Laura Cort

Genetic analysis of the influence of pertussis toxin on experimental allergic encephalomyelitis susceptibility: An environmental agent can override genetic checkpoints

Pertussis toxin (PTX) is a potent ancillary adjuvant used to elicit several different autoimmune diseases, including experimental allergic encephalomyelitis (EAE). To delineate the genetics of PTX effect in EAE, we mapped EAE-modifying (eae-m) loci in cohorts of backcross mice immunized with and without PTX. In this study, we analyzed the genetic basis of EAE susceptibility and severity and the intermediate phenotypes of mononuclear cell infiltration, suppuration, and demyelination. In animals immunized with PTX, one major locus, eae9, controls disease susceptibility and severity. Eae9 also regulates the extent of mononuclear cell infiltration of the spinal cord in male mice. Without PTX, five eae-m loci were noted, including three new loci in intervals on chromosomes 8 (eae14), 10 (eae17), and 18 (eae18). Taken together, these results suggest that eae9 controls the effects of PTX in EAE susceptibility, and is capable of overriding the other genetic checkpoints in the pathogenesis of this disease.

Secreted phospholipase A2 activity in experimental autoimmune encephalomyelitis and multiple sclerosis

BackgroundThere is increased interest in the contribution of the innate immune system to multiple sclerosis (MS), including the activity of acute inflammatory mediators. The purpose of this study was to test the involvement of systemic secreted phospholipase A2 (sPLA2) enzymes in experimental autoimmune encephalomyelitis (EAE), an MS model, and to determine if enzyme activity is elevated in MS patients.MethodsA non-invasive urinary assay was developed in order to monitor enzymatically active sPLA2 levels in Dark Agouti rats after induction of EAE. Some Rats were treated with nonapeptide CHEC-9, an uncompetitive sPLA2 enzyme inhibitor, during the initial rise in urinary enzyme levels. Body weight and clinical EAE score were measured for 18 days post immunization (PI), after which the rats were sacrificed for H&E and myelin staining, and for ED-1 immunocytochemistry, the latter to quantify macrophages and activated microglia. The urinary sPLA2 assay was also applied to un-timed samples collected from a cross section of 44 MS patients and 14 healthy controls.ResultsMean levels of enzymatically active sPLA2 in the urine increased following immunization and peaked between days 8–10 PI which was just prior to the onset of EAE symptoms. At this time, a transient attenuation of activity was detected in the urine of CHEC-9 treated rats consistent with the activity-dependent properties of the inhibitor. The peptide also reduced or abolished EAE symptoms compared to vehicle-injected controls. Histopathological changes in the spinal cords of the EAE rats correlated generally with clinical score including a significant reduction in ED-1+ cells after peptide treatment. Multiple Sclerosis patients also showed elevations in sPLA2 enzyme activity. Mean levels of sPLA2 were increased 6-fold in the urine of patients with active disease and 4-fold for patients in remission, regardless of immunomodulating therapy.ConclusionThe results suggest that sPLA2 enzymes, traditionally thought to be part the acute phase inflammatory response, are therapeutic targets for MS.

Prevention of type 1 diabetes in the rat with an allele-specific anti-T-cell receptor antibody: Vβ13 as a therapeutic target and biomarker.

In earlier studies of the Iddm14 diabetes susceptibility locus in the rat, we identified an allele of the T-cell receptor (TCR) β-chain, Tcrb-V13S1A1, as a candidate gene. To establish its importance, we treated susceptible rats with a depleting anti-rat Vβ13 monoclonal antibody and then exposed them to either polyinosinic:polycytidylic acid or a diabetogenic virus to induce diabetes. The overall frequency of diabetes in the controls was 74% (n = 50), compared with 17% (n = 30) in the anti-Vβ13–treated animals, with minimal islet pathology in nondiabetic treated animals. T cells isolated from islets on day 5 after starting induction showed a greater proportion of Vβ13+ T cells than did peripheral lymph node T cells. Vβ13 transcripts recovered from day 5 islets revealed focused Jβ usage and less CDR3 diversity than did transcripts from peripheral Vβ13+ T cells. CDR3 usage was not skewed in control Vβ16 CDR3 transcripts. Anti-rat Vβ13 antibody also prevented spontaneous diabetes in BBDP rats. The Iddm14 gene is likely to be Tcrb-V13, indicating that TCR β-chain usage is a determinant of susceptibility to autoimmune diabetes in rats. It may be possible to prevent autoimmune diabetes by targeting a limited element of the T-cell repertoire.

Refinement of the Iddm4 Diabetes Susceptibility Locus Reveals TCRVβ4 as a Candidate Gene

Abstract:  Iddm4 is a dominant non‐major histocompatibility complex (MHC) determinant of diabetes susceptibility in BBDR rats treated with poly I:C, plus depletion of regulatory T cells. In congenic MHC‐identical normal WF rats, Iddm4d sensitively and specifically predicts induced diabetes. We report a new diabetes‐susceptible subcongenic line that carries Iddm4 in a < 2.6 megabase interval. Candidate genes include the T cell receptor β chain variable (TCRVβ) family. We found that TCRVβ4 in WF rats contains a stop codon, whereas 5/5 diabetes‐susceptible rat strains express TCRVβ4. We conclude that Iddm4‐mediated diabetes resistance in rats may be due to a recessive protective mutation in TCRVβ4.

Genetic dissection reveals diabetes loci proximal to the gimap5 lymphopenia gene

Congenic DRF.(f/f) rats are protected from type 1 diabetes (T1D) by 34 Mb of F344 DNA introgressed proximal to the gimap5 lymphopenia gene. To dissect the genetic factor(s) that confer protection from T1D in the DRF.(f/f) rat line, DRF.(f/f) rats were crossed to inbred BBDR or DR.(lyp/lyp) rats to generate congenic sublines that were genotyped and monitored for T1D, and positional candidate genes were sequenced. All (100%) DR.(lyp/lyp) rats developed T1D by 83 days of age. Reduction of the DRF.(f/f) F344 DNA fragment by 26 Mb (42.52-68.51 Mb) retained complete T1D protection. Further dissection revealed that a 2 Mb interval of F344 DNA (67.41-70.17 Mb) (region 1) resulted in 47% protection and significantly delayed onset (P < 0.001 compared with DR.(lyp/lyp)). Retaining <1 Mb of F344 DNA at the distal end (76.49-76.83 Mb) (region 2) resulted in 28% protection and also delayed onset (P < 0.001 compared with DR.(lyp/lyp)). Comparative analysis of diabetes frequency in the DRF.(f/f) congenic sublines further refined the RNO4 region 1 interval to approximately 670 kb and region 2 to the 340 kb proximal to gimap5. All congenic DRF.(f/f) sublines were prone to low-grade pancreatic mononuclear cell infiltration around ducts and vessels, but <20% of islets in nondiabetic rats showed islet infiltration. Coding sequence analysis revealed TCR Vbeta 8E, 12, and 13 as candidate genes in region 1 and znf467 and atp6v0e2 as candidate genes in region 2. Our results show that spontaneous T1D is controlled by at least two genetic loci 7 Mb apart on rat chromosome 4.

Virus-induced autoimmune diabetes in the LEW.1WR1 rat requires Iddm14 and a genetic locus proximal to the major histocompatibility complex.

OBJECTIVE To identify genes that confer susceptibility to autoimmune diabetes following viral infection in the LEW.1WR1 rat. RESEARCH DESIGN AND METHODS About 2% of LEW.1WR1 rats develop spontaneous autoimmune diabetes. Immunological perturbants including viral infection increase both the frequency and tempo of diabetes onset. To identify diabetes susceptibility genes (LEW.1WR1 × WF), F2 rats were infected with Kilham rat virus following brief pretreatment with polyinosinic:polycytidylic acid. This treatment induces diabetes in 100% of parental LEW.1WR1 rats and 0% of parental WF rats. Linkage to diabetes was analyzed by genome-wide scanning. RESULTS Among 182 F2 rats, 57 (31%) developed autoimmune diabetes after a mean latency of 16 days. All diabetic animals and ∼20% of nondiabetic animals exhibited pancreatic insulitis. Genome-wide scanning revealed a requirement for the Iddm14 locus, long known to be required for diabetes in the BB rat. In addition, a new locus near the RT1 major histocompatibility complex (MHC) was found to be a major determinant of disease susceptibility. Interestingly, one gene linked to autoimmune diabetes in mouse and human, UBD, lies within this region. CONCLUSIONS The Iddm14 diabetes locus in the rat is a powerful determinant of disease penetrance in the LEW.1WR1 rat following viral infection. In addition, a locus near the MHC (Iddm37) conditions diabetes susceptibility in these animals. Other, as-yet-unidentified genes are required to convert latent susceptibility to overt diabetes. These data provide insight into the polygenic nature of autoimmune diabetes in the rat and the interplay of genetic and environmental factors underlying disease expression.

Fine-mapping quantitative trait loci affecting murine external ear tissue regeneration in the LG/J by SM/J advanced intercross line.

External ear hole closure in LG/J mice represents a model of regenerative response. It is accompanied by the formation of a blastema-like structure and the re-growth of multiple tissues, including cartilage. The ability to regenerate tissue is heritable. An F34 advanced intercross line of mice (Wustl:LG,SM-G34) was generated to identify genomic loci involved in ear hole closure over a 30-day healing period. We mapped 19 quantitative trait loci (QTL) for ear hole closure. Individual gene effects are relatively small (0.08 mm), and most loci have co-dominant effects with phenotypically intermediate heterozygotes. QTL support regions were limited to a median size of 2 Mb containing a median of 19 genes. Positional candidate genes were evaluated using differential transcript expression between LG/J and SM/J healing tissue, function analysis and bioinformatic analysis of single-nucleotide polymorphisms in and around positional candidate genes of interest. Analysis of the set of 34 positional candidate genes and those displaying expression differences revealed over-representation of genes involved in cell cycle regulation/DNA damage, cell migration and adhesion, developmentally related genes and metabolism. This indicates that the healing phenotype in LG/J mice involves multiple physiological mechanisms.

Microsatellite typing of CXB recombinant inbred and parental mouse strains

Recombinant inbred (RI) strains derive from crosses between two inbred mouse strains whose progeny are maintained under independent inbreeding from the F 2 generation onward (Bailey 1971). RI strains are one of the most useful tools in mammalian genetics for relatively rapid and efficient mapping of genetic loci that differ between the two progenitor strains (Taylor 1989). The distribution of parental phenotypes or genotypes among the RI strains is referred to as the strain distribution pattern (SDP). Statistical methods (Silver and Buckler 1986; Neumann 1991) and a computer program (Map Manager; Manly and Elliott 1991) are available for the purpose of determining linkage in RI strain sets. Numerous, well-distributed genetic markers in RI strains increase the probability that linkage can be detected for a new phenotype. The availability of these genetic markers is a major factor that determines the usefulness of an RI strain set. Inbred mouse strains vary in their susceptibility to E-55+ murine leukemia virus-induced lymphoma (Tumas et al. 1993), and BALB/c and C57BL/6J represent the type strains for the susceptibility and the resistance phenotype respectively. During our effort to map the genetic loci that control resistance to the development of lymphoma, the CXB RI strains were typed for lymphoma susceptibility and for simple sequence length polymorphisms (SSLPs). The CXB set consists of 13 strains that derived from the cross of parental BALB/cByJ (C) and C57BL/6ByJ (B) mice: CXBD, CXBE, CXBG, CXBH, CXBI, CXBJ, CXBK, CXBL, CXBN, CXBO, CXBP, CXBQ, and CXBR. At the same time, five inbred strains [BALB/cJ, BALB/cAnNCr, BALB/c -H-2 k (BALB.K), C57BL/lO-H-2 k (B10.BR), and C57BL/lO-H-2 d (B10.D2)] related to the CXB parental strains were also typed for the same SSLP markers. These markers are well spaced (approximately 10 cM apart) and span the mouse genome. Some SSLP mapping had already been accomplished by other groups (Taylor and Reifsnyder 1993; Taylor and Phillips 1995), but in most cases it was applied to the strains CXBD, CXBE, CXBG, CXBH, CXBI, CXBJ, and CXBK. Mapping of other loci (enzymes, endogenous retroviral sequences, etc.) was also mostly limited to these same seven strains. Our goal was to enrich the existing map and increase the usefulness of this RI set. The usefulness of an RI set is considered to be proportional to the genetic differences of the parental strains, the number of strains in the set, and the number and distribution of typed loci. The CXB RI set is biologically interesting since its parental strains show many differences in susceptibility to infection by Leishmania (ShartonKersten and Scott 1995), by cytomegalovirus (Price et al. 1993), by Candida (Afsman 1990), to cutaneous leishmaniasis (Howard et al. 1980), and murine leukemia virus-induced disease (Buller et al. 1990; Avidan et al. 1995). At least some of these traits may be related to the differential production of T helper cell subsets by BALB/c and C57BL/6 mice (Gtiler et al. 1996).

Diubiquitin (Ubd) is a susceptibility gene for virus-triggered autoimmune diabetes in rats.

Genetic studies of type 1 diabetes (T1D) have been advanced by comparative analysis of multiple susceptible and resistant rat strains with a permissive class II MHC haplotype, RT1u. LEW.1WR1 (but not resistant LEW.1W or WF) rats are susceptible to T1D induced by a TLR3 agonist polyinosinic:polycytidylic acid followed by infection with parvovirus. We have mapped genetic loci for virus-induced T1D susceptibility, identifying a major susceptibility locus (Iddm37) near the MHC. The Iddm37 homologs on mouse and human chromosomes are also diabetes linked. We report that a major effect gene within Iddm37 is diubiquitin (Ubd). Gene expression profiling of pancreatic lymph nodes in susceptible and resistant rats during disease induction showed differences in Ubd transcript abundance. The LEW.1WR1 Ubd promoter allele leads to higher inducible levels of UBD than that of LEW.1W or WF. Using zinc-finger nucleases , we deleted a segment of the LEW.1WR1 Ubd gene and eliminated its expression. UBD-deficient rats show substantially reduced diabetes after viral infection. Complementary studies show that there may be another diabetes gene in addition to Ubd in the Iddm37 interval. These data prove that Ubd is a diabetes susceptibility gene, providing insight into the interplay of multiple genes and environmental factors in T1D susceptibility.

Molecular characterization and chromosomal localization of mouse Pur? gene

Purα is a 39‐kDa sequence‐specific single‐stranded DNA/RNA binding protein with the ability to modulate transcription of several genes containing the Pur element in their promoter region. Human and mouse Purα exhibit an extraordinary degree of conservation with only two changes at amino acid residues 49 and 306. A 15‐kb genomic clone encompassing the mouse Purα gene was isolated by screening the mouse genomic library, using a PCR‐amplified fragment from human Purα cDNA. Results from sequencing analysis confirmed the isolated genomic clone to be Purα and not the other members of the Pur family, including Purβ. Characterization of the mouse Purα gene by restriction analysis/Southern blotting and sequencing revealed that the Purα gene contains only one intron within the 5′ UTR and the open reading frame was intact. Using chromosomal markers, the Purα gene was mapped to chromosome 18 in mouse and rat. J. Cell. Biochem. 77:1–5, 2000.