Spin labelling of Bacillus anthracis endospores: a model for in vivo tracking by EPR imaging

Anthrax is caused by the gram-negative bacterium, Bacillus anthracis. Infection by this microbe results from delivery of the endospore form of the bacillus through direct contact, either topical or inhalation. With regard to the latter route of administration, it is proposed that endospores of B. anthracis enter the lungs and are phagocytized by host alveolar macrophages. Thereafter, it is unclear as to how endospores travel to distal loci and what tissues are the targets. Herein, this study describes the spin labelling of endospores through two different approaches with various aminoxyls. Indeed, after exposure to RAW 264.7 cells, these aminoxyl-containing endospores were phagocytized, as demonstrated by EPR spectroscopy of the infected macrophage, thus providing a potential tool for EPR imaging in animals.     

Cell-penetrating nitroxides as molecular sensors for imaging of cancer in vivo, based on tissue redox activity

The present study shows that hydrophobic and cell-penetrating piperidine-type nitroxide radicals SLENU and TEMPOL, but not hydrophilic and partially penetrating or non-penetrating pyrrolidine-type nitroxides carbamoyl-PROXYL and carboxy-PROXYL, are appropriate contrast agents for magnetic resonance imaging (MRI) of cancer, based on its functionality - tissue redox activity. The experiments were conducted on anesthetized mice: healthy and neuroblastoma-bearing in a moderate stage of cancer development. The method is based on the nitroxide redox cycle, coupled with appearance or disappearance of the MRI signal. The half-life (τ(1/2)) of a nitroxide-enhanced MRI signal in the respective tissue was used as a marker to assess tissue redox activity to the nitroxide radical. In the case of SLENU and TEMPOL, there were large differences in the histograms between control and cancer-bearing mice. All tissues (cancer and non-cancer) of cancer-bearing organisms were characterized by a long-lived MRI signal (τ(1/2) > 14 min), indicating a high oxidative activity. The tissues of healthy organisms were characterized by a short-lived MRI signal (τ(1/2) = 1-3 min), indicating a high reducing activity. In the case of carbamoyl-PROXYL and carboxy-PROXYL, there was no difference in the histograms between control and cancer-bearing mice. The data show that the penetration of nitroxide in cells and tissues is obligatory for imaging of cancer, based on its redox activity. The principle of the method is applicable also to biopsy specimens, using MRI or EPR spectroscopy. We provide direct evidence that the nitroxide redox cycle could be used as a sensing platform for functional imaging of different pathologies, based on changes in cellular and tissue redox activity, as in the case of cancer.     

Metabolically labile cannabinoid esters: a 'soft drug' approach for the development of cannabinoid-based therapeutic drugs

Biphenylic ester derivatives, designed by using a 'soft-drug' approach, proved to possess good binding properties toward cannabinoid CB(1) and CB(2) receptors and, at the same time, their metabolically labile ester portion would promote a rapid systemic inactivation. This may constitute a possible solution to the psychotropic side effects encountered when cannabinoids are therapeutically employed as local analgesic or antiglaucoma agents.     

An in vivo model for monitoring trans-differentiation of bone marrow cells into functional hepatocytes

The plasticity of bone marrow cells (BMCs) remains controversial. The present study found that persistent injury induces efficient trans-differentiation of BMCs into functional hepatocytes. Mice with liver cirrhosis induced by carbon tetrachloride were injected with 1 x 10(5) non-treated green fluorescent protein (GFP)-positive BMCs via the tail vein. In these mice, transplanted GFP-positive BMCs efficiently migrated into the peri-portal area of liver lobules after one day, repopulating 25% of the recipient liver by 4 weeks. In contrast, no GFP-positive BMCs were detected following transplantation into control mice with undamaged livers. BMCs trans-differentiated into functional mature hepatocytes via immature hepatoblasts. Serum albumin levels were significantly elevated to compensate for chronic liver failure in BMC transplantation. These results reveal that recipient conditions and microenvironments represent key factors for successful cell therapy using BMCs.     

Optimization in the recovery of a labile intracellular enzyme

A high molecular weight intracellular enzyme of Bacillus brevis ATCC 9999 is released when the organism is disrupted by sonication of homogenization. However, both processes also degrade the enzyme. Assays for protein release and specific enzymatic activity of the released protein indicate that both release and degradation can be represented by first-order kinetic models. Utilization of the difference between the kinetics of release and degradation allows optimization in the recovery of this enzyme for both the sonication and homogenization processes.     

Development of nitroxides for selective localization inside cells

The use of nitroxides to measure intracellular phenomena, especially oxygen concentrations, is a new and potentially important approach to a number of physiological and pathophysiological studies. This study provides data indicating the feasibility of developing nitroxides that localize selectively in the intracellular compartment; it is based on the use of readily hydrolysed ester linkages, such that the nitroxides become converted intracellularly to ionic derivatives that do not cross cell membranes readily. Up to 120-fold increased concentrations of intracellular nitroxides (and their one electron reduction product, the hydroxylamines) were obtained. The ESR spectra of the intracellular nitroxides were consistent with their conversion to the ionic species. Preliminary studies indicate that these nitroxides have the properties needed for their use as probes of intracellular concentrations of oxygen and that it should be feasible to synthesize nitroxides that will be even more effective for this purpose.     

Phorbol esters modulate cyclic AMP accumulation in porcine thyroid cells

In cultured porcine thyroid cells, during 60 min incubation phorbol 12-myristate 13-acetate (PMA) had no effect on basal cyclic AMP accumulation and slightly stimulated cyclic AMP accumulation evoked by thyroid stimulating hormone (TSH) or forskolin. Cholera toxin-induced cyclic AMP accumulation was significantly stimulated by PMA. On the other hand, cyclic AMP accumulation evoked by prostaglandin E1 or E2 (PGE1 or PGE2) was markedly depressed by simultaneous addition of PMA. These opposing effects of PMA on cyclic AMP accumulation evoked by PGE and cholera toxin were observed in a dose-related fashion, with half-maximal effect of around 10(-9) M in either case. The almost same effects of PMA on cyclic AMP accumulation in basal and stimulated conditions were also observed in freshly prepared thyroid cells. The present study was performed in the presence of phosphodiesterase inhibitor, 3-iso-butyl-1-methylxanthine (IBMX), indicating that PMA affected adenylate cyclase activity. Therefore, it is suggested that PMA may modulate the production of cyclic AMP in response to different stimuli, possibly by affecting several sites in the adenylate cyclase complex in thyroid cells.     

In vivo imaging of autophagy in a mouse stroke model

Recent studies have suggested that autophagy is involved in a neural death pathway following cerebral ischemia. In vivo detection of autophagy could be important for evaluating ischemic neural cell damage for human stroke patients. Using novel green fluorescent protein (GFP)-fused microtubule-associated protein 1 light chain 3 (LC3) transgenic (Tg) mice, in vivo imaging of autophagy was performed at 1, 3 and 6 d after 60 min transient middle cerebral artery occlusion (tMCAO). Ex vivo imaging of autophagy, testing of the autophagy inhibitor 3-methyladenine (3-MA), estern blot analysis, immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) and fluorescent analyses were performed on brain sections following tMCAO. In vivo fluorescent signals were detected above the ischemic hemisphere through the skull bone at 1, 3 and 6 d after tMCAO, with a peak at 1 d. Similar results were obtained with ex vivo fluorescence imaging. western blot analysis revealed maximum LC3-I and LC3-II expression at 1 d after tMCAO and fluorescence immunohistochemistry demonstrated that GFP-LC3-positive cells were primarily neuronal, not astroglial or microglial, cells. The number of GFP-LC3/TUNEL double-positive cells was greater in the periischemic area than in the core. These results provided evidence of in vivo autophagy detection, with a peak at 1 d, in a live animal model following cerebral ischemia. This novel technique could be valuable for monitoring autophagic processes in vivo in live stroke patients, as well as for clarifying the detailed role of autophagy in the ischemic brain, as well as in other neurological diseases.     

A novel ascorbic acid-resistant nitroxide in fat emulsion is an efficient brain imaging probe for in vivo EPR imaging of mouse

Abstract

The loss of paramagnetism of nitroxide radicals due to reductant reactions in biological systems, places a fundamental time constraint on their application as an imaging probe in in vivo EPR imaging studies. However, in vitro studies of the newly synthesized tetraethyl-substituted piperidine nitroxide radical demonstrated high resistivity to paramagnetic reduction when exposed to ascorbic acid, a common reduction agent in biological systems. In this work we investigated the use of these nitroxides as an imaging probe in EPR imaging of small rodents. 2,2,6,6-Tetraethyl-piperidine nitroxide (TEEPONE) is not highly soluble in aqueous media, thus a lipid-based emulsion system of lecithin was used to solubilize TEEPONE. The obtained solution was homogenous and with low viscosity, allowing smooth intravenous injection into mice tail vein. Acquired three dimensional (3D) EPR images of mouse head clearly showed TEEPONE distributed in all tissues including brain tissues, with an average measurable signal half-life of more than 80 min, thus demonstrating high resistivity to reduction due to ascorbic acid in in vivo animal studies, and the potential for use of this compound in in vivo studies of animal model systems.     

Phorbol esters inhibit phosphate uptake in opossum kidney cells: a model of proximal renal tubular cells

The effects of phorbol esters and diacylglycerol on phosphate uptake in opossum kidney (OK) cells were investigated to assess the possible role of Ca2+-activated, phospholipid dependent protein kinase (protein kinase C) on renal phosphate handling. OK cells are widely used as a model of proximal renal tubular cells and are reported to possess a Na+-dependent phosphate transport system. Phorbol-12,13-dibutyrate (PDBu) inhibited phosphate uptake. This inhibitory effect was synergistically enhanced with A23187. 4 beta-phorbol 12,13-didecanoate inhibited phosphate uptake, while 4 alpha-phorbol 12,13-didecanoate did not. 1-oleoyl-2-acetyl-glycerol (OAG), a synthetic diacylglycerol, also exhibited an inhibitory effect on phosphate uptake. These data suggest the possible involvement of protein kinase C in proximal renal tubular phosphate transport.     

A novel ascorbic acid-resistant nitroxide in fat emulsion is an efficient brain imaging probe for in vivo EPR imaging of mouse

The loss of paramagnetism of nitroxide radicals due to reductant reactions in biological systems, places a fundamental time constraint on their application as an imaging probe in in vivo EPR imaging studies. However, in vitro studies of the newly synthesized tetraethyl-substituted piperidine nitroxide radical demonstrated high resistivity to paramagnetic reduction when exposed to ascorbic acid, a common reduction agent in biological systems. In this work we investigated the use of these nitroxides as an imaging probe in EPR imaging of small rodents. 2,2,6,6-Tetraethyl-piperidine nitroxide (TEEPONE) is not highly soluble in aqueous media, thus a lipid-based emulsion system of lecithin was used to solubilize TEEPONE. The obtained solution was homogenous and with low viscosity, allowing smooth intravenous injection into mice tail vein. Acquired three dimensional (3D) EPR images of mouse head clearly showed TEEPONE distributed in all tissues including brain tissues, with an average measurable signal half-life of more than 80 min, thus demonstrating high resistivity to reduction due to ascorbic acid in in vivo animal studies, and the potential for use of this compound in in vivo studies of animal model systems.     

In vivo imaging of immunotoxin treatment using Katushka-transfected A-431 cells in a murine xenograft tumour model

Purpose

Preclinical in vivo analyses of treatment responses are an important prerequisite to evaluate new therapeutics. Molecular in vivo imaging in the far red (FR)/near infra red (NIR) is a promising method, as it enables measurements at different time points in individual animals, thereby reducing the number of animals required, while increasing statistical significance. Here, we show the establishment of a method to monitor response to treatment using fluorescent cells, expressing the epidermal growth factor receptor (EGFR), a target already used in therapy.

Methods

We transfected A-431 tumour cells with the far red–emitting protein Katushka (Kat2), resulting in strong fluorescence allowing for the monitoring of tumour growth when implanted in BALB/c nu/nu mice with a CRi Maestro in vivo imager. We targeted A-431 cells with a previously reported immunotoxin (IT), consisting of the anti-EGFR antibody single-chain variable fragment (scFv) 425, fused to Pseudomonas aeruginosa Exotoxin A’ (ETA’). In addition, EGFR expression was verified using the 425(scFv) conjugated to a NIR dye BG-747 through a SNAP-tag linker.

Results

The results show the feasibility to evaluate response to treatment in vivo by FR imaging, while at the same location detecting EGFR expression. Treatment with 425(scFv)-ETA’ resulted in decelerated tumour growth, while not affecting the overall health of the animals. This is in contrast to treatment with Doxorubicin, which, although decreasing the tumour size, resulted in poor health.

Conclusions

We developed a novel method to non-invasively determine treatment responses by in vivo imaging of multiple parameters which showed the efficacy of 425(scFv)-ETA’.     

In vivo imaging platform for tracking immunotherapeutic cells

Cellular therapeutics show great promise for the treatment of disease, but few noninvasive techniques exist for monitoring the cells after administration. Here we present a magnetic resonance imaging (MRI) technology that uses perfluoropolyether (PFPE) agents to track cells in vivo. Fluorine MRI selectively images only the labeled cells, and a 'conventional' (1)H image places the cells in their anatomical context. We labeled phenotypically defined dendritic cells (DCs) with PFPE ex vivo and observed efficient intracellular uptake of the PFPE with little effect on DC function. We injected labeled DCs into tissue or intravenously in mice and then tracked the cells in vivo using (19)F MRI. Although we focused on DCs, which are being developed as immunotherapeutics for cancer and autoimmune diseases, this technology should be useful for monitoring a wide range of cell types in vivo.     

Early response to tendon fatigue damage accumulation in a novel in vivo model

This study describes the development and application of a novel rat patellar tendon model of mechanical fatigue for investigating the early in vivo response to tendon subfailure injury. Patellar tendons of adult female Sprague-Dawley rats were fatigue loaded between 1–35 N using a custom-designed loading apparatus. Patellar tendons were subjected to Low-, Moderate- or High-level fatigue damage, defined by grip-to-grip strain measurement. Molecular response was compared with that of a laceration-repair injury. Histological analyses showed that progression of tendon fatigue involves formation of localized kinked fiber deformations at Low damage, which increased in density with presence of fiber delaminations at Moderate damage, and fiber angulation and discontinuities at High damage levels. RT-PCR analysis performed at 1- and 3-day post-fatigue showed variable changes in type I, III and V collagen mRNA expression at Low and Moderate damage levels, consistent with clinical findings of tendon pathology and were modest compared with those observed at High damage levels, in which expression of all collagens evaluated were increased markedly. In contrast, only type I collagen expression was elevated at the same time points post-laceration. Findings suggest that cumulative fatigue in tendon invokes a different molecular response than laceration. Further, structural repair may not be initiated until reaching end-stage fatigue life, where the repair response may unable to restore the damaged tendon to its pre-fatigue architecture.     

Labeling of immune cells for in vivo imaging using magnetofluorescent nanoparticles

Observation of immune and stem cells in their native microenvironments requires the development of imaging agents to allow their in vivo tracking. We describe here the synthesis of magnetofluorescent nanoparticles for cell labeling in vitro and for multimodality imaging of administered cells in vivo. MION-47, a prototype monocrystalline iron oxide nanoparticle, was first converted to an intermediate bearing a fluorochrome and amine groups, then reacted with either HIV-Tat peptide or protamine to yield a nanoparticle with membrane-translocating properties. We describe how to assess optimal cell labeling with tests of cell phenotype and function. Synthesis of magnetofluorescent nanoparticles and cell-labeling optimization can be realized in 48 h, whereas nanoparticle uptakes and retention studies may generally take up to 120 h. Labeled cells can be detected by magnetic resonance imaging, fluorescence reflectance imaging, fluorescence-mediated tomography, confocal microscopy and flow cytometry, and can be purified based on their fluorescent or magnetic properties. The present protocol focuses on T-cell labeling but can be used for labeling a variety of circulating cells.     

Potential of albumin labeled with nitroxides as a contrast agent for magnetic resonance imaging and spectroscopy

The biological and physical properties of albumin and nitroxides make them attractive candidates as special purpose MRI contrast agents which could be used to study the intravascular compartment or specific targets in tissues. In this study, albumin-nitroxide complexes were prepared by reduction and alkylation of the disulfide bonds of the protein and characterized by electron spin resonance and ultraviolet absorption spectroscopy. An average of six nitroxides were bound covalently to each molecule of human serum albumin. The water proton relaxivity of the protein-bound nitroxide (at 20 MHz and 37 degrees C) was 4-fold greater than that of the free nitroxide. The digestion of the nitroxide-albumin complexes by cells or by trypsin decreased the relaxivity of the nitroxide-protein complex. The rate of reduction of albumin-bound nitroxide by cells was much slower than that of the free nitroxide but still was oxygen-sensitive (2-3-fold increase in the rate of reduction in the absence of oxygen).     

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.