The Expression Level of CB1 and CB2 Receptors Determines Their Efficacy at Inducing Apoptosis in Astrocytomas

Cannabinoids represent unique compounds for treating tumors, including astrocytomas. Whether CB(1) and CB(2) receptors mediate this therapeutic effect is unclear.We generated astrocytoma subclones that express set levels of CB(1) and CB(2), and found that cannabinoids induce apoptosis only in cells expressing low levels of receptors that couple to ERK1/2. In contrast, cannabinoids do not induce apoptosis in cells expressing high levels of receptors because these now also couple to the prosurvival signal AKT. Remarkably, cannabinoids applied at high concentration induce apoptosis in all subclones independently of CB(1), CB(2) and AKT, but still through a mechanism involving ERK1/2.The high expression level of CB(1) and CB(2) receptors commonly found in malignant astrocytomas precludes the use of cannabinoids as therapeutics, unless AKT is concomitantly inhibited, or cannabinoids are applied at concentrations that bypass CB(1) and CB(2) receptors, yet still activate ERK1/2

Loss of Sphingosine Kinase 1/S1P Signaling Impairs Cell Growth and Survival of Neurons and Progenitor Cells in the Developing Sensory Ganglia

Background: Lysophospholipids such as lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are important signaling molecules that can regulate a wide range of cellular responses. We discovered that Sphingosine kinase 1 (Sphk1), a key enzyme that converts sphingosine to S1P, is expressed in neurons and progenitor cells in nascent trigeminal and dorsal root ganglia during mouse embryogenesis. Methods and Findings: Sphk1 null mouse embryos do not display overt deficits owing to compensation by Sphk2. Thus, we analyzed embryos that are deficient in both Sphk1 and Sphk2 (which essentially eliminates S1P function) in order to investigate the role(s) of Sphk1 during sensory ganglia formation. While animals lacking 1–3 alleles of Sphk1 and Sphk2 had no obvious phenotype, embryos without both genes displayed clear developmental defects. The complete absence of Sphk1 and Sphk2 resulted in trigeminal and dorsal root ganglia with fewer neurons and progenitor cells. The profound loss in cell number could be attributed to a decrease in cell proliferation as well as an increase in apoptosis. Furthermore, Sphk1/ 2 double mutants displayed an overall reduction in other sphingolipids as well as an imbalance of S1P/sphingosine and S1P/ ceramide ratio, thereby favoring cell death and reducing cell growth. Conclusions: Together, these results provide strong in vivo evidence that sphingosine kinase/S1P signaling plays a

Protein-Protein Interaction Analysis Highlights Additional Loci of Interest for Multiple Sclerosis

PMCID: PMC3475710This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

An Optimized Pentaplex PCR for Detecting DNA Mismatch Repair-Deficient Colorectal Cancers

Microsatellite instability (MSI) is used to screen colorectal cancers (CRC) for Lynch Syndrome, and to predict outcome and response to treatment. The current technique for measuring MSI requires DNA from normal and neoplastic tissues, and fails to identify tumors with specific DNA mismatch repair (MMR) defects. We tested a panel of five quasi-monomorphic mononucleotide repeat markers amplified in a single multiplex PCR reaction (pentaplex PCR) to detect MSI.We investigated a cohort of 213 CRC patients, comprised of 114 MMR-deficient and 99 MMR-proficient tumors. Immunohistochemical (IHC) analysis evaluated the expression of MLH1, MSH2, PMS2 and MSH6. MSI status was defined by differences in the quasi-monomorphic variation range (QMVR) from a pool of normal DNA samples, and measuring differences in allele lengths in tumor DNA.Amplification of 426 normal alleles allowed optimization of the QMVR at each marker, and eliminated the requirement for matched reference DNA to define MSI in each sample. Using ≥2/5 unstable markers as the criteria for MSI resulted in a sensitivity of 95.6% (95% CI = 90.1–98.1%) and a positive predictive value of 100% (95% CI = 96.6%–100%). Detection of MSH6-deficiency was limited using all techniques. Data analysis with a three-marker panel (BAT26, NR21 and NR27) was comparable in sensitivity (97.4%) and positive predictive value (96.5%) to the five marker panel. Both approaches were superior to the standard approach to measuring MSI.An optimized pentaplex (or triplex) PCR offers a facile, robust, very inexpensive, highly sensitive, and specific assay for the identification of MSI in CRC

Imported Infections Versus Herd Immunity Gaps; A Didactic Demonstration of Compartment Models Through the Example of a Minor Measles Outbreak in Hungary

Introduction: In Hungary, where MMR vaccine coverage is 99%, in 2017, a minor measles epidemic started from imported cases due to two major factors – latent susceptible cohorts among the domestic population and the vicinity of measles-endemic countries. Suspended immunization activities due to the COVID-19 surge are an ominous precursor to a measles resurgence. This epidemiological demonstration is aimed at promoting a better public understanding of epidemiological data. Materials and Methods: Our previous MMR sero-epidemiological measurements (N of total measles cases = 3919, N of mumps cases = 2132, and N of rubella cases = 2132) were analyzed using open-source epidemiological data (ANTSZ) of a small-scale measles epidemic outbreak (2017, Hungary). A simplified SEIR model was applied in the analysis. Results: In case of measles, due to a cluster-specific inadequacy of IgG levels, the cumulative seropositivity ratios (measles = 89.97%) failed to reach the herd immunity threshold (HIT Measles = 92–95%). Despite the fact that 90% of overall vaccination coverage is just slightly below the HIT, unprotected individuals may pose an elevated epidemiological risk. According to the SEIR model, ≥74% of susceptible individuals are expected to get infected. Estimations based on the input data of a local epidemic may suggest an even lower effective coverage rate (80%) in certain clusters of the population. Conclusion: Serological survey-based, historical and model-computed results are in agreement. A practical demonstration of epidemiological events of the past and present may promote a higher awareness of infectious diseases. Because of the high R0 value of measles, continuous large-scale monitoring of humoral immunity levels is important

Secreted Gaussia Luciferase as a Biomarker for Monitoring Tumor Progression and Treatment Response of Systemic Metastases

Currently, only few techniques are available for quantifying systemic metastases in preclinical model. Thus techniques that can sensitively detect metastatic colonization and assess treatment response in real-time are urgently needed. To this end, we engineered tumor cells to express a naturally secreted Gaussia luciferase (Gluc), and investigated its use as a circulating biomarker for monitoring viable metastatic or primary tumor growth and their treatment responses.We first developed orthotopic primary and metastatic breast tumors with derivative of MDA-MB-231 cells expressing Gluc. We then correlated tumor burden with Gluc activity in the blood and urine along with bioluminescent imaging (BLI). Second, we utilized blood Gluc assay to monitor treatment response to lapatinib in an experimental model of systemic metastasis. We observed good correlation between the primary tumor volume and Gluc concentration in blood (R(2) = 0.84) and urine (R(2) = 0.55) in the breast tumor model. The correlation deviated as a primary tumor grew due to a reduction in viable tumor fraction. This was also supported by our mathematical models for tumor growth to compare the total and viable tumor burden in our model. In the experimental metastasis model, we found numerous brain metastases as well as systemic metastases including bone and lungs. Importantly, blood Gluc assay revealed early growth of metastatic tumors before BLI could visualize their presence. Using secreted Gluc, we localized systemic metastases by BLI and quantitatively monitored the total viable metastatic tumor burden by blood Gluc assay during the course of treatment with lapatinib, a dual tyrosine kinase inhibitor of EGFR and HER2.We demonstrated secreted Gluc assay accurately reflects the amount of viable cancer cells in primary and metastatic tumors. Blood Gluc activity not only tracks metastatic tumor progression but also serves as a longitudinal biomarker for tumor response to treatments

Reconstitution of Mammary Epithelial Morphogenesis by Murine Embryonic Stem Cells Undergoing Hematopoietic Stem Cell Differentiation

Background: Mammary stem cells are maintained within specific microenvironments and recruited throughout lifetime to reconstitute de novo the mammary gland. Mammary stem cells have been isolated through the identification of specific cell surface markers and in vivo transplantation into cleared mammary fat pads. Accumulating evidence showed that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary epithelial cells may be sequestered and reprogrammed to perform mammary epithelial cell functions and to adopt mammary epithelial characteristics during reconstruction of mammary epithelium in regenerating mammary tissue in vivo. Methodology/Principal Findings: To examine whether other types of progenitor cells are able to contribute to mammary branching morphogenesis, we examined the potential of murine embryonic stem (mES) cells, undergoing hematopoietic differentiation, to support mammary reconstitution in vivo. We observed that cells from day 14 embryoid bodies (EBs) under hematopoietic differentiation condition, but not supernatants derived from these cells, when transplanted into denuded mammary fat pads, were able to contribute to both the luminal and myoepithelial lineages in branching ductal structures resembling the ductal-alveolar architecture of the mammary tree. No teratomas were observed when these cells were transplanted in vivo. Conclusions/Significance: Our data provide evidence for the dominance of the tissue-specific mammary stem cell niche and its role in directing mES cells, undergoing hematopoietic differentiation, to reprogram into mammary epithelial cells and to promote mammary epithelial morphogenesis. These studies should also provide insights into regeneration of damaged mammary gland and the role of the mammary microenvironment in reprogramming cell fate. © 2010 Jiang et al

Transcriptional Networks in Epithelial-Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) changes polarized epithelial cells into migratory phenotypes associated with loss of cell-cell adhesion molecules and cytoskeletal rearrangements. This form of plasticity is seen in mesodermal development, fibroblast formation, and cancer metastasis.Here we identify prominent transcriptional networks active during three time points of this transitional process, as epithelial cells become fibroblasts. DNA microarray in cultured epithelia undergoing EMT, validated in vivo, were used to detect various patterns of gene expression. In particular, the promoter sequences of differentially expressed genes and their transcription factors were analyzed to identify potential binding sites and partners. The four most frequent cis-regulatory elements (CREs) in up-regulated genes were SRY, FTS-1, Evi-1, and GC-Box, and RNA inhibition of the four transcription factors, Atf2, Klf10, Sox11, and SP1, most frequently binding these CREs, establish their importance in the initiation and propagation of EMT. Oligonucleotides that block the most frequent CREs restrain EMT at early and intermediate stages through apoptosis of the cells.Our results identify new transcriptional interactions with high frequency CREs that modulate the stability of cellular plasticity, and may serve as targets for modulating these transitional states in fibroblasts

Novel Cell-Free Strategy for Therapeutic Angiogenesis: In Vitro Generated Conditioned Medium Can Replace Progenitor Cell Transplantation

BACKGROUND: Current evidence suggests that endothelial progenitor cells (EPC) contribute to ischemic tissue repair by both secretion of paracrine factors and incorporation into developing vessels. We tested the hypothesis that cell-free administration of paracrine factors secreted by cultured EPC may achieve an angiogenic effect equivalent to cell therapy. METHODOLOGY/PRINCIPAL FINDINGS: EPC-derived conditioned medium (EPC-CM) was obtained from culture expanded EPC subjected to 72 hours of hypoxia. In vitro, EPC-CM significantly inhibited apoptosis of mature endothelial cells and promoted angiogenesis in a rat aortic ring assay. The therapeutic potential of EPC-CM as compared to EPC transplantation was evaluated in a rat model of chronic hindlimb ischemia. Serial intramuscular injections of EPC-CM and EPC both significantly increased hindlimb blood flow assessed by laser Doppler (81.2+/-2.9% and 83.7+/-3.0% vs. 53.5+/-2.4% of normal, P<0.01) and improved muscle performance. A significantly increased capillary density (1.62+/-0.03 and 1.68+/-0.05/muscle fiber, P<0.05), enhanced vascular maturation (8.6+/-0.3 and 8.1+/-0.4/HPF, P<0.05) and muscle viability corroborated the findings of improved hindlimb perfusion and muscle function. Furthermore, EPC-CM transplantation stimulated the mobilization of bone marrow (BM)-derived EPC compared to control (678.7+/-44.1 vs. 340.0+/-29.1 CD34(+)/CD45(-) cells/1x10(5) mononuclear cells, P<0.05) and their recruitment to the ischemic muscles (5.9+/-0.7 vs. 2.6+/-0.4 CD34(+) cells/HPF, P<0.001) 3 days after the last injection. CONCLUSIONS/SIGNIFICANCE: Intramuscular injection of EPC-CM is as effective as cell transplantation for promoting tissue revascularization and functional recovery. Owing to the technical and practical limitations of cell therapy, cell free conditioned media may represent a potent alternative for therapeutic angiogenesis in ischemic cardiovascular diseases

Human Cord Blood-Derived AC133+ Progenitor Cells Preserve Endothelial Progenitor Characteristics after Long Term In Vitro Expansion

Stem cells/progenitors are central to the development of cell therapy approaches for vascular ischemic diseases. The crucial step in rescuing tissues from ischemia is improvement of vascularization that can be achieved by promoting neovascularization. Endothelial progenitor cells (EPCs) are the best candidates for developing such an approach due to their ability to self-renew, circulate and differentiate into mature endothelial cells (ECs). Studies showed that intravenously administered progenitors isolated from bone marrow, peripheral or cord blood home to ischemic sites. However, the successful clinical application of such transplantation therapy is limited by low quantities of EPCs that can be generated from patients. Hence, the ability to amplify the numbers of autologous EPCs by long term in vitro expansion while preserving their angiogenic potential is critically important for developing EPC based therapies. Therefore, the objective of this study was to evaluate the capacity of cord blood (CB)-derived AC133+ cells to differentiate, in vitro, towards functional, mature endothelial cells (ECs) after long term in vitro expansion.We systematically characterized the properties of CB AC133+ cells over the 30 days of in vitro expansion. During 30 days of culturing, CB AC133+ cells exhibited significant growth potential that was manifested as 148-fold increase in cell numbers. Flow cytometry and immunocytochemistry demonstrated that CB AC133+ cells' expression of endothelial progenitor markers was not affected by long term in vitro culturing. After culturing under EC differentiation conditions, cells exhibited high expression of mature ECs markers, such as CD31, VEGFR-2 and von Willebrand factor, as well as the morphological changes indicative of differentiation towards mature ECs. In addition, throughout the 30 day culture cells preserved their functional capacity that was demonstrated by high uptake of DiI fluorescently conjugated-acetylated-low density lipoprotein (DiI-Ac-LDL), in vitro and in vivo migration towards chemotactic stimuli and in vitro tube formation.These studies demonstrate that primary CB AC133+ culture contained mainly EPCs and that long term in vitro conditions facilitated the maintenance of these cells in the state of commitment towards endothelial lineage