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1.  Genomic locus on chromosome 1 regulates susceptibility to spontaneous arthritis in mice deficiency of IL-1RA 
BMC Immunology  2014;15(1):57.
To understand the role of genetic factors on chromosome 1 in the regulation of spontaneous arthritis in mice deficient in IL-1 receptor antagonist protein (IL_1RA), we previously used speed congenic breeding to transfer the QTL region from DBA/1−/− mice that are resistant to spontaneous arthritis into BALB/c−/− mice which are susceptible. We were able to establish two congenic strains which exhibited a delayed onset and reduced severity of disease. In this study, we asked a different set of questions. How will the QTL region from BALB/c−/− interact with the rest of the genome in the DBA/1−/− background? Will the DBA/1−/− mice become susceptible to spontaneous arthritis if the QTL genomic region on chromosome 1 was replaced with the genomic fragment of the same region from BALB/c−/−? We conducted the congenic breeding with the similar procedure as that of congenic strains with BALB/c−/− background.
Instead of BALB/c−/−, DBA/1−/− was used as the recurrent parent while BALB/c−/− was used as the donor parent. By the 6th generation we determined that all of the chromosomes in the progeny were of DBA/1−/− origin with the exception of the QTL portion of chromosome 1 which is heterozygous of BALB/c−/− and DBA/1−/− origin. We then intercrossed selected mice to produce homozygous strains containing the homozygous genomic region of BALB/c−/− on chromosome 1, while the rest of genome are homozygous DBA/1−/−. This strain was observed for the development of spontaneous arthritis. Up to 9 weeks of age, both congenic strain and DBA/1−/− did not develop arthritis. However, after 9 weeks, the congenic strain started to exhibit signs of arthritis, while the DBA/1−/− remained free from disease.
The result indicates a strong influence of genetic factor(s) on the QTL of chromosome 1 on the susceptibility to spontaneous arthritis. Identification of genetic factors within this QTL region in the future will significantly enhance our understanding of molecular mechanism of spontaneous arthritis.
PMCID: PMC4272550  PMID: 25488730
Arthritis; Congenic breeding; Mouse; QTL; DBA/1
3.  Effect of Fluorosis on Liver Cells of VC Deficient and Wild Type Mice 
The Scientific World Journal  2014;2014:287464.
For decades, mouse and other rodents have been used for the study of oxidative or related studies such as the effect of fluoride. It is known that rodents normally synthesize their own vitamin C (VC) due to the presence of a key enzyme in ascorbic acid synthesis, l-gulono-lactone-γ-oxidase (Gulo), while humans do not have the capacity of VC synthesis due to the deletion of most parts of the GULO gene. The spontaneous fracture (sfx) mouse recently emerged as a model for study of VC deficiency. We investigated the effect of fluoride on liver cells from wild type Balb/c and sfx mice. We found that activities of SOD, GPx, and CAT were reduced in both wild type and sfx mice; however, the amount of reduction in the sfx cells is more than that in Balb/c cells. In addition, while both cells increased MDA, the increase in the sfx cells is greater than that in Balb/c cells. Gene networks of Sod, Gpx, and Cat in the liver of humans and mice are also different. Our study suggests that reaction to fluoride in vitamin C deficient mice might be different from that of wild type mice.
PMCID: PMC3947854  PMID: 24693236
4.  Characterization of T cell phenotype and function in a double transgenic (collagen-specific TCR/HLA-DR1) humanized model of arthritis 
T cells orchestrate joint inflammation in rheumatoid arthritis (RA), yet they are difficult to study due to the small numbers of antigen-specific cells. The goal of this study was to characterize a new humanized model of autoimmune arthritis and to describe the phenotypic and functional changes that occur in autoimmune T cells following the induction of pathological events.
We developed a double transgenic mouse containing both the HLA-DR1 transgene and an HLA-DR1-restricted collagen-specific TCR in order to obtain large numbers of antigen-specific T cells that can be used for immunologic studies.
In vitro, CII-specific T cells from this mouse proliferated vigorously in response to the CII immunodominant peptide A2 and the cells altered their phenotype to become predominately CD62Llow and CD44high “activated” T cells. The response was accompanied by the production of Th1, Th2, and Th17-type cytokines. Following immunization with bovine CII/CFA, these mice develop an accelerated arthritis compared to single transgenic HLA-DR1 mice. On the other hand, when the mice were treated orally with the analog peptide A12, (a suppressive analog of collagen we have previously described), arthritis was significantly suppressed, despite the fact that >90% of the CD4+ T cells express the TCR Tg. In GALT tissues taken from the A12-treated mice, IL-2, IFN-γ, and IL-17 production to the autoimmune collagen determinant dropped while high levels of IL-10 and IL-4 were produced.
We have developed a humanized model of autoimmune arthritis that will be useful for the study of T cell directed therapies as well as T cell mediated mechanisms of autoimmune diseases.
PMCID: PMC3978884  PMID: 24405551
5.  Congenic Mice Provide Evidence for a Genetic Locus That Modulates Spontaneous Arthritis Caused by Deficiency of IL-1RA 
PLoS ONE  2013;8(6):e68158.
To understand the role of genetic factors involved in the development of spontaneous arthritis in mice deficient in IL-1 receptor antagonist protein (IL_1RA), we have identified a genomic region containing a major quantitative trait locus (QTL) for this disease. The QTL is on chromosome 1 and appears to be the strongest genetic region regulating arthritis. To confirm the importance of the QTL and to identify potential candidate genes within it, we conducted speed congenic breeding to transfer the QTL region from DBA/1 mice that are resistant to spontaneous arthritis into BALB/c−/− which are susceptible. Genetic markers along every chromosome were used to assist in the selection of progeny in each generation to backcross to BALB/c−/−. By the 6th generation we determined that all of the chromosomes in the progeny were of BALB/c origin with the exception of portions of chromosome 1. At this stage we intercrossed selected mice to produce homozygous strains containing the genomic background of BALB/c−/− except for the QTL region on chromosome 1, which was from DBA/1. We were able to establish two congenic strains with overlapping DBA/1 DNA segments. These strains were observed for the development of spontaneous arthritis. Both congenic strains were relatively resistant to spontaneous arthritis and had delayed onset and reduced severity of disease. The gene/s that regulates this major QTL would appear to be located in the region of the QTL that is shared by both strains. The common transferred region is between D1Mit110 and D1Mit209 on chromosome 1. We evaluated this region for candidate genes and have identified a limited number of candidates. Confirmation of the identity and precise role of the candidates will require additional study.
PMCID: PMC3695999  PMID: 23840826
6.  T Cells Stimulated by an Analog Peptide of Type II Collagen Require FcRγ to Secrete IL-4 and Suppress Autoimmune Arthritis 
Arthritis and rheumatism  2011;63(9):2661-2670.
Using the collagen-induced arthritis (CIA) model, we explored the characteristics of the T cell population which responds to an analog peptide (A9) of type II collagen (CII) and regulates autoimmunity.
A9 is a 26 amino acid peptide analogous to the sequence of a segment of CII (CII 245-270) but with substitutions made at amino acid positions 260 (alanine for isoleucine), 261 (hydroxyproline for alanine), and 263 (asparagine for phenylalanine). We have previously shown that A9 profoundly suppresses immunity to CII and CIA. In order to determine the mechanism of suppression, we used a transgenic mouse whose T cells express a CII specific receptor (TCR) and performed passive cell transfer experiments.
The results demonstrate that suppression of CIA by the A9 is dependent upon T cells. Using multiparameter flow cytometry, we determined that the cells responsible for suppression were CD4+ and expressed high levels of FcεRIγ(FcRγ). To establish the significance of this finding, we obtained mice genetically deficient in FcRγ to perform passive transfer experiments. The resulting FcRγ-/- CD4+ T cells when primed by culture with A9 could not transfer the suppression of arthritis nor secrete cytokines in response to A9.
Taken together, these data suggest that the suppression of arthritis and the Th2 cytokine profile elicited by A9 is dependent upon the presence of FcRγ in the T cells. These findings are novel and may have therapeutic potential for patients with autoimmune arthritis.
PMCID: PMC3575180  PMID: 21590683
7.  Genetic and Molecular Basis of Quantitative Trait Loci of Arthritis in Rat: Genes and Polymorphisms1 
Rheumatoid arthritis (RA) is an autoimmune disease, the pathogenesis of which is affected by multiple genetic and environmental factors. To understand the genetic and molecular basis of RA, a large number of quantitative trait loci (QTL) that regulate experimental autoimmune arthritis have been identified using various rat models for RA. However, identifying the particular responsible genes within these QTL remains a major challenge. Using currently available genome data and gene annotation information, we systematically examined RA-associated genes and polymorphisms within and outside QTL over the whole rat genome. By the whole genome analysis of genes and polymorphisms, we found that there are significantly more RA-associated genes in QTL regions as contrasted with non-QTL regions. Further experimental studies are necessary to determine whether these known RA-associated genes or polymorphisms are genetic components causing the QTL effect.
PMCID: PMC3466165  PMID: 18606636
8.  Characterization of inhibitory T cells induced by an analog of type II collagen in an HLA-DR1 humanized mouse model of autoimmune arthritis 
Arthritis Research & Therapy  2012;14(3):R107.
We used DR1 transgenic mice and covalently linked DR1 multimers to characterize analog-specific inhibitory T cells in collagen-induced arthritis (CIA). Because of the low numbers of antigen-specific T cells in wild-type mice, functional T-cell studies in autoimmune arthritis have been challenging. The use of T-cell receptor (TCR) transgenic mice has provided useful information, but such T cells may not represent the heterogeneous T-cell response that occurs in natural settings. Our focus was to develop tools to identify and characterize the population of immunoregulatory T cells induced in wild-type mice by an analog peptide of CII259-273, which contains amino acid substitutions at positions 263 (N) and 266 (D) (analog peptide A12).
DR1 multimers, developed by loading empty class II molecules with exogenous peptide, provide a method for visualizing antigen-specific T cells with flow cytometry. However, the low binding avidity of A12 for the major histocompatibility complex (MHC) made this strategy untenable. To overcome this problem, we generated DR1 multimers in which the analog peptide A12 was covalently linked, hoping that the low-avidity analog would occupy enough binding clefts to allow detection of the responsive T cells.
Staining with the tetramer revealed that A12-specific T cells were readily detectable at 10 days after immunization. These CD4(+) T cells are a highly selective subset of the TCR repertoire and have a limited clonality. Analysis of cytokine expression showed that cells detected by tetramer (A12) expressed primarily suppressive cytokines (interleukin-4 (IL-4) and IL-10) in response to collagen, compared with control cells. Although they did not express Fox-p3, they were extremely effective in preventing and suppressing inflammatory arthritis.
In summary, our studies showed that the use of covalently linked multimers allows characterization of analog-specific T cells that are otherwise difficult to detect. The suppressive character of the analog-specific T-cell response suggests that these cells attenuate autoimmunity and differ significantly in phenotype from the inflammatory T cells predominantly found in arthritic joints. Such reagents will become powerful tools to study T-cell responses in RA patients in upcoming clinical trials.
PMCID: PMC3446484  PMID: 22569209
9.  T Cell Receptor-Signaling Induced by an Analog Peptide of type II Collagen Requires Activation of Syk 
Clinical immunology (Orlando, Fla.)  2009;133(1):145-153.
We have previously described an analog peptide of type II collagen (CII) that can suppress collagen-induced arthritis (CIA). This analog peptide represents CII245-270, the immunodominant epitope of CII, but with substitutions at 260, 261, and 263 - CII245-270 (A260, B261, and N263) (A9). To elucidate the mechanisms responsible for suppression, we used mice transgenic for a collagen-specific T cell receptor (TCR). When we found that APCs pulsed with A9 failed to induce T cell phosphorylation of TCR-? and ZAP-70, we explored alternative signaling pathways. We determined that A9 instead induced phosphorylation of spleen tyrosine kinase (Syk). The importance of Syk was confirmed by the use of chemical Syk inhibitors, which blocked both cytokine secretion and activation of GATA-3 mediated by peptide A9. In summary, T cells use an alternative pathway in response to A9 that involves Syk. This novel T cell pathway may represent an important means for altering T cell phenotypes.
PMCID: PMC2756788  PMID: 19596610
Collagen II; T cells; altered peptide ligands; T cell signaling; Syk (spleen tyrosine kinase); autoimmunity
10.  Quantitative trait loci, genes, and polymorphisms that regulate bone mineral density in mouse 
Genomics  2009;93(5):401-414.
This is an in silico analysis of data available from genome-wide scans. Through analysis of QTL, genes and polymorphisms that regulate BMD, we identified 82 BMD QTL, 191 BMD-associated (BMDA) genes, and 83 genes containing known BMD-associated polymorphisms (BMDAP). The catalogue of all BMDA/BMDAP genes and relevant literatures are provided. In total, there are substantially more BMDA/BMDAP genes in regions of the genome where QTL have been identified than in non-QTL regions. Among 191 BMDA genes and 83 BMDAP genes, 133 and 58 are localized in QTL region, respectively. The difference was still noticeable for the chromosome distribution of these genes between QTL and non-QTL regions. These results have allowed us to generate an integrative profile of QTL, genes, polymorphisms that determine BMD. These data could facilitate more rapid and comprehensive identification of causal genes underlying the determination of BMD in mouse and provide new insights into how BMD is regulated in humans.
PMCID: PMC2901167  PMID: 19150398
BMD; QTL; gene; polymorphism; mouse
11.  Modulation of collagen-induced arthritis by adenovirus-mediated intra-articular expression of modified collagen type II 
Arthritis Research & Therapy  2010;12(4):R136.
Rheumatoid arthritis (RA) is a systemic disease manifested by chronic inflammation in multiple articular joints, including the knees and small joints of the hands and feet. We have developed a unique modification to a clinically accepted method for delivering therapies directly to the synovium. Our therapy is based on our previous discovery of an analog peptide (A9) with amino acid substitutions made at positions 260 (I to A), 261 (A to B), and 263 (F to N) that could profoundly suppress immunity to type II collagen (CII) and arthritis in the collagen-induced arthritis model (CIA).
We engineered an adenoviral vector to contain the CB11 portion of recombinant type II collagen and used PCR to introduce point mutations at three sites within (CII124-402, 260A, 261B, 263D), (rCB11-A9) so that the resulting molecule contained the A9 sequence at the exact site of the wild-type sequence.
We used this construct to target intra-articular tissues of mice and utilized the collagen-induced arthritis model to show that this treatment strategy provided a sustained, local therapy for individual arthritic joints, effective whether given to prevent arthritis or as a treatment. We also developed a novel system for in vivo bioimaging, using the firefly luciferase reporter gene to allow serial bioluminescence imaging to show that luciferase can be detected as late as 18 days post injection into the joint.
Our therapy is unique in that we target synovial cells to ultimately shut down T cell-mediated inflammation. Its effectiveness is based on its ability to transform potential inflammatory T cells and/or bystander T cells into therapeutic (regulatory-like) T cells which secrete interleukin (IL)-4. We believe this approach has potential to effectively suppress RA with minimal side effects.
PMCID: PMC2945026  PMID: 20615221
12.  Autoimmunity due to molecular mimicry as a cause of neurological disease 
Nature medicine  2002;8(5):509-513.
One hypothesis that couples infection with autoimmune disease is molecular mimicry. Molecular mimicry is characterized by an immune response to an environmental agent that cross-reacts with a host antigen, resulting in disease1,2. This hypothesis has been implicated in the pathogenesis of diabetes, lupus and multiple sclerosis (MS)1–4. There is limited direct evidence linking causative agents with pathogenic immune reactions in these diseases. Our study establishes a clear link between viral infection, autoimmunity and neurological disease in humans. As a model for molecular mimicry, we studied patients with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a disease that can be indistinguishable from MS (refs. 5–7). HAM/TSP patients develop antibodies to neurons8. We hypothesized these antibodies would identify a central nervous system (CNS) autoantigen. Immunoglobulin G isolated from HAM/TSP patients identified heterogeneous nuclear ribonuclear protein-A1 (hnRNP-A1) as the autoantigen. Antibodies to hnRNP-A1 cross-reacted with HTLV-1-tax, the immune response to which is associated with HAM/TSP (refs. 5,9). Immunoglobulin G specifically stained human Betz cells, whose axons are preferentially damaged7. Infusion of autoantibodies in brain sections inhibited neuronal firing, indicative of their pathogenic nature. These data demonstrate the importance of molecular mimicry between an infecting agent and hnRNP-A1 in autoimmune disease of the CNS.
PMCID: PMC2703733  PMID: 11984596
13.  Analog peptides of type II collagen can suppress arthritis in HLA-DR4 (DRB1*0401) transgenic mice 
Rheumatoid arthritis (RA) is an autoimmune disease associated with the recognition of self proteins secluded in diarthrodial joints. We have previously established that mice transgenic for the human DR genes associated with RA are susceptible to collagen-induced arthritis (CIA) and we have identified a determinant of type II collagen (CII263–270) that triggers T-cell immune responses in these mice. We have also determined that an analog of CII263–270 would suppress disease in DR1 transgenic mice. Because the immunodominant determinant is the same for both DR1 transgenic and DR4 transgenic mice, we attempted to determine whether the analog peptide that was suppressive in DR1 transgenic mice would also be effective in suppressing CIA in DR4 transgenic mice. We treated DR4 transgenic mice with two analog peptides of CII that contained substitutions in the core of the immunodominant determinant: CII256–276 (F263N, E266D) and CII256–270 (F263N, E266A). Mice were observed for CIA, and T-cell proliferative responses were determined. Either peptide administered at the time of immunization with CII significantly downregulated arthritis. Binding studies demonstrated that replacement of the phenylalanine residue in position 263 of the CII peptide with asparagine significantly decreased the affinity of the peptide for the DR4 molecule. In contrast, replacement of the glutamic acid residue in position 266 with aspartic acid or with alanine had differing results. Aspartic acid reduced the affinity (35-fold) whereas alanine did not. Both peptides were capable of suppressing CIA. With the use of either peptide, CII256–276 (F263N, E266D) or CII256–270 (F263N, E266A), the modulation of CIA was associated with an increase in T-cell secretion of IL-4 together with a decrease in IFN-γ. We have identified two analog peptides that are potent suppressors of CIA in DR4 transgenic mice. These experiments represent the first description of an analog peptide of CII recognized by T cells in the context of HLA-DR4 that can suppress autoimmune arthritis.
PMCID: PMC1779432  PMID: 16982003
14.  An HLA-DR1 Transgene Confers Susceptibility to Collagen-induced Arthritis Elicited with Human Type II Collagen 
The Journal of Experimental Medicine  1997;185(6):1113-1122.
Rheumatoid arthritis (RA) is an autoimmune disease that is strongly associated with the expression of several HLA-DR haplotypes, including DR1 (DRB1*0101). Although the antigen that initiates RA remains elusive, it has been shown that many patients have autoimmunity directed to type II collagen (CII). To test the hypothesis that HLA-DR1 is capable of mediating an immune response to CII, we have generated transgenic mice expressing chimeric (human/ mouse) HLA-DR1. When the DR1 transgenic mice were immunized with human CII (hCII), they developed a severe autoimmune arthritis, evidenced by severe swelling and erythema of the limbs and marked inflammation and erosion of articular joints. The development of the autoimmune arthritis was accompanied by strong DR1-restricted T and B cell responses to hCII. The T cell response was focused on a dominant determinant contained within CII(259–273) from which an eight amino acid core was defined. The B cell response was characterized by high titers of antibody specific for hCII, and a high degree of cross-reactivity with murine type II collagen. These data demonstrate that HLA-DR1 is capable of presenting peptides derived from hCII, and suggest that this DR1 transgenic model will be useful in the development of DR1-specific therapies for RA.
PMCID: PMC2196244  PMID: 9091584

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