-
Nature Biotechnology Aug 2020Chimeric antigen receptor (CAR) T cell therapy has shown promise in hematologic malignancies, but its application to solid tumors has been challenging. Given the unique...
Chimeric antigen receptor (CAR) T cell therapy has shown promise in hematologic malignancies, but its application to solid tumors has been challenging. Given the unique effector functions of macrophages and their capacity to penetrate tumors, we genetically engineered human macrophages with CARs to direct their phagocytic activity against tumors. We found that a chimeric adenoviral vector overcame the inherent resistance of primary human macrophages to genetic manipulation and imparted a sustained pro-inflammatory (M1) phenotype. CAR macrophages (CAR-Ms) demonstrated antigen-specific phagocytosis and tumor clearance in vitro. In two solid tumor xenograft mouse models, a single infusion of human CAR-Ms decreased tumor burden and prolonged overall survival. Characterization of CAR-M activity showed that CAR-Ms expressed pro-inflammatory cytokines and chemokines, converted bystander M2 macrophages to M1, upregulated antigen presentation machinery, recruited and presented antigen to T cells and resisted the effects of immunosuppressive cytokines. In humanized mouse models, CAR-Ms were further shown to induce a pro-inflammatory tumor microenvironment and boost anti-tumor T cell activity.
Topics: Animals; Cell Line, Tumor; Cell Survival; Humans; Immunotherapy; Immunotherapy, Adoptive; Lung Neoplasms; Macrophages; Mice; Microscopy, Video; Neoplasms; Neoplasms, Experimental
PubMed: 32361713
DOI: 10.1038/s41587-020-0462-y -
The European Respiratory Journal Mar 2019The lung is highly vulnerable during sepsis, yet its functional deterioration accompanied by disturbances in the pulmonary microcirculation is poorly understood. This...
The lung is highly vulnerable during sepsis, yet its functional deterioration accompanied by disturbances in the pulmonary microcirculation is poorly understood. This study aimed to investigate how the pulmonary microcirculation is distorted in sepsis-induced acute lung injury (ALI) and reveal the underlying cellular pathophysiologic mechanism.Using a custom-made intravital lung microscopic imaging system in a murine model of sepsis-induced ALI, we achieved direct real-time visualisation of the pulmonary microcirculation and circulating cells We derived the functional capillary ratio (FCR) as a quantitative parameter for assessing the fraction of functional microvasculature in the pulmonary microcirculation and dead space.We identified that the FCR rapidly decreases in the early stage of sepsis-induced ALI. The intravital imaging revealed that this decrease resulted from the generation of dead space, which was induced by prolonged neutrophil entrapment within the capillaries. We further showed that the neutrophils had an extended sequestration time and an arrest-like dynamic behaviour, both of which triggered neutrophil aggregates inside the capillaries and arterioles. Finally, we found that Mac-1 (CD11b/CD18) was upregulated in the sequestered neutrophils and that a Mac-1 inhibitor restored the FCR and improved hypoxaemia.Using the intravital lung imaging system, we observed that Mac-1-upregulated neutrophil aggregates led to the generation of dead space in the pulmonary microcirculation that was recovered by a Mac-1 inhibitor in sepsis-induced ALI.
Topics: Acute Lung Injury; Animals; Antibodies, Monoclonal; Capillaries; Disease Models, Animal; Lung; Macrophage-1 Antigen; Male; Mice; Mice, Inbred C57BL; Microcirculation; Microscopy, Video; Neutrophils; Sepsis
PubMed: 30635296
DOI: 10.1183/13993003.00786-2018 -
BMC Research Notes Apr 2016Bacterial infections are a common clinical problem in both acute and chronic wounds. With growing concerns over antibiotic resistance, treatment of bacterial infections...
BACKGROUND
Bacterial infections are a common clinical problem in both acute and chronic wounds. With growing concerns over antibiotic resistance, treatment of bacterial infections should only occur after positive diagnosis. Currently, diagnosis is delayed due to lengthy culturing methods which may also fail to identify the presence of bacteria. While newer costly bacterial identification methods are being explored, a simple and inexpensive diagnostic tool would aid in immediate and accurate treatments for bacterial infections. Histologically, hematoxylin and eosin (H&E) and Gram stains have been employed, but are far from optimal when analyzing tissue samples due to non-specific staining. The goal of the current study was to develop a modification of the Gram stain that enhances the contrast between bacteria and host tissue.
FINDINGS
A modified Gram stain was developed and tested as an alternative to Gram stain that improves the contrast between Gram positive bacteria, Gram negative bacteria and host tissue. Initially, clinically relevant strains of Pseudomonas aeruginosa and Staphylococcus aureus were visualized in vitro and in biopsies of infected, porcine burns using routine Gram stain, and immunohistochemistry techniques involving bacterial strain-specific fluorescent antibodies as validation tools. H&E and Gram stain of serial biopsy sections were then compared to a modification of the Gram stain incorporating a counterstain that highlights collagen found in tissue. The modified Gram stain clearly identified both Gram positive and Gram negative bacteria, and when compared to H&E or Gram stain alone provided excellent contrast between bacteria and non-viable burn eschar. Moreover, when applied to surgical biopsies from patients that underwent burn debridement this technique was able to clearly detect bacterial morphology within host tissue.
CONCLUSIONS
We describe a modification of the Gram stain that provides improved contrast of Gram positive and Gram negative microorganisms within host tissue. The samples used in this study demonstrate that this staining technique has laboratory and clinical applicability. This modification only adds minutes to traditional Gram stain with reusable reagents, and results in a cost- and time-efficient technique for identifying bacteria in any clinical biopsy containing connective tissue.
Topics: Animals; Bacterial Infections; Burns; Eosine Yellowish-(YS); Gentian Violet; Gram-Positive Bacteria; Hematoxylin; Host-Pathogen Interactions; Humans; Immunohistochemistry; Microscopy, Video; Phenazines; Pseudomonas aeruginosa; Reproducibility of Results; Sensitivity and Specificity; Skin; Staining and Labeling; Staphylococcus aureus; Swine
PubMed: 27071769
DOI: 10.1186/s13104-016-1902-0 -
The Journal of Cell Biology Mar 2016Mammalian stress granules (SGs) contain stalled translation preinitiation complexes that are assembled into discrete granules by specific RNA-binding proteins such as...
Mammalian stress granules (SGs) contain stalled translation preinitiation complexes that are assembled into discrete granules by specific RNA-binding proteins such as G3BP. We now show that cells lacking both G3BP1 and G3BP2 cannot form SGs in response to eukaryotic initiation factor 2α phosphorylation or eIF4A inhibition, but are still SG-competent when challenged with severe heat or osmotic stress. Rescue experiments using G3BP1 mutants show that phosphomimetic G3BP1-S149E fails to rescue SG formation, whereas G3BP1-F33W, a mutant unable to bind G3BP partner proteins Caprin1 or USP10, rescues SG formation. Caprin1/USP10 binding to G3BP is mutually exclusive: Caprin binding promotes, but USP10 binding inhibits, SG formation. G3BP interacts with 40S ribosomal subunits through its RGG motif, which is also required for G3BP-mediated SG formation. We propose that G3BP mediates the condensation of SGs by shifting between two different states that are controlled by the phosphorylation of S149 and by binding to Caprin1 or USP10.
Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Animals; Base Sequence; COS Cells; Carrier Proteins; Cell Cycle Proteins; Cell Line, Tumor; Chlorocebus aethiops; Cytoplasmic Granules; DNA Helicases; Eukaryotic Initiation Factor-2; Eukaryotic Initiation Factor-4A; Humans; Microscopy, Confocal; Microscopy, Video; Molecular Sequence Data; Mutation; Phosphorylation; Poly-ADP-Ribose Binding Proteins; Protein Binding; Protein Conformation; Protein Interaction Domains and Motifs; RNA Helicases; RNA Interference; RNA Recognition Motif Proteins; RNA-Binding Proteins; Ribosomal Proteins; Ribosome Subunits, Small, Eukaryotic; Signal Transduction; Structure-Activity Relationship; Transfection; Ubiquitin Thiolesterase
PubMed: 27022092
DOI: 10.1083/jcb.201508028 -
Biophysical Reports Jun 2024We present a method for tracking densely clustered, high-velocity, indistinguishable objects being spawned at a high rate and moving in a directed force field using only...
We present a method for tracking densely clustered, high-velocity, indistinguishable objects being spawned at a high rate and moving in a directed force field using only object centroids as inputs and no other image information. The algorithm places minimal restrictions on the velocities or accelerations of the objects being tracked and uses a methodology based on a scoring function and a backtracking refinement process. This combination leads to successful tracking of hundreds of particles in challenging environments even when the displacement of the individual objects at successive times approaches the separation between neighboring objects in any one frame. We note that these cases can be particularly difficult to handle by existing methods. The performance of the algorithm is methodically examined by comparison to simulated trajectories, which vary the temporal and spatial densities, velocities, and accelerations of the objects in motion, as well as the signal/noise ratio. Also, we demonstrate its capability by analyzing data from experiments with superparamagnetic microspheres moving in an inhomogeneous magnetic field in aqueous buffer at room temperature. Our method should be widely applicable since trajectory determination problems are ubiquitous in video microscopy applications in biology, materials science, physics, and engineering.
PubMed: 38505834
DOI: 10.1016/j.bpr.2024.100148 -
The Application of Clinical Genetics 2017Primary ciliary dyskinesia is a genetically heterogeneous disorder of motile cilia that is predominantly inherited in an autosomal-recessive fashion. It is associated... (Review)
Review
Primary ciliary dyskinesia is a genetically heterogeneous disorder of motile cilia that is predominantly inherited in an autosomal-recessive fashion. It is associated with abnormal ciliary structure and/or function leading to chronic upper and lower respiratory tract infections, male infertility, and situs inversus. The estimated prevalence of primary ciliary dyskinesia is approximately one in 10,000-40,000 live births. Diagnosis depends on clinical presentation, nasal nitric oxide, high-speed video-microscopy analysis, transmission electron microscopy, genetic testing, and immunofluorescence. Here, we review its clinical features, diagnostic methods, molecular basis, and available therapies.
PubMed: 29033599
DOI: 10.2147/TACG.S127129 -
Microcirculation (New York, N.Y. : 1994) Oct 2022The aim of this study was to describe possible remodeling (i.e., dilatation and elongation) of papillary capillaries induced by increased oxygen demand for the repair...
OBJECTIVE
The aim of this study was to describe possible remodeling (i.e., dilatation and elongation) of papillary capillaries induced by increased oxygen demand for the repair process following a skin wound.
METHODS
Computer-assisted video microscopy was used to examine 10 healthy volunteers before (baseline) and after (≈1 h and ≈24 h) an incision (5 mm long and 1 mm deep) on the forearm, 0-1 mm and 30 mm (control site) from the incision. We defined categories from 0 (low) to 3 (high) to grade dilatation and elongation of the nutritive papillary capillaries, as well as the visibility of the superficial vascular plexus. Approximately 10 000 capillaries from 200 films were scored.
RESULTS
The nutritive papillary capillaries were dilated and elongated (p < 0.01) after ≈24 h; that is, elongation (score 1.9 ± 0.9) vs baseline (score 0.9 ± 0.6), p < 0.01 and dilatation (score 2.2 ± 0.7) vs baseline (score 0.3 ± 0.3), p < 0.01. Superficial plexus visibility increased (p < 0.01) after ≈1 h (score 2.0 ± 0.7) and ≈24 h (score 2.7 ± 0.3) vs baseline (score 0.8 ± 0.4).
CONCLUSION
The superficial vascular skin plexus showed enhanced visibility already ≈1 h after the skin trauma. Morphological remodeling in the nutritive papillary capillaries-dilatation and elongation after ≈24 h-facilitate increased O supply.
Topics: Humans; Microcirculation; Capillaries; Skin; Microscopy, Video; Forearm
PubMed: 35231135
DOI: 10.1111/micc.12755 -
International Journal of Molecular... Jan 2023The radial spoke head protein 4 homolog A () gene is one of more than 50 genes that cause Primary ciliary dyskinesia (PCD), a rare genetic ciliopathy. Genetic mutations... (Review)
Review
The radial spoke head protein 4 homolog A () gene is one of more than 50 genes that cause Primary ciliary dyskinesia (PCD), a rare genetic ciliopathy. Genetic mutations in the gene alter an important protein structure involved in ciliary pathogenesis. Radial spoke proteins, such as RSPH4A, have been conserved across multiple species. In humans, ciliary function deficiency caused by pathogenic variants results in a clinical phenotype characterized by recurrent oto-sino-pulmonary infections. More than 30 pathogenic genetic variants have been associated with PCD. In Puerto Rican Hispanics, a founder mutation ( (c.921+3_921+6delAAGT (intronic)) has been described. The spectrum of the PCD phenotype does not include laterality defects, which results in a challenging diagnosis. PCD diagnostic tools can combine transmission electron microscopy (TEM), nasal nitric oxide (nNO), High-Speed Video microscopy Analysis (HSVA), and immunofluorescence. The purpose of this review article is to provide a comprehensive overview of current knowledge about the gene in PCD, ranging from basic science to human clinical phenotype.
Topics: Humans; Kartagener Syndrome; Cilia; Proteins; Mutation; Axoneme; Cytoskeletal Proteins
PubMed: 36768259
DOI: 10.3390/ijms24031936 -
BioTechniques Feb 2022The use of magnetic tweezers for single-molecule micromanipulation has evolved rapidly since its introduction approximately 30 years ago. Magnetic tweezers have... (Review)
Review
The use of magnetic tweezers for single-molecule micromanipulation has evolved rapidly since its introduction approximately 30 years ago. Magnetic tweezers have provided important insights into the dynamic activity of DNA-processing enzymes, as well as detailed, high-resolution information on the mechanical properties of DNA. These successes have been enabled by major advancements in the hardware and software components of these devices. These developments now allow for a much richer mechanistic understanding of the functions and mechanisms of DNA-binding enzymes. In this review, the authors briefly discuss the fundamental principles of magnetic tweezers and describe the advancements that have made it a superlative tool for investigating, at the single-molecule level, DNA and its interactions with DNA-binding proteins.
Topics: DNA; Magnetic Phenomena; Magnetics; Micromanipulation; Nanotechnology; Optical Tweezers
PubMed: 35037472
DOI: 10.2144/btn-2021-0104 -
The European Respiratory Journal Jan 2017The diagnosis of primary ciliary dyskinesia is often confirmed with standard, albeit complex and expensive, tests. In many cases, however, the diagnosis remains...
The diagnosis of primary ciliary dyskinesia is often confirmed with standard, albeit complex and expensive, tests. In many cases, however, the diagnosis remains difficult despite the array of sophisticated diagnostic tests. There is no "gold standard" reference test. Hence, a Task Force supported by the European Respiratory Society has developed this guideline to provide evidence-based recommendations on diagnostic testing, especially in light of new developments in such tests, and the need for robust diagnoses of patients who might enter randomised controlled trials of treatments. The guideline is based on pre-defined questions relevant for clinical care, a systematic review of the literature, and assessment of the evidence using the GRADE (Grading of Recommendations, Assessment, Development and Evaluation) approach. It focuses on clinical presentation, nasal nitric oxide, analysis of ciliary beat frequency and pattern by high-speed video-microscopy analysis, transmission electron microscopy, genotyping and immunofluorescence. It then used a modified Delphi survey to develop an algorithm for the use of diagnostic tests to definitively confirm and exclude the diagnosis of primary ciliary dyskinesia; and to provide advice when the diagnosis was not conclusive. Finally, this guideline proposes a set of quality criteria for future research on the validity of diagnostic methods for primary ciliary dyskinesia.
Topics: Cilia; Delphi Technique; Diagnosis, Differential; Europe; Fluorescent Antibody Technique; Genetic Testing; Humans; Kartagener Syndrome; Microscopy, Electron, Transmission; Microscopy, Video; Nitric Oxide; Review Literature as Topic; Societies, Medical
PubMed: 27836958
DOI: 10.1183/13993003.01090-2016