Stan Erlandsen, PhD
Areas of Research Strength:
Electron microscopy, immunocytochemistry, giardiasis, interaction of intestinal microbes with the intestinal mucosa, and high resolution imaging of cell surfaces (adhesion molecules) using cryofixation
and cryoSEM.
Research Interests:
High resolution imaging of cell adhesion molecules by cryoSEM. Two projects are being used as model systems for the imaging of cell adhesion molecules. In a eucaryotic model, a differential distribution of cell adhesion molecules to separate membrane domains (microvilli versus cell body) has been demonstrated in human leukocytes using colloidal gold immunocytochemistry and high resolution backscatter electron imaging (atomic number contrast). Selectin adhesion molecules have been shown to be confined to microvillus-like projections in neutrophils, lymphocytes, and transfected cells in which the genes have been altered to delete or switch the cytoplasmic portion of the receptor that is suspected of interacting with the cytoskeleton. A procaryotic model based on Enterococcus faecalis has been developed in which it has been possible to detect individual cell adhesion molecules by using cryofixation (high pressure freezing) and cryoSEM. The use of isogenic strains of E. faecalis which can be manipulated to constitutively express two surface molecules, one possessing a globular shape (Asc10) and the other a helical shape (Sec10), are being used as a model for investigating the high resolution SEM imaging of surface proteins. Development of methods for high resolution imaging of individual cell adhesion molecules may prove to be a powerful toll for examing cell adhesion molecules in migrating eucaryotic cells, such as leukocytes and embryonic cells.
Synthesis of specific rDNA probes to Giardia species in man and animals. Oligomeric rDNA probes (16-25 bp in length) to the variable regions in Giardia rRNA have been developed for use as primers for PCR amplification of rDNA from Giardia trophozoites isolated from various animals implicated in zoonotic or waterborne transmission, and for use as probes for in situ hydridization. Through use of these probes it has been possible to sequence the entire rRNA gene in three separate species of Giardia: a rodent isolate, G. muris; an isolate from the great blue heron, G. ardeae; and also to confirm the previously reported rRNA structure in G. lamblia (from man). In addition, we have shown that two major strains of Giardia exist in humans based on the molecular structure of their rRNAs.
Based on the known structure of the small subunit (SSU) of rDNA in these species, a series of universal primers have been developed for PCR amplification of the 5' ends of the SSU of rDNA from small numbers of cells (102-103) isolated from animals, birds or environmental samples. By applying these methods to characterizing the rDNA in samples of Giardia isolated from animals suspected of being involved in zoonotic or waterborne transmission (muskrats, beavers, birds), it should be possible to perform comparative studies at the molecular level of the nucleotide sequence for SSU rRNA. Sequence comparison of the molecular structure of rRNA has been widely used to investigate species in prokaryotes and should permit unambiguous identification of Giardia species. Using rDNA structure as a tool for Giardia species identification, it will be possible to determine which animals can harbor species of Giardia infective for man and identify those involved in environmental outbreaks of giardiasis. It is likely that commercial detection systems based on this same approach could prove to be useful in screening or analyzing environmental samples for the presence of those Giardia species having the potential for infecting humans.
Development of DNA probes for in situ hybridization. Probes specific for rRNA have been conjugated to fluorochromes having different emission spectra (fluorescein [520 nm], carboxymethylindocyanine dyes 3.18 [568 nm] and 5.18 [668 nm]) and also to haptens (biotin, digoxigenin) permitting detection by indirect methods using different detection systems (enzymes, fluorochromes, gold markers). Using laser scanning confocal microscopy, techniques have been developed using three-color fluorescence that permit the simultaneous detection of multiple species of Giardia within the same test or reference sample. The use of fluorescent in situ hybridization (FISH) has distinct advantages over isotopic or enzymatic methods in that it requires significantly less time and eliminates the use of harmful chemicals and isotopes. FISH has been successfully applied to the analysis of environmental samples for the detection of G. lamblia cysts in both human fecal samples and raw sewage. Further examination of environmental samples with gene probes may provide important information on whether or not multiple species of Giardia can infect a single host, or if one or more species may be involved in environmental outbreaks.
Selected Publications:
Erlandsen SL, CJ Kristich, GM Dunny, and CL Wells (2004). Hight resolution visualization of bacterial glycocalyx by low voltage SEM: Dependence on cationic probes. J. Histochem. Cytochem. 52:1427-1435.
Wells CL, Hess DJ, and Erlandsen SL. (2004). The impact of alterations in the indigenous flora in animal models of shock and sepsis. Shock . 22:562-568.
Erlandsen, SL, P. Russo, and JN, Turner (2004). New adhesion mechanism in Giardia: role of the ventrolateral flange in the attachment of trophozoites to rough and porous surfaces. Microscopy Today, May Issue.
Erlandsen SL, CJ Kristich, and GM Dunny (2004). Ultrastructure of Enterococcus faecalis biofilms. Biofilms 1:131-137.
Erlandsen SL, P. Russo, and JM Turner (2004). Evidence for adhesive activity of the ventrolateral flange in Giardia lamblia. J. Eukary. Microbiol. 51:73-80.
Erlandsen S, Y Chen, C Frethem, G. Dunny, and C. Wells (2003) High resoulution backscater electron imaging of colloidal gold in LVSEM. J. Microsec. 211:212-218.
Erlandsen, S, M. Lei, I. Martin-Lacave, G Dunny, and C Wells (2003). High resoulution cryoFESEM of microbial surfaces. Micros. Microanal. 9:1-6.
Kim AS, Garni RM, Henry-Stanley MJ, Bendel CM, Erlandsen SL, Wells CL (2003). Hypoxia and extraintestial dissemination of Candida albicans yeast forms. Shock 19:257-62.
Berlin, C., R. Bargatze, J.J. Campell, U.H. von Andrian, M.C. SZabo, S.R. Hasslen, R.D. Nelson, E.L. Berg, S.L. Erlandsen, and E.C. Butcher 1995 Integrin-mediated lymphocyte attachment and rolling under physiological flow. Cell 80:1-20.
Hasslen, S.H., U.H. von Andrian, E.C. Butcher, R.D. Nelson, and S.L. Erlandsen 1995 Spatial distribution of L-selectin (CD62L) on human lymphocytes and transfected murine L1-2 cells. Histochemical J. 27:547-554.
von Andrian, U.H., S.R. Hasslen, R.D. Nelson, S.L. Erlandsen, and E.C. Butcher 1995 A central role for microvillous receptor presentation in leukocyte adhesion under flow. Cell 82:1-20.
Erlandsen, S.L., R.D. Nelson, S.R. Hasslen, G.M. Dunny, S.B. Olmsted, C. Frethem, and C.L. Wells 1995 High resolution FESEM: Application of backscatter electron (BSE) imaging for biological samples. Workshop on Ultra High Resolution SEM, Oak Ridge National Lab.& University of Tennessee, D.Joy (ed.), Hitachi Instrument News, 27:10-15.
Erlandsen, S.L. and E. Rasch 1994 The DNA content of trophozoites and cysts of Giardia lamblia by microdensitometric quantitation of Feulgen staining and examination by laser scanning confocal microscopy. J. Histochemistry and Cytochemistry 42:1413-1416.
Erlandsen, S.L., S.R. Hasslen, and R.D. Nelson 1993 Detection and spatial distribution of the B2 integrin (Mac-1) and L-selectin (LECAM-1) adherence receptors on human neutrophils using high resolution field emission SEM. J. Histochem. Cytochem. 41:327-333.
van Keulen, H., R.R. Gutell, M.A. Gates, S.R. Campbell, S.L. Erlandsen, E.L. Jarroll, J. Kulda, and E.A. Meyer 1993 Unique phylogenetic position of Diplomonidida based on the complete small subunit ribosomal RNA sequence of Giardia ardeae, G. muris, G. duodenalis and Hexamita sp. FASEB J. 7:223-231.
Erlandsen, S.L., H. van Keulen, T. Brelje, A. Gurien, W. Jakubowksi, F.W. Schaefer III, P. Wallis, E. Jarroll. 1993 Molecular Approach to the Speciation and Detection of Giardia: Fluorochrome-rDNA Probes for Identification of Giardia lamblia, Giardia muris, and Giardia ardeae in Laboratory and Environmental Samples by In Situ Hybridization. In Giardia - From Molecules to Disease and Beyond. CAB International, pp.64-66.
Erlandsen,S.L. 1993 Biotic Tranmission - Is Giardiasis a Zoonosis? In: Giardia - From Molecules to Disease and Beyond. CAB International, pp.83-97.
Olmstead, S.B., S.L. Erlandsen, G.M. Dunny, and C.L. Wells 1993 High-resolution visualization by field emission scanning electron microscopy of Enterococcus faecalis cell surface proteins encoded by the pheromone-Induced conjugative plasmid pCF10. J. Bacteriol.175:6229-6237.
