Engineered exosomes for studies in tumor immunology.

Exosomes are a type of extracellular vesicle (EV) with diameters of 30-150 nm secreted by most of the cells into the extracellular spaces and can alter the microenvironment through cell-to-cell interactions by fusion with the plasma membrane and subsequent endocytosis and release of the cargo. Because of their biocompatibility, low toxicity and immunogenicity, permeability (even through the blood-brain barrier (BBB)), stability in biological fluids, and ability to accumulate in the lesions with higher specificity, investigators have started making designer's exosomes or engineered exosomes to carry biologically active protein on the surface or inside the exosomes as well as using exosomes to carry drugs, micro RNA, and other products to the site of interest. In this review, we have discussed biogenesis, markers, and contents of various exosomes including exosomes of immune cells. We have also discussed the current methods of making engineered and designer's exosomes as well as the use of engineered exosomes targeting different immune cells in the tumors, stroke, as well as at peripheral blood. Genetic engineering and customizing exosomes create an unlimited opportunity to use in diagnosis and treatment. Very little use has been discovered, and we are far away to reach its limits.

The Stroke Preclinical Assessment Network: Rationale, Design, Feasibility, and Stage 1 Results.

Cerebral ischemia and reperfusion initiate cellular events in brain that lead to neurological disability. Investigating these cellular events provides ample targets for developing new treatments. Despite considerable work, no such therapy has translated into successful stroke treatment. Among other issues-such as incomplete mechanistic knowledge and faulty clinical trial design-a key contributor to prior translational failures may be insufficient scientific rigor during preclinical assessment: nonblinded outcome assessment; missing randomization; inappropriate sample sizes; and preclinical assessments in young male animals that ignore relevant biological variables, such as age, sex, and relevant comorbid diseases. Promising results are rarely replicated in multiple laboratories. We sought to address some of these issues with rigorous assessment of candidate treatments across 6 independent research laboratories. The Stroke Preclinical Assessment Network (SPAN) implements state-of-the-art experimental design to test the hypothesis that rigorous preclinical assessment can successfully reduce or eliminate common sources of bias in choosing treatments for evaluation in clinical studies. SPAN is a randomized, placebo-controlled, blinded, multilaboratory trial using a multi-arm multi-stage protocol to select one or more putative stroke treatments with an implied high likelihood of success in human clinical stroke trials. The first stage of SPAN implemented procedural standardization and experimental rigor. All participating research laboratories performed middle cerebral artery occlusion surgery adhering to a common protocol and rapidly enrolled 913 mice in the first of 4 planned stages with excellent protocol adherence, remarkable data completion and low rates of subject loss. SPAN stage 1 successfully implemented treatment masking, randomization, prerandomization inclusion/exclusion criteria, and blinded assessment to exclude bias. Our data suggest that a large, multilaboratory, preclinical assessment effort to reduce known sources of bias is feasible and practical. Subsequent SPAN stages will evaluate candidate treatments for potential success in future stroke clinical trials using aged animals and animals with comorbid conditions.

CYP4A/20-HETE regulates ischemia-induced neovascularization via its actions on endothelial progenitor and preexisting endothelial cells.

20-Hydroxyeicosatetraenoic acid (20-HETE) was recently identified as a novel contributor of ischemia-induced neovascularization based on the key observation that pharmacological interferences of CYP4A/20-HETE decrease ischemic neovascularization. The objective of the present study is to examine whether the underlying cellular mechanisms involve endothelial progenitor cells (EPCs) and preexisting endothelial cells (ECs). We found that ischemia leads to a time-dependent increase of cyp4a12 expression and 20-HETE production, which are endothelial in origin, using immunofluorescent microscopy, Western blot analysis, and LC-MS/MS. This is accompanied by increases in the tissue stromal cell-derived factor-1α (SDF-1α) expressions as well as SDF-1α plasma levels, EPC mobilization from bone marrow, and subsequent homing to ischemic tissues. Pharmacological interferences of CYP4A/20-HETE with a 20-HETE synthesis inhibitor, dibromo-dodecenyl-methylsulfimide (DDMS), or a 20-HETE antagonist, N-(20-hydroxyeicosa-6(Z), 15(Z)-dienoyl) glycine (6, 15-20-HEDGE), significantly attenuated these increases. Importantly, we also determined that 20-HETE plays a novel role in maintaining EPC functions and increasing the expression of Oct4, Sox2, and Nanog, which are indicative of increased progenitor cell stemness. Flow cytometric analysis revealed that pharmacological interferences of CYP4A/20-HETE decrease the EPC population in culture, whereas 20-HETE increases the cultured EPC population. Furthermore, ischemia also markedly increased the proliferation, oxidative stress, and ICAM-1 expression in the preexisting EC in the hindlimb gracilis muscles. We found that these increases were markedly negated by DDMS and 6, 15-20-HEDGE. Taken together, CYP4A/20-HETE regulates ischemia-induced compensatory neovascularization via its combined actions on promoting EPC and local preexisting EC responses that are associated with increased neovascularization. NEW & NOTEWORTHY CYP4A/20-hydroxyeicosatetraenoic acid (20-HETE) was recently discovered as a novel contributor of ischemia-induced neovascularization. However, the underlying molecular and cellular mechanisms are completely unknown. Here, we show that CYP4A/20-HETE regulates the ischemic neovascularization process via its combined actions on both endothelial progenitor cells (EPCs) and preexisting endothelial cells. Moreover, this is the first study, to the best of our knowledge, that associates CYP4A/20-HETE with EPC differentiation and stemness.

Differential in vivo biodistribution of 131I-labeled exosomes from diverse cellular origins and its implication for theranostic application.

Exosomes are critical mediators of intercellular crosstalk and are regulator of the cellular/tumor microenvironment. Exosomes have great prospects for clinical application as a theranostic and prognostic probe. Nevertheless, the advancement of exosomes research has been thwarted by our limited knowledge of the most efficient isolation method and their in vivo trafficking. Here we have shown that a combination of two size-based methods using a 0.20 µm syringe filter and 100 k centrifuge membrane filter followed by ultracentrifugation yields a greater number of uniform exosomes. We also demonstrated the visual representation and quantification of the differential in vivo distribution of radioisotope 131I-labeled exosomes from diverse cellular origins, e.g., tumor cells with or without treatments, myeloid-derived suppressor cells and endothelial progenitor cells. We also determined that the distribution was dependent on the exosomal protein/cytokine contents. The applied in vivo imaging modalities can be utilized to monitor disease progression, metastasis, and exosome-based targeted therapy.

Glucocorticoid-Induced Leucine Zipper Promotes Neutrophil and T-Cell Polarization with Protective Effects in Acute Kidney Injury.

The glucocorticoid-induced leucine zipper (GILZ) mediates anti-inflammatory effects of glucocorticoids. Acute kidney injury (AKI) mobilizes immune/inflammatory mechanisms, causing tissue injury, but the impact of GILZ in AKI is not known. Neutrophils play context-specific proinflammatory [type 1 neutrophil (N1)] and anti-inflammatory [type 2 neutrophil (N2)] functional roles. Also, regulatory T lymphocytes (Tregs) and regulatory T-17 (Treg17) cells exert counterinflammatory effects, including the suppression of effector T lymphocytes [e.g., T-helper (Th) 17 cells]. Thus, utilizing cell preparations of mice kidneys subjected to AKI or sham operation, we determined the effects of GILZ on T cells and neutrophil subtypes in the context of its renoprotective effect; these studies used the transactivator of transcription (TAT)-GILZ or the TAT peptide. AKI increased N1 and Th-17 cells but reduced N2, Tregs, and Treg17 cells in association with increased interleukin (IL)-17+ but reduced IL-10+ cells accompanied with the disruption of mitochondrial membrane potential (ψ m) and increased apoptosis/necrosis compared with sham kidneys. TAT-GILZ, compared with TAT, treatment reduced N1 and Th-17 cells but increased N2 and Tregs, without affecting Treg17 cells, in association with a reduction in IL-17+ cells but an increase in IL-10+ cells; TAT-GILZ caused less disruption of ψ m and reduced cell death in AKI. Importantly, TAT-GILZ increased perfusion of the ischemic-reperfused kidney but reduced tissue edema compared with TAT. Utilizing splenic T cells and bone marrow-derived neutrophils, we further showed marked reduction in the proliferation of Th cells in response to TAT-GILZ compared with response to TAT. Collectively, the results indicate that GILZ exerts renoprotection accompanied by the upregulation of the regulatory/suppressive arm of immunity in AKI, likely via regulating cross talk between T cells and neutrophils.

Selective activation of cannabinoid receptor-2 reduces neuroinflammation after traumatic brain injury via alternative macrophage polarization.

Inflammation is an important mediator of secondary neurological injury after traumatic brain injury (TBI). Endocannabinoids, endogenously produced arachidonate based lipids, have recently emerged as powerful anti-inflammatory compounds, yet the molecular and cellular mechanisms underlying these effects are poorly defined. Endocannabinoids are physiological ligands for two known cannabinoid receptors, CB1R and CB2R. In the present study, we hypothesized that selective activation of CB2R attenuates neuroinflammation and reduces neurovascular injury after TBI. Using a murine controlled cortical impact (CCI) model of TBI, we observed a dramatic upregulation of CB2R within infiltrating myeloid cells beginning at 72 h. Administration of the selective CB2R agonist, GP1a (1-5 mg/kg), attenuated pro-inflammatory M1 macrophage polarization, increased anti-inflammatory M2 polarization, reduced edema development, enhanced cerebral blood flow, and improved neurobehavioral outcomes after TBI. In contrast, the CB2R antagonist, AM630, worsened outcomes. Taken together, our findings support the development of selective CB2R agonists as a therapeutic strategy to improve TBI outcomes while avoiding the psychoactive effects of CB1R activation.

Delineating Pro-Angiogenic Myeloid Cells in Cancer Therapy.

Recent evidence suggests that myeloid cells are critical in cancer development and therapy resistance processes. Pharmacological targeting of tumor-associated myeloid cells is an emerging approach among upcoming immune therapies. Surprisingly, myeloid cells are heterogeneous, including a subset of the myeloid cell displaying angiogenic properties in solid tumors. There is an urgent need to delineate angiogenic myeloid cell populations in order to facilitate specific targeting of protumor myeloid cells among heterogeneous pool. This review article is intended to compile all the relevant information in the literature for improved understanding of angiogenic myeloid cells and their role in tumor refractoriness to cancer therapy.

Arachidonic Acid Metabolite as a Novel Therapeutic Target in Breast Cancer Metastasis.

Metastatic breast cancer (BC) (also referred to as stage IV) spreads beyond the breast to the bones, lungs, liver, or brain and is a major contributor to the deaths of cancer patients. Interestingly, metastasis is a result of stroma-coordinated hallmarks such as invasion and migration of the tumor cells from the primary niche, regrowth of the invading tumor cells in the distant organs, proliferation, vascularization, and immune suppression. Targeted therapies, when used as monotherapies or combination therapies, have shown limited success in decreasing the established metastatic growth and improving survival. Thus, novel therapeutic targets are warranted to improve the metastasis outcomes. We have been actively investigating the cytochrome P450 4 (CYP4) family of enzymes that can biosynthesize 20-hydroxyeicosatetraenoic acid (20-HETE), an important signaling eicosanoid involved in the regulation of vascular tone and angiogenesis. We have shown that 20-HETE can activate several intracellular protein kinases, pro-inflammatory mediators, and chemokines in cancer. This review article is focused on understanding the role of the arachidonic acid metabolic pathway in BC metastasis with an emphasis on 20-HETE as a novel therapeutic target to decrease BC metastasis. We have discussed all the significant investigational mechanisms and put forward studies showing how 20-HETE can promote angiogenesis and metastasis, and how its inhibition could affect the metastatic niches. Potential adjuvant therapies targeting the tumor microenvironment showing anti-tumor properties against BC and its lung metastasis are discussed at the end. This review will highlight the importance of exploring tumor-inherent and stromal-inherent metabolic pathways in the development of novel therapeutics for treating BC metastasis.

Major Challenges and Potential Microenvironment-Targeted Therapies in Glioblastoma.

Glioblastoma (GBM) is considered one of the most malignant, genetically heterogeneous, and therapy-resistant solid tumor. Therapeutic options are limited in GBM and involve surgical resection followed by chemotherapy and/or radiotherapy. Adjuvant therapies, including antiangiogenic treatments (AATs) targeting the VEGF-VEGFR pathway, have witnessed enhanced infiltration of bone marrow-derived myeloid cells, causing therapy resistance and tumor relapse in clinics and in preclinical models of GBM. This review article is focused on gathering previous clinical and preclinical reports featuring major challenges and lessons in GBM. Potential combination therapies targeting the tumor microenvironment (TME) to overcome the myeloid cell-mediated resistance problem in GBM are discussed. Future directions are focused on the use of TME-directed therapies in combination with standard therapy in clinical trials, and the exploration of novel therapies and GBM models for preclinical studies. We believe this review will guide the future of GBM research and therapy.

20-HETE regulates the angiogenic functions of human endothelial progenitor cells and contributes to angiogenesis in vivo.

Circulating endothelial progenitor cells (EPC) contribute to postnatal neovascularization. We identified the cytochrome P450 4A/F-20-hydroxyeicosatetraenoic acid (CYP4A/F-20-HETE) system as a novel regulator of EPC functions associated with angiogenesis in vitro. Here, we explored cellular mechanisms by which 20-HETE regulates EPC angiogenic functions and assessed its contribution to EPC-mediated angiogenesis in vivo. Results showed that both hypoxia and vascular endothelial growth factor (VEGF) induce CYP4A11 gene and protein expression (the predominant 20-HETE synthases in human EPC), and this is accompanied by an increase in 20-HETE production by ~1.4- and 1.8-fold, respectively, compared with the control levels. Additional studies demonstrated that 20-HETE and VEGF have a synergistic effect on EPC proliferation, whereas 20-HETE antagonist 20-HEDGE or VEGF-neutralizing antibody negated 20-HETE- or VEGF-induced proliferation, respectively. These findings are consistent with the presence of a positive feedback regulation on EPC proliferation between the 20-HETE and the VEGF pathways. Furthermore, we found that 20-HETE induced EPC adhesion to fibronectin and endothelial cell monolayer by 40 ± 5.6 and 67 ± 10%, respectively, which was accompanied by a rapid induction of very late antigen-4 and chemokine receptor type 4 mRNA and protein expression. Basal and 20-HETE-stimulated increases in adhesion were negated by the inhibition of the CYP4A-20-HETE system. Lastly, EPC increased angiogenesis in vivo by 3.6 ± 0.2-fold using the Matrigel plug angiogenesis assay, and these increases were markedly reduced by the local inhibition of 20-HETE system. These results strengthened the notion that 20-HETE regulates the angiogenic functions of EPC in vitro and EPC-mediated angiogenesis in vivo.

Measurement of rat brain tumor kinetics using an intravascular MR contrast agent and DCE-MRI nested model selection.

PURPOSE: Using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in a rat glioma model, and nested model selection (NMS), to compare estimates of the pharmacokinetic parameters vp , K(trans) , and ve for two different contrast agents (CAs)-gadofosveset, which reversibly binds to human serum albumin, and gadopentetate dimeglumine, which does not. MATERIALS AND METHODS: DCE-MRI studies were performed on nine Fisher 344 rats inoculated intracerebrally with 9L gliosarcoma cells using both gadofosveset and gadopentetate. The parameters vp , K(trans) , and ve were estimated using NMS. RESULTS: K(trans) estimates using gadofosveset, compared to gadopentetate, differed in their means (gadofosveset 0.025 ± 0.008 min(-1) vs. gadopentetate 0.046 ± 0.011 min(-1) ; P = 0.0039). This difference notwithstanding, the intraclass correlation coefficient (ICC) for the two estimates of K(trans) showed nearly perfect linear dependence (ICC = 0.8479 by Pearson's r). Other estimates, ve (gadofosveset 22.7 ± 4.7% vs. gadopentetate 23.6 ± 5.6%; P = 0.4258) and vp (gadofosveset 1.5 ± 0.5% vs. gadopentetate 1.6 ± 0.4%; P = 0.25), were not different in their means between the two CAs, and there was almost perfect agreement for ve (ICC = 0.8798) and substantial agreement for vp (ICC = 0.7981) between the two CAs. CONCLUSION: Estimates of K(trans) were statistically different using gadofosveset and gadopentetate, whereas ve and vp were similar with two CAs. NMS produced robust estimates of pharmacokinetic parameters using DCE-MRI that show promise as important measures of tumor physiology and microenvironment.

Anticancer activity of pristimerin in ovarian carcinoma cells is mediated through the inhibition of prosurvival Akt/NF-κB/mTOR signaling.

Pristimerin isaquinonemethidetriterpenoidthathasshown anticancer activity against some cancer types. However, the antitumor effects of pristimerin (PM) in ovarian cancer cells have not been adequately studied. The objective of the present study was to determine the anticancer activity and its mechanism of action in human ovarian carcinoma cell lines. PM strongly inhibited the proliferation of ovarian cancer cells by inducing apoptosis characterized by increased annexin V-binding, cleavage of poly (ADP-ribose) polymerase (PARP-1) and procaspases-3, -8 and -9. Furthermore, PM caused mitochondrial depolarization. Western blot analysis showed inhibition of prosurvival phospho-AKT (p-AKT), nuclear factor kappa B (NF-κB) (p65) and phospho-mammalian target of rapamycin (p-mTOR) signaling proteins in cells treated with PM. Treatment with PM also inhibited the expression of NF-κB-regulated antiapoptotic Bcl-2, Bcl-xL, c-IAP1 and survivin. Thus, our data showing potent antiproliferative and apoptosis-inducing activity of PM in ovarian carcinoma cells through the inhibition of AKT/ NF-κB/ mTOR signaling pathway warrant further investigation of PM for the management of ovarian cancer.

Evidence that CXCL16 is a potent mediator of angiogenesis and is involved in endothelial progenitor cell chemotaxis : studies in mice with K/BxN serum-induced arthritis.

OBJECTIVE: To examine the possibility that CXCL16 recruits endothelial cells (ECs) to developing neovasculature in rheumatoid arthritis (RA) synovium. METHODS: We utilized the RA synovial tissue SCID mouse chimera system to examine human microvascular EC (HMVEC) and human endothelial progenitor cell (EPC) recruitment into engrafted human synovium that was injected intragraft with CXCL16-immunodepleted RA synovial fluid (SF). CXCR6-deficient and wild-type (WT) C57BL/6 mice were primed to develop K/BxN serum-induced arthritis and evaluated for angiogenesis. HMVECs and EPCs from human cord blood were also examined for CXCR6 expression, by immunofluorescence and assessment of CXCL16 signaling activity. RESULTS: CXCR6 was prominently expressed on human EPCs and HMVECs, and its expression on HMVECs could be up-regulated by interleukin-1ß. SCID mice injected with CXCL16-depleted RA SF exhibited a significant reduction in EPC recruitment. In experiments using the K/BxN serum-induced inflammatory arthritis model, CXCR6(-/-) mice showed profound reductions in hemoglobin levels, which correlated with reductions in monocyte and T cell recruitment to arthritic joint tissue compared to that observed in WT mice. Additionally, HMVECs and EPCs responded to CXCL16 stimulation, but exhibited unique signal transduction pathways and homing properties. CONCLUSION: These results indicate that CXCL16 and its receptor CXCR6 may be a central ligand/receptor pair that is closely associated with EPC recruitment and blood vessel formation in the RA joint.

Subcurative radiation significantly increases cell proliferation, invasion, and migration of primary glioblastoma multiforme in vivo.

Tumor cell proliferation, infiltration, migration, and neovascularization are known causes of treatment resistance in glioblastoma multiforme (GBM). The purpose of this study was to determine the effect of radiation on the growth characteristics of primary human GBM developed in a nude rat. Primary GBM cells grown from explanted GBM tissues were implanted orthotopically in nude rats. Tumor growth was confirmed by magnetic resonance imaging on day 77 (baseline) after implantation. The rats underwent irradiation to a dose of 50 Gy delivered subcuratively on day 84 postimplantation (n = 8), or underwent no radiation (n = 8). Brain tissues were obtained on day 112 (nonirradiated) or day 133 (irradiated). Immunohistochemistry was performed to determine tumor cell proliferation (Ki-67) and to assess the expression of infiltration marker (matrix metalloproteinase-2, MMP-2) and cell migration marker (CD44). Tumor neovascularization was assessed by microvessel density using von-Willebrand factor (vWF) staining. Magnetic resonance imaging showed well-developed, infiltrative tumors in 11 weeks postimplantation. The proportion of Ki-67-positive cells in tumors undergoing radiation was (71 +/- 15)% compared with (25 +/- 12)% in the nonirradiated group (P = 0.02). The number of MMP-2-positive areas and proportion of CD44-positive cells were also high in tumors receiving radiation, indicating great invasion and infiltration. Microvessel density analysis did not show a significant difference between nonirradiated and irradiated tumors. Taken together, we found that subcurative radiation significantly increased proliferation, invasion, and migration of primary GBM. Our study provides insights into possible mechanisms of treatment resistance following radiation therapy for GBM.

Isolation and immunosuppressive functions of myeloid-derived suppressor cell-derived exosomes.

Myeloid-derived suppressor cells (MDSCs) are an integral part of the tumor microenvironment (TME). MDSC's involvement in the TME starts as soon as the primary tumor starts to get its blood supply causing an immunosuppressive environment and tumor cell invasion, and then at the formation of premetastatic niche through full-blown metastasis in distal organs. All of these functions don't require physical interaction of MDSC as some of the MDSC's functions can be replicated by secreted exosomes (MDSC-derived exosomes), which can alter the microenvironment through cellular interaction by fusion with the plasma membrane and subsequent release of their cargo, consisting of proteins, soluble factors, lipids, DNAs, microRNAs (miRNAs), and RNAs. In this method paper, we explained how to isolate MDSC exosomes and how to use the exosome to observe immunosuppressive function. We also discussed how to measure the number of exosomes by nanoparticle tracking analysis. Additionally, we outlined how to measure the protein of exosomes as well as the types of protein by Bradford assay and membrane cytokine array respectively. We also provided instructions on how to utilize MDSC-derived exosomes to get knowledge about in vitro immune cell migration, scratch assay with the tumor cells, and in vivo effect of MDSC exosome along with T cell function and proliferation.

Exercise Improves Cerebral Blood Flow and Functional Outcomes in an Experimental Mouse Model of Vascular Cognitive Impairment and Dementia (VCID).

Vascular cognitive impairment and dementia (VCID) are a growing threat to public health without any known treatment. The bilateral common carotid artery stenosis (BCAS) mouse model is valid for VCID. Previously, we have reported that remote ischemic postconditioning (RIPostC) during chronic cerebral hypoperfusion (CCH) induced by BCAS increases cerebral blood flow (CBF), improves cognitive function, and reduces white matter damage. We hypothesized that physical exercise (EXR) would augment CBF during CCH and prevent cognitive impairment in the BCAS model. BCAS was performed in C57/B6 mice of both sexes to establish CCH. One week after the BCAS surgery, mice were randomized to treadmill exercise once daily or no EXR for four weeks. CBF was monitored with an LSCI pre-, post, and 4 weeks post-BCAS. Cognitive testing was performed for post-BCAS after exercise training, and brain tissue was harvested for histopathology and biochemical test. BCAS led to chronic hypoperfusion resulting in impaired cognitive function and other functional outcomes. Histological examination revealed that BCAS caused changes in neuronal morphology and cell death in the cortex and hippocampus. Immunoblotting showed that BCAS was associated with a significant downregulate of AMPK and pAMPK and NOS3 and pNOS3. BCAS also decreased red blood cell (RBC) deformability. EXR therapy increased and sustained improved CBF and cognitive function, muscular strength, reduced cell death, and loss of white matter. EXR is effective in the BCAS model, improving CBF and cognitive function, reducing white matter damage, improving RBC deformability, and increasing RBC NOS3 and AMPK. The mechanisms by which EXR improves CBF and attenuates tissue damage need further investigation.

Single episode of moderate to severe traumatic brain injury leads to chronic neurological deficits and Alzheimer's-like pathological dementia.

Traumatic brain injury (TBI) is one of the foremost causes of disability and mortality globally. While the scientific and medical emphasis is to save lives and avoid disability during acute period of injury, a severe health problem can manifest years after injury. For instance, TBI increases the risk of cognitive impairment in the elderly. Remote TBI history was reported to be a cause of the accelerated clinical trajectory of Alzheimer's disease-related dementia (ADRD) resulting in earlier onset of cognitive impairment and increased AD-associated pathological markers like greater amyloid deposition and cortical thinning. It is not well understood whether a single TBI event may increase the risk of dementia. Moreover, the cellular signaling pathways remain elusive for the chronic effects of TBI on cognition. We have hypothesized that a single TBI induces sustained neuroinflammation and disrupts cellular communication in a way that results later in ADRD pathology. To test this, we induced TBI in young adult CD1 mice and assessed the behavioral outcomes after 11 months followed by pathological, histological, transcriptomic, and MRI assessment. On MRI scans, these mice showed significant loss of tissue, reduced CBF, and higher white matter injury compared to sham mice. We found these brains showed progressive atrophy, markers of ADRD, sustained astrogliosis, loss of neuronal plasticity, and growth factors even after 1-year post-TBI. Because of progressive neurodegeneration, these mice had motor deficits, showed cognitive impairments, and wandered randomly in open field. We, therefore, conclude that progressive pathology after adulthood TBI leads to neurodegenerative conditions such as ADRD and impairs neuronal functions.

Altering biomolecular condensates as a potential mechanism that mediates cannabidiol effect on glioblastoma.

Glioblastoma (GBM) is an extremely aggressive primary brain tumor with poor prognosis, short survival time post-diagnosis and high recurrence. Currently, no cure for GBM exists. The identification of an effective therapeutic modality for GBM remains a high priority amongst medical professionals and researches. In recent studies, inhalant cannabidiol (CBD) has demonstrated promise in effectively inhibiting GBM tumor growth. However, exactly how CBD treatment affects the physiology of these tumor cells remains unclear. Stress granules (SG) (a sub-class of biomolecular condensates (BMC)) are dynamic, membrane-less intracellular microstructures which contain proteins and nucleic acids. The formation and signaling of SGs and BMCs plays a significant role in regulating malignancies. This study investigates whether inhaled CBD may play an intervening role towards SGs in GBM tumor cells. Integrated bioinformatics approaches were preformed to gain further insights. This includes use of Immunohistochemistry and flow cytometry to measure SGs, as well as expression and phosphorylation of eukaryotic initiation factor-2α (eIF2α). The findings of this study reveal that CBD receptors (and co-regulated genes) have the potential to play an important biological role in the formation of BMCs within GBM. In this experiment, CBD treatment significantly increased the volume of TIAR-1. This increase directly correlated with elevation in both eIF2α expression and p-eIF2α in CBD treated tissues in comparison to the placebo group (p < 0.05). These results suggest that inhalant CBD significantly up-regulated SGs in GBM, and thus support a theory of targeting BMCs as a potential therapeutic substrate for treating GBM.

Differential biodistribution of intravenously administered endothelial progenitor and cytotoxic T-cells in rat bearing orthotopic human glioma.

BACKGROUND: A major challenge in the development of cell based therapies for glioma is to deliver optimal number of cells (therapeutic dose) to the tumor. Imaging tools such as magnetic resonance imaging (MRI), optical imaging, positron emission tomography (PET) and single-photon emission computed tomography (SPECT) has been used in cell tracking and/or biodistribution studies. In this study, we evaluate the dynamic biodistribution of systemic injected labeled cells [human cord blood derived endothelial progenitor cells (EPCs) and cytotoxic T-cells (CTLs)] in rat glioma model with in vivo SPECT imaging. METHODS: Human cord blood EPCs, T-cells and CD14⁺ cells (monocytes/dendritic cells) were isolated using the MidiMACS system. CD14⁺ cells were converted to dendritic cells (DC) and also primed with U251 tumor cell line lysate. T-cells were co-cultured with irradiated primed DCs at 10:1 ratio to make CTLs. Both EPCs and CTLs were labeled with In-111-oxine at 37°C in serum free DMEM media. Glioma bearing animals were randomly assigned into three groups. In-111 labeled cells or In-111 oxine alone were injected through tail vein and SPECT imaging was performed on day 0, 1, and 3. In-111 oxine activity in various organs and tumor area was determined. Histochemical analysis was performed to further confirm the migration and homing of injected cells at the tumor site. RESULTS: EPCs and CTLs showed an In-111 labeling efficiency of 87.06 ± 7.75% and 70.8 ± 12.9% respectively. Initially cell migration was observed in lung following inravenous administration of In-111 labeled cells and decreased on day 1 and 3, which indicate re-distribution of labeled cells from lung to other organs. Relatively higher In-111 oxine activity was observed in tumor areas at 24 hours in animals received In-111 labeled cells (EPCs or CTLs). Histiological analysis revealed iron positive cells in and around the tumor area in animals that received labeled cells (CTLs and EPCs). CONCLUSION: We observed differential biodistribution of In-111-oxine labeled EPCs and CTLs in different organs and intracranial glioma. This study indicates In-111 oxine based SPECT imaging is an effective tool to study the biodistribution of therapeutically important cells.

Inhibition of telomerase activity by oleanane triterpenoid CDDO-Me in pancreatic cancer cells is ROS-dependent.

Methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me) is a synthetic derivative of oleanolic acid, a triterpene, with apoptosis-inducing activity in a wide range of cancer cells. Induction of apoptosis by CDDO-Me is associated with the generation of reactive oxygen species (ROS) and inhibition of telomerase activity. In the present study, we investigated the role of ROS in inhibition of telomerase by CDDO-me. Treatment of MiaPaCa-2 and Panc-1 pancreatic cancer cell lines with CDDO-Me induced the production of hydrogen peroxide and superoxide anions and inhibited the telomerase activity. Pretreatment of cells with N-acetylcycsteine, a general purpose antioxidant or overexpression of glutathione peroxidase (GPx) or superoxide dismutase-1 (SOD-1) blocked the telomerase inhibitory activity of CDDO-Me. Furthermore, blocking ROS generation also prevented the inhibition of hTERT gene expression, hTERT protein production and expression of a number of hTERT-regulatory proteins by CDDO-Me (e.g., c-Myc, Sp1, NF-κB and p-Akt). Data also showed that Akt plays an important role in the activation of telomerase activity. Together, these data suggest that inhibition of telomerase activity by CDDO-Me is mediated through a ROS-dependent mechanism; however, more work is needed to fully understand the role of ROS in down-regulation of hTERT gene and hTERT-regulatory proteins by CDDO-Me.