Dennis J Grab

Associate Professor

Academic Degrees

PhD, Cornell University 1977; AB, Hunter College 1969

School/Organization
School of Medicine

Department / Division
Pathology
Medical Microbiolgy

Joint Departmental Affiliations
Secondary in Pediatrics

Departmental Address
720 Rutland Avenue
Ross Building, Room 624

Email: dgrab1@jhmi.edu
Phone: 410-955-8654

Research and Professional Experience

• Human African trypanosomiasis (HAT), or sleeping sickness, occurs when single-cell trypanosome protozoan parasites spread from the blood to brain over the blood-brain barrier (BBB). This barrier is comprised of brain microvascular endothelial cells (BMEC) especially designed to keep pathogens out. Safe drugs for treating sleeping sickness are lacking and alternative treatments are urgently required. Using our human BMEC BBB model, we previously found that a parasite protease, brucipain, induced calcium activation signals that allowed this barrier to open-up to parasite crossing (J. Clin. Invest. 116:2739-2747, 2006). Because human BMEC express Protease-Activated Receptors (PARs) that trigger calcium signals in BMEC, we hypothesized, and later showed, a functional link between parasite brucipain and BMEC PARs (PLoS Negl. Trop. Dis. 3:e479, 2009). Utilizing RNA interference to block the production of one type of PAR called PAR-2, we hindered the ability of trypanosomes to both open-up and cross human BMEC. Using gene-profiling methods to interrogate candidate BMEC pathways specifically triggered by brucipain, several pathways that potentially link brain inflammatory processes were identified, a finding congruent with the known role of PAR-2 as a mediator of inflammation. Overall, our data support a role for parasite proteases and BMEC PAR in trypanosome BBB transmigration, and as potential triggers for brain inflammation associated with the disease. Furthermore, an understanding of these responses at a molecular level will help identify candidates for the early diagnosis, treatment, and prevention of CNS invasion with HAT.
• I am also interested in developing better HAT diagnostics based on two new recently introduce technologies. The first, Loop-Mediated Isothermal Amplification (or LAMP) is a recently introduced alternative to PCR for the detection of HAT that is based on Bst DNA polymerase and, compared to normal PCR, has advantages of reaction simplicity and detection sensitivity (J. Clin. Microbiol. 41:5517-24). Because LAMP requires 4 primers that identify 6 specific sites on the target DNA molecule, LAMP is also highly specific. Furthermore, since LAMP does not require complicated thermal cycling steps; a simple water bath (60-65oC) is sufficient to amplify the target DNA to detectable levels. A useful by-product of the reaction is the copious white precipitate of magnesium pyrophosphate, which allows easy and rapid visual confirmation that the target DNA was amplified by LAMP. It is important also to note that Bst DNA polymerase used in LAMP is not affected by blood-derived tissue components that can inactivate Taq DNA polymerase used in PCR. In the second procedure, using controlled site-specific chemistry, Ian Burbulis and colleagues (Nat. Methods 2:31, 2005) have been able to covalently tag a ‘DNA barcode’ to a single site on a protein, they call a "tadpole". Because the DNA barcode contains a region for PCR amplification, this affords a range of ultra-sensitive analyses should have an important potential application for pathogen detection, disease diagnosis and environmental monitoring. Alone, the above two procedures are clearly very powerful. However, we are currently looking into the possibility of usng the Bst DNA polymerase procedure of ‘LAMP’ of replace the PCR of ‘tadpole’, to create even more powerful technique methods for not only HAT, but other tropical diseases (Nat Meth 2:635, 2005).

Honors and Awards

• Monbusho Foreign Associate Professor, Faculty of Agriculture, Saga University, Japan: From the Ministry of Education, Science, Sports and Culture. Government of Japan. [January 1998 to March 2000].

Research Projects
Blood-Brain Barrier Traversal By African Trypanosomes
The neurological complications of Human African trypanosomiasis, or sleeping sickness, caused by Trypanosoma brucei gambiense and T. b. rhodesiense are attributed to the penetration of the central nervous system by trypanosomes. Yet, how the single-cell trypanosome protozoan parasites spread from...

LAMP and Tadpoles for the field diagnosis of HAT
Trypanosoma brucei gambiense and T. b. rhodesiense are pathogens responsible for human African trypanosomiasis (HAT; or sleeping sickness). Death from HAT is inevitable if untreated. Recently, HAT has reached epidemic proportions with more that 40,000 people dying every year in Africa. While...



Selected Publications

• 1. Grab, D.J., Garcia-Garcia, J.C., Nikolskaia, O.V., Kim, Y.V., Brown, A., Pardo, C.A., Zhang, Y., Becker, K.G., Wilson, B.A., Scharfstein, J. Lima, A.P.C.A., Dumler, J.S. Protease activated receptor signaling is required for African trypanosome traversal of human brain microvascular endothelial cells. PLoS Negl. Trop. Dis. 3:e479, 2009.
• 2. Yamage, M., Yoshiyama, Grab, D.J., M., Kubo, M., Iwasaki, T., Kitani, H., Ishibashi, J., Yamakawa, M. Characteristics of novel insect defensin-based membrane-disrupting trypanocidal peptides. Biosci. Biotechnol. Biochem. 73:1520-1527, 2009.
• 3. Grab, D.J., Nyarko, E., Nikolskaia, O.V., Kim, Y.V., Dumler, J.S. Blood-brain traversal by Borrelia burgdorferi requires calcium signaling. Clin. Microbiol. Infect. 15:422-426, 2009.
• 4. Grab, D.J. , Kennedy, P.G.E. Traversal of human and animal trypanosomes across the blood-brain barrier. J. Neurovirol. 14:344-351, 2008.
• 5. Abdulla, M.H., O’Brien, T., Mackay, Z.B., Grab, D.J., McKerrow, J.H. 2008. In vivo RNAi of Trypanosoma brucei cathepsin B and L clarifies their contribution to disease progression. PLoS Negl. Trop. Dis. 2:e298, 2008.
• 6. Grab, D.J., Nyarko, E., Barat, N.C., Nikolskaia, O.V., Dumler, J.S. Chemokine, cytokine and matrix metalloproteinase expression by human brain microvascular endothelial cells is enhanced with Anaplasma phagocytophilum-Borrelia burgdorferi coinfection. Clin. Vac. Immunol. 14:1420-1424, 2007
• 7. Nikolskaia, O.V., Lima, A.P.C.A, Kim, Y.V., Lonsdale-Eccles, J.D., Fukuma, T., Scharfstein, J., Grab, D.J. Blood-brain barrier traversal by African trypanosome requires calcium signaling induced by parasite cysteine protease. J. Clin. Invest. 116:2739-2747, 2006. Erratum in: J. Clin. Invest. 118:1974, 2008
• 8. Nikolskaia, O.V., Kim, Y.V., Kovbasnjuk, O., Kim, K.J., Grab, D.J. Entry of Trypanosoma brucei gambiense into microvascular endothelial cells of the human blood-brain barrier. For Int. J. Parasitol. Thematic isssue Int. J. Parasitol. 36:513-519, 2006.
• 9. Nyarko, E.K., Grab, D.J., Dumler, J.S. Anaplasma phygocytophilum-infected neutrophils enhance transmigration of Borrelia burgdorferi across the human blood-brain barrier. Thematic isssue Int. J. Parasitol. 36:601-605, 2006.
• 10. Dumler, J.S., Choi, K,S., Garcia-Garcia, J.C., NBarat, N,S, Scorpio, D.G., Garyu, J.W., Grab, D.J., Bakken. J.S. Human Granulocytic Anaplasmosis and Anaplasma phagocytophilum. Emerging Inf. Dis. 11 (12):1828-1834, 2005
• 11. Grab, D.J., Lonsdale-Eccles, J.D., Inoue, N. Lamp for tadpoles. Nature Methods 2: 635, 2005.
• 12. Grab, D.J., Perides, G., Dumler, J.S., Kim, K.J., Park, J., Kim, YV., Nikolskaia, O, Choi, K.S., Stins, M.F., Kim, K.S. Borrelia burgdorferi traversal of an in vitro human blood-brain barrier model requires host-derived proteases. Infect. Immun. 73(2):1014-22, 2005.
• 13. Justin W.A. Garyu, J.W.A., Choi , K.S., Grab, D.J., Dumler, J.S., Defective phagocytosis in Anaplasma phagocytophilum-infected neutrophils. Infection. Immun. 73(2):1187-90, 2005.
• 14. Grab, D.J., Nikolskaia, O., Kim, Y.V., Lonsdale-Eccles, J.D., SIto, S., Hara, T., Fukuma, T., Nyarko, E., Kim, K.J., Stins, M., Delannoy, M.J., Rodgers, J., Kim, K.S. African Trypanosome Interactions with an in vitro model of the Human Blood-Brain Barrier. J. Parasitology 90: 970-979, 2004.
• 15. Nyarko E., Hara, T., Grab, D.J., Kim, Y.V., Nikolskaia, O., Fukuma, T., Tabata, M. In vitro toxicity of Pd(II) and Au(III) porphyrins and their aqueous metal ion counterparts on Trypanosoma brucei brucei growth. Chem. Biol. Interactions 148: 19-25, 2004.
• 16. Park, J., Grab, D.J., J. S. Dumler. Anaplasma phagocytophilum AnkA binds to granulocyte DNA and nuclear proteins. Cell. Microbiol. 6: 743-51, 2004.
• 17. Choi, K.S., Grab, D.J., Dumler, J.S. Anaplasma phagocytophilum infection induces protracted neutrophil degranulation. Infect. Immun.72: 3680-3683, 2004.
• 18. Grab, D.J., Salem, M.L., Dumler, J.S., Bucala, R. A Role for the Peripheral Blood Fibrocyte in Leishmania? Trends Parasitol. 20: 12, 2004.
• 19. Kuboki, N., Inoue, N., Sakurai, T., Di Cello, F., Grab, D.J., Suzuki, H., Sugimoto, C., Igarashi, I. Loop-mediated Isothermal Amplification (LAMP) and its application for the detection African Trypanosomes. J. Clin. Microbiol. 4: 5517-5524, 2003.
• 20. Lonsdale-Eccles, Grab, D.J. Trypanosome hydrolases and the Blood-brain barrier. Trends Parasitol., 18: 17-19. 2002.

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