Expression in Saccharomyces boulardii of recombinant Toxin-coregulated pilus A subunit (TcpA) of Vibrio cholerae O1

Cholera is endemic in over 50 countries with an estimated mortality of 100,000-120,000. Vaccination is considered the complementary key to prevent and control cholera; therefore, alternative vaccine preparations are needed. Toxin Co-regulated Pilus is part of the toxR virulence regulon, which is necessary for colonization in the intestinal mucosa. In order to express Vibrio cholerae TcpA protein in Saccharomyces boulardii, the expression plasmid pYES2 was constructed by inserting tcpA gene isolated from local Vibrio cholerae Eltor Inaba isolates. The new construct was transferred into Saccharomyces boulardii cells and the expression of tcpA gene was induced from the GAL1 promoter by adding galactose to the medium. The SDS-PAGE and Western blot analysis showed the presence of TcpA in yeast. These results showed that Saccharomyces boulardii is a promising host to express Vibrio cholerae toxin TcpA as the first step in attempt to produce an oral Vibrio cholerae vaccine(AU)

El cólera es endémico en más de 50 países. Se estima una mortalidad entre 100.000 - 120.000 debido a esta enfermedad. La vacunación se considera una medida complementaria para prevenir y controlar el cólera, por lo tanto, se necesitan preparaciones vacunales alternativas a las existentes. El Pili corregulado con la toxina, es parte del regulón de virulencia toxR, y es necesario para la colonización en la mucosa intestinal. Para expresar la proteína tcpA de Vibrio cholerae en Saccharomyces boulardii, se construyó el plásmido de expresión pYES2 insertando el gen tcpA obtenido a partir de aislamientos locales de Vibrio cholerae El Tor Inaba. La nueva construcción se transfirió a las células de Saccharomyces boulardii y se indujo la expresión del gen tcpA a partir del promotor GAL1 mediante la adición de galactosa al medio. El análisis mediante SDS-PAGE y Western blot demostró la presencia de TcpA en levaduras. Los resultados demostraron que Saccharomyces boulardii es un hospedero prometedor para expresar el gen tcpA de Vibrio cholerae como el primer paso en el intento de producir una vacuna oral contra Vibrio cholerae(AU)

Induction of mice adult bone marrow mesenchymal stem cells into functional motor neuron-like cells.

The differentiation of mesenchymal stem cells (MSC) into acetylcholine secreted motor neuron-like cells, followed by elongation of the cell axon, is a promising treatment for spinal cord injury and motor neuron cell dysfunction in mammals. Differentiation is induced through a pre-induction step using Beta- mercaptoethanol (BME) followed by four days of induction with retinoic acid and sonic hedgehog. This process results in a very efficient differentiation of BM-MSCs into motor neuron-like cells. Immunocytochemistry showed that these treated cells had specific motor neural markers: microtubule associated protein-2 and acetylcholine transferase. The ability of these cells to function as motor neuron cells was assessed by measuring acetylcholine levels in a culture media during differentiation. High-performance liquid chromatography (HPLC) showed that the differentiated cells were functional. Motor neuron axon elongation was then induced by adding different concentrations of a nerve growth factor (NGF) to the differentiation media. Using a collagen matrix to mimic the natural condition of neural cells in a three-dimensional model showed that the MSCs were successfully differentiated into motor neuron-like cells. This process can efficiently differentiate MSCs into functional motor neurons that can be used for autologous nervous system therapy and especially for treating spinal cord injuries.

Live attenuated measles virus vaccine therapy for locally established malignant glioblastoma tumor cells.

Glioblastoma multiforme is the most aggressive malignant primary brain tumor in humans, with poor prognosis. A new glioblastoma cell line (ANGM5) was established from a cerebral glioblastoma multiforme in a 72-year-old Iraqi man who underwent surgery for an intracranial tumor. This study was carried out to evaluate the antitumor effect of live attenuated measles virus (MV) Schwarz vaccine strain on glioblastoma multiforme tumor cell lines in vitro. Live attenuated MV Schwarz strain was propagated on Vero, human rhabdomyosarcoma, and human glioblastoma-multiform (ANGM5) cell lines. The infected confluent monolayer appeared to be covered with syncytia with granulation and vacuolation, as well as cell rounding, shrinkage, and large empty space with cell debris as a result of cell lysis and death. Cell lines infected with virus have the ability for hemadsorption to human red blood cells after 72 hours of infection, whereas no hemadsorption of uninfected cells is seen. Detection of MV hemagglutinin protein by monoclonal antibodies in infected cells of all cell lines by immunocytochemistry assay gave positive results (brown color) in the cytoplasm of infected cells. Cell viability was measured after 72 hours of infection by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results showed a significant cytotoxic effect for MV (P≤0.05) on growth of ANGM5 and rhabdomyosarcoma cell lines after 72 hours of infection. Induction of apoptosis by MV was assessed by measuring mitochondrial membrane potentials in tumor cells after 48, 72, and 120 hours of infection. Apoptotic cells were counted, and the mean percentage of dead cells was significantly higher after 48, 72, and 120 hours of infection compared with control cells. This study concludes that live attenuated MV Schwarz vaccine induces the oncolytic effect in Iraqi tumor cell line ANGM5 and in the rhabdomyosarcoma cell line through syncytia in tumor cells, which is one of the causes of cell death. The MV vaccine strain has the ability to insert its hemagglutinin protein into the tumor cell surface, leading to modification of the antigenic surface of tumor cells that may induce an antitumor immune response, MV vaccine strain induced cell killing by direct cytolysis and apoptosis induction. These antitumor features may indicate the use of MV in the treatment of glioblastoma.