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Background: Species-specific differences in astrocytes and their Alzheimer disease-associated pathology may influence cellular responses to other insults. Herein, human glial chimeric mice were generated to evaluate how Alzheimer disease predisposing genetic background in human astrocytes contributes to behavioral outcome and brain pathology after cortical photothrombotic ischemia. Methods: Neonatal (P0) immunodeficient mice of both sexes were transplanted with induced pluripotent stem cell–derived astrocyte progenitors from Alzheimer disease patients carrying PSEN1 exon 9 deletion (PSEN1 ΔE9), with isogenic controls, with cells from a healthy donor, or with mouse astrocytes or vehicle. After 14 months, a photothrombotic lesion was produced with Rose Bengal in the motor cortex. Behavior was assessed before ischemia and 1 and 4 weeks after the induction of stroke, followed by tissue perfusion for histology. Results: Open field, cylinder, and grid-walking tests showed a persistent locomotor and sensorimotor impairment after ischemia and female mice had larger infarct sizes; yet, these were not affected by astrocytes with PSEN1 ΔE9 background. Staining for human nuclear antigen confirmed that human cells successfully engrafted throughout the mouse brain. However, only a small number of human cells were positive for astrocytic marker GFAP (glial fibrillary acidic protein), mostly located in the corpus callosum and retaining complex human-specific morphology with longer processes compared with host counterparts. While host astrocytes formed the glial scar, human astrocytes were scattered in small numbers close to the lesion boundary. Aβ (beta-amyloid) deposits were not present in PSEN1 ΔE9 astrocyte-transplanted mice. Conclusions: Transplanted human cells survived and distributed widely in the host brain but had no impact on severity of ischemic damage after cortical photothrombosis in chimeric mice. Only a small number of transplanted human astrocytes acquired GFAP-positive glial phenotype or migrated toward the ischemic lesion forming glial scar. PSEN1 ΔE9 astrocytes did not impair behavioral recovery after experimental stroke.

Breast cancer cell lines BT-474, SKBR3, JIMT-1, and MDA-MB-436 were infected with indicated viruses at increasing plaque-forming units per cell (pfu/cell). The pfu-titers were first determined for each virus in a standard tissue culture infectious dose 50 (TCID50) assay. Ad5/3-Î"24-tras mediated significantly higher cancer cell killing as compared to its oncolytic control virus Ad5/3-Î"24 (P

To confirm that observed ADCC mediated by virus-produced trastuzumab (Fig. 3) requires immune cells, supernatants from mock or Ad5/3-Î"24-tras infected 293 cells were purified and concentrated (virus-free, antibody-containing), and then added on N87 HER2+ gastric cancer cells without human peripheral blood mononuclear cells, followed by measuring lactate dehydrogenase (LDH) release after 6 hours as in Figure 3. As expected when lacking the effect of immune cells, neither virus-produced trastuzumab, mock-treated supernatant, nor irrelevant IgG control antibody showed cell killing (LDH release) at the 6-hour timepoint. Of note, similarly treated HER2-positive N87 cells showed dose-dependent susceptibility to commercial and virus-produced trastuzumab after a longer, 6-day incubation (Supplementary Fig. S1). Maximum LDH Release reagent was used as a positive control.

3-4 week-old female nude/NMRI mice (Harlan Laboratories) were xenografted in both flanks with MDA-MB-436 triple-negative breast cancer cells (6Ã-106 cells/tumor). When tumors reached 5 mm in diameter, mice were randomized and treated intratumorally with 2Ã-109 VP of Ad5/3-Î"24 or Ad5/3-Î"24-tras, or mock-treated (PBS only) on indicated days, and tumor growth was monitored. Trastuzumab-coding Ad5/3-â^†24-tras virus treatment resulted in significant tumor-growth inhibition as compared mock (P

A) Breast cancer BT-474, SKBR3, JIMT-1, and MDA-MB-436 cells, B) ovarian cancer OVCAR3, SKOV3, CaOV3, and HEY cells, and C) gastric N87, MKN-28, MKN-45 and esophageal cancer OE19 cells were treated with commercial or virus-produced trastuzumab (N87 cell line) in escalating doses (left panels), or infected with indicated viruses at increasing virus particle per cell (VP/cell) titers (right panels). Three HER2-positive (HER2+) cancer cell lines, BT-474, N87 and OE19, showed dose-dependent susceptibility to anti-HER2 antibody treatment with IC50-value at 80.3 ng/mL, 196.3 ng/mL, and 239.1 ng/mL on day 6 post-treatment, respectively. Equivalent doses of virus-produced trastuzumab purified from supernatant of Ad5/3-Î"24-tras infected 293 cells resulted in comparable cytotoxicity of HER2-positive N87 gastric cancer (C, top left, dashed line: data from identical separate experiment). Trastuzumab-coding virus Ad5/3-Î"24-tras and the oncolytic control virus Ad5/3-Î"24 showed significant cytotoxicity in all tested cancer cell lines (right panels, A-C), while non-replicating control viruses lacked efficacy. Potency of Ad5/3-Î"24-tras virus in vitro was pronounced in cell lines expressing HER2 (comparable to oncolytic control virus in BT-474, SKBR3, OVCAR3 and N87), while HER2-negative MDA-MB-436 cells were also found equally susceptible to oncolytic viruses. Importantly, both oncolytic viruses were able to mediate total cytotoxicity at the relevant 100 VP/cell titer. Of note, because Ad5/3-Î"24 lacks a transgene and its genome is therefore smaller, the virus packages more efficiently leading to a lower VP/pfu ratio (24 versus 203 in Ad5/3-Î"24-tras). Therefore cytotoxicity was also tested according to functional titers, which rendered higher potency for the recombinant Ad5/3-Î"24-tras virus (see Supplementary Fig. S2). IC50, half-maximal inhibitory concentration.

3-4 week-old male nude/NMRI mice (Taconic) bearing HER2-expressing N87 gastric cancer xenografts were treated in a mechanism of action experiment as in figure 4, but with adjusted doses: 3Ã-108 VP/tumor or saline on days 0, 4, 8, and 15, while one saline-treated group also received 5.0 µg/g of systemic commercial trastuzumab intraperitoneally (tras). On day 20, tumors, draining lymph nodes (DLNs) and spleens were collected, homogenized and analyzed by flow cytometry with CD49b NK-cell marker. Overall absolute levels of NK-cells did not differ between groups in any compartment. However, when distribution of NK cells into draining lymph nodes (left panel) and tumors (right panel) over the spleens of the animals was assessed, the oncolytic trastuzumab-coding virus (OV-tras) showed the highest ratio of NK cells in both target tissues; In particular, NK-cells showed homing towards the draining lymph nodes where they have been reported to e.g. mediate DC-editing and polarize activated CD4+ T-cells into T-helper 1 phenotype (36). Ratio of NK cells between compartments was calculated by dividing the average NK-cell frequency in tumors/lymph nodes by the average of NK-cells in spleens, each per 5Ã-104 cells. n=3 mice per group. Mock, animals treated with intratumoral saline injections; DLN, draining lymph node; OV-tras, oncolytic adenovirus coding for trastuzumab; OV, oncolytic control virus; tras, systemic trastuzumab therapy.

PDF file - 100K, PBMCs were isolated from patients before and circa one month after treatment and pulsed with an adenovirus-derived peptide pool and analyzed with IFN-gamma ELISPOT.

Purpose: Multiple injections of oncolytic adenovirus could enhance immunologic response. In the first part of this article, the focus was on immunologic aspects. Sixty patients previously naïve to oncolytic virus and who had white blood cells available were treated. Thirty-nine of 60 were assessed after a single virus administration, whereas 21 of 60 received a “serial treatment” consisting of three injections within 10 weeks. In the second part, we focused on 115 patients treated with a granulocyte macrophage colony-stimulating factor (GM–CSF)–coding capsid chimeric adenovirus, CGTG-102.Results: Following serial treatment, both increase and decrease in antitumor T cells in blood were seen more frequently, findings which are compatible with induction of T-cell immunity and trafficking of T cells to tumors, respectively. Safety was good in both groups. In 115 patients treated with CGTG-102 (Ad5/3-D24-GMCSF), median overall survival was 111 days following single and 277 days after serial treatment in nonrandomized comparison. Switching the virus capsid for avoiding neutralizing antibodies in a serial treatment featuring three different viruses did not impact safety or efficacy. A correlation between antiviral and antitumor T cells was seen (P = 0.001), suggesting that viral oncolysis can result in epitope spreading and breaking of tumor-associated immunologic tolerance. Alternatively, some patients may be more susceptible to induction of T-cell immunity and/or trafficking.Conclusions: These results provide the first human data linking antiviral immunity with antitumor immunity, implying that oncolytic viruses could have an important role in cancer immunotherapy. Clin Cancer Res; 19(10); 2734–44. ©2013 AACR.

PDF file - 62K, Adverse reactions of all CGTG-102 patients.

PDF file - 67K, Adverse reactions in patients na�ve to oncolytic adenovirus.

PDF file - 77K

Purpose: Twenty-one patients with cancer were treated with a single round of oncolytic adenovirus ICOVIR-7.Experimental Design: ICOVIR-7 features an RGD-4C modification of the fiber HI-loop of serotype 5 adenovirus for enhanced entry into tumor cells. Tumor selectivity is mediated by an insulator, a modified E2F promoter, and a Rb-binding site deletion of E1A, whereas replication is optimized with E2F binding hairpins and a Kozak sequence. ICOVIR-7 doses ranged from 2 × 1010 to 1 × 1012 viral particles. All patients had advanced and metastatic solid tumors refractory to standard therapies.Results: ICOVIR-7 treatment was well tolerated with mild to moderate fever, fatigue, elevated liver transaminases, chills, and hyponatremia. One patient had grade 3 anemia but no other serious side effects were seen. At baseline, 9 of 21 of patients had neutralizing antibody titers against the ICOVIR-7 capsid. Treatment resulted in neutralizing antibody titer induction within 4 weeks in 16 of 18 patients. No elevations of serum proinflammatory cytokine levels were detected. Viral genomes were detected in the circulation in 18 of 21 of patients after injection and 7 of 15 of the samples were positive 2 to 4 weeks later suggesting viral replication.Conclusions: Overall, objective evidence of antitumor activity was seen in 9 of 17 evaluable patients. In radiological analyses, 5 of 12 evaluable patients had stabilization or reduction in tumor size. These consisted of one partial response, two minor responses and two cases of stable disease, all occurring in patients who had progressive disease before treatment. In summary, ICOVIR-7 treatment is apparently safe, resulting in anticancer activity, and is therefore promising for further clinical testing. Clin Cancer Res; 16(11); 3035–43. ©2010 AACR.

PDF file - 889K, Individual anti-tumor (survivin) and anti-viral (Ad5) ELISPOT results in single (A) and serial (B) treated patients.

PDF file - 71K, Characteristics of patients na�ve to oncolytic adenovirus.

PDF file - 94K, The phenotypic panel of circulating white blood cells.

PDF file - 95K, A panel of proinflammatory cytokines was analyzed to evaluate the safety of treatment.

Supplementary Data from Oncolytic Adenovirus ICOVIR-7 in Patients with Advanced and Refractory Solid Tumors

PDF file - 141K, Example of progressive inflammation in serial treatment.

PDF file - 48K, Description of disease at the time of first treatment.

Supplementary Figures 1-6, Tables 1-3 from Oncolytic Adenovirus Coding for Granulocyte Macrophage Colony-Stimulating Factor Induces Antitumoral Immunity in Cancer Patients

PDF file - 1MB, PBMCs of a healthy donor was stimulated with supernatant collected from virus (CGTG-401 or control virus not coding CD40L) infected A549 lung cancer cells and IL-8 production was assessed 12, 24, 48, and 72 h later with FACSArray. The increase demonstrated at later time points indicates biological activity of the virus produced CD40L.

PDF file - 1.1MB, (A) Patient serum samples were analyzed for Th1 induced cytokines interferon-� (IFN-�), tumor necrosis factor-� (TNF-�) and interleukin-2 (IL-2) and Th2 cytokines interleukin-4 (IL-4), interleukin-5 (IL-5) and Interleukin 10 (IL-10). The ratio between Th1 and Th2 type cytokines was assessed. Cytokines levels are reported relative to their baseline level, which was set as 1 and a ratio between Th1/Th2 was calculated for each time point. (B) Malignant ascites of patient R8 and pleural effusion of patient T181 were analyzed for Th1 induced cytokines IFN-� and IL-2, and for Th2 induced IL-10 before and after treatment.

PDF file - 1.2MB, Blood samples for all 9 patients were collected before (baseline) and at several time points after the treatment. Soluble CD40L (sCD40L) and RANTES concentrations in the serum were assessed. Data are presented as median � SD.

Granulocyte macrophage colony-stimulating factor (GMCSF) can mediate antitumor effects by recruiting natural killer cells and by induction of tumor-specific cytotoxic T-cells through antigen-presenting cells. Oncolytic tumor cell–killing can produce a potent costimulatory danger signal and release of tumor epitopes for antigen-presenting cell sampling. Therefore, an oncolytic adenovirus coding for GMCSF was engineered and shown to induce tumor-specific immunity in an immunocompetent syngeneic hamster model. Subsequently, 20 patients with advanced solid tumors refractory to standard therapies were treated with Ad5-D24-GMCSF. Of the 16 radiologically evaluable patients, 2 had complete responses, 1 had a minor response, and 5 had disease stabilization. Responses were frequently seen in injected and noninjected tumors. Treatment was well tolerated and resulted in the induction of both tumor-specific and virus-specific immunity as measured by ELISPOT and pentamer analysis. This is the first time that oncolytic virus–mediated antitumor immunity has been shown in humans. Ad5-D24-GMCSF is promising for further clinical testing. Cancer Res; 70(11); 4297–309. ©2010 AACR.

PDF file - 64K, Adverse reactions. 9 patients total, 6 received concomitant low-lose cyclophosphamide 50 mg/d, 3 did not. Numbers of patients experiencing the reaction at any time during their treatment (including 28d follow-up) is shown. The data is shown for the two subgroups separately in parentheses (cyclophosphamide/none).

PDF file - 41K, Response evaluation of injected and non-injected lesions.

PDF file - 58K, Prior therapies.

PDF file - 2MB, Serum levels for (A) IL-6, (B) IL-8, (C) IL-10, (D) IL-12, (E) TNF-alpha, and (F) IFN-gamma was assessed before and after the treatment. Data are presented separately for patients receiving virus treatment with or without concomitant low dose cyclophosphamide (50 mg/d) and shown as median � SD. NA, sample not available.

PDF file - 69K, Description of historical controls (Pesonen et al. Gene Ther 2010;17:892-904) treated with unarmed oncolytic adenovirus Ad5/3-cox2L-D24.

Summary Multiple protocols have been published for generation of iMGLs from hESCs/iPSCs. To date, there are no guides to assist researchers to determine the most appropriate methodology for microglial studies. To establish a framework to facilitate future microglial studies, we first performed a comparative transcriptional analysis between iMGLs derived using three published datasets, which allowed us to establish the baseline protocol that is most representative of bona fide human microglia. Secondly, using CRISPR to tag the classic microglial marker CX3CR1 with nanoluciferase and tdTomato, we generated and functionally validated a reporter ESC line. Finally, using this cell line, we demonstrated that co-culture of iMGL precursors with human glia and neurons enhanced transcriptional resemblance of iMGLs to ex vivo microglia. Together, our comprehensive molecular analysis and reporter cell line are a useful resource for neurobiologists seeking to use iMGLs for disease modeling and drug screening studies., Highlights • Integrated molecular characterization reveals differences in four iMGL protocols • A dual tdTomato and nanoluciferase reporter tool was generated to track iMGLs via CX3CR1 • Kinetics of surface marker expression during iMGL differentiation • Co-culture with glia/neurons restores ex vivo microglial transcription factors, In this article, Jose Polo and colleagues molecularly compare existing microglia differentiation methods from stem cells, generate a new dual fluorescent and enzymatic reporter tool to study microglia, and show that human glia/neuron co-culture pushes the regulatory landscape of in vitro microglia-like cells to more closely resemble bona fide microglia.

BackgroundOncolytic viruses are a potent form of active immunotherapy, capable of invoking antitumor T-cell responses. Meanwhile, less is known about their effects on immune checkpoints, the main targets for passive immunotherapy of cancer. T-cell immunoglobulin and mucin domain-3 (TIM-3) is a coinhibitory checkpoint driving T-cell exhaustion in cancer. Here we investigated the effects of oncolytic adenovirus on the TIM-3 checkpoint on tumor-infiltrating immune cells and clinical impact in patients with cancer receiving oncolytic immunotherapy.MethodsModulation of TIM-3 expression on tumor-infiltrating immune cells was studied preclinically in B16 melanoma following intratumoral treatment with Ad5/3∆24-granulocyte-macrophage colony-stimulating factor oncolytic adenovirus. We conducted a retrospective longitudinal analysis of 15 patients with advanced-stage cancer with tumor-site biopsies before and after oncolytic immunotherapy, treated in the Advanced Therapy Access Program (ISRCTN10141600, April 5, 2011). Following patient stratification with regard to TIM-3 (increase vs decrease in tumors), overall survival and imaging/marker responses were evaluated by log-rank and Fisher’s test, while coinhibitory receptors/ligands, transcriptomic changes and tumor-reactive and tumor-infltrating immune cells in biopsies and blood samples were studied by microarray rank-based statistics and immunoassays.ResultsPreclinically, TIM-3+ tumor-infiltrating lymphocytes (TILs) in B16 melanoma showed an exhausted phenotype, whereas oncolytic adenovirus treatment significantly reduced the proportion of TIM-3+ TIL subset through recruitment of less-exhausted CD8+ TIL. Decrease of TIM-3 was observed in 60% of patients, which was associated with improved overall survival over TIM-3 increase patients (p=0.004), together with evidence of clinical benefit by imaging and blood analyses. Coinhibitory T-cell receptors and ligands were consistently associated with TIM-3 changes in gene expression data, while core transcriptional exhaustion programs and T-cell dysfunction were enriched in patients with TIM-3 increase, thus identifying patients potentially benefiting from checkpoint blockade. In striking contrast, patients with TIM-3 decrease displayed an acute inflammatory signature, redistribution of tumor-reactive CD8+ lymphocytes and higher influx of CD8+ TIL into tumors, which were associated with the longest overall survival, suggesting benefit from active immunotherapy.ConclusionsOur results indicate a key role for the TIM-3 immune checkpoint in oncolytic adenoviral immunotherapy. Moreover, our results identify TIM-3 as a potential biomarker for oncolytic adenoviruses and create rationale for combination with passive immunotherapy for a subset of patients.

Oncolytic viruses are a potent form of active immunotherapy, capable of invoking antitumor T-cell responses. Meanwhile, less is known about their effects on immune checkpoints, the main targets for passive immunotherapy of cancer. T-cell immunoglobulin and mucin domain-3 (TIM-3) is a coinhibitory checkpoint driving T-cell exhaustion in cancer. Here we investigated the effects of oncolytic adenovirus on the TIM-3 checkpoint on tumor-infiltrating immune cells and clinical impact in patients with cancer receiving oncolytic immunotherapy.Modulation of TIM-3 expression on tumor-infiltrating immune cells was studied preclinically in B16 melanoma following intratumoral treatment with Ad5/3∆24-granulocyte-macrophage colony-stimulating factor oncolytic adenovirus. We conducted a retrospective longitudinal analysis of 15 patients with advanced-stage cancer with tumor-site biopsies before and after oncolytic immunotherapy, treated in the Advanced Therapy Access Program (ISRCTN10141600, April 5, 2011). Following patient stratification with regard to TIM-3 (increase vs decrease in tumors), overall survival and imaging/marker responses were evaluated by log-rank and Fisher's test, while coinhibitory receptors/ligands, transcriptomic changes and tumor-reactive and tumor-infltrating immune cells in biopsies and blood samples were studied by microarray rank-based statistics and immunoassays.Preclinically, TIM-3Our results indicate a key role for the TIM-3 immune checkpoint in oncolytic adenoviral immunotherapy. Moreover, our results identify TIM-3 as a potential biomarker for oncolytic adenoviruses and create rationale for combination with passive immunotherapy for a subset of patients.

Astrocytes are the most abundant cell type in the brain. They were long considered only as passive support for neuronal cells. However, recent data have revealed many active roles for these cells both in maintenance of the normal physiological homeostasis in the brain as well as in neurodegeneration and disease. Moreover, human astrocytes have been found to be much more complex than their rodent counterparts, and to date, astrocytes are known to actively participate in a multitude of processes such as neurotransmitter uptake and recycling, gliotransmitter release, neuroenergetics, inflammation, modulation of synaptic activity, ionic balance, maintenance of the blood–brain barrier, and many other crucial functions of the brain. This review focuses on the role of astrocytes in human neurodegenerative disease and the potential of the novel stem cell-based platforms in modeling astrocytic functions in health and in disease.

We describe a methodology to measure ballistocardiography (BCG) signals from the body surface, using body-worn digital accelerometers to extract medically relevant information for Pervasive Healthcare. We are able to measure measuring heart rate with an 95% accuracy as well as other cardiac metrics, such as the S1-S2 interval, deviating from ECG by only 1.3%. Our results show that BCG can be a viable alternative to an electrocardiogram to provide complementary information on the heart’s condition in mobile and pervasive use cases. We further show that BCG information can be detected from arm as reliably as from chest, which is especially convenient for measuring from supine positions in Pervasive healthcare applications.

A double mutation (KM670/671NL) in amyloid precursor protein gene (APP) is causative for familial Alzheimer's disease and has been shown to increase the total Aβ burden. Here we report the generation and characterization of an iPSC line from a fAD patient carrying APP KM670/671NL. The generated iPSCs retained the mutation, expressed pluripotency markers, showed a normal karyotype and differentiated into all three germ layers. This iPSC line can be used, for example, in disease modeling and mechanistic studies.Resource table.Unlabelled TableUnique stem cell line identifierUEFi002-AAlternative name(s) of stem cell lineLL008 1.4InstitutionA.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, FinlandContact information of distributorProf. Jari Koistinaho(jari.koistinaho@uef.fi; jari.koistinaho@helsinki.fi)Type of cell lineiPSCOriginHumanAdditional origin infoAge: 58Sex: FemaleEthnicity: ScandinavianCell SourceSkin fibroblastsClonalityClonalMethod of reprogrammingSendai virus carrying OCT3/4, SOX2, KLF4 and c-MYC(CytoTune 1.0)Genetic ModificationNoType of ModificationN/AAssociated diseaseFamilial Alzheimer's diseaseGene/locusAPP (MIM # 104760) located on the long arm of chromosome 21 at position 21q21.3; 27,269,939 G > T, 27269938 A > C (rs63751263, rs63750445)Method of modificationN/AName of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock dateMarch 2016Cell line repository/bankN/AEthical approvalEthical license number 2017/834–31/1 (The ethical review board of Karolinska Institutet/Karolinska University Hospital)

The first US Food and Drug Administration (FDA)- and EMA-approved oncolytic virus has been available since 2015. However, there are no markers available that would predict benefit for the individual patient. During 2007–2012, we treated 290 patients with advanced chemotherapy-refractory cancers, using 10 different oncolytic adenoviruses. Treatments were given in a Finnish Medicines Agency (FIMEA)-regulated individualized patient treatment program (the Advanced Therapy Access Program [ATAP]), which required long-term follow-up of patients, which is presented here. Focusing on the longest surviving patients, some key clinical and biological features are presented as “oncograms.” Some key attributes that could be captured in the oncogram are suggested to predict treatment response and survival after oncolytic adenovirus treatment. The oncogram includes immunological laboratory parameters assessed in peripheral blood (leukocytes, neutrophil-to-lymphocyte ratio, interleukin-8 [IL-8], HMGB1, anti-viral neutralizing antibody status), features of the patient (gender, performance status), tumor features (histological tumor type, tumor load, region of metastases), and oncolytic virus-specific features (arming of the virus). The retrospective approach used here facilitates verification in a prospective controlled trial setting. To our knowledge, the oncogram is the first holistic attempt to identify the patients most likely to benefit from adenoviral oncolytic virotherapy. Keywords: oncolytic adenovirus, oncolytic virotherapy, immunotherapy, cancer, immunostimulation, anti-cancer, adenovirus, oncoimmunology, immunogram, oncogram

Summary The blood-brain barrier (BBB) presents a barrier for circulating factors, but simultaneously challenges drug delivery. How the BBB is altered in Alzheimer disease (AD) is not fully understood. To facilitate this analysis, we derived brain endothelial cells (iBECs) from human induced pluripotent stem cells (hiPSCs) of several patients carrying the familial AD PSEN1 mutation. We demonstrate that, compared with isogenic PSEN1 corrected and control iBECs, AD-iBECs exhibit altered tight and adherens junction protein expression as well as efflux properties. Furthermore, by applying focused ultrasound (FUS) that transiently opens the BBB and achieves multiple therapeutic effects in AD mouse models, we found an altered permeability to 3–5 kDa dextran as a model cargo and the amyloid-β (Aβ) peptide in AD-iBECs compared with control iBECs. This presents human-derived in vitro models of the BBB as a valuable tool to understand its role and properties in a disease context, with possible implications for drug delivery., Highlights • iBECs with familial AD mutation express altered levels of tight junction proteins • AD-iBECs exhibit altered efflux transporter expression and function to control iBECs • Focused ultrasound disrupts iBEC monolayer indicating effects of BBB opening • AD-iBECs respond differently to control iBECs to effects of focused ultrasound, In this article, Oikari and colleagues use patient-derived iPSCs with a familial Alzheimer disease (AD) mutation to study the phenotypical and functional differences of brain endothelial cells (iBECs) in AD. In addition, the authors study the effects of focused ultrasound on the iBEC monolayer to model blood-brain barrier opening and demonstrate key differences between AD and control iBECs.

Astrocytes are the gatekeepers of neuronal energy supply. In neurodegenerative diseases, bio-energetics demand increases and becomes reliant upon fatty acid oxidation as a source of energy. Defective fatty acid oxidation and mitochondrial dysfunctions correlate with hippocampal neurodegeneration and memory deficits in Alzheimer’s disease (AD), but it is unclear whether energy metabolism can be targeted to prevent or treat the disease. Here we show for the first time an impairment in fatty acid oxidation in human astrocytes derived from induced pluripotent stem cells of AD patients. The impairment was corrected by treatment with a synthetic peroxisome proliferator activated receptor delta (PPARβ/δ) agonist GW0742 which acts to regulate an array of genes governing cellular metabolism. GW0742 enhanced the expression of CPT1a, the gene encoding for a rate-limiting enzyme of fatty acid oxidation. Similarly, treatment of a mouse model of AD, the APP/PS1-mice, with GW0742 increased the expression of Cpt1a and concomitantly reversed memory deficits in a fear conditioning test. Although the GW0742-treated mice did not show altered astrocytic glial fibrillary acidic protein-immunoreactivity or reduction in amyloid beta (Aβ) load, GW0742 treatment increased hippo-campal neurogenesis and enhanced neuronal differentiation of neuronal progenitor cells. Furthermore, GW0742 prevented Aβ-induced impairment of long-term potentiation in hippocampal slices. Collectively, these data suggest that PPARβ/δ-agonism alleviates AD related deficits through increasing fatty acid oxidation in astrocytes and improves cognition in a transgenic mouse model of AD.

Monoclonal anti-HER2 antibody trastuzumab has significantly improved the survival of patients with HER2-overexpressing tumors. Nevertheless, systemic antibody therapy is expensive, limited in efficacy due to physical tumor barriers, and carries the risk of severe side effects such as cardiomyopathy. Oncolytic viruses mediate cancer-selective transgene expression, kill infected cancer cells while mounting antitumor immune responses, and have recently demonstrated promising efficacy in combination treatments. Here, we armed an oncolytic adenovirus with full-length trastuzumab to achieve effective in situ antibody production coupled with progressive oncolytic cancer cell killing. We constructed an infectivity-enhanced serotype 5 oncolytic adenovirus, Ad5/3-Δ24-tras, coding for human trastuzumab antibody heavy- and light-chain genes, connected by an internal ribosome entry site. Infected cancer cells were able to assemble full-length functional antibody, as confirmed by Western blot, ELISA, and antibody-dependent cell-mediated cytotoxicity assay. Importantly, oncolysis was required for release of the antibody into tumors, providing additional spatial selectivity. Ad5/3-Δ24-tras showed potent in vitro cytotoxicity and enhanced antitumor efficacy over oncolytic control virus Ad5/3-Δ24 or commercial trastuzumab in HER2-positive cancer models in vivo (both P < 0.05). Furthermore, Ad5/3-Δ24-tras resulted in significantly higher tumor-to-systemic antibody concentrations (P < 0.001) over conventional delivery. Immunological analyses revealed dendritic cell activation and natural killer cell accumulation in tumor-draining lymph nodes. Thus, Ad5/3-Δ24-tras is an attractive anticancer approach combining oncolytic immunotherapy with local trastuzumab production, resulting in improved in vivo efficacy and immune cell activation in HER2-positive cancer. Moreover, the finding that tumor cells can produce functional antibody as directed by oncolytic virus could lead to many valuable antitumor approaches. Mol Cancer Ther; 15(9); 2259–69. ©2016 AACR.

In clinical trials with oncolytic adenoviruses, there has been no mortality associated with treatment vectors. Likewise, in the Advanced Therapy Access Program (ATAP), where 290 patients were treated with 10 different viruses, no vector-related mortality was observed. However, as the patient population who received adenovirus treatments in ATAP represented heavily pretreated patients, often with very advanced disease, some patients died relatively soon after receiving their virus treatment mandating autopsy to investigate cause of death. Eleven such autopsies were performed and confirmed disease progression as the cause of death in each case. The regulatory requirement for investigating the safety of advanced therapy medical products presented a unique opportunity to study tissue samples collected as a routine part of the autopsies. Oncolytic adenoviral DNA was recovered in a wide range of tissues, including injected and noninjected tumors and various normal tissues, demonstrating the ability of the vector to disseminate through the vascular route. Furthermore, we recovered and cultured viable virus from samples of noninjected brain metastases of an intravenously treated patient, confirming that oncolytic adenovirus can reach tumors through the intravascular route. Data presented here give mechanistic insight into mode of action and biodistribution of oncolytic adenoviruses in cancer patients.