaired, plaques were more diffuse, and the Aß42:Aß40 ratio and amount of soluble, fibrillar Aß oligomers were elevated in Trem2-deficient brains. These results suggest that the Trem2-dependent compaction of Aß into dense plaques is a protective microglial activity, limiting the exposure of neurons to toxic Aß species.


Subject(s)
Alzheimer Disease/genetics , Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Axons/pathology , Dendritic Spines/pathology , Membrane Glycoproteins/genetics , Peptide Fragments/metabolism , Plaque, Amyloid/genetics , Receptors, Immunologic/genetics , Trefoil Factor-1/metabolism , Animals , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microglia/pathology , Neurites/pathology , Neurofilament Proteins/cerebrospinal fluid , Plaque, Amyloid/pathology
5.
Proc Natl Acad Sci U S A ; 112(18): 5679-84, 2015 May 05.
Article in English | MEDLINE | ID: mdl-25902490

ABSTRACT

TNF superfamily death ligands are expressed on the surface of immune cells and can trigger apoptosis in susceptible cancer cells by engaging cognate death receptors. A recombinant soluble protein comprising the ectodomain of Apo2 ligand/TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) has shown remarkable preclinical anticancer activity but lacked broad efficacy in patients, possibly owing to insufficient exposure or potency. We observed that antibody cross-linking substantially enhanced cytotoxicity of soluble Apo2L/TRAIL against diverse cancer cell lines. Presentation of the ligand on glass-supported lipid bilayers enhanced its ability to drive receptor microclustering and apoptotic signaling. Furthermore, covalent surface attachment of Apo2L/TRAIL onto liposomes--synthetic lipid-bilayer nanospheres--similarly augmented activity. In vivo, liposome-displayed Apo2L/TRAIL achieved markedly better exposure and antitumor activity. Thus, covalent synthetic-membrane attachment of a cell-surface ligand enhances efficacy, increasing therapeutic potential. These findings have translational implications for liposomal approaches as well as for Apo2L/TRAIL and other clinically relevant TNF ligands.


Subject(s)
Antineoplastic Agents/chemistry , Cell Membrane/metabolism , TNF-Related Apoptosis-Inducing Ligand/metabolism , Animals , Apoptosis , Biotinylation , CD27 Ligand/metabolism , Caspase 8/metabolism , Caspases/metabolism , Cell Line, Tumor , Epitopes/chemistry , Fas Ligand Protein/metabolism , Humans , Immune System , Immunotherapy/methods , Inhibitory Concentration 50 , Ligands , Liposomes/chemistry , Mice , Mice, Nude , Microscopy, Fluorescence , Neoplasm Transplantation , Neoplasms/immunology , Neoplasms/metabolism , Recombinant Proteins/metabolism
6.
J Neurosci ; 34(24): 8277-88, 2014 Jun 11.
Article in English | MEDLINE | ID: mdl-24920631

ABSTRACT

Extensive evidence implicates GluN2B-containing NMDA receptors (GluN2B-NMDARs) in excitotoxic-insult-induced neurodegeneration and amyloid ß (Aß)-induced synaptic dysfunction. Therefore, inhibiting GluN2B-NMDARs would appear to be a potential therapeutic strategy to provide neuroprotection and improve cognitive function in Alzheimer's disease (AD). However, there are no reports of long-term in vivo treatment of AD mouse models with GluN2B antagonists. We used piperidine18 (Pip18), a potent and selective GluN2B-NMDAR antagonist with favorable pharmacokinetic properties, for long-term dosing in AD mouse models. Reduced freezing behavior in Tg2576 mice during fear conditioning was partially reversed after subchronic (17 d) Pip18 treatment. However, analysis of freezing behavior in different contexts indicated that this increased freezing likely involves elevated anxiety or excessive memory generalization in both nontransgenic (NTG) and Tg2576 mice. In PS2APP mice chronically fed with medicated food containing Pip18 for 4 months, spatial learning and memory deficits were not rescued, plaque-associated spine loss was not affected, and synaptic function was not altered. At the same time, altered open field activity consistent with increased anxiety and degraded performance in an active avoidance task were observed in NTG after chronic treatment. These results indicate that long-term treatment with a GluN2B-NMDAR antagonist does not provide a disease-modifying benefit and could cause cognitive liabilities rather than symptomatic benefit in AD mouse models. Therefore, these results challenge the expectation of the therapeutic potential for GluN2B-NMDAR antagonists in AD.


Subject(s)
Alzheimer Disease/complications , Attention Deficit and Disruptive Behavior Disorders/chemically induced , Memory Disorders/etiology , Memory Disorders/pathology , Synapses/pathology , Alzheimer Disease/genetics , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Animals , Behavior, Animal/drug effects , Disease Models, Animal , Female , HEK293 Cells , Humans , In Vitro Techniques , Male , Maze Learning/drug effects , Maze Learning/physiology , Membrane Potentials/drug effects , Membrane Potentials/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutation/genetics , Piperidines/pharmacology
7.
J Neurosci ; 34(19): 6425-37, 2014 May 07.
Article in English | MEDLINE | ID: mdl-24806669

ABSTRACT

Recent studies implicate death receptor 6 (DR6) in an amyloid precursor protein (APP)-dependent pathway regulating developmental axon pruning, and in a pruning pathway operating during plastic rearrangements in adult brain. DR6 has also been suggested to mediate toxicity in vitro of Aß peptides derived from APP. Given the link between APP, Aß, and Alzheimer's disease (AD), these findings have raised the possibility that DR6 contributes to aspects of neurodegeneration in AD. To test this possibility, we have used mouse models to characterize potential function(s) of DR6 in the adult CNS and in AD-related pathophysiology. We show that DR6 is broadly expressed within the adult CNS and regulates the density of excitatory synaptic connections onto pyramidal neurons in a genetic pathway with APP. DR6 knock-out also gives rise to behavioral abnormalities, some of which are similar to those previously documented in APP knock-out animals. However, in two distinct APP transgenic models of AD, we did not observe any alteration in the formation of amyloid plaques, gliosis, synaptic loss, or cognitive behavioral deficits with genetic deletion of DR6, though we did observe a transient reduction in the degree of microglial activation in one model. Our results support the view that DR6 functions with APP to modulate synaptic density in the adult CNS, but do not provide evidence for a role of DR6 in the pathophysiology of AD.


Subject(s)
Alzheimer Disease/physiopathology , Amyloid beta-Protein Precursor/physiology , Central Nervous System/cytology , Receptors, Tumor Necrosis Factor/physiology , Signal Transduction/physiology , Synapses/physiology , Alzheimer Disease/pathology , Animals , Avoidance Learning/physiology , Central Nervous System/growth & development , Conditioning, Operant/physiology , Dendritic Spines/physiology , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Fear/psychology , Gliosis/pathology , Humans , In Situ Hybridization , Maze Learning/physiology , Mice , Mice, Inbred C57BL , Motor Activity/physiology , Neural Pathways/physiology , Plaque, Amyloid/pathology
8.
Cell Rep Methods ; 4(1): 100673, 2024 Jan 22.
Article in English | MEDLINE | ID: mdl-38171361

ABSTRACT

While antisense oligonucleotides (ASOs) are used in the clinic, therapeutic development is hindered by the inability to assay ASO delivery and activity in vivo. Accordingly, we developed a dual-fluorescence, knockin mouse model that constitutively expresses mKate2 and an engineered EGFP that is alternatively spliced in the presence of ASO to induce expression. We first examined free ASO activity in the brain following intracerebroventricular injection revealing EGFP splice-switching is both ASO concentration and time dependent in major central nervous system cell types. We then assayed the impact of lipid nanoparticle delivery on ASO activity after intravenous administration. Robust EGFP fluorescence was observed in the liver and EGFP+ cells were successfully isolated using fluorescence-activated cell sorting. Together, these results show the utility of this animal model in quantifying both cell-type- and organ-specific ASO delivery, which can be used to advance ASO therapeutics for many disease indications.


Subject(s)
Oligonucleotides, Antisense , Oligonucleotides , Mice , Animals , Liver/metabolism , Administration, Intravenous , Coloring Agents/metabolism
9.
J Neurosci ; 32(28): 9677-89, 2012 Jul 11.
Article in English | MEDLINE | ID: mdl-22787053

ABSTRACT

Passive immunization against ß-amyloid (Aß) has become an increasingly desirable strategy as a therapeutic treatment for Alzheimer's disease (AD). However, traditional passive immunization approaches carry the risk of Fcγ receptor-mediated overactivation of microglial cells, which may contribute to an inappropriate proinflammatory response leading to vasogenic edema and cerebral microhemorrhage. Here, we describe the generation of a humanized anti-Aß monoclonal antibody of an IgG4 isotype, known as MABT5102A (MABT). An IgG4 subclass was selected to reduce the risk of Fcγ receptor-mediated overactivation of microglia. MABT bound with high affinity to multiple forms of Aß, protected against Aß1-42 oligomer-induced cytotoxicity, and increased uptake of neurotoxic Aß oligomers by microglia. Furthermore, MABT-mediated amyloid plaque removal was demonstrated using in vivo live imaging in hAPP((V717I))/PS1 transgenic mice. When compared with a human IgG1 wild-type subclass, containing the same antigen-binding variable domains and with equal binding to Aß, MABT showed reduced activation of stress-activated p38MAPK (p38 mitogen-activated protein kinase) in microglia and induced less release of the proinflammatory cytokine TNFα. We propose that a humanized IgG4 anti-Aß antibody that takes advantage of a unique Aß binding profile, while also possessing reduced effector function, may provide a safer therapeutic alternative for passive immunotherapy for AD. Data from a phase I clinical trial testing MABT is consistent with this hypothesis, showing no signs of vasogenic edema, even in ApoE4 carriers.


Subject(s)
Alzheimer Disease/therapy , Amyloid beta-Peptides/immunology , Immunoglobulin G/pharmacology , Microglia/drug effects , Microglia/metabolism , Neuroprotective Agents/pharmacology , Peptide Fragments/metabolism , Aged , Aged, 80 and over , Alzheimer Disease/blood , Alzheimer Disease/immunology , Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Animals , Animals, Newborn , CX3C Chemokine Receptor 1 , Cells, Cultured , Cerebral Cortex/cytology , Disease Models, Animal , Dose-Response Relationship, Drug , Dose-Response Relationship, Immunologic , Double-Blind Method , Enzyme-Linked Immunosorbent Assay , Female , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Gene Expression Regulation/immunology , Green Fluorescent Proteins/genetics , Hippocampus/cytology , Humans , Immunoglobulin G/metabolism , Male , Mice , Mice, Transgenic , Microscopy, Confocal , Middle Aged , Mutation/genetics , Neurons/drug effects , Neurons/metabolism , Neuroprotective Agents/metabolism , Plaque, Amyloid/immunology , Plaque, Amyloid/metabolism , Plaque, Amyloid/pathology , Presenilin-1/genetics , Protein Binding/drug effects , Rats , Rats, Sprague-Dawley , Receptors, Chemokine/genetics , Statistics, Nonparametric , Time Factors , Tumor Necrosis Factor-alpha/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism
10.
Nat Commun ; 14(1): 4703, 2023 08 05.
Article in English | MEDLINE | ID: mdl-37543621

ABSTRACT

TGFß signaling is associated with non-response to immune checkpoint blockade in patients with advanced cancers, particularly in the immune-excluded phenotype. While previous work demonstrates that converting tumors from excluded to inflamed phenotypes requires attenuation of PD-L1 and TGFß signaling, the underlying cellular mechanisms remain unclear. Here, we show that TGFß and PD-L1 restrain intratumoral stem cell-like CD8 T cell (TSCL) expansion and replacement of progenitor-exhausted and dysfunctional CD8 T cells with non-exhausted T effector cells in the EMT6 tumor model in female mice. Upon combined TGFß/PD-L1 blockade IFNγhi CD8 T effector cells show enhanced motility and accumulate in the tumor. Ensuing IFNγ signaling transforms myeloid, stromal, and tumor niches to yield an immune-supportive ecosystem. Blocking IFNγ abolishes the anti-PD-L1/anti-TGFß therapy efficacy. Our data suggest that TGFß works with PD-L1 to prevent TSCL expansion and replacement of exhausted CD8 T cells, thereby maintaining the T cell compartment in a dysfunctional state.


Subject(s)
B7-H1 Antigen , Breast Neoplasms , CD8-Positive T-Lymphocytes , Immune Checkpoint Inhibitors , Transforming Growth Factor beta , Female , Animals , Mice , Cell Differentiation , CD8-Positive T-Lymphocytes/immunology , Stem Cells , B7-H1 Antigen/antagonists & inhibitors , Transforming Growth Factor beta/antagonists & inhibitors , Interferon-gamma/immunology , T-Cell Exhaustion , Immune Checkpoint Inhibitors/pharmacology , Mice, Inbred BALB C , Cell Line, Tumor , Breast Neoplasms/drug therapy , Breast Neoplasms/immunology , RNA-Seq
11.
Arterioscler Thromb Vasc Biol ; 31(2): 270-9, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21071686

ABSTRACT

OBJECTIVE: To quantitatively compare aortic curvature and motion with resulting aneurysm location, direction of expansion, and pathophysiological features in experimental abdominal aortic aneurysms (AAAs). METHODS AND RESULTS: MRI was performed at 4.7 T with the following parameters: (1) 3D acquisition for vessel geometry and (2) 2D cardiac-gated acquisition to quantify luminal motion. Male 24-week-old mice were imaged before and after AAA formation induced by angiotensin II (AngII)-filled osmotic pump implantation or infusion of elastase. AngII-induced AAAs formed near the location of maximum abdominal aortic curvature, and the leftward direction of expansion was correlated with the direction of suprarenal aortic motion. Elastase-induced AAAs formed in a region of low vessel curvature and had no repeatable direction of expansion. AngII significantly increased mean blood pressure (22.7 mm Hg, P<0.05), whereas both models showed a significant 2-fold decrease in aortic cyclic strain (P<0.05). Differences in patterns of elastin degradation and localization of fluorescent signal from protease-activated probes were also observed. CONCLUSIONS: The direction of AngII aneurysm expansion correlated with the direction of motion, medial elastin dissection, and adventitial remodeling. Anterior infrarenal aortic motion correlated with medial elastin degradation in elastase-induced aneurysms. Results from both models suggest a relationship between aneurysm pathological features and aortic geometry and motion.


Subject(s)
Angiotensin II/adverse effects , Aorta, Abdominal/pathology , Aorta, Abdominal/physiopathology , Aortic Aneurysm, Abdominal/chemically induced , Aortic Aneurysm, Abdominal/physiopathology , Animals , Aorta, Abdominal/diagnostic imaging , Aortic Aneurysm, Abdominal/pathology , Biomechanical Phenomena , Blood Pressure/physiology , Disease Progression , Elastin/metabolism , Hypertension/physiopathology , Magnetic Resonance Imaging , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Animal , Models, Cardiovascular , Ultrasonography
12.
Cell Rep Med ; 2(8): 100381, 2021 08 17.
Article in English | MEDLINE | ID: mdl-34467254

ABSTRACT

Anti-integrins are therapeutically effective for inflammatory bowel disease, yet the relative contribution of α4ß7 and αEß7 to gut lymphocyte trafficking is not fully elucidated. Here, we evaluate the effect of α4ß7 and αEß7 blockade using a combination of murine models of gut trafficking and longitudinal gene expression analysis in etrolizumab-treated patients with Crohn's disease (CD). Dual blockade of α4ß7 and αEß7 reduces CD8+ T cell accumulation in the gut to a greater extent than blockade of either integrin alone. Anti-αEß7 reduces epithelial:T cell interactions and promotes egress of activated T cells from the mucosa into lymphatics. Inflammatory gene expression is greater in human intestinal αEß7+ T cells. Etrolizumab-treated patients with CD display a treatment-specific reduction in inflammatory and cytotoxic intraepithelial lymphocytes (IEL) genes. Concurrent blockade of α4ß7 and αEß7 promotes reduction of cytotoxic IELs and inflammatory T cells in the gut mucosa through a stepwise inhibition of intestinal tissue entry and retention.


Subject(s)
Inflammatory Bowel Diseases/immunology , Integrins/metabolism , Lymphocytes/immunology , Animals , Antibodies, Monoclonal, Humanized/pharmacology , Biopsy , CD8-Positive T-Lymphocytes , Cadherins/metabolism , Cell Communication , Cell Movement , Colon/pathology , Epitopes/immunology , Female , Gene Expression Regulation/drug effects , Inflammation/complications , Inflammation/pathology , Inflammatory Bowel Diseases/complications , Inflammatory Bowel Diseases/pathology , Intestinal Mucosa/drug effects , Intestinal Mucosa/immunology , Intestinal Mucosa/pathology , Lymph Nodes/pathology , Mice, Inbred C57BL , Mice, Transgenic , T-Lymphocytes, Cytotoxic/drug effects
13.
J Exp Med ; 218(4)2021 04 05.
Article in English | MEDLINE | ID: mdl-33620419

ABSTRACT

Despite the development of effective therapies, a substantial proportion of asthmatics continue to have uncontrolled symptoms, airflow limitation, and exacerbations. Transient receptor potential cation channel member A1 (TRPA1) agonists are elevated in human asthmatic airways, and in rodents, TRPA1 is involved in the induction of airway inflammation and hyperreactivity. Here, the discovery and early clinical development of GDC-0334, a highly potent, selective, and orally bioavailable TRPA1 antagonist, is described. GDC-0334 inhibited TRPA1 function on airway smooth muscle and sensory neurons, decreasing edema, dermal blood flow (DBF), cough, and allergic airway inflammation in several preclinical species. In a healthy volunteer Phase 1 study, treatment with GDC-0334 reduced TRPA1 agonist-induced DBF, pain, and itch, demonstrating GDC-0334 target engagement in humans. These data provide therapeutic rationale for evaluating TRPA1 inhibition as a clinical therapy for asthma.


Subject(s)
Asthma/drug therapy , Neurogenic Inflammation/drug therapy , Pain/drug therapy , Pruritus/drug therapy , Pyridines/pharmacology , Pyridines/therapeutic use , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , TRPA1 Cation Channel/antagonists & inhibitors , Adolescent , Adult , Animals , Cohort Studies , Disease Models, Animal , Dogs , Double-Blind Method , Female , Guinea Pigs , Healthy Volunteers , Humans , Isothiocyanates/administration & dosage , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Pain/chemically induced , Pruritus/chemically induced , Rats , Rats, Sprague-Dawley , TRPA1 Cation Channel/deficiency , Treatment Outcome , Young Adult
14.
Commun Biol ; 3(1): 687, 2020 11 19.
Article in English | MEDLINE | ID: mdl-33214666

ABSTRACT

Vascular leakage, or edema, is a serious complication of acute allergic reactions. Vascular leakage is triggered by the release of histamine and serotonin from granules within tissue-resident mast cells. Here, we show that expression of Neutrophil Serine Protease 4 (NSP4) during the early stages of mast cell development regulates mast cell-mediated vascular leakage. In myeloid precursors, the granulocyte-macrophage progenitors (GMPs), loss of NSP4 results in the decrease of cellular levels of histamine, serotonin and heparin/heparan sulfate. Mast cells that are derived from NSP4-deficient GMPs have abnormal secretory granule morphology and a sustained reduction in histamine and serotonin levels. Consequently, in passive cutaneous anaphylaxis and acute arthritis models, mast cell-mediated vascular leakage in the skin and joints is substantially reduced in NSP4-deficient mice. Our findings reveal that NSP4 is required for the proper storage of vasoactive amines in mast cell granules, which impacts mast cell-dependent vascular leakage in mouse models of immune complex-mediated diseases.


Subject(s)
Mast Cells/enzymology , Serine Proteases/metabolism , Adoptive Transfer , Animals , Antigen-Antibody Complex , Gene Expression Regulation, Enzymologic , Histamine/metabolism , Mice , Mice, Knockout , Neutrophils , Serine Proteases/genetics , Serotonin/metabolism
15.
Cancer Res ; 67(10): 4924-32, 2007 May 15.
Article in English | MEDLINE | ID: mdl-17510422

ABSTRACT

MUC16 is a well-validated cell surface marker for serous adenocarcinomas of the ovary and other gynecologic malignancies that is distinguished by highly repetitive sequences ("mucin repeats") in the extracellular domain (ECD). We produced and compared two monoclonal antibodies: one (11D10) recognizing a unique, nonrepeating epitope in the ECD and another (3A5) that recognizes the repeats and binds multiple sites on each MUC16 protein. 3A5 conjugated to cytotoxic drugs exhibited superior toxicity against tumor cells in vitro and in tumor xenograft models compared with antibody-drug conjugates of 11D10. Importantly, drug conjugates of 3A5 were well tolerated in primates at levels in excess of therapeutic doses. Additionally, the presence of circulating CA125 in a rat model did not exacerbate the toxicity of 3A5 drug conjugates. We conclude that targeting the repeat MUC16 domains, thereby increasing cell-associated levels of drug-conjugated antibody, provides superior efficacy in vitro and in vivo without compromising safety.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , CA-125 Antigen/immunology , Membrane Proteins/immunology , Ovarian Neoplasms/immunology , Ovarian Neoplasms/therapy , Animals , Binding Sites, Antibody , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred ICR , Mice, SCID , Rats , Xenograft Model Antitumor Assays
16.
Cell Rep ; 28(8): 2111-2123.e6, 2019 08 20.
Article in English | MEDLINE | ID: mdl-31433986

ABSTRACT

Complement pathway overactivation can lead to neuronal damage in various neurological diseases. Although Alzheimer's disease (AD) is characterized by ß-amyloid plaques and tau tangles, previous work examining complement has largely focused on amyloidosis models. We find that glial cells show increased expression of classical complement components and the central component C3 in mouse models of amyloidosis (PS2APP) and more extensively tauopathy (TauP301S). Blocking complement function by deleting C3 rescues plaque-associated synapse loss in PS2APP mice and ameliorates neuron loss and brain atrophy in TauP301S mice, improving neurophysiological and behavioral measurements. In addition, C3 protein is elevated in AD patient brains, including at synapses, and levels and processing of C3 are increased in AD patient CSF and correlate with tau. These results demonstrate that complement activation contributes to neurodegeneration caused by tau pathology and suggest that blocking C3 function might be protective in AD and other tauopathies.


Subject(s)
Alzheimer Disease/immunology , Amyloidosis/immunology , Complement C3/metabolism , Nerve Degeneration/immunology , Tauopathies/immunology , Alzheimer Disease/genetics , Animals , Atrophy , Behavior, Animal , Biomarkers/metabolism , Brain/pathology , Complement C1q/metabolism , Complement C3/cerebrospinal fluid , Complement C3/genetics , Disease Models, Animal , Female , Gene Deletion , Gene Expression Regulation , Humans , Male , Mice, Transgenic , Nerve Degeneration/genetics , Neurons/metabolism , Neurons/pathology , Plaque, Amyloid/metabolism , Synapses/metabolism
17.
Cancer Res ; 66(2): 999-1006, 2006 Jan 15.
Article in English | MEDLINE | ID: mdl-16424035

ABSTRACT

The usual paradigm for developing kinase inhibitors in oncology is to use a high-affinity proof-of-concept inhibitor with acceptable metabolic properties for key target validation experiments. This approach requires substantial medicinal chemistry and can be confounded by drug toxicity and off-target activities of the test molecule. As a better alternative, we have developed inducible short-hairpin RNA xenograft models to examine the in vivo efficacy of inhibiting oncogenic BRAF. Our results show that tumor regression resulting from BRAF suppression is inducible, reversible, and tightly regulated in these models. Analysis of regressing tumors showed the primary mechanism of action for BRAF to be increased tumor cell proliferation and survival. In a metastatic melanoma model, conditional BRAF suppression slowed systemic tumor growth as determined by in vivo bioluminescence imaging. Taken together, gain-of-function BRAF signaling is strongly associated with in vivo tumorigenicity, confirming BRAF as an important target for small-molecule and RNA interference-based therapeutics.


Subject(s)
Melanoma/pathology , Proto-Oncogene Proteins B-raf/biosynthesis , Proto-Oncogene Proteins B-raf/physiology , Skin Neoplasms/pathology , Animals , Cell Proliferation , Disease Models, Animal , Down-Regulation , Female , Humans , Melanoma/genetics , Mice , Mice, Nude , Neoplasm Metastasis , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , RNA Interference , Signal Transduction , Skin Neoplasms/genetics , Transplantation, Heterologous
18.
BMC Biotechnol ; 7: 61, 2007 Sep 26.
Article in English | MEDLINE | ID: mdl-17897455

ABSTRACT

BACKGROUND: Conditional expression vectors have become a valuable research tool to avoid artefacts that may result from traditional gene expression studies. However, most systems require multiple plasmids that must be independently engineered into the target system, resulting in experimental delay and an increased potential for selection of a cell subpopulation that differs significantly from the parental line. We have therefore developed pHUSH, an inducible expression system that allows regulated expression of shRNA, miRNA or cDNA cassettes on a single viral vector. RESULTS: Both Pol II and Pol III promoters have been successfully combined with a second expression cassette containing a codon-optimized tetracycline repressor and selectable marker. We provide examples of how pHUSH has been successfully employed to study the function of target genes in a number of cell types within in vitro and in vivo assays, including conditional gene knockdown in a murine model of brain cancer. CONCLUSION: We have successfully developed and employed a single vector system that enables Doxycycline regulated RNAi or transgene expression in a variety of in vitro and in vivo model systems. These studies demonstrate the broad application potential of pHUSH for conditional genetic engineering in mammalian cells.


Subject(s)
Gene Expression/genetics , Gene Targeting/methods , Genetic Vectors/genetics , Protein Engineering/methods , Recombinant Proteins/genetics , Transfection/methods
19.
Sci Transl Med ; 9(403)2017 Aug 16.
Article in English | MEDLINE | ID: mdl-28814543

ABSTRACT

Hallmarks of chronic neurodegenerative disease include progressive synaptic loss and neuronal cell death, yet the cellular pathways that underlie these processes remain largely undefined. We provide evidence that dual leucine zipper kinase (DLK) is an essential regulator of the progressive neurodegeneration that occurs in amyotrophic lateral sclerosis and Alzheimer's disease. We demonstrate that DLK/c-Jun N-terminal kinase signaling was increased in mouse models and human patients with these disorders and that genetic deletion of DLK protected against axon degeneration, neuronal loss, and functional decline in vivo. Furthermore, pharmacological inhibition of DLK activity was sufficient to attenuate the neuronal stress response and to provide functional benefit even in the presence of ongoing disease. These findings demonstrate that pathological activation of DLK is a conserved mechanism that regulates neurodegeneration and suggest that DLK inhibition may be a potential approach to treat multiple neurodegenerative diseases.


Subject(s)
Leucine Zippers , MAP Kinase Kinase Kinases/metabolism , Neurodegenerative Diseases/enzymology , Neurodegenerative Diseases/pathology , Signal Transduction , Alzheimer Disease/enzymology , Alzheimer Disease/pathology , Amyotrophic Lateral Sclerosis/enzymology , Amyotrophic Lateral Sclerosis/pathology , Animals , Disease Models, Animal , Gene Deletion , Gene Expression Regulation/drug effects , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , MAP Kinase Signaling System , Mice, Transgenic , Neuroprotection , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Spinal Cord/enzymology , Spinal Cord/pathology , Superoxide Dismutase/metabolism
20.
Brain Res ; 945(1): 139-43, 2002 Jul 26.
Article in English | MEDLINE | ID: mdl-12113962

ABSTRACT

Aversion to capsaicin (0.1-10 ppm) was assessed using a two-bottle paired preference paradigm, before and after intracisternal injection of substance P conjugated to saporin (SP-SAP) to ablate neurons in superficial medullary and cervical dorsal horn that express NK-1 receptors. Before SP-SAP, there was a concentration-dependent decrease in consumption of capsaicin with a threshold of 0.1-0.3 ppm. Following SP-SAP, significantly more capsaicin solution was consumed at 1- and 10-ppm concentrations. These results support a role for substance P in the mediation of high, but not low, levels of capsaicin-induced oral irritation.


Subject(s)
Capsaicin/administration & dosage , Irritants/administration & dosage , Medulla Oblongata/physiopathology , Neurons/physiology , Pain/chemically induced , Pain/physiopathology , Receptors, Neurokinin-1/metabolism , Administration, Oral , Animals , Cervical Vertebrae , Injections, Intraventricular , Male , Medulla Oblongata/pathology , Pain Threshold/physiology , Rats , Rats, Sprague-Dawley , Solutions , Spinal Cord/pathology , Spinal Cord/physiopathology , Substance P/administration & dosage , Substance P/pharmacology
SELECTION OF CITATIONS
SEARCH DETAIL