Malaria and the Blood-Brain Barrier

 EPCR and ICAM Receptors

Neuromics Astrocytes and Pericytes were used in this important study-Yvonne Adams, Rebecca W. Olsen, Anja Bengtsson, Nanna Dalgaard, Mykola Zdioruk, Sanghamitra Satpathi, Prativa K. Behera, Praveen K. Sahu, Sean E. Lawler, Klaus Qvortrup, Samuel C. Wassmer, and Anja T.R. Jensen. (2021). Plasmodium Falciparum Erythrocyte Membrane Protein 1 Variants Induce Cell Swelling and Disrupt the Blood–Brain Barrier in Cerebral Malaria. Journal of Experimental Medicine, 218 (3). doi: 10.1084/jem.20201266.

This is the first study showing malaria pericytes interacting with human endothelial cells. Please note we have the complete BBB Model that can be used for studies like these.

METHOD OF TREATING CENTRAL NERVOUS SYSTEM DISEASE

 Blood-Brain Barrier (BBB) Permeability Assay

Yes, These are troubled times. With our current focus on eradicating COVID-19, we forget that many of the world's most insidious and costly diseases have limited treatments and no cures.

That is why Neuromics continues to develop its human based 3-D Cell-Based Assays. At the center if our offerings is our BBB Model.

Here's a recent patent application that includes a permeability assay using our model-Sookhee Bang, Jeong Kuen Song, Seung-wook Shin, Kwan Hee Lee, and Ho June Lee. (2020). Method of Treating Central Nervous System Disease. United States Patent Application 20200230218

BBB Permeability Assay of AL04 The in vitro human BBB model (Neuromics, USA) was established using co-cultures of primary Human brain endothelial cells (HBEC), Human brain pericytes (HBPC), and Human brain astrocytes (HBAC). In vitro human BBB model kit has two sides (luminal, blood/abluminal, brain) with 12 transwell inserts (polyester membrane, 0.4 um pore, diameter 12 mm, insert growth area: 1.12 cm2). HBPC were grown on the bottom side of the inserts, HBEC were monolayered on the upper side of the inserts, and HBAC were grown on the bottom of the 12-well culture plate (Neuromics, USA). In vitro BBB model was activated according to the manufacturer's instructions for 4 days. Briefly, the medium from both luminal and abluminal (lower, brain) sides of the transwell insert was changed every other day. Before the transport experiment, the abluminal side was filled with the permeability assay medium. For the permeability assay, purified AL04 or recombinant HSA (Sigma, USA) were added to the luminal side (0.3 ml) of the transwell insert to yield a final concentration of 1 or 10 uM. Incubations were performed on orbital shaker (100 rpm) at 37° C. Samples (150 μl) were collected from the abluminal side (1.2 ml) at 60, 120 and 240 min and immediately replaced with fresh permeability assay medium. The concentrations of transported AL04 or rHSA were measured by Human albumin quantitation ELISA kit (Bethyl laboratory, USA) and analyzed using the standard curve method. Permeability coefficients (Pe, cm s−1) were calculated using the equation: Pe=(VA/(A×c0))×(dQ/dt), where VA is volume of assay buffer in blood side (inside of the insert), A is the surface area of the insert (1.12 cm2), c0 the initial concentration of protein sample added into blood side, dQ/dt the concentration of transported protein sample in brain side in a defined time period. The permeability coefficients (Pe, cm s−1) for the purified AL04 or recombinant HSA were calculated as previously described (Prades, 2015; Nakagawa, 2009).

If you have interest and want to learn more, please email Rose Ludescher, VP, Customer Satisfaction-rose@neuromics.com

Be safe; be healthy.

Human Primary Cells Trifecta

Tri-culture of Neuromics' Human Primary Cells in 3-D BBB Model

I mentioned in an earlier post that we take Researchers questioning the validity of our Human Primary Cells very seriously. We follow up with our clients to make sure our cells are working as expected. If they are not 100% happy, we offer a free replacement or full refund.

...So it is encouraging to see Researchers using our Human Pericytes,  Brain Microvascular Endothelial Cells (HBMES) and Astrocytes to build a static 3-D Blood-Brain Barrier Model.

Ece Bayir, M. Mert Celtikoglu, Aylin Sendemira. The use of bacterial cellulose as a basement membrane improves the plausibility of the static in vitro blood-brain barrier model. https://doi.org/10.1016/j.ijbiomac.2018.12.257.

Image: Pericytes, HBMECS, and Astrocytes 5 days after culturing. Live cells (green) and dead cells (red).

The investigators concluded, "Caffeine and sucrose permeability values obtained from all models were close to literature data and physiological values."

Primary Neurons vs PC12 cells for Compound Testing

This publication compares PC12 Cells vs E18 Primary Cortical Neurons. The cells showed permeability to some key compounds where the Neurons did not. This demonstrates the importance of including primary neurons in compound testing assays for Neuro-disease research: Wei Zhang , Radhia Benmohamed, Anthony C. Arvanites, Richard I. Morimoto, Robert J. Ferrante, Donald R. Kirsch, Richard B. Silverman. Cyclohexane 1,3-diones and their inhibition of mutant SOD1-dependent protein aggregation and toxicity in PC12 cells. Bioorganic & Medicinal Chemistry. Elsevier Ltd. All rights reserved.doi:10.1016/j.bmc.2011.11.039.
Abstract: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. Currently, there is only one FDA-approved treatment for ALS (riluzole), and that drug only extends life, on average, by 2–3 months. Mutations in Cu/Zn superoxide dismutase (SOD1) are found in familial forms of the disease and have played an important role in the study of ALS pathophysiology. On the basis of their activity in a PC12-G93A-YFP high-throughput screening assay, several bioactive compounds have been identified and classified as cyclohexane-1,3-dione (CHD) derivatives. A concise and efficient synthetic route has been developed to provide diverse CHD analogs. The structural modification of the CHD scaffold led to the discovery of a more potent analog (26) with an EC50 of 700 nM having good pharmacokinetic properties, such as high solubility, low human and mouse metabolic potential, and relatively good plasma stability. It was also found to efficiently penetrate the blood–brain barrier. However, compound 26 did not exhibit any significant life span extension in the ALS mouse model. It was found that, although 26 was active in PC12 cells, it had poor activity in other cell types, including primary cortical neurons, indicating that it can penetrate into the brain, but is not active in neuronal cell potentially due to poor selective cell penetration. Further structural modification of the CHD scaffold was aimed at improving global cell activity as well as maintaining potency. Two new analogs (71 and 73)
were synthesized, which had significantly enhanced cortical neuronal cell permeability, as well as similar
potency to that of 26 in the PC12-G93A assay. These CHD analogs are being investigated further as novel
therapeutic candidates for ALS.

see: Bioorg. Med. Chem. 2011, 19, 613. and J. Med. Chem. 2012, in press

Related Links: Primary Neurons and Astrocytes-Primary human, rat and mouse neurons and astrocytes.

Image: E18 hippocampal neurons stained with Tau (red) and Doublecortin (green). The two proteins overlap in the proximal dendrites (yellow) Axons (low doublecortin content) are red. Blue staining is the nuclear DNA.