ONCR-177, an Oncolytic HSV-1 Designed to Potently Activate Systemic Antitumor Immunity.

ONCR-177 is an engineered recombinant oncolytic herpes simplex virus (HSV) with complementary safety mechanisms, including tissue-specific miRNA attenuation and mutant UL37 to inhibit replication, neuropathic activity, and latency in normal cells. ONCR-177 is armed with five transgenes for IL12, FLT3LG (extracellular domain), CCL4, and antagonists to immune checkpoints PD-1 and CTLA-4. In vitro assays demonstrated that targeted miRNAs could efficiently suppress ONCR-177 replication and transgene expression, as could the HSV-1 standard-of-care therapy acyclovir. Although ONCR-177 was oncolytic across a panel of human cancer cell lines, including in the presence of type I IFN, replication was suppressed in human pluripotent stem cell-derived neurons, cardiomyocytes, and hepatocytes. Dendritic cells activated with ONCR-177 tumor lysates efficiently stimulated tumor antigen-specific CD8+ T-cell responses. In vivo, biodistribution analyses suggested that viral copy number and transgene expression peaked approximately 24 to 72 hours after injection and remained primarily within the injected tumor. Intratumoral administration of ONCR-177 mouse surrogate virus, mONCR-171, was efficacious across a panel of syngeneic bilateral mouse tumor models, resulting in partial or complete tumor regressions that translated into significant survival benefits and to the elicitation of a protective memory response. Antitumor effects correlated with local and distant intratumoral infiltration of several immune effector cell types, consistent with the proposed functions of the transgenes. The addition of systemic anti-PD-1 augmented the efficacy of mONCR-171, particularly for abscopal tumors. Based in part upon these preclinical results, ONCR-177 is being evaluated in patients with metastatic cancer (ONCR-177-101, NCT04348916).

Cytomegalovirus promotes murine glioblastoma growth via pericyte recruitment and angiogenesis.

Cytomegalovirus (CMV) has been implicated in glioblastoma (GBM); however, a mechanistic connection in vivo has not been established. The purpose of this study is to characterize the effects of murine CMV (MCMV) on GBM growth in murine models. Syngeneic GBM models were established in mice perinatally infected with MCMV. We found that tumor growth was markedly enhanced in MCMV+ mice, with a significant reduction in overall survival compared with that of controls (P < 0.001). We observed increased angiogenesis and tumor blood flow in MCMV+ mice. MCMV reactivation was observed in intratumoral perivascular pericytes and tumor cells in mouse and human GBM specimens, and pericyte coverage of tumor vasculature was strikingly augmented in MCMV+ mice. We identified PDGF-D as a CMV-induced factor essential for pericyte recruitment, angiogenesis, and tumor growth. The antiviral drug cidofovir improved survival in MCMV+ mice, inhibiting MCMV reactivation, PDGF-D expression, pericyte recruitment, and tumor angiogenesis. These data show that MCMV potentiates GBM growth in vivo by increased pericyte recruitment and angiogenesis due to alterations in the secretome of CMV-infected cells. Our model provides evidence for a role of CMV in GBM growth and supports the application of antiviral approaches for GBM therapy.

Expression patterns of heat shock protein genes in Rita rita from natural riverine habitat as biomarker response against environmental pollution.

River pollution is one of the principal environmental concerns and biomonitoring tools can play an important role in pollution assessment in the riverine environment. Heat shock proteins (Hsps) have been found to be suitable tools for monitoring stress response. In the present study, expression analyses of hsp genes (hsp27, hsp47, hsp60, hsp70, hsc70, and hsp90) and selected hsp-regulatory genes (hsf1, hyou1, ask1, jnk) were carried out by RT-qPCR in catfish Rita rita collected from selected stretches of river Ganga to investigate changes in their expression patterns as biomarker response. Water quality characteristics were measured in terms of physico-chemical characteristics (DO, BOD, COD, pH, conductivity), element profile (arsenic, mercury, cadmium, lead, chromium, zinc, copper) and persistent organic pollutants (POPs; HCH, DDT, aldrin, endosulphan, heptachlor). Water quality index was calculated and sampling sites were categorized as good/medium/bad. Multivariate analysis was carried out taking the water quality parameters and the fold changes in hsp gene expression as variables, which showed that hsp47 and hsp70b correlated well with BOD, an indicator of organic pollution. To identify the organic pollutant(s) which could be influencing the expression of hsps, again multivariate analysis was employed taking concentration of POPs and fold changes of hsps, which showed up-regulation of hsp47 and hsp70b (HSP72i) correlated well with concentrations of aldrin and HCH. Synergistic effects of these POPs could be responsible for the up-regulation of said hsps, although individually present in low concentration; thus, indicating synergistic effect of the POPs on hsp47 and hsp70b up-regulation as biomarker response.

Development of a Function-Blocking Antibody Against Fibulin-3 as a Targeted Reagent for Glioblastoma.

Purpose: We sought a novel approach against glioblastomas (GBM) focused on targeting signaling molecules localized in the tumor extracellular matrix (ECM). We investigated fibulin-3, a glycoprotein that forms the ECM scaffold of GBMs and promotes tumor progression by driving Notch and NFκB signaling.Experimental Design: We used deletion constructs to identify a key signaling motif of fibulin-3. An mAb (mAb428.2) was generated against this epitope and extensively validated for specific detection of human fibulin-3. mAb428.2 was tested in cultures to measure its inhibitory effect on fibulin-3 signaling. Nude mice carrying subcutaneous and intracranial GBM xenografts were treated with the maximum achievable dose of mAb428.2 to measure target engagement and antitumor efficacy.Results: We identified a critical 23-amino acid sequence of fibulin-3 that activates its signaling mechanisms. mAb428.2 binds to that epitope with nanomolar affinity and blocks the ability of fibulin-3 to activate ADAM17, Notch, and NFκB signaling in GBM cells. mAb428.2 treatment of subcutaneous GBM xenografts inhibited fibulin-3, increased tumor cell apoptosis, and enhanced the infiltration of inflammatory macrophages. The antibody reduced tumor growth and extended survival of mice carrying GBMs as well as other fibulin-3-expressing tumors. Locally infused mAb428.2 showed efficacy against intracranial GBMs, increasing tumor apoptosis and reducing tumor invasion and vascularization, which are enhanced by fibulin-3.Conclusions: To our knowledge, this is the first rationally developed, function-blocking antibody against an ECM target in GBM. Our results offer a proof of principle for using "anti-ECM" strategies toward more efficient targeted therapies for malignant glioma. Clin Cancer Res; 24(4); 821-33. ©2017 AACR.

BKM-120 (Buparlisib): A Phosphatidyl-Inositol-3 Kinase Inhibitor with Anti-Invasive Properties in Glioblastoma.

Glioblastoma is an aggressive, invasive tumor of the central nervous system (CNS). There is a widely acknowledged need for anti-invasive therapeutics to limit glioblastoma invasion. BKM-120 is a CNS-penetrant pan-class I phosphatidyl-inositol-3 kinase (PI3K) inhibitor in clinical trials for solid tumors, including glioblastoma. We observed that BKM-120 has potent anti-invasive effects in glioblastoma cell lines and patient-derived glioma cells in vitro. These anti-migratory effects were clearly distinguishable from cytostatic and cytotoxic effects at higher drug concentrations and longer durations of drug exposure. The effects were reversible and accompanied by changes in cell morphology and pronounced reduction in both cell/cell and cell/substrate adhesion. In vivo studies showed that a short period of treatment with BKM-120 slowed tumor spread in an intracranial xenografts. GDC-0941, a similar potent and selective PI3K inhibitor, only caused a moderate reduction in glioblastoma cell migration. The effects of BKM-120 and GDC-0941 were indistinguishable by in vitro kinase selectivity screening and phospho-protein arrays. BKM-120 reduced the numbers of focal adhesions and the velocity of microtubule treadmilling compared with GDC-0941, suggesting that mechanisms in addition to PI3K inhibition contribute to the anti-invasive effects of BKM-120. Our data suggest the CNS-penetrant PI3K inhibitor BKM-120 may have anti-invasive properties in glioblastoma.

Polydimethylsiloxane Core-Polycaprolactone Shell Nanofibers as Biocompatible, Real-Time Oxygen Sensors.

Real-time, continuous monitoring of local oxygen contents at the cellular level is desirable both for the study of cancer cell biology and in tissue engineering. In this paper, we report the successful fabrication of polydimethylsiloxane (PDMS) nanofibers containing oxygen-sensitive probes by electrospinning and the applications of these fibers as optical oxygen sensors for both gaseous and dissolved oxygen. A protective 'shell' layer of polycaprolactone (PCL) not only maintains the fiber morphology of PDMS during the slow curing process but also provides more biocompatible surfaces. Once this strategy was perfected, tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) (Ru(dpp)) and platinum octaethylporphyrin (PtOEP) were dissolved in the PDMS core and the resulting sensing performance established. These new core-shell sensors containing different sensitivity probes showed slight variations in oxygen response but all exhibited excellent Stern-Volmer linearity. Due in part to the porous nature of the fibers and the excellent oxygen permeability of PDMS, the new sensors show faster response (<0.5 s) -4-10 times faster than previous reports - than conventional 2D film-based oxygen sensors. Such core-shell fibers are readily integrated into standard cell culture plates or bioreactors. The photostability of these nanofiber-based sensors was also assessed. Culture of glioma cell lines (CNS1, U251) and glioma-derived primary cells (GBM34) revealed negligible differences in biological behavior suggesting that the presence of the porphyrin dyes within the core carries with it no strong cytotoxic effects. The unique combination of demonstrated biocompatibility due to the PCL 'shell' and the excellent oxygen transparency of the PDMS core makes this particular sensing platform promising for sensing in the context of biological environments.

Strategies in gene therapy for glioblastoma.

Glioblastoma (GBM) is the most aggressive form of brain cancer, with a dismal prognosis and extremely low percentage of survivors. Novel therapies are in dire need to improve the clinical management of these tumors and extend patient survival. Genetic therapies for GBM have been postulated and attempted for the past twenty years, with variable degrees of success in pre-clinical models and clinical trials. Here we review the most common approaches to treat GBM by gene therapy, including strategies to deliver tumor-suppressor genes, suicide genes, immunomodulatory cytokines to improve immune response, and conditionally-replicating oncolytic viruses. The review focuses on the strategies used for gene delivery, including the most common and widely used vehicles (i.e., replicating and non-replicating viruses) as well as novel therapeutic approaches such as stem cell-mediated therapy and nanotechnologies used for gene delivery. We present an overview of these strategies, their targets, different advantages, and challenges for success. Finally, we discuss the potential of gene therapy-based strategies to effectively attack such a complex genetic target as GBM, alone or in combination with conventional therapy.

Serum Homocysteine, Dehydroepiandrosterone Sulphate and Lipoprotein (a) in Alzheimer's Disease and Vascular Dementia.

Alzheimer's disease (AD) and vascular dementia (VAD) are the major forms of dementia affecting elderly people, in which the levels of many metabolites are altered in cerebrospinal fluid (CSF) and serum. These metabolites could be risk factors or potential biomarkers, but the significance of some of these are not clearly understood in the context of the disease pathogenesis. In the present study serum levels of homocysteine, dehydroepiandrosterone sulphate (DHEA-S) and lipoprotein (a) or Lp(a) have been measured by ELISA using commercial kits in AD (n = 40), VAD (n = 40) and age matched control subjects (n = 40). The data are compared by ANOVA and post-hoc analysis. The serum homocysteine is markedly elevated compared to control both in AD and VAD subjects, but to a significantly higher extent in the latter. Lp(a) is increased in the serum of VAD subjects only compared to control. Likewise, serum DHEA-S level is lowered in AD but not in VAD compared to control. The analysis of the present data and those published by others suggest that alterations in homocysteine and Lp(a) in serum are indicators of vascular pathology in AD or VAD, while the lowering of serum DHEA-S is a consequence of AD pathology.

Rapid response oxygen-sensing nanofibers.

Molecular oxygen has profound effects on cell and tissue viability. Relevant sensor forms that can rapidly determine dissolved oxygen levels under biologically relevant conditions provide critical metabolic information. Using 0.5 µm diameter electrospun polycaprolactone (PCL) fiber containing an oxygen-sensitive probe, tris (4,7-diphenyl-1,10-phenanthroline) ruthenium(II) dichloride, we observed a response time of 0.9±0.12 s while the t95 for the corresponding film was more than two orders of magnitude greater. Interestingly, the response and recovery times of slightly larger diameter PCL fibers were 1.79±0.23 s and 2.29±0.13 s, respectively, while the recovery time was not statistically different likely due to the more limited interactions of nitrogen with the polymer matrix. A more than 10-fold increase in PCL fiber diameter reduces oxygen sensitivity while having minor effects on response time; conversely, decreases in fiber diameter to less than 0.5 µm would likely decrease response times even further. In addition, a 50°C heat treatment of the electrospun fiber resulted in both increased Stern-Volmer slope and linearity likely due to secondary recrystallization that further homogenized the probe microenvironment. At exposure times up to 3600 s in length, photobleaching was observed but was largely eliminated by the use of either polyethersulfone (PES) or a PES-PCL core-shell composition. However, this resulted in 2- and 3-fold slower response times. Finally, even the non-core shell compositions containing the Ru oxygen probe result in no apparent cytotoxicity in representative glioblastoma cell populations.

Aging promotes amyloid-ß peptide induced mitochondrial dysfunctions in rat brain: a molecular link between aging and Alzheimer's disease.

The entangled relationship of brain aging, mitochondrial dysfunction, and amyloid-ß peptide (Aß42) toxicity occupies the center stage in the pathogenesis of Alzheimer's disease (AD). The present study examines some of the toxic effects of Aß42 on brain mitochondria and provides evidence that aged brain mitochondria are significantly more vulnerable to Aß42 toxicity. In particular, the study has shown that the aggregated, but not the monomeric, form of Aß42 in varying concentrations (10-40 µM) during in vitro incubation causes a loss of mitochondrial membrane potential, a decrease in phosphorylation capacity and ATP synthesis, and the release of cytochrome c from the mitochondria but without any noticeable change in the activities of respiratory chain complexes. Such effects of Aß42 are strikingly more conspicuous on aged rat (22-24 months) brain mitochondria compared to that on brain mitochondria of young rats (4-6 months). More interestingly is the observation that in contrast to young rat brain mitochondria, a significantly higher level of Aß42 remains associated with aged brain mitochondria under basal incubation condition as well as after exposure to exogenously added peptide. Extrapolated to an in vivo scenario, the results have clear implications in AD pathogenesis and also partly explain why brain aging is a dominant risk factor for this disease condition.

Mitochondrial bioenergetics is not impaired in nonobese subjects with type 2 diabetes mellitus.

Although mitochondrial dysfunction has been well documented in obese people with type 2 diabetes mellitus, its presence or absence in nonobese subjects with type 2 diabetes mellitus has not been well studied so far. The aim of the present study was to assess the status of mitochondrial oxidative phosphorylation in subcutaneous adipose tissue of nonobese type 2 diabetes mellitus subjects in comparison to control, obese nondiabetic, and obese type 2 diabetes mellitus subjects. Mitochondria were isolated from subcutaneous white adipose tissue obtained from the abdominal region of control, obese nondiabetic, nonobese type 2 diabetes mellitus, and obese type 2 diabetes mellitus subjects. The activities of complex I, I to III, II to III, and IV; transmembrane potential; and inorganic phosphate utilization of mitochondria from different groups were measured. Mitochondrial transmembrane potential, inorganic phosphate utilization, and the activities of respiratory chain complexes were significantly reduced in obese nondiabetic and obese type 2 diabetes mellitus patients compared with those in control subjects. No detectable change in mitochondrial functional parameters was observed in case of nonobese type 2 diabetes mellitus subjects compared with control subjects. Furthermore, a significant difference was noticed in mitochondrial phosphate utilization and activities of respiratory complexes, for example, I, I to III, and II to III, between obese type 2 diabetes mellitus subjects and obese nondiabetic subjects. Obesity modulates mitochondrial dysfunction associated with type 2 diabetes mellitus.

Age-related oxidative decline of mitochondrial functions in rat brain is prevented by long term oral antioxidant supplementation.

A combination of antioxidants (N-acetyl cysteine, α-lipoic acid, and α-tocopherol) was selected for long term oral supplementation study in rats for protective effects on age-related mitochondrial alterations in the brain. Four groups of rats were chosen: young control (6-7 months); aged rats (22-24 months); aged rats (22-24 months) on daily antioxidant supplementation from 18 month onwards and young rats (6-7 months) on daily antioxidant supplementation from 2 month onwards. The brain mitochondrial functional parameters, status of antioxidant enzymes and accumulation of oxidative damage markers were measured in the four groups of rats. A significant decrease in complex IV activity and a loss of transmembrane potential and phosphorylation capacity along with an increased accumulation of oxidative damage markers and compromised antioxidant enzyme status were noticed in aged rat brain mitochondria as compared to that in young controls, but in aged rats supplemented with oral antioxidants the mitochondrial alterations were largely prevented. Antioxidant supplementation in young rats had no effect on mitochondrial parameters investigated in this study. The results have implications in biochemical and functional deficits of brain during aging as well as in neurodegenerative disorders.