Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 13 de 13
Filter
Add more filters










Publication year range
1.
Cancers (Basel) ; 16(2)2024 Jan 12.
Article in English | MEDLINE | ID: mdl-38254822

ABSTRACT

Treatment options for ovarian cancer patients are limited, and a high unmet clinical need remains for targeted and long-lasting, efficient drugs. Genetically modified T cells expressing chimeric antigen receptors (CAR), are promising new drugs that can be directed towards a defined target and have shown efficient, as well as persisting, anti-tumor responses in many patients. We sought to develop novel CAR T cells targeting ovarian cancer and to assess these candidates preclinically. First, we identified potential CAR targets on ovarian cancer samples. We confirmed high and consistent expressions of the tumor-associated antigen FOLR1 on primary ovarian cancer samples. Subsequently, we designed a series of CAR T cell candidates against the identified target and demonstrated their functionality against ovarian cancer cell lines in vitro and in an in vivo xenograft model. Finally, we performed additional in vitro assays recapitulating immune suppressive mechanisms present in solid tumors and developed a process for the automated manufacturing of our CAR T cell candidate. These findings demonstrate the feasibility of anti-FOLR1 CAR T cells for ovarian cancer and potentially other FOLR1-expressing tumors.

2.
Brain ; 144(10): 3226-3238, 2021 11 29.
Article in English | MEDLINE | ID: mdl-33964142

ABSTRACT

Axonal degeneration is an early and ongoing event that causes disability and disease progression in many neurodegenerative disorders of the peripheral and central nervous systems. Chemotherapy-induced peripheral neuropathy (CIPN) is a major cause of morbidity and the main cause of dose reductions and discontinuations in cancer treatment. Preclinical evidence indicates that activation of the Wallerian-like degeneration pathway driven by sterile alpha and TIR motif containing 1 (SARM1) is responsible for axonopathy in CIPN. SARM1 is the central driver of an evolutionarily conserved programme of axonal degeneration downstream of chemical, inflammatory, mechanical or metabolic insults to the axon. SARM1 contains an intrinsic NADase enzymatic activity essential for its pro-degenerative functions, making it a compelling therapeutic target to treat neurodegeneration characterized by axonopathies of the peripheral and central nervous systems. Small molecule SARM1 inhibitors have the potential to prevent axonal degeneration in peripheral and central axonopathies and to provide a transformational disease-modifying treatment for these disorders. Using a biochemical assay for SARM1 NADase we identified a novel series of potent and selective irreversible isothiazole inhibitors of SARM1 enzymatic activity that protected rodent and human axons in vitro. In sciatic nerve axotomy, we observed that these irreversible SARM1 inhibitors decreased a rise in nerve cADPR and plasma neurofilament light chain released from injured sciatic nerves in vivo. In a mouse paclitaxel model of CIPN we determined that Sarm1 knockout mice prevented loss of axonal function, assessed by sensory nerve action potential amplitudes of the tail nerve, in a gene-dosage-dependent manner. In that CIPN model, the irreversible SARM1 inhibitors prevented loss of intraepidermal nerve fibres induced by paclitaxel and provided partial protection of axonal function assessed by sensory nerve action potential amplitude and mechanical allodynia.


Subject(s)
Armadillo Domain Proteins/antagonists & inhibitors , Axons/drug effects , Cytoskeletal Proteins/antagonists & inhibitors , Paclitaxel/toxicity , Peripheral Nervous System Diseases/chemically induced , Peripheral Nervous System Diseases/drug therapy , Thiazoles/therapeutic use , Animals , Antineoplastic Agents, Phytogenic/toxicity , Armadillo Domain Proteins/deficiency , Armadillo Domain Proteins/genetics , Axons/metabolism , Cells, Cultured , Cytoskeletal Proteins/deficiency , Cytoskeletal Proteins/genetics , Dose-Response Relationship, Drug , HEK293 Cells , Humans , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Mice , Mice, Knockout , Peripheral Nervous System Diseases/genetics , Peripheral Nervous System Diseases/metabolism , Thiazoles/pharmacology
3.
Biotechnol Rep (Amst) ; 17: 110-116, 2018 Mar.
Article in English | MEDLINE | ID: mdl-29541604

ABSTRACT

Polysialic acid (polySia) is a promising molecule for various medical applications (e.g., treatment of inflammatory neurodegenerative diseases). In this study a complete production process for human-identical α-(2,8)-linked polySia was developed using a disposable bioreactor for cultivation of Escherichia coli K1 and single-use membrane adsorbers for downstream processing (DSP). The cultivation process was optimized to minimize complex media components and a maturation process after cultivation was established. The maturation led to further product release from the cell surface into the supernatant. Afterwards DSP was established using sodium hydroxide treatment combined with anion exchange membrane adsorbers for endotoxin and DNA depletion. After downstream processing the final product had neither detectable protein nor DNA contamination. Endotoxin content was below 3 EU mg-1. Investigation of the maximal chain length showed no effect of the harsh sodium hydroxide treatment during DSP on the stability of the polySia. Maximal chain length was ∼98 degree of polymerization.

4.
Eng Life Sci ; 17(7): 723-731, 2017 Jul.
Article in English | MEDLINE | ID: mdl-32624817

ABSTRACT

Polysialic acid (polySia), consisting of α-(2,8)-linked N-acetylneuraminic acid monomers plays a crucial role in many biological processes. This study presents a novel process for the production of endogenous polySia using Escherichia coli K1 in a disposable bag reactor with wave-induced mixing. Disposable bag reactors provide easy and fast production in terms of regulatory requirements as GMP, flexibility, and can easily be adjusted to larger production capacities not only by scale up but also by parallelization. Due to the poor oxygen transfer rate compared to a stirred tank reactor, pure oxygen was added during the cultivation to avoid oxygen limitation. During the exponential growth phase the growth rate was 0.61 h-1. Investigation of stress-related product release from the cell surface showed no significant differences between the disposable bag reactor with wave-induced mixing and the stirred tank reactor. After batch cultivation a cell dry weight of 6.8 g L-1 and a polySia concentration of 245 mg L-1 were reached. The total protein concentration in the supernatant was 132 mg L-1. After efficient and time-saving downstream processing characterization of the final product showed a protein content of below 0.04 mgprotein/gpolySia and a maximal chain length of ∼90 degree of polymerization.

5.
EMBO Mol Med ; 9(2): 154-166, 2017 02.
Article in English | MEDLINE | ID: mdl-28003336

ABSTRACT

Age-related macular degeneration (AMD) is a major cause of blindness in the elderly population. Its pathophysiology is linked to reactive oxygen species (ROS) and activation of the complement system. Sialic acid polymers prevent ROS production of human mononuclear phagocytes via the inhibitory sialic acid-binding immunoglobulin-like lectin-11 (SIGLEC11) receptor. Here, we show that low-dose intravitreal injection of low molecular weight polysialic acid with average degree of polymerization 20 (polySia avDP20) in humanized transgenic mice expressing SIGLEC11 on mononuclear phagocytes reduced their reactivity and vascular leakage induced by laser coagulation. Furthermore, polySia avDP20 prevented deposition of the membrane attack complex in both SIGLEC11 transgenic and wild-type animals. In vitro, polySia avDP20 showed two independent, but synergistic effects on the innate immune system. First, polySia avDP20 prevented tumor necrosis factor-α, vascular endothelial growth factor A, and superoxide production by SIGLEC11-positive phagocytes. Second, polySia avDP20 directly interfered with complement activation. Our data provide evidence that polySia avDP20 ameliorates laser-induced damage in the retina and thus is a promising candidate to prevent AMD-related inflammation and angiogenesis.


Subject(s)
Choroidal Neovascularization/prevention & control , Complement Activation , Immunologic Factors/administration & dosage , Phagocytes/drug effects , Phagocytes/immunology , Retina/injuries , Sialic Acids/administration & dosage , Animals , Humans , Lasers , Lectins/genetics , Lectins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice, SCID , Mice, Transgenic
6.
Glia ; 64(4): 635-49, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26683584

ABSTRACT

Microglia, innate immune cells of the CNS, sense infection and damage through overlapping receptor sets. Toll-like receptor (TLR) 4 recognizes bacterial lipopolysaccharide (LPS) and multiple injury-associated factors. We show that its co-receptor CD14 serves three non-redundant functions in microglia. First, it confers an up to 100-fold higher LPS sensitivity compared to peripheral macrophages to enable efficient proinflammatory cytokine induction. Second, CD14 prevents excessive responses to massive LPS challenges via an interferon ß-mediated feedback. Third, CD14 is mandatory for microglial reactions to tissue damage-associated signals. In mice, these functions are essential for balanced CNS responses to bacterial infection, traumatic and ischemic injuries, since CD14 deficiency causes either hypo- or hyperinflammation, insufficient or exaggerated immune cell recruitment or worsened stroke outcomes. While CD14 orchestrates functions of TLR4 and related immune receptors, it is itself regulated by TLR and non-TLR systems to thereby fine-tune microglial damage-sensing capacity upon infectious and non-infectious CNS challenges.


Subject(s)
Brain Injuries/immunology , Brain Ischemia/immunology , Escherichia coli Infections/metabolism , Lipopolysaccharide Receptors/metabolism , Microglia/immunology , Stroke/immunology , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Brain/immunology , Brain/pathology , Brain Injuries/complications , Brain Injuries/pathology , Brain Ischemia/pathology , Cells, Cultured , Disease Models, Animal , Escherichia coli , Escherichia coli Infections/complications , Escherichia coli Infections/pathology , Feedback, Physiological/physiology , Infarction, Middle Cerebral Artery , Interferon-beta/metabolism , Lipopolysaccharide Receptors/genetics , Lipopolysaccharides/toxicity , Macrophages/immunology , Male , Mice, Inbred C57BL , Mice, Knockout , Neuroimmunomodulation , Stroke/pathology , Toll-Like Receptor 4/agonists , Toll-Like Receptor 4/antagonists & inhibitors , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism
7.
Brain ; 139(Pt 2): 338-45, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26685157

ABSTRACT

Isolated cytochrome c oxidase (complex IV) deficiency is one of the most frequent respiratory chain defects in humans and is usually caused by mutations in proteins required for assembly of the complex. Mutations in nuclear-encoded structural subunits are very rare. In a patient with Leigh-like syndrome presenting with leukodystrophy and severe epilepsy, we identified a homozygous splice site mutation in COX8A, which codes for the ubiquitously expressed isoform of subunit VIII, the smallest nuclear-encoded subunit of complex IV. The mutation, affecting the last nucleotide of intron 1, leads to aberrant splicing, a frame-shift in the highly conserved exon 2, and decreased amount of the COX8A transcript. The loss of the wild-type COX8A protein severely impairs the stability of the entire cytochrome c oxidase enzyme complex and manifests in isolated complex IV deficiency in skeletal muscle and fibroblasts, similar to the frequent c.845_846delCT mutation in the assembly factor SURF1 gene. Stability and activity of complex IV could be rescued in the patient's fibroblasts by lentiviral expression of wild-type COX8A. Our findings demonstrate that COX8A is indispensable for function of human complex IV and its mutation causes human disease.


Subject(s)
Electron Transport Complex IV/genetics , Epilepsy/diagnosis , Epilepsy/genetics , Leigh Disease/diagnosis , Leigh Disease/genetics , Protein Subunits/genetics , Child , Electron Transport Complex IV/physiology , Epilepsy/complications , Fatal Outcome , Female , Humans , Leigh Disease/complications , Mutation/genetics
8.
Sci Rep ; 5: 16800, 2015 Nov 19.
Article in English | MEDLINE | ID: mdl-26582367

ABSTRACT

Oligosialic and polysialic acid (oligoSia and polySia) of the glycocalyx of neural and immune cells are linear chains, in which the sialic acid monomers are α2.8-glycosidically linked. Sialic acid-binding immunoglobulin-like lectin-11 (SIGLEC-11) is a primate-lineage specific receptor of human tissue macrophages and microglia that binds to α2.8-linked oligoSia. Here, we show that soluble low molecular weight polySia with an average degree of polymerization 20 (avDP20) interacts with SIGLEC-11 and acts anti-inflammatory on human THP1 macrophages involving the SIGLEC-11 receptor. Soluble polySia avDP20 inhibited the lipopolysaccharide (LPS)-induced gene transcription and protein expression of tumor necrosis factor-α (Tumor Necrosis Factor Superfamily Member 2, TNFSF2). In addition, polySia avDP20 neutralized the LPS-triggered increase in macrophage phagocytosis, but did not affect basal phagocytosis or endocytosis. Moreover, polySia avDP20 prevented the oxidative burst of human macrophages triggered by neural debris or fibrillary amyloid-ß1-42. In a human macrophage-neuron co-culture system, polySia avDP20 also reduced loss of neurites triggered by fibrillary amyloid-ß1-42. Thus, treatment with polySia avDP20 might be a new anti-inflammatory therapeutic strategy that also prevents the oxidative burst of macrophages.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Macrophages/metabolism , Sialic Acids/pharmacology , Amyloid beta-Peptides/metabolism , Cell Line , Chromatography, High Pressure Liquid , Homeostasis/drug effects , Humans , Lectins/metabolism , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Membrane Proteins/metabolism , Microspheres , Molecular Weight , Neuroprotective Agents/pharmacology , Phagocytosis/drug effects , Polymerization , Respiratory Burst/drug effects , Tumor Necrosis Factor-alpha/biosynthesis
9.
J Neurosci ; 34(25): 8546-56, 2014 Jun 18.
Article in English | MEDLINE | ID: mdl-24948809

ABSTRACT

Systemic inflammatory reactions have been postulated to exacerbate neurodegenerative diseases via microglial activation. We now demonstrate in vivo that repeated systemic challenge of mice over four consecutive days with bacterial LPS maintained an elevated microglial inflammatory phenotype and induced loss of dopaminergic neurons in the substantia nigra. The same total cumulative LPS dose given within a single application did not induce neurodegeneration. Whole-genome transcriptome analysis of the brain demonstrated that repeated systemic LPS application induced an activation pattern involving the classical complement system and its associated phagosome pathway. Loss of dopaminergic neurons induced by repeated systemic LPS application was rescued in complement C3-deficient mice, confirming the involvement of the complement system in neurodegeneration. Our data demonstrate that a phagosomal inflammatory response of microglia is leading to complement-mediated loss of dopaminergic neurons.


Subject(s)
Complement Activation/physiology , Complement C3/metabolism , Complement System Proteins/physiology , Dopaminergic Neurons/metabolism , Microglia/metabolism , Nerve Degeneration/metabolism , Phagosomes/physiology , Animals , Dopaminergic Neurons/pathology , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Microglia/pathology , Nerve Degeneration/pathology , Neural Pathways/physiology , Phagosomes/metabolism , Phagosomes/pathology
10.
Glycobiology ; 24(9): 794-9, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24833613

ABSTRACT

Microglia are the resident immune cells of the central nervous system. They can sense intact or lesioned cells and then respond in an appropriate way. Therefore, microglia need recognition receptors that lead to either the activation or the inhibition of the immune response pathways. Most Siglecs contain an immunoreceptor tyrosine based inhibition motif and its signaling leads to the termination of signals emerging from immunoreceptor tyrosine-based activation motif-signaling receptors. Pro-inflammatory immune responses and phagocytosis are turned down in microglia by inhibitory Siglec signaling. Recently, it was demonstrated that inhibitory Siglecs have neuroprotective effects on cultured neurons by preventing the phagocytosis-associated oxidative burst. Furthermore, microglial mouse Siglec-E and human Siglec-11 have been shown to prevent neurotoxicity via interaction with sialic acid exposed on the neuronal glycocalyx. Thus, Siglecs sensing the intact glycocalyx of neighboring cells keep microglia in a silent homeostatic status.


Subject(s)
Microglia/metabolism , Sialic Acid Binding Immunoglobulin-like Lectins/metabolism , Signal Transduction , Animals , Humans , Microglia/immunology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Receptors, Immunologic/metabolism
11.
Glia ; 61(7): 1122-33, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23633299

ABSTRACT

Sialic-acid-binding immunoglobulin-like lectin-h (Siglec-h) is a recently identified mouse-specific CD33-related Siglec that signals via DAP12/TYROBP. Expression of Siglec-h has been observed on plasmacytoid dendritic cells and microglia, but the ligand and the function of Siglec-h remained elusive. Here, we demonstrate gene transcription and protein expression of Siglec-h by mouse microglia after interferon-γ treatment or polarization into a M1-subtype. Microglial Siglec-h acted as phagocytosis receptor since targeting of microsphere beads to Siglec-h triggered their uptake into the microglia. The extracellular domain of Siglec-h protein bound to mouse glioma lines, but not to astrocytes or other normal mouse cells. Microglial cells stimulated to express Siglec-h engulfed intact glioma cells without prior induction of apoptosis and slightly reduced glioma cell number in culture. Phagocytosis of glioma cells by activated microglia was dependent on Siglec-h and its adapter molecule DAP12. Thus, data show that M1-polarized microglial cells can engulf glioma cells via a DAP12-mediated Siglec-h dependent mechanism.


Subject(s)
Glioma/metabolism , Neuroglia/metabolism , Sialic Acid Binding Ig-like Lectin 1/metabolism , Animals , Animals, Newborn , Annexin A5/metabolism , Apoptosis/genetics , Apoptosis/physiology , Brain/cytology , Cell Polarity/drug effects , Cell Polarity/genetics , Cells, Cultured , Coculture Techniques , Cricetulus , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Glioma/pathology , Humans , Interferon-gamma/pharmacology , Mice , Mice, Inbred C57BL , Neoplasm Invasiveness/genetics , Neuroglia/drug effects , Phagocytosis/physiology , Protein Binding/drug effects , Protein Binding/genetics , Sialic Acid Binding Ig-like Lectin 1/genetics
12.
J Neurosci ; 32(3): 946-52, 2012 Jan 18.
Article in English | MEDLINE | ID: mdl-22262892

ABSTRACT

Microglial cells are professional phagocytes of the CNS responsible for clearance of unwanted structures. Neuronal processes are marked by complement C1 before they are removed in development or during disease processes. Target molecules involved in C1 binding and mechanisms of clearance are still unclear. Here we show that the terminal sugar residue sialic acid of the mouse neuronal glycocalyx determines complement C1 binding and microglial-mediated clearance function. Several early components of the classical complement cascade including C1q, C1r, C1s, and C3 were produced by cultured mouse microglia. The opsonin C1q was binding to neurites after enzymatic removal of sialic acid residues from the neuronal glycocalyx. Desialylated neurites, but not neurites with intact sialic acid caps, were cleared and taken up by cocultured microglial cells. The removal of the desialylated neurites was mediated via the complement receptor-3 (CR3; CD11b/CD18). Data demonstrate that mouse microglial cells via CR3 recognize and remove neuronal structures with an altered neuronal glycocalyx lacking terminal sialic acid.


Subject(s)
Complement C1q/metabolism , Glycocalyx/metabolism , Microglia/metabolism , Neurons/cytology , Receptors, Complement/metabolism , Sialic Acids/metabolism , Analysis of Variance , Animals , Animals, Newborn , Antigens, CD/metabolism , Brain/cytology , CD11b Antigen/metabolism , Cells, Cultured , Coculture Techniques , Complement C1q/pharmacology , Complement C3/genetics , Complement C3/metabolism , Cytokines/pharmacology , Embryo, Mammalian , Female , Flow Cytometry , Gene Expression Regulation/drug effects , Glycocalyx/drug effects , Green Fluorescent Proteins/genetics , Humans , Lipopolysaccharides/pharmacology , Male , Mice , Mice, Inbred C57BL , Microglia/drug effects , Neuraminidase/pharmacology , Neurons/drug effects , Protein Binding/drug effects , Protein Binding/physiology , Receptors, Complement/genetics , Transfection , Tubulin/metabolism
13.
Br Med Bull ; 95: 47-61, 2010.
Article in English | MEDLINE | ID: mdl-20643690

ABSTRACT

INTRODUCTION: Lung cancer is the commonest fatal malignancy in the developed world. Survival rates for lung cancer have not changed significantly over the past 30 years. Sources of data This report is a systematic review of the literature on our current understanding of lung cancer biology. Searches were carried out using PUBMED. 1990-2010. AREAS OF AGREEMENT: A concerted effort to reduce cigarette smoking and nicotine addiction is required. A better understanding of the biology of lung cancer will lead to the identification of earlier diagnostic markers and improved therapy. AREAS OF CONTROVERSY: How chronic inflammatory disorders such as COPD and lung fibrosis contribute to lung cancer development is incompletely understood. GROWING POINTS: Developing novel biological agents to target lung cancer. New microarray-based technologies provide new methods for predicting prognosis and response to treatment. AREAS TIMELY FOR DEVELOPING RESEARCH: Developing strategies to target lung cancer stem cells may provide a novel approach for treating drug resistant disease.


Subject(s)
Anticarcinogenic Agents/therapeutic use , Carcinoma, Bronchogenic/therapy , Lung Neoplasms/therapy , Signal Transduction/genetics , Smoking/adverse effects , Carcinoma, Bronchogenic/genetics , Cell Adhesion , Drug Resistance, Neoplasm/genetics , Humans , Lung Neoplasms/genetics
SELECTION OF CITATIONS
SEARCH DETAIL
...