Is oncolytic adenoviral-mediated immunotherapy through p53-overexpression the solution to refractory pancreatic ductal adenocarcinoma?

Evaluation of: Araki H, Tazawa H, Kanaya N, et al. Oncolytic virus-mediated p53 overexpression promotes immunogenic cell death and efficacy of PD-1 blockade in pancreatic cancer. Mol Ther Oncolytics. 2022;27:3-13.Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with poor prognosis. PDAC has a dense, desmoplastic stroma and immunosuppressive microenvironment, which impedes current treatment options. Immunotherapy delivered via oncolytic virotherapy is one potential solution to these barriers. Immune checkpoint inhibitors may facilitate immunogenic cell death by improving immune cell infiltration in cancer cells. PD-1 blockade shows better clinical outcomes for certain cancers. The addition of p53 to stimulate cell cycle arrest remains a novel field of research. The evaluated article by Araki et al. explores the efficacy of PD-1 blockade with oncolytic adenovirus platforms on immunogenic cell death and the possibility of combining PD-1 blockade and p53-activation. In vitro analysis showed increased cell death in multiple cell lines infected with AdV mediating p53 expression. The underlying process may attribute to apoptosis and autophagy, with evidence of increased immunogenic cell death. In vivo models demonstrated improved efficacy of p53-expressing AdV, particularly with the addition of PD-1 blockade which appears to be related to CD8+ cell infiltration.

Assessing the effect of hypoxia on cardiac metabolism using hyperpolarized 13 C magnetic resonance spectroscopy.

Hypoxia plays a role in many diseases and can have a wide range of effects on cardiac metabolism depending on the extent of the hypoxic insult. Noninvasive imaging methods could shed valuable light on the metabolic effects of hypoxia on the heart in vivo. Hyperpolarized carbon-13 magnetic resonance spectroscopy (HP 13 C MRS) in particular is an exciting technique for imaging metabolism that could provide such information. The aim of our work was, therefore, to establish whether hyperpolarized 13 C MRS can be used to assess the in vivo heart's metabolism of pyruvate in response to systemic acute and chronic hypoxic exposure. Groups of healthy male Wistar rats were exposed to either acute (30 minutes), 1 week or 3 weeks of hypoxia. In vivo MRS of hyperpolarized [1-13 C] pyruvate was carried out along with assessments of physiological parameters and ejection fraction. Hematocrit was elevated after 1 week and 3 weeks of hypoxia. 30 minutes of hypoxia resulted in a significant reduction in pyruvate dehydrogenase (PDH) flux, whereas 1 or 3 weeks of hypoxia resulted in a PDH flux that was not different to normoxic animals. Conversion of hyperpolarized [1-13 C] pyruvate into [1-13 C] lactate was elevated following acute hypoxia, suggestive of enhanced anaerobic glycolysis. Elevated HP pyruvate to lactate conversion was also seen at the one week timepoint, in concert with an increase in lactate dehydrogenase (LDH) expression. Following three weeks of hypoxic exposure, cardiac metabolism of pyruvate was comparable with that observed in normoxia. We have successfully visualized the effects of systemic hypoxia on cardiac metabolism of pyruvate using hyperpolarized 13 C MRS, with differences observed following 30 minutes and 1 week of hypoxia. This demonstrates the potential of in vivo hyperpolarized 13 C MRS data for assessing the cardiometabolic effects of hypoxia in disease.

Investigating mitochondrial metabolism in contracting HL-1 cardiomyocytes following hypoxia and pharmacological HIF activation identifies HIF-dependent and independent mechanisms of regulation.

Hypoxia is a consequence of cardiac disease and downregulates mitochondrial metabolism, yet the molecular mechanisms through which this occurs in the heart are incompletely characterized. Therefore, we aimed to use a contracting HL-1 cardiomyocyte model to investigate the effects of hypoxia on mitochondrial metabolism. Cells were exposed to hypoxia (2% O2) for 6, 12, 24, and 48 hours to characterize the metabolic response. Cells were subsequently treated with the hypoxia inducible factor (HIF)-activating compound, dimethyloxalylglycine (DMOG), to determine whether hypoxia-induced mitochondrial changes were HIF dependent or independent, and to assess the suitability of this cultured cardiac cell line for cardiovascular pharmacological studies. Hypoxic cells had increased glycolysis after 24 hours, with glucose transporter 1 and lactate levels increased 5-fold and 15-fold, respectively. After 24 hours of hypoxia, mitochondrial networks were more fragmented but there was no change in citrate synthase activity, indicating that mitochondrial content was unchanged. Cellular oxygen consumption was 30% lower, accompanied by decreases in the enzymatic activities of electron transport chain (ETC) complexes I and IV, and aconitase by 81%, 96%, and 72%, relative to controls. Pharmacological HIF activation with DMOG decreased cellular oxygen consumption by 43%, coincident with decreases in the activities of aconitase and complex I by 26% and 30%, indicating that these adaptations were HIF mediated. In contrast, the hypoxia-mediated decrease in complex IV activity was not replicated by DMOG treatment, suggesting HIF-independent regulation of this complex. In conclusion, 24 hours of hypoxia increased anaerobic glycolysis and decreased mitochondrial respiration, which was associated with changes in ETC and tricarboxylic acid cycle enzyme activities in contracting HL-1 cells. Pharmacological HIF activation in this cardiac cell line allowed both HIF-dependent and independent mitochondrial metabolic changes to be identified.

Metabolic adaptation to chronic hypoxia in cardiac mitochondria.

Chronic hypoxia decreases cardiomyocyte respiration, yet the mitochondrial mechanisms remain largely unknown. We investigated the mitochondrial metabolic pathways and enzymes that were decreased following in vivo hypoxia, and questioned whether hypoxic adaptation was protective for the mitochondria. Wistar rats were housed in hypoxia (7 days acclimatisation and 14 days at 11% oxygen), while control rats were housed in normoxia. Chronic exposure to physiological hypoxia increased haematocrit and cardiac vascular endothelial growth factor, in the absence of weight loss and changes in cardiac mass. In both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria isolated from hypoxic hearts, state 3 respiration rates with fatty acid were decreased by 17-18%, and with pyruvate were decreased by 29-15%, respectively. State 3 respiration rates with electron transport chain (ETC) substrates were decreased only in hypoxic SSM, not in hypoxic IFM. SSM from hypoxic hearts had decreased activities of ETC complexes I, II and IV, which were associated with decreased reactive oxygen species generation and protection against mitochondrial permeability transition pore (MPTP) opening. In contrast, IFM from hypoxic hearts had decreased activity of the Krebs cycle enzyme, aconitase, which did not modify ROS production or MPTP opening. In conclusion, cardiac mitochondrial respiration was decreased following chronic hypoxia, associated with downregulation of different pathways in the two mitochondrial populations, determined by their subcellular location. Hypoxic adaptation was not deleterious for the mitochondria, in fact, SSM acquired increased protection against oxidative damage under the oxygen-limited conditions.

Reversible vacuolation of T-tubules in skeletal muscle: mechanisms and implications for cell biology.

The majority of investigations of the transverse tubules (T-system) of skeletal muscle have been devoted to their role in excitation-contraction coupling, with particular reference to contact with the sarcoplasmic reticulum and the mechanism of Ca2- release. By contrast, this review is concerned with structural and functional aspects of the vacuolation of T-tubules. It covers experimental procedures used in reversible vacuolation induced by the efflux-influx of glycerol and other small nonelectrolytes, sugars, and ions. The characteristics of the phenomenon, associated alterations in muscle function, and the swelling of analogous structures in nonmuscle cells are considered. Possible functions of reversible vacuolation in water balance, transport, membrane repair, muscle pathology, and fatigue are considered, and the potential application of reversible vacuolation in the transfection of skeletal muscle is discussed. In relation to the possible mechanisms involved in reversible vacuolation, particular attention is given to the dynamic and structural aspects of the opening and closing of T-tubules, the origin of vacuolar membranes, and the localized character of tubular swelling.

Phosphatidylserine-dependent adhesion of T cells to endothelial cells.

Phosphatidylserine (PS) was exposed at the surface of human umbilical vein endothelial cells (HUVECs) and cultured cell lines by agonists that increase cytosolic Ca(2+), and factors governing the adhesion of T cells to the treated cells were investigated. Thrombin, ionophore A23187 and the Ca(2+)-ATPase inhibitor 2, 5-di-tert-butyl-1,4-benzohydroquinone each induced a PS-dependent adhesion of Jurkat T cells. A23187, which was the most effective agonist in releasing PS-bearing microvesicles, was the least effective in inducing the PS-dependent adhesion of Jurkat cells. Treatment of ECV304 and EA.hy926 cells with EGTA, followed by a return to normal medium, resulted in an influx of Ca(2+) and an increase in adhering Jurkat cells. Oxidised low-density lipoprotein induced a procoagulant response in cultured ECV304 cells and increased the number of adhering Jurkat cells, but adhesion was not inhibited by pretreating ECV304 cells with annexin V. PS was not significantly exposed on untreated Jurkat cells, as determined by flow cytometry with annexin V-FITC. However, after adhesion to thrombin-treated ECV304 cells for 10 min followed by detachment in 1 mM EDTA, there was a marked exposure of PS on the Jurkat cells. Binding of annexin V-FITC to the detached cells was inhibited by pretreating them with unlabelled annexin V. Contact with thrombin-treated ECV304 cells thus induced the exposure of PS on Jurkat cells and, as Jurkat cells were unable to adhere to thrombin-treated ECV304 cells in the presence of EGTA, the adhesion of the two cell types may involve a Ca(2+) bridge between PS on both cell surfaces. The number of T cells from normal, human peripheral blood that adhered to ECV304 cells was not increased by treating the latter with thrombin. However, findings made with several T cell lines were generally, but not completely, consistent with the possibility that adhesion to surface PS on endothelial cells may be a feature of T cells that express both CD4(+) and CD8(+) antigens. Possible implications for PS-dependent adhesion of T cells to endothelial cells in metastasis, and early in atherogenesis, are discussed.

Accessibility of T-tubule vacuoles to extracellular dextran and DNA: mechanism and potential application of vacuolation.

In addition to its function in excitation-contraction coupling, the ability of the T-system of skeletal muscle fibres to undergo reversible vacuolation indicates that it may play a role in volume regulation. The mechanism of reversible vacuolation has been investigated by confocal microscopy using fluorescein dextran to probe the accessibility of T-tubules to the extracellular environment. Vacuolation was induced by loading the fibres with 60-100 nM glycerol for 30 minutes and then returning them to glycerol-free medium. Devacuolation was subsequently induced by the reentry of glycerol. During their formation from T-tubules, the vacuoles filled with fluorescent dextran from the extracellular medium. The inaccessibility of the vacuoles to extracellular ferritin observed in a previous study raised the possibility that the vacuoles may be detached from the surface membrane after their formation. However, it is apparent from the present work that, although the tubules of the treated fibres are constricted, the vacuoles maintain a open connection with the external medium for smaller macromolecules. In the light of these experiments, it is proposed that vacuolation is caused by water moving into T-tubules from the cytoplasm faster than it can exit to the extracellular space during a decrease in fibre volume. Since T-tubules have been implicated in the transfection of skeletal muscle by direct injection, the accessibility of plasmid DNA to T-tubules has also been investigated. DNA penetrated into the vacuoles from the extracellular medium less well than dextran, but many vacuoles containing fluorescent DNA were observed in the superficial layers of vacuolated fibres, and it is suggested that T-tubule vacuolation might be used to improve the efficiency of the transfection of skeletal muscle by direct injection.

[Functional role of vacuolization of the T-system of skeletal muscle fibers]. / Funktsional'naia rol' vakuolizatsii T-sistemy skeletnykh myshechnykh volokon.

T-tubules of skeletal muscle fibres easily transform into large vacuoles under the influence of various factors. These include osmotic shock produced by the efflux of small molecular weight molecules (e.g. glycerol), hypertonic shock, muscle fatigue and muscle damage. In most cases, vacuolation is reversible but the molecular mechanisms involved are not clear. Also, the functional role of reversible vacuolation has not been established. However, three possibilities may be considered. (1) Redistribution of ions and water between the cytoplasm and the extracellular space comprised by the T-system. Thus, the formation of large vacuoles may be a mechanisms for rapid osmoregulation that corresponds to regulated volume decrease in other types of cell. However, in our hands, inhibitors of various pathways that participate in volume regulation had no effect on reversible vacuolation. (2) Resealing of mechanical damage of the plasma membrane. This is usually accompanied by the development near the damaged membrane of numerous vacuoles which we have observed by confocal microscopy and use of a hydrophobic dye (RH414), to arise in part from T-tubules. (3) By confocal microscopy, it has also been shown that extracellular fluorescein dextran (Mr = 10,000), and both plasmid DNA (pUC18) and sonicated high molecular weight DNA stained with YOYO, enter vacuoles derived from T-tubules. This finding may indicate that reversible vacuolation, in the absence of membrane damage, could provide a pathway from the extracellular environment to the cytoplasm that is additional or complimentary to endocytosis; it may also be particularly relevant to the ability of muscle to be transfected by the direct injection of DNA. These several observations strongly indicate that the function of the T-system in skeletal muscle fibers is not restricted to excitation-contraction coupling.

Phosphatidylserine-mediated adhesion of T-cells to endothelial cells.

Phosphatidylserine was exposed on the surface of human umbilical endothelial cells (ECV304) a few minutes after adding thrombin in vitro, as monitored by prothrombinase assays with and without annexin V. Jurkat T cells adhered to the thrombin-treated cells. The adhesion was inhibited by annexin V, indicating that it was mediated by exposed phosphatidylserine on the endothelial cells.

Reversible vacuolation of the transverse tubules of frog skeletal muscle: a confocal fluorescence microscopy study.

A confocal microscope was used to investigate the reversible vacuolation of frog skeletal muscle fibres produced by the efflux and entry of glycerol (80-100 mM). The formation, development and disappearance of vacuoles was observed in the fibres by staining simultaneously with two fluorescent membrane probes, RH414 and DiOC6(3). The styryl dye, RH414, stains only the plasmalemma and the membranes of the transverse tubules. In normal and glycerol-loaded fibres, RH414 revealed regular, narrow dotted bands located at the position of the Z-lines. Glycerol removal produced, within 2-10 min, many empty round vacuoles (0.4-1.5 microns in diameter) that were apparently anchored to the stained bands. Later on, individual vacuoles tended to enlarge and align into longitudinal chains of vacuoles. Neighbouring vacuoles that contacted each other fused to form large vacuoles up to several sarcomeres in length. Neither the T-tubules, nor the vacuoles, were stained by DiOC6(3). However, glycerol efflux was also accompanied by a redistribution of sarcoplasmic reticulum membranes and by changes in mitochondria that were revealed on staining the same fibres with the carbocyanine dye, DiOC6(3). The alterations in staining patterns revealed by RH414 and DiOC6(3) were completely reversible. Within 5-10 min after a second application of glycerol, the pattern of staining returned to normal with the exception of very bright, spots stained with RH414, which appeared in place of many but not all of the vacuoles, and probably correspond to the irregular nets of T-tubules observed under the electron microscope in such fibres. They are considered to be defects in regeneration of the T-system after vacuolation. The vacuolation/devacuolation cycle could be repeated several times following glycerol efflux and entry. The development and disappearance of vacuoles then mainly involved conversion of bright spots to large vacuoles and vice versa. Some possible mechanisms of vacuole formation and disappearance are discussed, and it is suggested that vacuolation of the T-system may be important in relation to regulating the volume of skeletal muscle cells.

Spatial and temporal distribution of [Ca2+]i in normal human myotubes. A fura-2 imaging study.

The spatio-temporal distribution of intracellular, free calcium ions, [Ca2+]i, induced in human myotubes by electrical stimulation typically showed a relatively large increase of [Ca2+]i in the vicinity of the plasmalemma. The similarity of this distribution, with that observed after the application of caffeine, and the lack of any effect of lanthanum, strongly suggest that the main source of Ca2+ participating in the electrically induced transient is the sarcoplasmic reticulum. Aneurally cultured human myotubes therefore display a 'skeletal muscle type' coupling between membrane depolarization and calcium release. However, the relatively slow time course of the electrically induced transients compared to rat and mouse myotubes, together with the inability of Ca2+ released from the sarcoplasmic reticulum to activate the contractile machinery, implies that aneurally cultured human myotubes achieve only a limited degree of differentiation. The relevance this may have to an apparent delay between the electrically induced rise in intranuclear Ca2+ relative to cytosolic Ca2+ remains to be determined but, at this stage of differentiation, there appears to be some form of barrier to free diffusion between the two cellular compartments.

A spectroscopic study of the mitochondrial transit peptide of rat malate dehydrogenase.

A peptide corresponding to the N-terminal sequence of the rat malate dehydrogenase, comprising the transit sequence and two residues of the mature protein (MLSALARPVGAALR-RSFSTSAQNNAK) has been chemically synthesized, and its structural characteristics investigated by Fourier-transform i.r. (FT-IR), c.d. and 1H-n.m.r. spectroscopy. FT-IR and c.d. spectra of the peptide were recorded in a variety of environments (aqueous solution, trifluoroethanol) and after incorporation into phospholipid bilayers. The peptide was found to be mainly in aperiodic or undefined conformation in aqueous solution. However, in trifluoroethanol a marked increase in alpha-helical content was observed. An increase in alpha-helical content was also observed in negatively charged lipids (dimyristoylphosphatidylglycerol and cardiolipin). However, when reconstituted in a zwitterionic phospholipid (dimyristoylphosphatidylcholine), no alpha-helical structure was observed. N.m.r. spectroscopy was used to characterize the helical structure in greater detail in trifluoroethanol. The 1H-n.m.r. spectrum of the peptide in this solvent was assigned using standard homonuclear two-dimensional methods. The observed patterns of nuclear Overhauser enhancements confirmed the deductions obtained from c.d. and FT-1R spectroscopy concerning the solution conformation, suggesting a region of flexible nascent helix between Ala-4 and Ser-18. This structure is discussed in terms of the possible function of the peptide.

Interactions between metal ions and poly(ethylene glycol) in the fusion of human erythrocytes.

Diffusion of the fluorescent membrane probe, Dil-C16 (3), from labelled to unlabelled human erythrocytes has been employed to monitor hemi-fusion (membrane fusion) in monolayers of cells exposed to poly(ethylene glycol) (PEG). Diffusion of the cytoplasmic probe, 6-carboxyfluorescein, was used similarly to monitor cell fusion (cytoplasmic mixing). Hemi-fusion, which is normally seen when erythrocytes are exposed to dehydrating concentrations of commercial PEG 6000, did not occur when the PEG was pretreated with Chelex 100 resin to remove metal ions. Cytoplasmic mixing, which is normally observed when the dehydrated erythrocytes are substantially rehydrated, also failed to occur when both PEG 6000 and the rehydrating buffer had been treated with Chelex 100. The re-addition to Chelex-treated PEG of components removed by the resin, and the addition of 10 mu mM concentrations of La3+ or Al3+, restored its ability to induce hemi-fusion and cell fusion. Higher concentrations of several other metals, including Ca2+, were also effective. These observations show that metal ions are required for hemi-fusion with erythrocytes in the presence of PEG, and that dehydration alone is insufficient to induce hemi-fusion. Phosphatidylserine was apparently not accessible in erythrocytes treated with PEG 6000 until the cells were rehydrated. This indicates that metal ions do not assist the hemi-fusion of erythrocytes by forming trans complexes with surface phosphatidylserine when the cells are dehydrated by PEG.(ABSTRACT TRUNCATED AT 250 WORDS)

Dystrophin as a mechanochemical transducer in skeletal muscle.

This review is primarily concerned with two key issues in research on dystrophin: (1) how the protein interacts with the plasma membrane of skeletal muscle fibres and (2) how an absence of dystrophin gives rise to Duchenne muscular dystrophy. In relation to the first point, we suggest that the post-translational acylation of dystrophin may contribute to its interaction with the plasma membrane. Regarding the second point, it is generally considered that an absence of dystrophin makes the plasma membrane susceptible to damage by contraction/relaxation cycles. In this connection, we propose that the progressive nature of Duchenne dystrophy, and the phenotypic characteristics of mdx mice, are more consistent with dystrophin functioning as a mechanical transducer that transmits growth stimuli from the enlarging skeleton to the muscle. On the basis of this hypothesis, dystrophin-deficient muscles would be unable to grow at the same rate as the skeleton.

Divalent cations, phospholipid asymmetry and osmotic swelling in electrically-induced lysis, cell fusion and giant cell formation with human erythrocytes.

We have previously reported that acidic phospholipids are exposed at the surface of human erythrocytes when the cells are subjected to electrical breakdown. It has now been shown that the prothrombinase assay, which was used previously for the determination of acidic phospholipids, is specific for phosphatidylserine under the conditions of our experiments. In the light of this finding, we have investigated and characterised factors that govern cell lysis, cell fusion, and the formation of giant cells induced by electrical breakdown with human erythrocytes in media of low ionic strength. Divalent cations (1.1 mM) protected the cells against haemolysis, in the order Mn2+ > Ca2+ > Ba2+ > Mg2+ >> Zn2+, whereas about 99% of the cells lysed immediately on breakdown in the presence of Na+ or K+ (2.1 mM), or Al3+ (0.95 mM). The lengths of pearl chains of fused erythrocytes formed was similarly greatest with Mn2+ and decreased progressively with Ba2+, Zn2+, Ca2+ and Mg2+. No cell fusion occurred with Na+, K+, or Al3+. It is suggested that interactions with phosphatidylserine, which is exposed at the cell surface by electrical breakdown, may enable Mn2+, Ba2+ and Ca2+ ions to inhibit cell lysis (via membrane resealing) and facilitate cell fusion. Following electrically-induced cell fusion, erythrocytes round-up into giant cells. It has previously been proposed that Ca2+ ions accelerate the rounding-up process. However, data are presented which show that, as with erythrocytes treated with Sendai virus, the formation of rounded, giant cells following cell fusion depends on the osmotic swelling properties of permeabilised erythrocytes. Osmotic swelling may also have induced any hemi-fused cells present to fuse completely. Zn2+ ions anomalously enabled erythrocytes to round-up very rapidly into giant cells following electrical breakdown. This phenomenon may result from an interaction of Zn2+ ions with cysteine groups in membrane proteins, which decreases the immediate loss of ions that occurs when erythrocytes are subjected to electrical breakdown in low-ionic-strength media.

Relationships between the surface exposure of acidic phospholipids and cell fusion in erythrocytes subjected to electrical breakdown.

The procoagulant activity of human erythrocytes, which provides a measure of the translocation of acidic phospholipids from the inner to the outer monolayer of the plasma membrane, has been compared with the percentage cell fusion in experiments on the effects of electrical breakdown pulses under differing experimental conditions. After treatment with breakdown pulses of 20 microseconds or longer (5 kV cm-1), the plasma membranes of erythrocytes in 250 mM sucrose exhibited an almost complete loss of asymmetry with respect to acidic phospholipids. As the breakdown voltage was increased from 2 to 5 kV cm-1 (with breakdown pulses of 99 microseconds), the surface exposure of acidic phospholipids and cell fusion increased approximately in parallel. Furthermore, with 99 microseconds pulses and a voltage of 3 kV cm-1, a decrease in the osmolarity from 250 to 150 mM of the sucrose medium was accompanied by an increase in both the surface exposure of acidic phospholipids and the extent of cell fusion. Breakdown pulses of 2-5 microseconds were sufficient to cause a marked loss of asymmetry, but no cell fusion was observed unless the pulse length was at least 20 microseconds. Kinetic experiments indicated that exposure of the acidic phospholipids at the cell surface was more likely to be due to a direct effect of the electric field pulses on plasma membrane structure than to secondary effects, such as the action of endogenous proteinases on the membrane skeleton. It seems possible that a localised, surface exposure of acidic phospholipids may contribute to the 'long-lived fusogenic state' (Sowers, A.E. (1986) J. Cell Biol. 102, 1358-1362) and the 'transient permeant structures' (Teissié, J. and Rols, M.P. (1986) Biochem. Biophys. Res. Commun. 140, 258-266) that enable cell fusion to occur when contact between cells is established after they have been subjected to field pulses. Our observations also provide circumstantial support for the concept that changes in the phospholipid asymmetry of membranes may be important in physiologically-occurring instances of biomembrane fusion.