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2.
Blood ; 119(11): e86-95, 2012 Mar 15.
Article in English | MEDLINE | ID: mdl-22262767

ABSTRACT

The low incidence of CFU-F significantly complicates the isolation of homogeneous populations of mouse bone marrow stromal cells (BMSCs), a common problem being contamination with hematopoietic cells. Taking advantage of burgeoning evidence demonstrating the perivascular location of stromal cell stem/progenitors, we hypothesized that a potential reason for the low yield of mouse BMSCs is the flushing of the marrow used to remove single-cell suspensions and the consequent destruction of the marrow vasculature, which may adversely affect recovery of BMSCs physically associated with the abluminal surface of blood vessels. Herein, we describe a simple methodology based on preparation and enzymatic disaggregation of intact marrow plugs, which yields distinct populations of both stromal and endothelial cells. The recovery of CFU-F obtained by pooling the product of each digestion (1631.8 + 199) reproducibly exceeds that obtained using the standard BM flushing technique (14.32 + 1.9) by at least 2 orders of magnitude (P < .001; N = 8) with an accompanying 113.95-fold enrichment of CFU-F frequency when plated at low oxygen (5%). Purified BMSC populations devoid of hematopoietic contamination are readily obtained by FACS at P0 and from freshly prepared single-cell suspensions. Furthermore, this population demonstrates robust multilineage differentiation using standard in vivo and in vitro bioassays.


Subject(s)
Bone Marrow Cells/cytology , Endothelium, Vascular/cytology , Stem Cells/cytology , Stromal Cells/cytology , Animals , Cell Differentiation , Cells, Cultured , Colony-Forming Units Assay , Flow Cytometry , Fluorescent Antibody Technique , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID
3.
Infect Immun ; 76(10): 4737-44, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18678660

ABSTRACT

Microsporidia are obligate intracellular parasites that are ubiquitous in nature and have been recognized as causing an important emerging disease among immunocompromised individuals. Limited knowledge exists about the immune response against these organisms, and virtually nothing is known about the receptors involved in host recognition. Toll-like receptors (TLR) are pattern recognition receptors that bind to specific molecules found on pathogens and signal a variety of inflammatory responses. In this study, we show that both Encephalitozoon cuniculi and Encephalitozoon intestinalis are preferentially recognized by TLR2 and not by TLR4 in primary human macrophages. This is the first demonstration of host receptor recognition of any microsporidian species. TLR2 ligation is known to activate NF-kappaB, resulting in inflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha) and interleukin-8 (IL-8). We found that the infection of primary human macrophages leads to the nuclear translocation of NF-kappaB in as early as 1 h and the subsequent production of TNF-alpha and IL-8. To verify the direct role of TLR2 parasite recognition in the production of these cytokines, the receptor was knocked down in primary human macrophages using small interfering RNA. This knockdown resulted in decreases in both the nuclear translocation of NF-kappaB and the levels of TNF-alpha and IL-8 after challenge with spores. Taken together, these experiments directly link the initial inflammatory response induced by Encephalitozoon spp. to TLR2 stimulation in human macrophages.


Subject(s)
Encephalitozoon/immunology , Inflammation/immunology , NF-kappa B/metabolism , Toll-Like Receptor 2/immunology , Cell Nucleus/chemistry , Cells, Cultured , Gene Silencing , Humans , Interleukin-8/metabolism , Macrophages/immunology , Macrophages/microbiology , RNA, Small Interfering/genetics , Time Factors , Toll-Like Receptor 4/immunology , Tumor Necrosis Factor-alpha/metabolism
4.
Infect Immun ; 75(4): 1619-25, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17178789

ABSTRACT

Microsporidians are a group of emerging pathogens typically associated with chronic diarrhea in immunocompromised individuals. The number of reports of infections with these organisms and the disseminated pathology is growing as diagnostic tools become more readily available. However, little is known about the innate immune response induced by and generated against these parasites. Using a coculture chemotaxis system, primary human macrophages were infected with Encephalitozoon cuniculi or Encephalitozoon intestinalis, and the recruitment of naïve monocytes was monitored. Encephalitozoon spp. induced an average threefold increase in migration of naïve cells 48 h postinfection, which corresponded to optimal infection of monocyte-derived-macrophages. A limited microarray analysis of infected macrophages revealed several chemokines involved in the inflammatory responses whose expression was upregulated, including CCL1, CCL2, CCL3, CCL4, CCL7, CCL15, CCL20, CXCL1, CXCL2, CXCL3, CXCL5, and CXCL8. The levels of 6 of 11 chemokines also present in the microarray were confirmed to be elevated by protein profiling. Kinetic studies confirmed that secreted CCL2, CCL3, and CCL4 were expressed as early as 6 h postinfection, with peak expression at 12 to 24 h and expression remaining until 48 h postinfection. Neutralization of these chemokines, specifically CCL4, significantly reduced the number of migrating cells in vitro, indicating their role in the induction of monocyte migration. This mechanism of recruitment not only supports the evidence that in vivo cellular infiltration occurs but also provides new hosts for the parasites, which escape macrophages by rupturing the host cell. To our knowledge, this is the first documentation that chemokine production is induced by microsporidian infections in human macrophages.


Subject(s)
Chemokines/metabolism , Chemotaxis, Leukocyte , Encephalitozoon/immunology , Macrophages/immunology , Macrophages/microbiology , Monocytes/immunology , Cells, Cultured , Chemokine CCL4 , Chemokines/genetics , Chemokines, CC/metabolism , Coculture Techniques , Encephalitozoon cuniculi/immunology , Gene Expression Regulation , Humans , Monocytes/physiology , Oligonucleotide Array Sequence Analysis , Proteome/analysis , RNA, Messenger/analysis , Receptors, Chemokine/genetics , Receptors, Chemokine/metabolism , Up-Regulation
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