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Anatomical Record (Hoboken, N.J. : 2007) Aug 2014In situ hybridization is a technique that is used to detect nucleotide sequences in cells, tissue sections, and even whole tissue. This method is based on the... (Review)
Review
In situ hybridization is a technique that is used to detect nucleotide sequences in cells, tissue sections, and even whole tissue. This method is based on the complementary binding of a nucleotide probe to a specific target sequence of DNA or RNA. These probes can be labeled with either radio-, fluorescent-, or antigen-labeled bases. Depending on the probe used, autoradiography, fluorescence microscopy, or immunohistochemistry, respectively, are used for visualization. In situ hybridization is extensively used in research, as well as clinical applications, especially for diagnostic purposes. This review discusses the basic technique of in situ hybridization. The standard in situ hybridization process is reviewed, and different types of in situ hybridization, their applications, and advantages and disadvantages are discussed.
Topics: Autoradiography; Humans; In Situ Hybridization; Isotope Labeling; Microscopy, Fluorescence; Paraffin Embedding; RNA Probes; Tissue Fixation
PubMed: 24810158
DOI: 10.1002/ar.22944 -
European Journal of Nuclear Medicine... Sep 2022Fibroblast activation protein (FAP) is a membrane-bound protease that has limited expression in normal adult tissues but is highly expressed in the tumor...
PURPOSE
Fibroblast activation protein (FAP) is a membrane-bound protease that has limited expression in normal adult tissues but is highly expressed in the tumor microenvironment of many solid cancers. FAP-2286 is a FAP-binding peptide coupled to a radionuclide chelator that is currently being investigated in patients as an imaging and therapeutic agent. The potency, selectivity, and efficacy of FAP-2286 were evaluated in preclinical studies.
METHODS
FAP expression analysis was performed by immunohistochemistry and autoradiography on primary human cancer specimens. FAP-2286 was assessed in biochemical and cellular assays and in in vivo imaging and efficacy studies, and was further evaluated against FAPI-46, a small molecule-based FAP-targeting agent.
RESULTS
Immunohistochemistry confirmed elevated levels of FAP expression in multiple tumor types including pancreatic, breast, and sarcoma, which correlated with FAP binding by FAP-2286 autoradiography. FAP-2286 and its metal complexes demonstrated high affinity to FAP recombinant protein and cell surface FAP expressed on fibroblasts. Biodistribution studies in mice showed rapid and persistent uptake of Ga-FAP-2286, In-FAP-2286, and Lu-FAP-2286 in FAP-positive tumors, with renal clearance and minimal uptake in normal tissues. Lu-FAP-2286 exhibited antitumor activity in FAP-expressing HEK293 tumors and sarcoma patient-derived xenografts, with no significant weight loss. In addition, FAP-2286 maintained longer tumor retention and suppression in comparison to FAPI-46.
CONCLUSION
In preclinical models, radiolabeled FAP-2286 demonstrated high tumor uptake and retention, as well as potent efficacy in FAP-positive tumors. These results support clinical development of Ga-FAP-2286 for imaging and Lu-FAP-2286 for therapeutic use in a broad spectrum of FAP-positive tumors.
Topics: Adult; Animals; Cell Line, Tumor; Fibroblasts; Gallium Radioisotopes; HEK293 Cells; Humans; Mice; Radionuclide Imaging; Sarcoma; Tissue Distribution; Tumor Microenvironment
PubMed: 35608703
DOI: 10.1007/s00259-022-05842-5 -
Molecular Biology of the Cell Aug 2021With No Lysine (K) WNK kinases regulate electro-neutral cotransporters that are controlled by osmotic stress and chloride. We showed previously that autophosphorylation...
With No Lysine (K) WNK kinases regulate electro-neutral cotransporters that are controlled by osmotic stress and chloride. We showed previously that autophosphorylation of WNK1 is inhibited by chloride, raising the possibility that WNKs are activated by osmotic stress. Here we demonstrate that unphosphorylated WNK isoforms 3 and 1 autophosphorylate in response to osmotic pressure in vitro, applied with the crowding agent polyethylene glycol (PEG)400 or osmolyte ethylene glycol (EG), and that this activation is opposed by chloride. Small angle x-ray scattering of WNK3 in the presence and absence of PEG400, static light scattering in EG, and crystallography of WNK1 were used to understand the mechanism. Osmosensing in WNK3 and WNK1 appears to occur through a conformational equilibrium between an inactive, unphosphorylated, chloride-binding dimer and an autophosphorylation-competent monomer. An improved structure of the inactive kinase domain of WNK1, and a comparison with the structure of a monophosphorylated form of WNK1, suggests that large cavities, greater hydration, and specific bound water may participate in the osmosensing mechanism. Our prior work showed that osmolytes have effects on the structure of phosphorylated WNK1, suggestive of multiple stages of osmotic regulation in WNKs.
Topics: Autoradiography; Chromatography, Gel; Ethylene Glycol; Osmotic Pressure; Phosphorylation; Polyethylene Glycols; Protein Conformation; Protein Kinases; Protein Multimerization; Scattering, Small Angle; WNK Lysine-Deficient Protein Kinase 1; Water; X-Ray Diffraction
PubMed: 33689398
DOI: 10.1091/mbc.E20-01-0089 -
Biomolecules Sep 2021Many diseases, including cancer, can lead to neuropathic pain (NP). NP is one of the accompanying symptoms of suffering in many conditions and the life quality of NP... (Review)
Review
Many diseases, including cancer, can lead to neuropathic pain (NP). NP is one of the accompanying symptoms of suffering in many conditions and the life quality of NP patient is seriously affected. Due to complex causes, the effects of clinical treatments have been very unsatisfactory. Many experts have found that neuron-microglia interaction plays an essential role in NP occurrence and development. Therefore, the activation of microglia, related inflammatory mediators and molecular and cellular signaling pathways have become the focus of NP research. With the help of modern functional imaging technology, advanced pre-and clinical studies have been carried out and NP interventions have been attempted by using the different pharmaceuticals and the extracted active components of various traditional herbal medicines. In this communication, we review the mechanism of microglia on NP formation and treatment and molecular imaging technology's role in the clinical diagnosis and evaluation of NP therapies.
Topics: Animals; Autoradiography; Brain; Humans; Microglia; Models, Biological; Neuralgia; Neuroimaging
PubMed: 34572554
DOI: 10.3390/biom11091343 -
Biomedicines Sep 2022Pharmacokinetic assessment of drug disposition processes in vivo is critical in predicting pharmacodynamics and toxicology to reduce the risk of inappropriate drug... (Review)
Review
Pharmacokinetic assessment of drug disposition processes in vivo is critical in predicting pharmacodynamics and toxicology to reduce the risk of inappropriate drug development. The blood-brain barrier (BBB), a special physiological structure in brain tissue, hinders the entry of targeted drugs into the central nervous system (CNS), making the drug concentrations in target tissue correlate poorly with the blood drug concentrations. Additionally, once non-CNS drugs act directly on the fragile and important brain tissue, they may produce extra-therapeutic effects that may impair CNS function. Thus, an intracerebral pharmacokinetic study was developed to reflect the disposition and course of action of drugs following intracerebral absorption. Through an increasing understanding of the fine structure in the brain and the rapid development of analytical techniques, cerebral pharmacokinetic techniques have developed into non-invasive imaging techniques. Through non-invasive imaging techniques, molecules can be tracked and visualized in the entire BBB, visualizing how they enter the BBB, allowing quantitative tools to be combined with the imaging system to derive reliable pharmacokinetic profiles. The advent of imaging-based pharmacokinetic techniques in the brain has made the field of intracerebral pharmacokinetics more complete and reliable, paving the way for elucidating the dynamics of drug action in the brain and predicting its course. The paper reviews the development and application of imaging technologies for cerebral pharmacokinetic study, represented by optical imaging, radiographic autoradiography, radionuclide imaging and mass spectrometry imaging, and objectively evaluates the advantages and limitations of these methods for predicting the pharmacodynamic and toxic effects of drugs in brain tissues.
PubMed: 36289709
DOI: 10.3390/biomedicines10102447 -
Pharmaceuticals (Basel, Switzerland) Dec 2020Understanding pharmacokinetics and biodistribution of antibody-drug conjugates (ADCs) is a one of the critical steps enabling their successful development and... (Review)
Review
Understanding pharmacokinetics and biodistribution of antibody-drug conjugates (ADCs) is a one of the critical steps enabling their successful development and optimization. Their complex structure combining large and small molecule characteristics brought out multiple bioanalytical methods to decipher the behavior and fate of both components in vivo. In this respect, these methods must provide insights into different key elements including half-life and blood stability of the construct, premature release of the drug, whole-body biodistribution, and amount of the drug accumulated within the targeted pathological tissues, all of them being directly related to efficacy and safety of the ADC. In this review, we will focus on the main strategies enabling to quantify and characterize ADCs in biological matrices and discuss their associated technical challenges and current limitations.
PubMed: 33327644
DOI: 10.3390/ph13120462 -
Medical Physics Jun 2017Conventional charged-particle imaging techniques - such as autoradiography - provide only two-dimensional (2D) black ex vivo images of thin tissue slices. In order to...
PURPOSE
Conventional charged-particle imaging techniques - such as autoradiography - provide only two-dimensional (2D) black ex vivo images of thin tissue slices. In order to get volumetric information, images of multiple thin slices are stacked. This process is time consuming and prone to distortions, as registration of 2D images is required. We propose a direct three-dimensional (3D) autoradiography technique, which we call charged-particle emission tomography (CPET). This 3D imaging technique enables imaging of thick tissue sections, thus increasing laboratory throughput and eliminating distortions due to registration. CPET also has the potential to enable in vivo charged-particle imaging with a window chamber or an endoscope.
METHODS
Our approach to charged-particle emission tomography uses particle-processing detectors (PPDs) to estimate attributes of each detected particle. The attributes we estimate include location, direction of propagation, and/or the energy deposited in the detector. Estimated attributes are then fed into a reconstruction algorithm to reconstruct the 3D distribution of charged-particle-emitting radionuclides. Several setups to realize PPDs are designed. Reconstruction algorithms for CPET are developed.
RESULTS
Reconstruction results from simulated data showed that a PPD enables CPET if the PPD measures more attributes than just the position from each detected particle. Experiments showed that a two-foil charged-particle detector is able to measure the position and direction of incident alpha particles.
CONCLUSIONS
We proposed a new volumetric imaging technique for charged-particle-emitting radionuclides, which we have called charged-particle emission tomography (CPET). We also proposed a new class of charged-particle detectors, which we have called particle-processing detectors (PPDs). When a PPD is used to measure the direction and/or energy attributes along with the position attributes, CPET is feasible.
Topics: Algorithms; Imaging, Three-Dimensional; Tomography, Emission-Computed
PubMed: 28370094
DOI: 10.1002/mp.12245 -
Neuroscience Letters Jan 2019Dopamine D3 receptors have key roles in behavioral reward, addiction, Parkinson's disease, and schizophrenia, and there is interest in studying their role in these... (Review)
Review
Dopamine D3 receptors have key roles in behavioral reward, addiction, Parkinson's disease, and schizophrenia, and there is interest in studying their role in these disorders using PET. However, current PET radiotracers for studying D3 receptors in humans all bind to both D2 and D3 due to similarities between the two receptors. Selective D2 and D3 radioligands would aid investigation of the differences between D2 and D3 circuitry in the central nervous system. While there are currently in vitro measures of ligand D3/D2 selectivity, there is a need for an in vivo PET measure of D3/D2 selectivity. This review discusses current PET imaging of dopamine D2/D3 receptors and proposes methodology for quantitating in vivo selectivity of probes for PET imaging of dopamine D3 receptors.
Topics: Autoradiography; Carbon Radioisotopes; Fluorine Radioisotopes; Humans; Ligands; Positron-Emission Tomography; Receptors, Dopamine D2; Receptors, Dopamine D3
PubMed: 29518538
DOI: 10.1016/j.neulet.2018.03.006 -
Theranostics 2022The accelerated approval of the monoclonal antibody (mAb) aducanumab as a treatment option for Alzheimer's Disease and the continued discussions about its efficacy have...
The accelerated approval of the monoclonal antibody (mAb) aducanumab as a treatment option for Alzheimer's Disease and the continued discussions about its efficacy have shown that a better understanding of immunotherapy for the treatment of neurodegenerative diseases is needed. Zr-immuno-PET could be a suitable tool to open new avenues for the diagnosis of CNS disorders, monitoring disease progression, and assessment of novel therapeutics. Herein, three different Zr-labeling strategies and direct radioiodination with I of a bispecific anti-amyloid-beta aducanumab derivate, consisting of aducanumab with a C-terminal fused anti-transferrin receptor binding single chain Fab fragment derived from 8D3 (Adu-8D3), were compared and with regard to brain uptake and target engagement in an APP/PS1 Alzheimer's disease mouse model and wild type animals. Adu-8D3 and a negative control antibody, based on the HIV specific B12 antibody also carrying C-terminal fused 8D3 scFab (B12-8D3), were each conjugated with NCS-DFO, NCS-DFO*, or TFP--suc-DFO-Fe-ester, followed by radiolabeling with Zr. I was used as a substitute for I for labeling of both antibodies. 30 µg of radiolabeled mAb, corresponding to approximately 6 MBq Zr or 2.5 MBq I, were injected per mouse. PET imaging was performed 1, 3 and 7 days post injection (p.i.). All mice were sacrificed on day 7 p.i. and subjected to biodistribution and brain autoradiography. Immunostaining on brain tissue was performed after autoradiography for further validation. biodistribution revealed that the brain uptake of [Zr]Zr-DFO*-NCS-Adu-8D3 (2.19 ±0.12 %ID/g) was as high as for its I-analog (2.21 ±0.15 %ID/g). [Zr]Zr-DFO-NCS-Adu-8D3 and [Zr]Zr-DFO--suc-Adu-8D3 showed significantly lower uptake (< 0.65 %ID/g), being in the same range as for the Zr-labeled controls (B12-8D3). Autoradiography of [Zr]Zr-DFO*-NCS-Adu-8D3 and [I]I-Adu-8D3 showed an amyloid-beta related granular uptake pattern of radioactivity. In contrast, the [Zr]Zr-DFO-conjugates and the control antibody groups did not show any amyloid-beta related uptake pattern, indicating that DFO is inferior for Zr-immuno-PET imaging of the brain in comparison to DFO* for Adu-8D3. This was confirmed by day 7 PET images showing only amyloid-beta related brain uptake for [Zr]Zr-DFO*-NCS-Adu-8D3. In wild type animals, such an uptake was not observed. Immunostaining showed a co-localization of all administered Adu-8D3 conjugates with amyloid-beta plaques. We successfully demonstrated that Zr-immuno-PET is suitable for imaging and quantifying amyloid-beta specific brain uptake using a bispecific aducanumab brain shuttling antibody, Adu-8D3, but only when using the novel chelator DFO*, and not DFO, for labeling with Zr.
Topics: Animals; Mice; Iodine Radioisotopes; Chelating Agents; Deferoxamine; Zirconium; Tissue Distribution; Alzheimer Disease; Cell Line, Tumor; Positron-Emission Tomography; Antibodies, Monoclonal; Amyloid beta-Peptides; Antibodies, Bispecific; Immunoglobulin Fab Fragments; Esters
PubMed: 36276653
DOI: 10.7150/thno.73509 -
BioRxiv : the Preprint Server For... Sep 2023Positron Emission Tomography (PET) ligands have advanced Alzheimer's disease (AD) diagnosis and treatment. Using autoradiography and cryo-EM, we identified AD brain...
Positron Emission Tomography (PET) ligands have advanced Alzheimer's disease (AD) diagnosis and treatment. Using autoradiography and cryo-EM, we identified AD brain tissue with elevated tau burden, purified filaments, and determined the structure of second-generation high avidity PET ligand MK-6240 at 2.31 Å resolution, which bound at a 1:1 ratio within the cleft of tau paired-helical filament (PHF), engaging with glutamine 351, lysine K353, and isoleucine 360. This information elucidates the basis of MK-6240 PET in quantifying PHF deposits in AD and may facilitate the structure-based design of superior ligands against tau amyloids.
PubMed: 37790438
DOI: 10.1101/2023.09.22.558671