Risk factors of postoperative complications after radical cystectomy with continent or conduit urinary diversion in Armenia

To estimate the surgical volume and the incidence of in-hospital complications of RC in Armenia from 2005 to 2012, and to investigate potential risk factors of complications. The study utilized a retrospective chart review in a cohort of patients who had RC followed by either continent or conduit urinary diversion in all hospitals of Armenia from 2005 to 2012. A detailed chart review was conducted abstracting information on baseline demographic and clinical characteristics, surgical procedural details, postoperative management and in-hospital complications. Multivariable logistic regression analysis was applied to estimate the independent risk factors for developing ‘any postoperative complication’. The total study sample included 273 patients (mean age = 58.5 years, 93.4 % men). Overall, 28.9 % (n = 79) of patients had at least one in-hospital complication. The hospital mortality rate was 4.8 % (n = 13). The most frequent types of complications were wound-related (10.3 %), gastrointestinal (9.2 %) and infectious (7.0 %). The ischemic heart disease (OR = 3.3, 95 % CI 1.5–7.4), perioperative transfusion (OR = 2.0, 1.1–3.6), glucose level [OR = 0.71 (0.63–0.95)], and hospital type (OR = 2.3, 95 % CI 1.1–4.7) were independent predictors of postoperative complications. The rate of RC complications in Armenia was similar to those observed in other countries. Future prospective studies should evaluate the effect of RC complications on long-term outcomes and costs in Armenia. Policy recommendations should address the issues regarding surgeon training and hospital volume to decrease the risk of RC complications

Quantitative Evaluation of Intraventricular Delivery of Therapeutic Neural Stem Cells to Orthotopic Glioma

Neural stem cells (NSCs) are inherently tumor-tropic, which allows them to migrate through normal tissue and selectively localize to invasive tumor sites in the brain. We have engineered a clonal, immortalized allogeneic NSC line (HB1.F3.CD21; CD-NSCs) that maintains its stem-like properties, a normal karyotype and is HLA Class II negative. It is genetically and functionally stable over time and multiple passages, and has demonstrated safety in phase I glioma trials. These properties enable the production of an “off-the-shelf” therapy that can be readily available for patient treatment. There are multiple factors contributing to stem cell tumor-tropism, and much remains to be elucidated. The route of NSC delivery and the distribution of NSCs at tumor sites are key factors in the development of effective cell-based therapies. Stem cells can be engineered to deliver and/or produce many different therapeutic agents, including prodrug activating enzymes (which locally convert systemically administered prodrugs to active chemotherapeutic agents); oncolytic viruses; tumor-targeted antibodies; therapeutic nanoparticles; and extracellular vesicles that contain therapeutic oligonucleotides. By targeting these therapeutics selectively to tumor foci, we aim to minimize toxicity to normal tissues and maximize therapeutic benefits. In this manuscript, we demonstrate that NSCs administered via intracerebral/ventricular (IVEN) routes can migrate efficiently toward single or multiple tumor foci. IVEN delivery will enable repeat administrations for patients through an Ommaya reservoir, potentially resulting in improved therapeutic outcomes. In our preclinical studies using various glioma lines, we have quantified NSC migration and distribution in mouse brains and have found robust migration of our clinically relevant HB1.F3.CD21 NSC line toward invasive tumor foci, irrespective of their origin. These results establish proof-of-concept and demonstrate the potential of developing a multitude of therapeutic options using modified NSCs

Risk factors of postoperative complications after radical cystectomy with continent or conduit urinary diversion in Armenia

Abstract To estimate the surgical volume and the incidence of in-hospital complications of RC in Armenia from 2005 to 2012, and to investigate potential risk factors of complications. The study utilized a retrospective chart review in a cohort of patients who had RC followed by either continent or conduit urinary diversion in all hospitals of Armenia from 2005 to 2012. A detailed chart review was conducted abstracting information on baseline demographic and clinical characteristics, surgical procedural details, postoperative management and in-hospital complications. Multivariable logistic regression analysis was applied to estimate the independent risk factors for developing ‘any postoperative complication’. The total study sample included 273 patients (mean age = 58.5 years, 93.4 % men). Overall, 28.9 % (n = 79) of patients had at least one in-hospital complication. The hospital mortality rate was 4.8 % (n = 13). The most frequent types of complications were wound-related (10.3 %), gastrointestinal (9.2 %) and infectious (7.0 %). The ischemic heart disease (OR = 3.3, 95 % CI 1.5–7.4), perioperative transfusion (OR = 2.0, 1.1–3.6), glucose level [OR = 0.71 (0.63–0.95)], and hospital type (OR = 2.3, 95 % CI 1.1–4.7) were independent predictors of postoperative complications. The rate of RC complications in Armenia was similar to those observed in other countries. Future prospective studies should evaluate the effect of RC complications on long-term outcomes and costs in Armenia. Policy recommendations should address the issues regarding surgeon training and hospital volume to decrease the risk of RC complications

Detection and development of a quantitation method for undeclared compounds in antidiabetic biologically active additives and its validation by high performance liquid chromatography

An isocratic, high-performance liquid chromatography (HPLC) quantitation method was developed for the quantitative determination of metformin, glibenclamide, gliclazide, glimepiride in some antidiabetic biologically active additives. A Nucleosil C18, 5 μm, 4.6 mm × 150 mm, column with mobile phase containing buffer (10 mm Na2HPO4, 10 mm sodium dodecyl sulfate): acetonitrile = 68 : 32 (V/V), pH = 7.5 was used. The flow rate was 1.0 mL/min, and effluents were monitored at 226 nm. The retention times of gliclazide glibenclamide, glimepiride and metformin, were 2.203, 4.587, 5.667 and 10.182 min, respectively. Linearity was studied by preparing standard solutions of gliclazide, glibenclamide, glimepiride and metformin at the concentration range of 50% to 150% of working concentration from a stock solution. The method was successfully applied to the estimation of gliclazide, glibenclamide, glimepiride and metformin in some antidiabetic biologically active additives. This method was validated to confirm its system suitability, selectivity, linearity, precision and accuracy according to international conference on harmonization (ICH) guidelines

Intranasally Administered L-Myc-Immortalized Human Neural Stem Cells Migrate to Primary and Distal Sites of Damage after Cortical Impact and Enhance Spatial Learning

As the success of stem cell-based therapies is contingent on efficient cell delivery to damaged areas, neural stem cells (NSCs) have promising therapeutic potential because they inherently migrate to sites of central nervous system (CNS) damage. To explore the possibility of NSC-based therapy after traumatic brain injury (TBI), isoflurane-anesthetized adult male rats received a controlled cortical impact (CCI) of moderate severity (2.8 mm deformation at 4 m/s) or sham injury (i.e., no cortical impact). Beginning 1-week post-injury, the rats were immunosuppressed and 1×106 human NSCs (LM-NS008.GFP.fLuc) or vehicle (VEH) (2% human serum albumen) were administered intranasally (IN) on post-operative days 7, 9, 11, 13, 15, and 17. To evaluate the spatial distributions of the LM-NSC008 cells, half of the rats were euthanized on day 25, one day after completion of the cognitive task, and the other half were euthanized on day 46. 1 mm thick brain sections were optically cleared (CLARITY), and volumes were imaged by confocal microscopy. In addition, LM-NSC008 cell migration to the TBI site by immunohistochemistry for human-specific Nestin was observed at day 39. Acquisition of spatial learning was assessed in a well-established Morris water maze task on six successive days beginning on post-injury day 18. IN administration of LM-NSC008 cells after TBI (TBI+NSC) significantly facilitated spatial learning relative to TBI+VEH rats (p<0.05) and had no effect on sham+NSC rats. Overall, these data indicate that IN-administered LM-NSC008 cells migrate to sites of TBI damage and that their presence correlates with cognitive improvement. Future studies will expand on these preliminary findings by evaluating other LM-NSC008 cell dosing paradigms and evaluating mechanisms by which LM-NSC008 cells contribute to cognitive recovery

Long-term stability and computational analysis of migration patterns of L-<i>MYC</i> immortalized neural stem cells in the brain

<div><p>Background</p><p>Preclinical studies indicate that neural stem cells (NSCs) can limit or reverse central nervous system (CNS) damage through delivery of therapeutic agents for cell regeneration. Clinical translation of cell-based therapies raises concerns about long-term stability, differentiation and fate, and absence of tumorigenicity of these cells, as well as manufacturing time required to produce therapeutic cells in quantities sufficient for clinical use. Allogeneic NSC lines are in growing demand due to challenges inherent in using autologous stem cells, including production costs that limit availability to patients.</p><p>Methods/Principal findings</p><p>We demonstrate the long-term stability of L-<i>MYC</i> immortalized human NSCs (LM-NSC008) cells <i>in vivo</i>, including engraftment, migration, and absence of tumorigenicity in mouse brains for up to nine months. We also examined the distributions of engrafted LM-NSC008 cells within brain, and present computational techniques to analyze NSC migration characteristics in relation to intrinsic brain structures.</p><p>Conclusions/Significance</p><p>This computational analysis of NSC distributions following implantation provides proof-of-concept for the development of computational models that can be used clinically to predict NSC migration paths in patients. Previously, models of preferential migration of malignant tumor cells along white matter tracts have been used to predict their final distributions. We suggest that quantitative measures of tissue orientation and white matter tracts determined from MR images can be used in a diffusion tensor imaging tractography-like approach to describe the most likely migration routes and final distributions of NSCs administered in a clinical setting. Such a model could be very useful in choosing the optimal anatomical locations for NSC administration to patients to achieve maximum therapeutic effects.</p></div

Computational analysis of distribution of LM-NSC008 cells.

<p><b>(A)</b> Distances of LM-NSC008 cell clusters from injection sites at 3, 6, and 9 months post-injection. Distances were calculated as Euclidean distances in the 2-dimensional plane. <b>(B, C)</b> Cumulative probability distributions of distances of LM-NSC008 clusters from the injection site in WM and GM at 3, 6 and 9 months post-injection. The 9 month curve reflecting a greater distance from the injection center as compared to 3 month curve indicates the migration of NSCs away from the injection site. <b>(D)</b> Percentage of LM-NSC008 cells identified in the WM at 3, 6, and 9 months post-injection. <b>(E, F)</b> Cumulative probability distribution of distance from white matter/grey matter (WM/GM) interface. The 9 month curve being closer to the WM/GM interface as compared to the 3 month curve indicates that LM-NSC008 cells increasingly aggregated near the WM/GM interface over time.</p

Characterization of LM-NSC008 cells.

<p>Visualization of LM-NSC008 cells in naïve non-tumor bearing mouse brain 6 and 9 months post administration. <b>(A)</b> Immunohistochemistry (IHC) staining of LM-NSC008 cells (stably expressing eGFP/Ffluc) with anti-HNA antibodies (NSC injection site, white arrows indicate the LM-NSC008 cells, scale bar 100 μm). <b>(1a-1d)</b> Insets show LM-NSC008 cells co-expressing HNA and eGFP proteins (inset scale bars [1a-1f] 2 μm). <b>(B)</b> IHC staining for huNestin (green) in mouse brain sections from 6 months after administration, scale bar 20 μm; <b>(C)</b> IHC staining for Pax6 (red)/eGFP in LM-NSC008 cells, scale bar 50 μm. Inset <b>(1e</b>) shows enlarged area of neuronal cells co-expressing Pax6 and eGFP. <b>(D)</b> Bright-field image of LM-NSC008 cells stained with Stem123 and contrastained with hematoxylin, scale bar 200 μm. Inset <b>(1f)</b> enlarged LM-NSC008 cell, expressing the glial marker Stem123. Arrows (black) show LM-NSC008s (brown) in the corpus callosum. <b>(E)</b> IHC for Ki-67, showing negative staining (red) of LM-NSC008 cells expressing eGFP (injection site), scale bar 20 μm. Fluorescent-stained slides were counterstained with DAPI (blue) to visualize nuclei. <b>(F)</b> PCR analysis of genomic DNA derived from LM-NSC008 cells at every 5th passage <i>in vitro</i> (p5-p50). Controls were: L-<i>MYC</i> plasmid, DNA derived from untransduced NSC008 cells, no DNA template. (<b>G</b>) Protein expression profiles from cell lysates (G1 and G3) and CM (G2 and G4) from LM-NSC008 cells at passages 5 and 45. Protein array analysis was conducted using Cytokine Antibody Array V from RayBiotech.</p

Computational analysis of LM-NSC008 cells and tissue orientation.

<p><b>(A)</b> DiI myelin-stained cross-section defining regions of WM. <b>(B)</b> The corresponding relative tissue anisotropy (coherence) image generated by the OrientationJ plugin to FIJI with 1 pixel Gaussian kernel. A higher pixel intensity indicates higher anisotropy and vice-versa. <b>(C)</b> Orientation vectors of WM overlaid on the coherence image. The angle θ_WM is the orientation of the WM. The red dots and the yellow lines indicate the ellipses and the dominant eigenvector calculated via structure tensor analysis (see Supplementary Materials). <b>(D)</b> Stochastic simulations of 500 NSC migration paths overlaid on the coherence map evaluated using 5 pixel Gaussian kernel. Preferential migration along the corpus callosum is evident. Inset shows the paths near the seed initialization region (analogous to injection site in a biological experiment) contrasting the directional motility in the WM and GM. <b>(E)</b> Nestin-stained cross-section adjacent to the section shown in (A) of naïve mouse brain 9 months after injection of NSCs (brown). <b>(F)</b> NSC clusters were identified using color and intensity thresholds. These clusters are dilated and eroded to create a map of NSC density. <b>(G)</b> NSC density overlaid on the DiI-defined WM boundaries, excluding the injection site. The green curve through the corpus callosum shows the curve fit through one NSC coalesced region. The angle θ_NSC is the orientation angle of an NSC cluster. <b>(H)</b> Scatter plot showing the orientation of NSC clusters (θ_NSC) against the orientation of WM (θ_WM) weighted by cluster size. The slope of the line indicates the slope of the fit through the scatter plot. Note that the NSCs clusters analyzed above include clusters in both WM and GM. Separate scatter plots for NSCs in WM and GM are shown in the supplemental data.</p

L-MYC Expression Maintains Self-Renewal and Prolongs Multipotency of Primary Human Neural Stem Cells

Pre-clinical studies indicate that neural stem cells (NSCs) can limit or reverse CNS damage through direct cell replacement, promotion of regeneration, or delivery of therapeutic agents. Immortalized NSC lines are in growing demand due to the inherent limitations of adult patient-derived NSCs, including availability, expandability, potential for genetic modifications, and costs. Here, we describe the generation and characterization of a new human fetal NSC line, immortalized by transduction with L-MYC (LM-NSC008) that in vitro displays both self-renewal and multipotent differentiation into neurons, oligodendrocytes, and astrocytes. These LM-NSC008 cells were non-tumorigenic in vivo, and migrated to orthotopic glioma xenografts in immunodeficient mice. When administered intranasally, LM-NSC008 distributed specifically to sites of traumatic brain injury (TBI). These data support the therapeutic development of immortalized LM-NSC008 cells for allogeneic use in TBI and other CNS diseases