The authors would like to thank Prof. Hartwig Wolburg for performing the electron microscopy.

1. Prepare Cell Culture Filter Inserts for Seeding HIBCPP Cells in an Inverted Model System
<ol>
	<li>Pre-warm DMEM/ F12 (Ham) supplemented with 5 &#181;g/ml insulin, 100 U/ml penicillin, 100 &#181;g/ml streptomycin and 10% fetal calf serum (FCS).</li>
	<li>Use sterile forceps to place 0.33 cm&#178; growth area cell culture filter inserts with a pore size of 3 &#181;m upside down into a 12-well plate (Figure 1E).</li>
	<li>Fill medium into the lower compartment of the cell culture filter insert (about 3 ml) and 100 &#181;l on top of the filter insert. Flood the plate as well as the lower compartment with medium. Aspirate the excessive medium in such a way that the lower compartment of the filter insert stays filled (Figure 1E). 
	Note: It is recommended to use a serological pipette for this step.</li>
	<li>Cover 12-well plate with the lid and transfer the prepared cell culture filter inserts to the incubator, 37 &#176;C, 5% CO2 until addition of cells.</li>
</ol>
2. Cultivation and Passaging of HIBCPP Cells
<ol>
	<li>Prepare DMEM/ F12 (Ham) supplemented with 5 &#181;g/ml, 100 U/ml penicillin, 100 &#181;g/ml streptomycin and 10% FCS.</li>
	<li>Pre-warm medium and PBS in a 37 &#176;C water bath. Aspirate medium from flask. Add 10 ml PBS to the flask and swirl. Repeat this step once.</li>
	<li>Add 3 ml 0.25% trypsin-EDTA to the flask and swirl. Place into the incubator, 37 &#176;C, 5% CO2.</li>
	<li>After approximately 20 min remove the flask from the incubator. Ensure that the cells are detached from the bottom of the flask and display a round shape when surveyed with the microscope. 
	Note: The cells do not detach completely from each other and are often found in agglomerates. It is recommended to use the cells up to passage 38.</li>
	<li>To stop trypsinization add 17 ml medium. Resuspend the cells by pipetting up and down and transfer the suspension into a 50 ml tube. Centrifuge at 50 x g for 10 min at room temperature.</li>
	<li>Resuspend the cells in an appropriate volume of medium and count the cells by using a hemocytometer. 
	Note: The concentration of resuspended HIBCPP cells should be 1 x 106 cells/ml. For maintenance of HIBCPP cells it is suggested to transfer an amount of 1-6 x 106 cells in 10 ml medium to a T75-flask. Change medium every second day.</li>
</ol>
3. Seeding Inverted Cell Culture Filter Inserts with HIBCPP Cells
<ol>
	<li>On top of each inverted filter insert (i.e. the bottom side of the filter) add 80 &#181;l of cell suspension (i.e. 8 x 104 cells) (Figure 1E). 
	Note: Make sure that the cells are evenly distributed in the suspension by inverting the tube before seeding.</li>
	<li>Cover the seeded-cell culture filter inserts with the lid of the 12-well plate and transfer to the incubator, 37 &#176;C, 5% CO2.</li>
	<li>On the first day, fill 1 ml medium into the wells of a 24-well plate. Lift the cell culture filter inserts by using forceps out of the 12-well plate, discard the medium inside, turn the filter inserts and place them in the standard orientation into the prepared 24-well plate (Figure 1E).</li>
	<li>Place the cells into fresh medium every second day. Prepare 24-wells with fresh medium and transfer the filter inserts to it. Fill inserts with 0.5 ml fresh medium. Check TEER every day as described in section 4.</li>
	<li>When TEER values of HIBCPP seeded-cells on cell culture inserts exceeds 70 &#937; x cm&#178; (approximately 4 days after seeding), then continue cell culture in medium containing 1% FCS and 5 &#181;g/ml insulin. Prepare 24-well plates with 1 ml medium for each well. Transfer filter inserts to the prepared wells and exchange medium in the upper compartment. 
	Note: This serum withdrawal after confluency leads to the formation of a higher membrane potential.</li>
	<li>Aspirate medium from filter compartment, transfer to the prepared well and fill with 500 &#181;l medium. Repeat this step once. Put the cells overnight into the incubator, 37 &#176;C, 5% CO2.</li>
</ol>
4. Measurement of Transepithelial Electrical Resistance (TEER)
<ol>
	<li>Immerse the electrode tips of epithelial tissue voltohmmeter for 15 min in 80% ethanol. Transfer voltohmmeter under the hood and let electrode dry for a moment. Place electrode into respective culture medium used for the cells for another 15 min to equilibrate.</li>
	<li>Perform measurements by positioning the longer arm of the electrode so that it touches the bottom of the lower compartment each time, place the shorter arm into the filter insert compartment. 
	Note: Resistance values of cell culture filter inserts without cells in medium should be used as blank values ((measured value (&#8486;) &#8211; blank value (&#8486;)) x filter surface (i.e. 0.33 cm&#178;)).</li>
	<li>After measuring place the electrode back into 80% ethanol for 15 min. Store in a dry tube.</li>
</ol>
5. Determination of Paracellular Permeability
<ol>
	<li>Dissolve 1 g FITC-Inulin in 200 ml culture medium supplemented with 5 &#181;g/ml insulin 1% FCS. Apply 50 &#181;g/ml of the solution into the upper filter compartment before infection of the cells.</li>
	<li>After infection collect medium samples from the lower well to determine how much Inulin passed from the filter compartment through the cell layer.</li>
	<li>Prepare a FITC-Inulin standard solution and perform 1:2 dilutions, 10 times (100%, 50%, 25%, 12.5%, 6.25%, 3.13%, 1.56%, 0.78%, 0.39%, 0.2% and 0%). Determine fluorescence by measurement of all samples in microplate reader.</li>
</ol>
6. Preparation of Bacteria for Infection of HIBCPP Cells on Cell Culture Filter Inserts
<ol>
	<li>One day prior to the experiment, scrape the frozen N. meningitidis strains off a glycerol-stock and streak out on Chocolate Agar with Vitox. Grow overnight in the incubator, at 37 &#176;C, with 5% CO2.</li>
	<li>Extract 20-30 colonies from the overnight culture and transfer into a tube containing 8 ml Proteose Peptone Medium supplemented with 0.042% NaHCO3, 0.01 M MgCl2 and 1% Polyvitex.</li>
	<li>Shake the bacteria for 1.25 hr at 220 rpm, 37 &#176;C. Pellet the bacteria by centrifugation at 2,684 g for 10 min at room temperature. Discard supernatant and resuspend the bacterial pellet with 8 ml serum-free medium. Vortex to ensure an even suspension.</li>
	<li>To determine culture density, measure a dilution of 1:10 at an optical density (OD) of 600 nm. Adjust the bacterial suspension to an OD600 of 0.1. 
	Note: This suspension contains approx. 1 x 108 CFU/ml.</li>
	<li>To confirm the bacterial cell concentration, dilute the suspension stepwise (1:10) up to a dilution of 10-5 and plate onto Chocolate Agar plates. 
	Note: Similar serial dilutions need to be performed to calculate bacterial growth curves.</li>
</ol>
7. Infection of HIBCPP Cells on Cell Culture Filter Inserts and Determination of Bacterial Invasion by Double Immunofluorescence
<ol>
	<li>After continuing cell culture in medium containing 1% FCS and 5 &#181;g/ml insulin, measure cells every day using an epithelial tissue voltohmmeter. If cells have reached a TEER of around 500 &#937; x cm&#178; , carry out the infection.</li>
	<li>Infect the cells with the prepared bacterial suspension at a multiplicity of infection (MOI) of 10. Store the infected cells in the incubator, at 37 &#176;C, with 5% CO2 for the indicated period of time. 
	Note: The MOI can be calculated by taking into account the number of cells per cell culture filter insert at confluence (1.21 x 106 cells/cm2).</li>
	<li>Stop the infection by washing three times with 500 &#181;l serum-free medium (SFM) containing 1% bovine serum albumin (BSA) applied to the filter compartment, 1 ml to the lower compartment.</li>
	<li>Block with 500 &#181;l SFM containing 1% BSA buffer in the filter compartment and 1 ml in the lower compartment for 20 min at room temperature to prevent adherence of antibodies to unspecific binding sites.</li>
	<li>Incubate with 100 &#181;l primary antibody anti N. meningtidis &#945;-OMP (1:200) in the filter compartment and 500 &#181;l in the lower compartment for 20 min at room temperature. 
	Note: If required, the antibody concentration needs to be titrated.</li>
	<li>Wash the cells by aspirating the medium from the filter compartment, transfer the filter insert to a prepared well with 1 ml SFM containing 1% BSA and fill the emptied filter compartment with 500 &#181;l SFM containing 1% BSA. Repeat this step twice.</li>
	<li>Fix cells with 500 &#181;l 4% formaldehyde in the filter compartment, 1 ml in the lower compartment for 10 min at room temperature.</li>
	<li>Wash the cells by aspirating the medium from the filter compartment, transfer the filter insert to a prepared well with 1 ml PBS and fill the emptied filter compartment with 500 &#181;l PBS. Repeat this step once. 
	Note: Samples can be stored overnight in PBS at 4 &#176;C.</li>
	<li>Cut fixed cell culture filters out of the inserts and wash with 250 &#181;l PBS containing 1% BSA. Incubate cells for 15 min at room temperature with 250 &#181;l fluorescent labeled (excitation wavelength 594 nm) secondary antibody chicken anti rabbit (1:500) to stain extracellular bacteria. 
	Note: If required, the antibody concentration needs to be titrated.</li>
	<li>Permeabilize cells with 250 &#181;l PBS containing 1% BSA and 0.5% Triton X-100 for 1 hr at room temperature. Wash cells three times with 250 &#181;l PBS containing 1% BSA.</li>
	<li>Incubate with 250 &#181;l primary antibody anti N. meningitidis &#945;-OMP (1:200) for 30 min to stain extra- and intracellular bacteria. Wash cells three times with 250 &#181;l PBS containing 1% BSA.</li>
	<li>Apply 250 &#181;l of fluorescent labeled (excitation wavelength 488 nm) secondary antibody (1:500), fluorescent labeled (excitation wavelength 660 nm) Phalloidin (1:250) and 4&#180;,6-diamidino-2-phenylindole dihydrochloride (DAPI) (1:50,000) for 1 hr at room temperature to stain extra- and intracellular bacteria, actin cytoskeleton and nuclei. 
	Note: If required, the antibody concentration needs to be titrated.</li>
	<li>Wash cells three times with 250 &#181;l PBS containing 1% BSA. Embed the cells in mounting medium and store at 4 &#176;C until examination via microscope.</li>
	<li>Determine number of invaded bacteria per predefined field. Do this by counting 20 fields per filter membrane. Calculate the percentage of invaded bacteria. 
	Note: Multiply the mean bacterial count of the 20 microscopic fields with an area coefficient. The result expresses the amount of total bacteria present in a 0.33 cm&#178; cell culture filter insert. Divide this value by the amount of bacteria grown in media during the duration of infection.</li>
</ol>

<ol>
	<li>Abott, N. J., Patabendige, A. A. K., Dolman, D. E. M., Yusof, S. R., Begley, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+the+blood-brain+barrier.">Structure and function of the blood-brain barrier.</a> Neurobiol Dis. 37, 13-25 (2009).</li><li>Wolburg, H., Paulus, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choroid+plexus:+biology+and+pathology.">Choroid plexus: biology and pathology.</a> Acta Neuropathol. 119, 75-88 (2010).</li><li>Engelhardt, B., Sorokin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+and+the+blood-cerebrospinal+fluid+barriers:+function+and+dysfunction.">The blood-brain and the blood-cerebrospinal fluid barriers: function and dysfunction.</a> Semin Imunopathol. 31, 497-511 (2009).</li><li>Ishiwata, I., Ishiwata, C., Ishiwata, E., Sato, Y., Kiguchi, K., Tachibana, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+a+human+malignant+choroid+plexus+papilloma+cell+line+(HIBCPP).">Establishment and characterization of a human malignant choroid plexus papilloma cell line (HIBCPP).</a> Hum Cell. 18, 67-72 (2005).</li><li>Schwerk, C., Papandreou, T., Schuhmann, D., Nickol, L., Borkowski, J., Steinmann, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polar+invasion+and+translocation+of+Neisseria+meningitidis+and+Streptococcus+suis+in+anovel+human+model+of+the+blood-cerebrospinal+fluid+barrier.">Polar invasion and translocation of Neisseria meningitidis and Streptococcus suis in anovel human model of the blood-cerebrospinal fluid barrier.</a> PloS One. 7, e30069 (2012).</li><li>Bernd, A., Ott, M., Ishikawa, H., Schroten, H., Schwerk, C., Fricker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+efflux+transport+proteins+of+the+human+choroid+plecus+papilloma+cell+line+HIBCPP,+a+functional+in+vitro+model+of+the+blood-cerebrospinal+fluid+barrier.">Characterization of efflux transport proteins of the human choroid plecus papilloma cell line HIBCPP, a functional in vitro model of the blood-cerebrospinal fluid barrier.</a> Pharm Res. , (2014).</li><li>Gr&#252;ndler, T., Quednau, N., Stump, C., Orian-Rousseau, V., Ishikawa, H., Wolburg, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+surface+proteins+InlA+and+InlB+are+interdependently+required+for+polar+basolateral+invasion+by+Listeria+monocytogenes+in+a+human+model+of+the+blood-cerebrospinal+fluid+barrier.">The surface proteins InlA and InlB are interdependently required for polar basolateral invasion by Listeria monocytogenes in a human model of the blood-cerebrospinal fluid barrier.</a> Microbes Infect. 15, 291-301 (2013).</li><li>Schwerk, C., Tenenbaum, T., Kwang, S. K., Schroten, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+choroid+plexus+-+a+multi-role+player+during+infectious+diseases+of+the+CNS.">The choroid plexus - a multi-role player during infectious diseases of the CNS.</a> Front Cell Neurosci. 9, 80 (2015).</li><li>Pron, B., Taha, M. K., Rambaud, C., Fournet, J. C., Pattey, N., Monnet, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+Neisseria+meningtidis+with+the+components+of+the+blood-brain+barrier+correlates+with+increased+expression+of+PilC.">Interaction of Neisseria meningtidis with the components of the blood-brain barrier correlates with increased expression of PilC.</a> J Infect Dis. 176, 1285-1292 (1997).</li><li>Guarner, J., Greer, P. W., Whitney, A., Shieh, W. J., Fischer, M., White, E. H., Carlone, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+and+diagnosis+of+human+meningococcal+disease+using+immunohistochemical+and+PCR+assays+assays.">Pathogenesis and diagnosis of human meningococcal disease using immunohistochemical and PCR assays assays.</a> Am J Clin Pathol. 122, 754-764 (2004).</li><li>Pizarro-Cerda, J., Kuhbacher, A., Cossart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+of+Listeria+monocytogenes+in+mammalian+epithelial+cells:+an+updated+view.">Entry of Listeria monocytogenes in mammalian epithelial cells: an updated view.</a> Cold Spring Harb Perspect Med. 2, (2012).</li><li>Beutler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbe+sensing,+positive+feedback+loops+and+the+pathogenesis+of+inflammatory+diseases.">Microbe sensing, positive feedback loops and the pathogenesis of inflammatory diseases.</a> Immunol. Rev. 227, 248-263 (2009).</li><li>Wilson, E. H., Weninger, W., Hunter, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trafficking+of+immune+cells+in+the+central+nervous+system.">Trafficking of immune cells in the central nervous system.</a> J Clin Invest. 120, 1368-1379 (2010).</li><li>Meeker, R. B., Williams, K., Killebrew, D. A., Hudson, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+trafficking+through+the+choroid+plexus.">Cell trafficking through the choroid plexus.</a> Cell Adh Migr. 6, 390-396 (2012).</li><li>Borkowski, J., Li, L., Steinmann, U., Quednau, N., Stump-Guthier, C., Weiss, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neisseria+meningitidis+elicits+a+pro-inflammatory+response+involving+I+kappa+B+zeta+in+a+human+blood-cerebrospinal+fluid+barrier+model.">Neisseria meningitidis elicits a pro-inflammatory response involving I kappa B zeta in a human blood-cerebrospinal fluid barrier model.</a> J Neuroinflammation. 11, 163 (2014).</li><li>Steinmann, U., Borkowski, J., Wolburg, H., Schroppel, B., Findeisen, P., Weiss, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmigration+of+polymorphnuclear+neutrophils+and+monocytes+through+the+human+blood-cerebrospinal+fluid+barrier+after+bacterial+infection+in+vitro.">Transmigration of polymorphnuclear neutrophils and monocytes through the human blood-cerebrospinal fluid barrier after bacterial infection in vitro.</a> J Neuroinflammation. 10, 30 (2013).</li><li>McGuiness, B. T., Clarke, I. N., Lambden, P. R., Barlow, A. K., Poolman, J. T., Heckels, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Point+mutation+in+meningococcal+por+A+gene+associated+with+increased+endemic+disease.">Point mutation in meningococcal por A gene associated with increased endemic disease.</a> Lancet. 337, 514-517 (1991).</li><li>Ram, S., Cox, A. D., Wright, J. C., Vogel, U., Getzlaff, S., Boden, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neisserial+lipopolysaccharide+is+a+target+for+complement+component+C4b.+inner+core+phosphoethanolamine+residues+define+C4b+linkage+specificity.">Neisserial lipopolysaccharide is a target for complement component C4b. inner core phosphoethanolamine residues define C4b linkage specificity.</a> J Biol Chem. 278, 50853-50862 (2003).</li><li>Claus, H., Maiden, M. C., Maag, R., Frosch, M., Vogel, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Many+carried+meningococci+lack+the+genes+required+for+capsule+synthesis+and+transport.">Many carried meningococci lack the genes required for capsule synthesis and transport.</a> Microbiology. 148, 1813-1819 (2002).</li><li>Claus, H., Maiden, M. C., Wilson, D. J., Mccarthy, N. D., Jolley, K. A., Urwin, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+meningococci+carried+by+children+and+young+adults.">Genetic analysis of meningococci carried by children and young adults.</a> J Infect Dis. 191, 1263-1271 (2005).</li><li>Tenenbaum, T., Papandreou, T., Gellrich, D., Friedrichs, U., Seibt, A., Adam, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polar+bacterial+invasion+and+translocation+of+Streptococcus+suis+across+the+blood-cerebrospinal+fluid+barrier+in+vitro.">Polar bacterial invasion and translocation of Streptococcus suis across the blood-cerebrospinal fluid barrier in vitro.</a> Cell Microbiol. 11, 323-336 (2009).</li><li>Laflamme, N., Echchannaoui, H., Landmann, R., Rivest, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperation+between+toll-like+receptor+2+and+4+in+the+brain+of+mice+challenged+with+cell+wall+components+derived+from+gram-negative+and+gram-positive+bacteria.">Cooperation between toll-like receptor 2 and 4 in the brain of mice challenged with cell wall components derived from gram-negative and gram-positive bacteria.</a> Eur J Immunol. 33, 1127-1138 (2003).</li><li>Laflamme, S., Rivest, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+4:+the+missing+link+of+the+cerebral+innate+immune+response+triggered+by+circulating+gram-negative+bacterial+cell+wall+components.">Toll-like receptor 4: the missing link of the cerebral innate immune response triggered by circulating gram-negative bacterial cell wall components.</a> FASEB J. 15, 155-163 (2001).</li><li>Zughaier, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neisseria+meningitidis+capsular+polysaccharides+indice+inflammatory+responses+via+TLR2+and+TLR4-MD-2.">Neisseria meningitidis capsular polysaccharides indice inflammatory responses via TLR2 and TLR4-MD-2.</a> J Leukoc Biol. 89, 469-480 (2011).</li><li>Yamamoto, M., Yamazaki, S., Uematsu, S., Sato, S., Hemmi, M., Hoshino, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Toll/IL-1-receptor+-mediated+gene+expression+by+the+inducible+nuclear+protein+IkappaBzeta.">Regulation of Toll/IL-1-receptor -mediated gene expression by the inducible nuclear protein IkappaBzeta.</a> Nature. 430, 218-222 (2004).</li><li>Lorenz, J., Zahlten, J., Pollok, I., Lippmann, J., Scharf, S., N'Guessan, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Legionella+pheumophila-induced+IkappaBzeta-dependent+expression+of+interleukin-6+in+lung+epithelium.">Legionella pheumophila-induced IkappaBzeta-dependent expression of interleukin-6 in lung epithelium.</a> Eur Respir J. 37, 648-657 (2011).</li><li>Jaerve, A., Muller, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+in+CNS+injury+and+repair.">Chemokines in CNS injury and repair.</a> Cell Tissue Res. 349, 229-248 (2012).</li><li>Schneider, H., Weber, C. E., Schoeller, J., Steinmann, U., Borkowski, J., Ishikawa, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+of+T-cells+after+infection+of+human+choroid+plexus+papilloma+cells+with+Echovirus+30+in+an+in+vitro+model+of+the+blood-cerebrospinal+fluid+barrier.">Chemotaxis of T-cells after infection of human choroid plexus papilloma cells with Echovirus 30 in an in vitro model of the blood-cerebrospinal fluid barrier.</a> Virus Res. 170, 66-74 (2012).</li><li>Chodobski, A., Szmydynger-Chodobska, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choroid+plexus:+Target+for+polypeptides+and+site+of+their+synthesis.">Choroid plexus: Target for polypeptides and site of their synthesis.</a> Microsc. Res. Tech. 52, 65-82 (2001).</li><li>Dickson, P. W., Schreiber, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+levels+of+messenger+RNA+for+transthyretin+(prealbumin)+in+human+choroid+plexus.">High levels of messenger RNA for transthyretin (prealbumin) in human choroid plexus.</a> Neurosci. Lett. 66, 311-315 (1986).</li><li>Stylianopoulou, F., Herbert, J., Soares, M. B., Efstratiadis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+insulin-like+growth+factor+II+gene+in+the+choroid+plexus+and+the+leptomeninges+of+the+adult+rat+central+nervous+system.">Expression of the insulin-like growth factor II gene in the choroid plexus and the leptomeninges of the adult rat central nervous system.</a> Proc Natl Acad Sci USA. 85, 141-145 (1988).</li><li>Lim, L., Zhou, H., Costa, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+winged+helix+transcription+factor+HFH-4+is+expressed+during+choroid+plexus+epithelial+development+in+the+mouse+embryo.">The winged helix transcription factor HFH-4 is expressed during choroid plexus epithelial development in the mouse embryo.</a> Proc. Natl Acad. Sci. USA. 94, 3094-3099 (1997).</li><li>Vandenhaute, E., Stump-Guthier, C., Lasierra Losada, M., Tenenbaum, T., Rudolph, H., Ishikawa, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+choroid+plexus+may+be+an+underestimated+site+of+tumor+invasion+to+the+brain:+an+in+vitro+study+using+neuroblastoma+cell+lines.">The choroid plexus may be an underestimated site of tumor invasion to the brain: an in vitro study using neuroblastoma cell lines.</a> Cancer Cell Int. , 15-102 (2015).</li></ol>

The authors have nothing to disclose.

The epithelial cells of the CP form the BCSFB that separates the CSF from the blood2,3. We recently established the HIBCPP cell line as a functional human model of the BCSFB. The cells display important barrier functions of the BCSFB in vitro, including the development of a high membrane potential, a low permeability for macromolecules, as well as the presence of continuous strands of TJs5. The TJ proteins contribute to an apical/basolateral polarity of the cells. The polarity is of high im...

The blood-cerebrospinal fluid barrier (BCSFB) is one of the three barrier sites between the blood and the brain1. Its morphological correlate are the epithelial cells of the choroid plexus (CP)2,3, an endothelial-epithelial convolute, which is strongly vascularized and located in the ventricles of the brain. The CP serves to produce the cerebrospinal fluid (CSF) as well as to separate the latter from the blood. In order to achieve barrier function, the CP epithelial cells show a low pinocytotic activity, express specific transporters, and are densely connected by a continuous network of tight junctions (TJs)2,3.
Human choroid plexus papilloma (HIBCPP) cells, derived from a malignant choroid plexus papilloma of a Japanese woman4, were used to construct a functional in vitro model of the BCSFB. HIBCPP cells show a couple of characteristics of a functional BCSFB as the formation of TJ strands, the development of a high transepithelial membrane potential that can be determined as transepithelial electrical resistance (TEER), and minor permeabilities for macromolecules. Moreover, HIBCPP cells express characteristic transporters, which may serve to regulate the ionic microenvironment, and show apical/basolateral polarity5,6,7.
The BCSFB has been shown to function as an entry site for pathogens (bacteria, viruses, and fungi) into the central nervous system (CNS)8. The invasion of pathogens, including Neisseria meningitidis (N. meningitidis), a Gram-negative bacterium, can cause severe diseases like meningitis. Evidence that it overcomes the protective epithelial barrier of the CP is supported by histopathological observations in patients with meningococcal disease exhibiting increased amounts of meningococci in the vessels and CP epithelial cells9,10. To gain entry into host cells bacteria often hijack endocytotic mechanisms, which are mediated or triggered by specific surface receptors located on the host cells. Since interactions of pathogens with these receptors can be species specific11, animal models can only be consulted to a restricted extent. The HIBCPP cell line provides the opportunity to study the invasion process as well as the underlying molecular mechanisms in a human model system. Employing cell culture inserts enables us to analyze interactions of pathogens with host cells from two distinct cell sides. Many bacteria, including N. meningitidis, are strongly subject to the impact of gravity during infection assays. For optimal interaction of pathogens with the HIBCPP cells during the assays, the bacteria are initially added into the upper compartment of the cell culture filter insert system. To enable infection from the apical or the basolateral cell side, respectively, two variations of the in vitro system have been established: In the standard system HIBCPP cells are seeded into the upper compartment of the filter insert, mimicking the situation when microorganisms are located on the CSF-side and get into contact with the apical side of the cells (Figure 1A, C). In contrast, using the HIBCPP cells in an inverted cell culture filter insert system reflects the conditions when bacteria have entered the blood stream. Microorganisms disseminate in the blood and encounter CP epithelial cells from the basolateral side (Figure 1B, D). Noteworthy, in this model system it has been shown that bacteria invade HIBCPP cells in a polar fashion specifically from the basolateral cell side5,7.
Subsequently to infection of the CP, the invaded pathogens can be recognized by the innate immune system through ligation to pattern-recognition receptors (PRRs). Well-described members of the PRRs belong to the Toll-like receptor (TLR) family. TLRs can bind to characteristic structures of infectious microorganisms, which are termed pathogen-associated molecular patterns (PAMPs). Ligation of the receptors leads to activation of host cell signaling cascades that trigger expression of cytokines and chemokines12, which in turn stimulate transmigration of immune cells across the BCSFB13,14. It has been shown that HIBCPP cells express several TLRs at mRNA level and that infection with N. meningitidis results in secretion of several cytokines and chemokines, including CXCL1-3, IL6, IL8 and TNF&#945;15,16.
Here, we describe cultivation and infection of the human cell line HIBCPP in an inverted cell culture insert system that mimics the BCSFB. This model system enables to study interactions of pathogens with the in vivo relevant basolateral cell side as well as the subsequent cellular response.

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			<td height="21" style="height:21px;width:216px;">0.25% Trypsin-EDTA</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#180;,6 diamidino-2-phenylindole (DAPI)</td>
			<td>Life Technologies</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12-well plates</td>
			<td>Starlab</td>
			<td>CC7682-7512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well plates</td>
			<td>Starlab</td>
			<td>CC7682-7524</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti Neisseria meningitidis &#945;-OMP</td>
			<td></td>
			<td></td>
			<td>This antibody was a gift from Drs. H. Claus and U. Vogel (University of W&#252;rzburg, Germany)</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 (chicken anti rabbit)</td>
			<td>Invitrogen</td>
			<td>A21441</td>
			<td></td>
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			<td height="21" style="height:21px;">Alexa Fluor 594 (chicken anti rabbit)</td>
			<td>Invitrogen</td>
			<td>A21442</td>
			<td></td>
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			<td height="21" style="height:21px;">Alexa Fluor 660 Phalloidin</td>
			<td>Invitrogen</td>
			<td>A22285</td>
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			<td height="21" style="height:21px;">Bovine serum albumine (BSA)</td>
			<td>Calbiochem</td>
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			<td height="21" style="height:21px;">Chocolate agar plates</td>
			<td>Biomerieux</td>
			<td>43109</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Cytochalasin D</td>
			<td>Sigma</td>
			<td>C8273</td>
			<td></td>
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			<td height="21" style="height:21px;">DMEM/F12 + L-Glut + 15 mM HEPES</td>
			<td>Gibco</td>
			<td>31330-095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12 + L-Glut + 15 mM HEPES w/o Phenolred</td>
			<td>Gibco</td>
			<td>11039-047</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal calf serum (FCS)</td>
			<td>Life Technologies</td>
			<td>10270106</td>
			<td></td>
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			<td height="21" style="height:21px;">FITC-Inulin</td>
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			<td>F3272</td>
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			<td height="21" style="height:21px;">Insulin</td>
			<td>Sigma</td>
			<td>19278</td>
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			<td height="21" style="height:21px;">MgCl2</td>
			<td>Sigma</td>
			<td>2393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaHCO3</td>
			<td>Sigma</td>
			<td>55761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS + Mg +Ca</td>
			<td>Gibco</td>
			<td>14040-174</td>
			<td></td>
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			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>MP Biomedicals</td>
			<td>1670049</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Polyvitex</td>
			<td>Biomerieux</td>
			<td>55651</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Proteose peptone</td>
			<td>BD</td>
			<td>211684</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Serum-free medium</td>
			<td>Gibco</td>
			<td>10902-096</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Thincert cell culture inserts for 24-well plates, pore size 3 &#181;m</td>
			<td>Greiner</td>
			<td>662630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue culture flask 75 cm&#178; red cap sterile</td>
			<td>Greiner</td>
			<td>658175</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Volt-Ohm Meter Millicell-ERS2 with MERSSTX01 electrode</td>
			<td>Millipore</td>
			<td>MERSSTX00</td>
			<td></td>
		</tr>
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a choroid plexus epithelial cell-based model of the human blood-cerebrospinal fluid barrier to study bacterial infection from the basolateral side

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB functions in separating the cerebrospinal fluid (CSF) from the blood and restricting the molecular exchange to a minimum extent. An in vitro model of the BCSFB is based on cells derived from a human choroid plexus papilloma (HIBCPP). HIBCPP cells display typical barrier functions including formation of tight junctions (TJs), development of a transepithelial electrical resistance (TEER), as well as minor permeabilities for macromolecules. There are several pathogens that can enter the central nervous system (CNS) via the BCSFB and subsequently cause severe disease like meningitis. One of these pathogens is Neisseria meningitidis (N. meningitidis), a human-specific bacterium. Employing the HIBCPP cells in an inverted cell culture filter insert system enables to study interactions of pathogens with cells of the BCSFB from the basolateral cell side, which is relevant in vivo. In this article, we describe seeding and culturing of HIBCPP cells on cell culture inserts. Further, infection of the cells with N. meningitidis along with analysis of invaded and adhered bacteria via double immunofluorescence is demonstrated. As the cells of the CP are also involved in other diseases, including neurodegenerative disorders like Alzheimer`s disease and Multiple Sclerosis, as well as during the brain metastasis of tumor cells, the model system can also be applied in other fields of research. It provides the potential to decipher molecular mechanisms and to identify novel therapeutic targets.

Here we describe culturing and infection of HIBCPP cells in an inverted cell culture insert system. This model allows us to study invasion mechanisms and the underlying molecular signaling pathways from the basolateral cell side, reproducing a physiological situation of bacteria disseminating and entering epithelial cells via the blood stream (Figure 1).
The HIBCPP cells display certain barrier functions, which ...

Watch this Scientific Journal Video about A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side at JoVE.com

A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side

The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The ...

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14.1K Views.  Heidelberg University. The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

Video: A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel stenting are crucial for translational approaches1,2.
The commonly used animal models include mice, rats, rabbits, and pigs3-5. However, the translation of these models into clinical settings remains difficult, since those biological processes are already studied in animal vessels but never performed before in human research models6,7. In this video we demonstrate a new humanized model to overcome this translational gap. The shown procedure is reproducible, easy, and fast to perform and is suitable to study the development of intimal hyperplasia and the applicability of diverse stents.
This video shows how to perform the stent technique in human vessels followed by transplantation into immunodeficient rats, and identifies the origin of proliferating cells as human.

Human Internal Mammary Artery IMA Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

This video shows a model to study the development of intimal hyperplasia after stent deployment using a human vessel (IMA) in an immunodeficient rat model.

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic lesions. Mouse models are an important tool to investigate inflammatory processes in atherogenesis, but these models suffer from the phenotypic and functional differences between the murine and human immune system. In vitro cell experiments are used to specifically evaluate cell type-dependent changes caused by a substance of interest, but culture-dependent variations and the inability to analyze the influence of specific molecules in the context of the inflammatory compound in atherosclerotic lesions limit the impact of the results. In addition, measuring levels of a molecule of interest in human blood helps to further investigate its clinical relevance, but this represents systemic and not local inflammation. Therefore, we here describe a plaque culture model to study human atherosclerotic lesion biology ex vivo. In short, fresh plaques are obtained from patients undergoing endarterectomy or coronary artery bypass grafting and stored in RPMI medium on ice until usage. The specimens are cut into small pieces followed by random distribution into a 48-well plate, containing RPMI medium in addition to a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. After incubation, the plaque pieces can be shock frozen for mRNA isolation, embedded in Paraffin or OCT for immunohistochemistry staining or smashed and lysed for western blotting. Furthermore, cells may be isolated from the plaque for flow cytometry analysis. In addition, supernatants can be collected for protein measurement by ELISA. In conclusion, the presented ex vivo model opens the possibility to further study inflammatory lesional biology, which may result in identification of novel disease mechanisms and therapeutic targets.

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory process. This manuscript illustrates an easy to use ex vivo model to investigate fresh carotid or coronary artery plaques. The ex vivo model allows for the investigation of potential substances on the inflammatory milieu in human atherosclerotic lesions and results can be analyzed by various methods.

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...