Doctor of Philosophy, University of British Columbia (2009)
Paul Utz, Postdoctoral Faculty Sponsor
Autoantibodies against cytokines, chemokines, and growth factors inhibit normal immunity and are implicated in inflammatory autoimmune disease and diseases of immune deficiency. In an effort to evaluate serum from autoimmune and immunodeficient patients for Abs against cytokines, chemokines, and growth factors in a high-throughput and unbiased manner, we constructed a multiplex protein microarray for detection of serum factor-binding Abs and used the microarray to detect autoantibody targets in SLE. We designed a nitrocellulose-surface microarray containing human cytokines, chemokines, and other circulating proteins and demonstrated that the array permitted specific detection of serum factor-binding probes. We used the arrays to detect previously described autoantibodies against cytokines in samples from individuals with autoimmune polyendocrine syndrome type 1 and chronic mycobacterial infection. Serum profiling from individuals with SLE revealed that among several targets, elevated IgG autoantibody reactivity to B cell-activating factor (BAFF) was associated with SLE compared with control samples. BAFF reactivity correlated with the severity of disease-associated features, including IFN-α-driven SLE pathology. Our results showed that serum factor protein microarrays facilitate detection of autoantibody reactivity to serum factors in human samples and that BAFF-reactive autoantibodies may be associated with an elevated inflammatory disease state within the spectrum of SLE.
View details for DOI 10.1172/JCI70231
View details for Web of Science ID 000327826100020
View details for PubMedID 24270423
Hereditary spherocytosis (HS) is a heterogeneous group of spontaneously arising and inherited red blood cell disorders ranging from very mild subclinical cases to severe and life-threatening cases, with symptoms linked directly to the severity of the mutation at the molecular level. We investigated a novel mouse model in which the heterozygotes present with the diagnostic hallmarks of mild HS and surviving homozygotes phenocopy severe hemolytic HS.We used N-ethyl-N-nitrosourea mutagenesis to generate random point mutations in the mouse genome and a dominant screen to identify mouse models of human hematopoietic disease. Gene mapping of the HS strain revealed a unique in-frame nonsense mutation arising from a single base transversion in exon 27 of Ank1 (strain designation: Ank1(E924X)). Employing conventional hematopoietic, pathological, biochemical, and cell biology assays, we characterized heterozygous and homozygous Ank1(E924X) mice at the biochemical, cellular, and pathophysiological levels.Although Ank1(E924X/E924X) red blood cell ghosts lack abundant full-length ankyrin-1 isoforms, N-terminal epitope ankyrin-1 antibodies reveal a band consistent with the theoretical size of a truncated mutant ankyrin-1. Using domain-specific antibodies, we further show that this protein lacks both a spectrin-binding domain and a C-terminal regulatory domain. Finally, using antisera that detect C-terminal residues of the products of alternative Ank1 transcripts, we find unique immunoreactive bands not observed in red blood cell ghosts from wild-type or Ank1(E924X) heterozygous mice, including a band similar in size to full-length ankyrin-1.The Ank1(E924X) strain provides a novel tool to study Ank1 and model HS.
View details for DOI 10.1016/j.exphem.2010.12.009
View details for Web of Science ID 000288288200005
View details for PubMedID 21193012
In this study, we compared hematopoietic stem cells and mast cells, using microarray expression analysis, and identified the cellular prion protein (PrP(C)) as a potentially novel marker of mast cells. On further investigation, we found that PrP(C) is expressed on the surface of human and mouse mast cells, is rapidly released by mast cells upon activation, and is released in response to mast cell-dependent allergic inflammation in vivo. Because mast cells are long lived and traffic to the brain and central nervous system, our observations could have important implications for the transmission and pathology of prion diseases.
View details for DOI 10.1086/605022
View details for Web of Science ID 000268683900022
View details for PubMedID 19642931
SHIP1 inhibits immune receptor signaling through hydrolysis of the PI3K product phosphatidylinositol 3,4,5-trisphosphate, forming phosphatidylinositol 3,4-bisphosphate. In mast cells, SHIP1 represses FcepsilonRI- and cytokine-mediated activation in vitro, but little is known regarding the function of SHIP1 in mast cells in vivo or the susceptibility of Ship1(-/-) mice to mast cell-associated diseases. In this study, we found that Ship1(-/-) mice have systemic mast cell hyperplasia, increased serum levels of IL-6, TNF, and IL-5, and heightened anaphylactic response. Further, by reconstituting mast cell-deficient mice with Ship1(+/+) or Ship1(-/-) mast cells, we found that the above defects were due to loss of SHIP1 in mast cells. Additionally, we found that mice reconstituted with Ship1(-/-) mast cells suffered worse allergic asthma pathology than those reconstituted with Ship1(+/+) mast cells. In summary, our data show that SHIP1 represses allergic inflammation and mast cell hyperplasia in vivo and exerts these effects specifically in mast cells.
View details for DOI 10.4049/jimmunol.0900427
View details for Web of Science ID 000275119400026
View details for PubMedID 19542434
Asthma is a pulmonary inflammatory disease dependent on eosinophil and mast cell infiltration into the lung. CD34 is a sialomucin expressed by both of these cell types, and we have used CD34(-/-) mice and a standard mouse model of asthma to evaluate the importance of CD34 expression on disease development. In comparison with wild-type (wt) mice, CD34(-/-) mice exhibited a dramatic reduction in all hallmarks of allergic asthma, including lowered airway inflammatory cell infiltration, airway hyperresponsiveness, and mast-cell recruitment. Bone marrow transplantation experiments confirmed that these defects are due to CD34 expression by bone marrow-derived cells. This was not, however, due to an inability to respond to antigen as, on a per cell basis, wt and CD34(-/-) inflammatory cells exhibit identical responses in cytokine production. We found a striking reduction in mobility of CD34(-/-) eosinophils in vitro, the major component of inflammatory infiltrates, which was consistent with proposed models for CD34 as an inhibitor of cell-cell adhesion. In summary, our data suggest that CD34 enhances mast-cell and eosinophil invasiveness and that its expression by these cells is a prerequisite for development of allergic asthma.
View details for DOI 10.1182/blood-2006-12-062448
View details for Web of Science ID 000249671700045
View details for PubMedID 17557898