Senolytic CAR T cells reverse senescence-associated pathologies.

Cellular senescence is characterized by stable cell-cycle arrest and a secretory program that modulates the tissue microenvironment1,2. Physiologically, senescence serves as a tumour-suppressive mechanism that prevents the expansion of premalignant cells3,4 and has a beneficial role in wound-healing responses5,6. Pathologically, the aberrant accumulation of senescent cells generates an inflammatory milieu that leads to chronic tissue damage and contributes to diseases such as liver and lung fibrosis, atherosclerosis, diabetes and osteoarthritis1,7. Accordingly, eliminating senescent cells from damaged tissues in mice ameliorates the symptoms of these pathologies and even promotes longevity1,2,8-10. Here we test the therapeutic concept that chimeric antigen receptor (CAR) T cells that target senescent cells can be effective senolytic agents. We identify the urokinase-type plasminogen activator receptor (uPAR)11 as a cell-surface protein that is broadly induced during senescence and show that uPAR-specific CAR T cells efficiently ablate senescent cells in vitro and in vivo. CAR T cells that target uPAR extend the survival of mice with lung adenocarcinoma that are treated with a senescence-inducing combination of drugs, and restore tissue homeostasis in mice in which liver fibrosis is induced chemically or by diet. These results establish the therapeutic potential of senolytic CAR T cells for senescence-associated diseases.

Evaluation of a photodynamic therapy agent using a canine prostate cancer model.

BACKGROUND: Male dogs can develop spontaneous prostate cancer, which is similar physiologically to human disease. Recently, Tweedle and coworkers have developed an orthotopic canine prostate model allowing implanted tumors and therapeutic agents to be tested in a more translational large animal model. We used the canine model to evaluate prostate-specific membrane antigen (PSMA)-targeted gold nanoparticles as a theranostic approach for fluorescence (FL) imaging and photodynamic therapy (PDT) of early stage prostate cancer. METHODS: Dogs (four in total) were immunosuppressed with a cyclosporine-based immunosuppressant regimen and their prostate glands were injected with Ace-1-hPSMA cells using transabdominal ultrasound (US) guidance. Intraprostatic tumors grew in 4-5 weeks and were monitored by ultrasound (US). When tumors reached an appropriate size, dogs were injected intravenously (iv) with PSMA-targeted nano agents (AuNPs-Pc158) and underwent surgery 24 h later to expose the prostate tumors for FL imaging and PDT. Ex vivo FL imaging and histopathological studies were performed to confirm PDT efficacy. RESULTS: All dogs had tumor growth in the prostate gland as revealed by US. Twenty-four hours after injection of PSMA-targeted nano agents (AuNPs-Pc158), the tumors were imaged using a Curadel FL imaging device. While normal prostate tissue had minimal fluorescent signal, the prostate tumors had significantly increased FL. PDT was activated by irradiating specific fluorescent tumor areas with laser light (672 nm). PDT bleached the FL signal, while fluorescent signals from the other unexposed tumor tissues were unaffected. Histological analysis of tumors and adjacent prostate revealed that PDT damaged the irradiated areas to a depth of 1-2 mms with the presence of necrosis, hemorrhage, secondary inflammation, and occasional focal thrombosis. The nonirradiated areas showed no visible damages by PDT. CONCLUSION: We have successfully established a PSMA-expressing canine orthotopic prostate tumor model and used the model to evaluate the PSMA-targeted nano agents (AuNPs-Pc158) in the application of FL imaging and PDT. It was demonstrated that the nano agents allowed visualization of the cancer cells and enabled their destruction when they were irradiated with a specific wavelength of light.

Characterization of Cerebral Hemodynamics with TCD in Patients Undergoing VA-ECMO and VV-ECMO: a Prospective Observational Study.

BACKGROUND: Extracorporeal membrane oxygenation has a high risk of acute brain injury and resultant mortality. Transcranial Doppler characterizes cerebral hemodynamics in real time, but limited data exist on its interpretation in ECMO. Here, we report TCD mean flow velocity and pulsatility index in a large ECMO population. METHODS: This was a prospective cohort study at a tertiary care center. The patients were adults on venoarterial ECMO or venovenous ECMO undergoing TCD studies. RESULTS: A total of 135 patients underwent a total of 237 TCD studies while on VA-ECMO (n = 95, 70.3%) or VV-ECMO (n = 40, 29.6%). MFVs were captured reliably (approximately 90%) and were similar to a published healthy cohort in all vessels except the internal carotid artery. Presence of a recordable PI was strongly associated with ECMO mode (57% in VA vs. 95% in VV, p < 0.001). Absence of TCD pulsatility was associated with intraparenchymal hemorrhage (14.7 vs. 1.6%, p = 0.03) in VA-ECMO patients. CONCLUSIONS: Transcranial Doppler analysis in a single-center cohort of VA-ECMO and VV-ECMO patients demonstrates similar MFVs and PIs. Absence of PIs was associated with a higher frequency of intraparenchymal hemorrhage and a composite bleeding event. However, cautious interpretation and external validation is necessary for these findings with a multicenter study with a larger sample size.

Options for imaging cellular therapeutics in vivo: a multi-stakeholder perspective.

Cell-based therapies have been making great advances toward clinical reality. Despite the increase in trial activity, few therapies have successfully navigated late-phase clinical trials and received market authorization. One possible explanation for this is that additional tools and technologies to enable their development have only recently become available. To support the safety evaluation of cell therapies, the Health and Environmental Sciences Institute Cell Therapy-Tracking, Circulation and Safety Committee, a multisector collaborative committee, polled the attendees of the 2017 International Society for Cell & Gene Therapy conference in London, UK, to understand the gaps and needs that cell therapy developers have encountered regarding safety evaluations in vivo. The goal of the survey was to collect information to inform stakeholders of areas of interest that can help ensure the safe use of cellular therapeutics in the clinic. This review is a response to the cellular imaging interests of those respondents. The authors offer a brief overview of available technologies and then highlight the areas of interest from the survey by describing how imaging technologies can meet those needs. The areas of interest include imaging of cells over time, sensitivity of imaging modalities, ability to quantify cells, imaging cellular survival and differentiation and safety concerns around adding imaging agents to cellular therapy protocols. The Health and Environmental Sciences Institute Cell Therapy-Tracking, Circulation and Safety Committee believes that the ability to understand therapeutic cell fate is vital for determining and understanding cell therapy efficacy and safety and offers this review to aid in those needs. An aim of this article is to share the available imaging technologies with the cell therapy community to demonstrate how these technologies can accomplish unmet needs throughout the translational process and strengthen the understanding of cellular therapeutics.

Erratum to: Interaction of the signaling state analog and the apoprotein form of the orange carotenoid protein with the fluorescence recovery protein.

In Fig. 1a in the original article, the amino acid side chains were incorrectly labeled in the structure representation of the orange carotenoid protein (OCP). The corrected figure is printed in this erratum.

Interaction of the signaling state analog and the apoprotein form of the orange carotenoid protein with the fluorescence recovery protein.

Photoprotection in cyanobacteria relies on the interplay between the orange carotenoid protein (OCP) and the fluorescence recovery protein (FRP) in a process termed non-photochemical quenching, NPQ. Illumination with blue-green light converts OCP from the basic orange state (OCPO) into the red-shifted, active state (OCPR) that quenches phycobilisome (PBs) fluorescence to avoid excessive energy flow to the photosynthetic reaction centers. Upon binding of FRP, OCPR is converted to OCPO and dissociates from PBs; however, the mode and site of OCPR/FRP interactions remain elusive. Recently, we have introduced the purple OCPW288A mutant as a competent model for the signaling state OCPR (Sluchanko et al., Biochim Biophys Acta 1858:1-11, 2017). Here, we have utilized fluorescence labeling of OCP at its native cysteine residues to generate fluorescent OCP proteins for fluorescence correlation spectroscopy (FCS). Our results show that OCPW288A has a 1.6(±0.4)-fold larger hydrodynamic radius than OCPO, supporting the hypothesis of domain separation upon OCP photoactivation. Whereas the addition of FRP did not change the diffusion behavior of OCPO, a substantial compaction of the OCPW288A mutant and of the OCP apoprotein was observed. These results show that sufficiently stable complexes between FRP and OCPW288A or the OCP apoprotein are formed to be detected by FCS. 1:1 complex formation with a micromolar apparent dissociation constant between OCP apoprotein and FRP was confirmed by size-exclusion chromatography. Beyond the established OCP/FRP interaction underlying NPQ cessation, the OCP apoprotein/FRP interaction suggests a more general role of FRP as a scaffold protein for OCP maturation.

Ornithine Decarboxylase Is Sufficient for Prostate Tumorigenesis via Androgen Receptor Signaling.

Increased polyamine synthesis is known to play an important role in prostate cancer. We aimed to explore its functional significance in prostate tumor initiation and its link to androgen receptor (AR) signaling. For this purpose, we generated a new cell line derived from normal epithelial prostate cells (RWPE-1) with overexpression of ornithine decarboxylase (ODC) and used it for in vitro and in vivo experiments. We then comprehensively analyzed the expression of the main metabolic enzymes of the polyamine pathway and spermine abundance in 120 well-characterized cases of human prostate cancer and high-grade prostate intraepithelial neoplasia (HGPIN). Herein, we show that the ODC-overexpressing prostate cells underwent malignant transformation, revealing that ODC is sufficient for de novo tumor initiation in 94% of injected mice. This oncogenic capacity was acquired through alteration of critical signaling networks, including AR, EIF2, and mTOR/MAPK. RNA silencing experiments revealed the link between AR signaling and polyamine metabolism. Human prostate cancers consistently demonstrated up-regulation of the main polyamine enzymes analyzed (ODC, polyamine oxidase, and spermine synthase) and reduction of spermine. This phenotype was also dominant in HGPIN, rendering it a new biomarker of malignant transformation. In summary, we report that ODC plays a key role in prostate tumorigenesis and that the polyamine pathway is altered as early as HGPIN.

Multiparticle Brownian dynamics simulation of experimental kinetics of cytochrome bf oxidation and photosystem I reduction by plastocyanin.

A model of electron transport from cytochrome f to photosystem I mediated by plastocyanin was designed on the basis of the multiparticle Brownian dynamics method. The model combines events which occur over a wide time range, including protein diffusion along the thylakoid membrane, long-distance interactions between proteins, formation of a multiprotein complex, electron transfer within a complex and complex dissociation. Results of the modeling were compared with the experimental kinetics measured in chloroplast thylakoids. Computer simulation demonstrated that the complex interior of the photosynthetic membrane, electrostatic interactions and Brownian diffusion provide physical conditions for the directed electron flow along the photosynthetic electron transport chain.

Nuclear-Based Labeling of Cellular Immunotherapies: A Simple Protocol for Preclinical Use.

Labeling and tracking existing and emerging cell-based immunotherapies using nuclear imaging is widely used to guide the preclinical phases of development and testing of existing and new emerging off-the-shelf cell-based immunotherapies. In fact, advancing our knowledge about their mechanism of action and limitations could provide preclinical support and justification for moving towards clinical experimentation of newly generated products and expedite their approval by the Food and Drug Administration (FDA).Here we provide the reader with a ready to use protocol describing the labeling methodologies and practical procedures to render different candidate cell therapies in vivo traceable by nuclear-based imaging. The protocol includes sufficient practical details to aid researchers at all career stages and from different fields in familiarizing with the described concepts and incorporating them into their work.

Transport of the Tumen River water to the Far Eastern Marine Reserve (Posyet Bay) based on in situ, satellite data and Lagrangian modeling using ROMS current velocity output.

We study physical mechanisms of the Tumen River water transport in the area of the Posyet Bay (Peter the Great Bay, Sea of Japan). This study is based on the satellite and in situ measurements, and numerical simulation of advection of river water by the current velocity simulated by Regional Ocean Model System (ROMS). The importance of this study is in identification of the reasons of the transport of pollutants into the area of the Far Eastern Marine Reserve. The results of the study showed that such reasons are wind currents and mesoscale cyclonic eddies. These eddies were originally detected on satellite imagery and CTD and bio-optical measurements. The anomalies in the form of spots of the chlorophyll a (CHL) increased concentration were detected on satellite images in fall 2009. The oceanographic sections of CTD and bio-optical measurements through the anomalies show that they are cyclonic eddies. These eddies consist of two cores - upper and lower. The upper core is filled with river waters with low salinity, high values of CHL and colored dissolved organic matter content (CDOM). The lower core is filled with cold saline waters. The ROMS results show that eddies are generated as a result of symmetrical and centrifugal instabilities.

Vascularized tumor on a microfluidic chip to study mechanisms promoting tumor neovascularization and vascular targeted therapies.

The cascade of events leading to tumor formation includes induction of a tumor supporting neovasculature as a primary hallmark of cancer. Developing vasculature is difficult to evaluate in vivo but can be captured using microfluidic chip technology and patient derived cells. Herein, we established an on chip approach to investigate the mechanisms promoting tumor vascularization and vascular targeted therapies via co-culture of metastatic renal cell carcinoma spheroids and endothelial cells in a 3D environment. Our model permitted real-time, high-resolution observation and assessment of tumor-induced angiogenesis, where endothelial cells sprout towards the tumor and mimic a vascular network. Bevacizumab, an angiogenic inhibitor, disrupted interactions between vessels and tumors, destroying the vascular network. The on chip approach enabled assessment of endothelial cell biology, vessel's functionality, drug delivery, and molecular expression of PSMA. Finally, observations in the vascularized tumor on chip permitted direct and conclusive quantification of this therapy in weeks as opposed to months in a comparable animal model. Teaser: Vascularized tumor on microfluidic chip provides opportunity to study targeted therapies and improves preclinical drug discovery.

Simultaneous quantitative imaging of two PET radiotracers via the detection of positron-electron annihilation and prompt gamma emissions.

In conventional positron emission tomography (PET), only one radiotracer can be imaged at a time, because all PET isotopes produce the same two 511 keV annihilation photons. Here we describe an image reconstruction method for the simultaneous in vivo imaging of two PET tracers and thereby the independent quantification of two molecular signals. This method of multiplexed PET imaging leverages the 350-700 keV range to maximize the capture of 511 keV annihilation photons and prompt γ-ray emission in the same energy window, hence eliminating the need for energy discrimination during reconstruction or for signal separation beforehand. We used multiplexed PET to track, in mice with subcutaneous tumours, the biodistributions of intravenously injected [124I]I-trametinib and 2-deoxy-2-[18F]fluoro-D-glucose, [124I]I-trametinib and its nanoparticle carrier [89Zr]Zr-ferumoxytol, and the prostate-specific membrane antigen (PSMA) and infused PSMA-targeted chimaeric antigen receptor T cells after the systemic administration of [68Ga]Ga-PSMA-11 and [124I]I. Multiplexed PET provides more information depth, gives new uses to prompt γ-ray-emitting isotopes, reduces radiation burden by omitting the need for an additional computed-tomography scan and can be implemented on preclinical and clinical systems without any modifications in hardware or image acquisition software.

Mechanism of cell death mediated by a BF2-chelated tetraaryl-azadipyrromethene photodynamic therapeutic: dissection of the apoptotic pathway in vitro and in vivo.

Photodynamic therapy (PDT) is an established treatment modality for cancer. ADPM06 is an emerging non-porphyrin PDT agent which has been specifically designed for therapeutic application. Recently, we have demonstrated that ADPM06-PDT is well tolerated in vivo and elicits impressive complete response rates in various models of cancer when a short drug-light interval is applied. Herein, the mechanism of action of ADPM06-PDT in vitro and in vivo is outlined. Using a drug and light combination that reduces the clonogenicity of MDA-MB-231 cells by >90%, we detected a well-orchestrated apoptotic response accompanied by the activation of various caspases in vitro. The generation of reactive oxygen species (ROS) upon photosensitizer irradiation was found to be the key instigator in the observed apoptotic response, with the endoplasmic reticulum (ER) found to be the intracellular site of initial PDT damage, as determined by induction of a rapid ER stress response post-PDT. PDT-induced apoptosis was also found to be independent of p53 tumor suppressor status. A robust therapeutic response in vivo was demonstrated, with a substantial reduction in tumor proliferation observed, as well as a rapid induction of apoptosis and initiation of ER stress, mirroring numerous aspects of the mechanism of action of ADPM06 in vitro. Finally, using a combination of (18) F-labeled 3'-deoxy-3'-fluorothymidine ((18) F-FLT) nuclear and optical imaging, a considerable decrease in tumor proliferation over 24-hr in two models of human cancer was observed. Taken together, this data clearly establishes ADPM06 as an exciting novel PDT agent with significant potential for further translational development.

Addictive behavior among young people in Ukraine: a pilot study.

The AUDIT-like tests system was created for complex assessment and evaluation of the addictive status of adolescents in a Ukrainian population. The AUDIT-like tests system has been created from the Alcohol Use Disorders Identification Test (AUDIT) developed by the World Health Organization. The AUDIT-like tests were minimally modified from the original AUDIT. Attention was brought to similarities between stages of different addictions (TV, computer games, the Internet, etc.) and alcohol addiction. Seventeen AUDIT-like tests were created to detect the different types of chemical and non-chemical addictions.

Imaging cellular immunotherapies and immune cell biomarkers: from preclinical studies to patients.

Cellular immunotherapies have emerged as a successful therapeutic approach to fight a wide range of human diseases, including cancer. However, responses are limited to few patients and tumor types. An in-depth understanding of the complexity and dynamics of cellular immunotherapeutics, including what is behind their success and failure in a patient, the role of other immune cell types and molecular biomarkers in determining a response, is now paramount. As the cellular immunotherapy arsenal expands, whole-body non-invasive molecular imaging can shed a light on their in vivo fate and contribute to the reliable assessment of treatment outcome and prediction of therapeutic response. In this review, we outline the non-invasive strategies that can be tailored toward the molecular imaging of cellular immunotherapies and immune-related components, with a focus on those that have been extensively tested preclinically and are currently under clinical development or have already entered the clinical trial phase. We also provide a critical appraisal on the current role and consolidation of molecular imaging into clinical practice.

Transcranial Doppler microemboli and acute brain injury in extracorporeal membrane oxygenation: A prospective observational study.

Objective: Extracorporeal membrane oxygenation (ECMO) carries a high morbidity of acute brain injury (ABI) with resultant mortality increase. Transcranial Doppler (TCD) allows real-time characterization of regional cerebral hemodynamics, but limited data exist on the interpretation of microembolic signals (MES) in ECMO. Methods: This prospective cohort study was conducted at a single tertiary care center, November 2017 through February 2022, and included all adult patients receiving venoarterial (VA) and venovenous (VV) ECMO undergoing TCD examinations, which all included MES monitoring. Results: Of 145 patients on ECMO who underwent at least 1 TCD examination, 100 (68.9%) patients on VA-ECMO received 187 examinations whereas 45 (31.1%) patients on VV-ECMO received 65 examinations (P = .81). MES were observed in 35 (35.0%) patients on VA-ECMO and 2 (4.7%) patients on VV-ECMO (P < .001), corresponding to 46 (24.6%) and 2 (3.1%) TCD examinations, respectively. MES were present in 29.4% of patients on VA-ECMO without additional cardiac support, compared with 38.1% with intra-aortic balloon pump and 57.1% with left ventricular assist device, but these differences were not statistically significant (P = .39; P = .20, respectively). Presence or number of MES was not associated with VA-ECMO cannulation mode (23.4% MES presence in peripheral cannulation vs 25.8% in central cannulation, P = .80). In both VA- and VV-ECMO, MES presence or number was not associated with presence of clot or fibrin in the ECMO circuit or with any studied hemodynamic, laboratory, or ECMO parameters at the time of TCD. ABI occurred in 38% and 31.1% of patients on VA- and VV-ECMO, respectively. In multivariable logistic regression analyses, neither ABI nor a composite outcome of arterial thromboembolic events was associated with presence or number of MES in VA- ECMO. Conclusions: TCD analysis in a large cohort of patients on ECMO demonstrates a significant number of MES, especially in patients on VA-ECMO with intra-aortic balloon pump, and/or left ventricular assist device. However, clinical associations and significance of TCD MES remain unresolved and warrant further correlation with systematic imaging and long-term neurologic follow-up.

Distal Acquired Demyelinating Symmetric Neuropathy Associated with Decreased Electrical Excitability of the Femoral Nerves.

Introduction: There are many phenotypic variants of chronic inflammatory demyelinating polyneuropathy. Methods:An Ancient Greek aryvallos painted c. 480-450 BC, now on display at the Louvre museum, was meticulously studied regarding its painted surface, which presents an outpatient clinic in Ancient Greece. Other Ancient Greek works of art presenting medical activities have been also evaluated in order to reach informed conclusions regarding medical practice of that period. Case report: We report a rare case of the distal phenotype of chronic inflammatory demyelinating polyneuropathy with a subacute onset and rapidly progressive course. A 58-year-old male had distal, symmetric, predominantly motor impairment without ataxia and tremor. After a three-month duration of the disease, the patient had already complete paresis of the feet with absence of compound muscle action potentials (CMAPs) over the feet and lower leg muscles, but preserved proprioception and sural sensory nerve action potential. Cerebrospinal fluid protein level was elevated to 3.4 g/L. Demyelinating neuropathy was predominantly in the proximal segment of the nerves. Low amplitude of CMAPs was recorded hardly over the vastus medialis and rectus femoris muscles, while weakness and atrophy in these muscles were not. The patient was refractory to treatment. He died three years after disease onset. Conclusion:We described a new clinical-electrophysiological phenomenon, which was characterized as a decrease in the evoked electrical excitability at the femoral nerve stimulation site (decreased CMAP), while the natural physiological conduction of the impulse from the motor neuron to the muscle was not blocked (preserved muscle strength).

Predicting CAR-T cell Immunotherapy Success through ImmunoPET.

Chimeric antigen receptor (CAR)-T cell therapy has generated unprecedented advances in the treatment of hematologic cancers, but readily translatable imaging approaches to visualize the in vivo dynamics of CAR-T cells are lacking. Noninvasive PET imaging is the ideal tool to monitor CAR-T cells.See related article by Simonetta et al., p. 1058.

Applications of nuclear-based imaging in gene and cell therapy: probe considerations.

Several types of gene- and cell-based therapeutics are now emerging in the cancer immunotherapy, transplantation, and regenerative medicine landscapes. Radionuclear-based imaging can be used as a molecular imaging tool for repetitive and non-invasive visualization as well as in vivo monitoring of therapy success. In this review, we discuss the principles of nuclear-based imaging and provide a comprehensive overview of its application in gene and cell therapy. This review aims to inform investigators in the biomedical field as well as clinicians on the state of the art of nuclear imaging, from probe design to available radiopharmaceuticals and advances of direct (probe-based) and indirect (transgene-based) strategies in both preclinical and clinical settings. Notably, as the nuclear-based imaging toolbox is continuously expanding, it will be increasingly incorporated into the clinical setting where the distribution, targeting, and persistence of a new generation of therapeutics can be imaged and ultimately guide therapeutic decisions.