Polymorphisms in the taste receptor gene (Tas1r3) region are associated with saccharin preference in 30 mouse strains.

The results of recent studies suggest that the mouse Sac (saccharin preference) locus is identical to the Tas1r3 (taste receptor) gene. The goal of this study was to identify Tas1r3 sequence variants associated with saccharin preference in a large number of inbred mouse strains. Initially, we sequenced approximately 6.7 kb of the Tas1r3 gene and its flanking regions from six inbred mouse strains with high and low saccharin preference, including the strains in which the Sac alleles were described originally (C57BL/6J, Sac(b); DBA/2J, Sac(d)). Of the 89 sequence variants detected among these six strains, eight polymorphic sites were significantly associated with preferences for 1.6 mm saccharin. Next, each of these eight variant sites were genotyped in 24 additional mouse strains. Analysis of the genotype-phenotype associations in all 30 strains showed the strongest association with saccharin preference at three sites: nucleotide (nt) -791 (3 bp insertion/deletion), nt +135 (Ser45Ser), and nt +179 (Ile60Thr). We measured Tas1r3 gene expression, transcript size, and T1R3 immunoreactivity in the taste tissue of two inbred mouse strains with different Tas1r3 haplotypes and saccharin preferences. The results of these experiments suggest that the polymorphisms associated with saccharin preference do not act by blocking gene expression, changing alternative splicing, or interfering with protein translation in taste tissue. The amino acid substitution (Ile60Thr) may influence the ability of the protein to form dimers or bind sweeteners. Here, we present data for future studies directed to experimentally confirm the function of these polymorphisms and highlight some of the difficulties of identifying specific DNA sequence variants that underlie quantitative trait loci.

Positional cloning of the mouse saccharin preference (Sac) locus.

Differences in sweetener intake among inbred strains of mice are partially determined by allelic variation of the saccharin preference (Sac) locus. Genetic and physical mapping limited a critical genomic interval containing Sac to a 194 kb DNA fragment. Sequencing and annotation of this region identified a gene (Tas1r3) encoding the third member of the T1R family of putative taste receptors, T1R3. Introgression by serial backcrossing of the 194 kb chromosomal fragment containing the Tas1r3 allele from the high-sweetener-preferring C57BL/6ByJ strain onto the genetic background of the low-sweetener-preferring 129P3/J strain rescued its low-sweetener-preference phenotype. Polymorphisms of Tas1r3 that are likely to have functional significance were identified using analysis of genomic sequences and sweetener-preference phenotypes of genealogically distant mouse strains. Tas1r3 has two common haplotypes, consisting of six single nucleotide polymorphisms: one haplotype was found in mouse strains with elevated sweetener preference and the other in strains relatively indifferent to sweeteners. This study provides compelling evidence that Tas1r3 is equivalent to the Sac locus and that the T1R3 receptor responds to sweeteners.

Cutting edge: introduction of an endopeptidase cleavage motif into a determinant flanking region of hen egg lysozyme results in enhanced T cell determinant display.

The choice of which determinants of a whole Ag will be presented on cell surface MHC class II molecules after uptake and processing by APC is the result of the interplay between structural characteristics of the Ag and the processing machinery of the APC. In this study, we demonstrate that introduction of a dibasic motif adjacent to a subdominant determinant enhances the presentation of this determinant from the whole molecule. This is the first report showing that a single amino acid substitution in a whole Ag, designed to introduce an endopeptidase recognition site, enhances display of class II-restricted determinants, most likely by creating a peptide chain cleavage in the antigenic molecule. Our findings have important implications for the understanding of immunodominance and for vaccine design.

Mouse genetics/genomics: an effective approach for drug target discovery and validation.

The mouse has become the premier mammalian system for the identification of the genetic basis of both mono- and oligogenic disorders, as well as the understanding of complex diseases with gene-gene and gene-environment interactions. The similarity between human and mouse genetic disease is sometimes striking, while in other cases the phenotypes are less similar. The ability to genetically map and then clone single gene disorders rapidly, and the emerging technologies that will allow the economical identification of the polygenes controlling quantitative traits further demonstrate the utility of the mouse as a model for gene discovery. Additionally, the ability to genetically manipulate the mouse through transgenesis and gene targeting allows for the testing of hypotheses regarding specific gene function and their role in disease. The utility of the mouse extends beyond being just a gene discovery tool to provide prevalidated targets. It can also be used for the development of animal models, and the testing of compounds in specifically constructed transgenic and knockout strains to further define the target and pathway of a therapeutic compound.

A genome-wide search identifies potential new susceptibility loci for Crohn's disease.

Chronic inflammatory bowel disease (IBD) presents as two major clinical forms, Crohn's disease (CD) and ulcerative colitis (UC). Genetic epidemiological studies and animal models suggest that inherited factors play significant roles in the susceptibility to both forms of IBD. From four genome-wide scans, putative susceptibility loci on chromosome 16 (IBD1 for CD), and on chromosomes 1, 3, 4, 6, 7, 10, and 12 for IBD, have been identified. Several other groups, including ours, have confirmed linkage to the loci on chromosomes 12 and 16. The aim of this study is to identify other potential susceptibility loci for CD with a genome-wide search approach. In our sample of 222 individuals from 46 families (20 Jewish and 26 non-Jewish), with a total of 65 sibpairs diagnosed with CD, we observed a novel locus with suggestive linkage [multipoint logarithm of the odds score (Mlod) > 2] at chromosome 14q11.2 (Mlod = 2.8, p = 0.0002). In addition, suggestive linkage was observed in our Jewish families at chromosome 17q21-q23 (Mlod = 2.1, p = 0.01) and chromosome 5q33-q35 (Mlod = 2.2, p = 0.0003). The syntenic regions of the latter locus are mapped within two putative loci on mouse chromosomes 11 and 18, which were identified in a mouse IBD model induced by dextran sulfate sodium (29). Our preliminary results provide potential evidence for several susceptibility loci contributing to the risk of CD. The observation of man-mouse synteny may accelerate the identification of CD susceptibility gene(s) on human chromosome 5.

Additional evidence of linkage between Crohn's disease and a putative locus on chromosome 12.

PURPOSE: The inflammatory bowel diseases (IBD), Crohn's disease (CD), and ulcerative colitis (UC) are chronic intestinal disorder of unknown etiology. Genetic factors play an important role in the pathogenesis of these diseases, but with a complex pattern of inheritance. A number of genome-wide scans have identified several putative susceptibility loci for both CD and UC, including a locus on chromosome 12 reported in a set of British families. We aim to evaluate the linkage between CD or UC and this chromosome 12 locus in an independent set of U.S. Caucasian families (36% being of Ashkenazi Jewish origin). METHODS: Microsatellite markers along chromosome 12 spaced at approximately 10 cm intervals were used to test the putative loci in CD only families (65 sib pairs from 46 families). Regions with positive linkage for CD were then tested in a panel of UC and mixed families (44 sib pairs from 29 families). Two point linkage analysis was performed with SIBPAL. Multipoint linkage analysis was carried out with MAPMAKER/SIBS. RESULTS: We observed evidence of linkage between the region on chromosome 12 and Crohn's disease, because there was a significant excess of allele sharing in CD sib pairs (pi = 0.62, p = 0.0004 from two-point linkage; and logarithm of the odds score (LOD) = 2.0 from multipoint linkage). However, we did not observe the same linkage in UC and mixed families (p = 0.48; not significant [ns]). CONCLUSION: Our data provided further evidence that the region on chromosome 12 is likely to contain a gene predisposing to CD.

Determinants of T cell reactivity to the Mycobacterium leprae GroES homologue.

The 10-kDa protein Ag of Mycobacterium leprae, a human GroES hsp10 cognate, is a major T cell Ag in human leprosy infection. We investigated the mechanism for T cell responsiveness to this Ag according to the trimolecular interaction between T cell, peptide, and Ag-presenting element. This research was accomplished by mapping T cell epitopes in leprosy patients and correlating these responses with peptide-MHC binding affinities. We found that the majority of tuberculoid leprosy patients responded to peptides corresponding to residues 25-39 and 28-42. Truncation analysis of these peptides mapped the exact epitope to be within the overlapping region comprising residues 28-39. Responsiveness was correlated with the HLA-DRB5*0101 allele, which bound the peptides with moderate affinity. This allele is linked to HLA-DR2, which is associated with the resistant form of leprosy. Therefore, T cell responsiveness in tuberculoid leprosy may be mediated by the ability of HLA-DRB5*0101 to bind and present peptides of the immunodominant 10-kDa Ag.

Susceptibility locus for inflammatory bowel disease on chromosome 16 has a role in Crohn's disease, but not in ulcerative colitis.

In the Western world, chronic inflammatory bowel disease (IBD) presents as two major clinical forms, Crohn's disease (CD) and ulcerative colitis (UC) [Targan, S.R. and Shanahan, F. (1994). In Retford, D.C (ed.), Inflammatory Bowel Disease: From Bench to Bedside. Williams and Wilkins, Baltimore]. Genetic epidemiological studies, the occurrence of rare syndromes associated with IBD, and animal models suggest that inherited factors play significant roles in the susceptibility to both forms of IBD [Yang, H.-Y. and Rotter, J.I. (1995) In Kirsner, J.B. and Shorter, R.G. (eds). Genetic Aspects of Idiopathic Inflammatory Bowel Disease. Williams and Wilkins, Baltimore, pp.301-331]. Recently, a genome-wide search on European families with multiple affected members with CD identified a putative susceptibility locus in the centromeric region of chromosome 16 [Hugot, J.-P. et al. (1996) Nature, 379, 821-823]. We have now tested this region in an independent set of US families, confirmed that this region is likely to contain a gene predisposing to CD, and further refined the chromosomal location of this gene. Most importantly with respect to this locus, our data also seem to indicate that there is heterogeneity both within the CD group, and between the CD and UC groups with respect to this locus. The susceptibility locus appears to be involved only in non-Jewish CD sibpairs and not in our Ashkenazi Jewish CD sibpairs. Additionally, we have tested sibpairs having either only UC or both UC and CD for involvement of this locus, and have found no evidence that this region predisposes to IBD in these patients.

Overexpression of IL-10 in atopic dermatitis. Contrasting cytokine patterns with delayed-type hypersensitivity reactions.

The skin lesions of patients with atopic dermatitis provide a model to study immunoregulation in human allergy. To determine the local cytokine pattern of cells present (both endogenous and recruited) at the site of disease, we extracted RNA from skin biopsy specimens from patients with atopic dermatitis, allergic contract dermatitis, and positive tuberculin reactions and used PCR to assay for cytokine mRNA. cDNAs were normalized to the intensity of the CD3 delta PCR product as a marker of T cell mRNA. We found overexpression of IL-10 mRNA in atopic dermatitis lesions, in comparison with allergic contact dermatitis lesions and tuberculin reactions. In contrast, IL-4 mRNA was most strongly expressed in allergic contact dermatitis lesions and IFN-gamma mRNA was the predominant cytokine in tuberculin reactions. Using an anti-IL-10 mAb with immunoperoxidase, we localized IL-10 protein to large mononuclear cells in the dermal infiltrate of atopic lesions. After immunomagnetic sorting of mononuclear cell populations from PBMC of atopic dermatitis subjects, IL-10 mRNA as measured by PCR was found to be strongly expressed in CD14+ cells. Spontaneous release of IL-10 from PBMC-derived adherent cells was greater in atopic dermatitis donors than normal controls. We therefore renormalized skin biopsy cDNA according to the level of beta-actin PCR product, as a marker of total cellular mRNA, and found by PCR that IL-10 was nevertheless greatest in atopic dermatitis subjects. We conclude that the relative overexpression of IL-10 in atopic dermatitis greatest in atopic dermatitis subjects. We conclude that the relative overexpression of IL-10 in atopic dermatitis may contribute to the up-regulation of humoral responses and the down-regulation of Th1 responses.

Multiple sclerosis: limited diversity of the V delta 2-J delta 3 T-cell receptor in chronic active lesions.

T lymphocytes bearing the gamma delta T-cell receptor have been found in the central nervous system of patients with multiple sclerosis in association with demyelinated lesions. Although the biological function of these cells remains to be established, it has been proposed that they are involved in the response to highly conserved antigens, such as heat shock proteins (hsp), expressed during tissue damage and thus may contribute to the development of an autoimmune response. Using polymerase chain reaction, we probed for the presence of T-cell receptor gamma delta cells in fresh-frozen early autopsy brain tissue from patients with multiple sclerosis and patients with non-multiple sclerosis conditions. The results demonstrated the presence of two major V-J combinations of the T-cell receptor delta chain--V delta 2-J delta 3, V delta 2-J delta 1--and we used a direct sequencing technique to determine whether this gamma delta T-cell population was clonal or diverse. In chronic-active plaques from 9 patients with multiple sclerosis, we found a striking predominant gene rearrangement within the V delta 2-J delta 3 T-cell receptor population that was not present in central nervous system tissue from patients with other neurological diseases. In contrast, within the V delta 2-J delta 1 T-cell receptor population, a predominant rearrangement pattern was detected in only 1 of the multiple sclerosis patients. The sequence of the predominant V delta 2-J delta 3 gene rearrangement was confirmed by cloning and sequencing the gene products from 1 multiple sclerosis patient.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective accumulation of T cells according to T-cell receptor V beta gene usage in skin cancer.

To investigate whether specific T-cell populations are overrepresented in tumor-infiltrating lymphocytes (TIL) in skin cancer, we determined the T-cell receptor (TCR) diversity in biopsy specimens of basal cell carcinoma and squamous cell carcinoma. Immunostaining of tissue sections indicated that the majority of T cells expressed alpha beta TCRs. To assess diversity of the TCR beta chain, RNA was isolated directly from the tumor specimens and peripheral blood mononuclear cells (PBMC) from the same patient, cDNA was synthesized, and variable (V) beta chain gene usage was determined by the polymerase chain reaction (PCR). In each basal cell (n = 11) and squamous cell (n = 7) carcinoma studied, several V beta families were overrepresented in TIL versus PBMC, in that they accounted for greater than 5% of the repertoire in TIL and were at least 2% higher in TIL than in PBMC. The predominant V beta gene segments overrepresented in TIL generally differed from individual to individual. Simultaneous comparison of the V beta repertoire of TIL to that of uninvolved skin and PBMC from the same individual revealed preferential expression of V beta families within the TIL in three of five basal cell and four of four squamous cell carcinomas. Again, the predominant V beta s differed from individual to individual. Comparison of the TCR repertoire in uninvolved skin versus PBMC did indicate that some V beta families were overexpressed in the resident T-cell compartment in skin, although the overrepresented families were not constant from individual to individual. These data indicate the selective concentration of T cells bearing specific alpha beta TCRs in the local immune response to basal cell and squamous cell carcinomas.

Evidence for a superantigen in human tuberculosis.

T cells are not only required for resistance to tuberculosis, but they likely contribute to the tissue damage characteristic of the disease. To define better the T cell populations that contribute to the immunopathogenesis of human tuberculosis, we investigated the T cell receptor (TCR) beta chain repertoire expressed in patients with tuberculous pleuritis. Analysis by polymerase chain reaction and flow cytometry indicated an expansion of V beta 8+ T cells at the site of disease in some donors, suggesting the possibility that Mycobacterium tuberculosis contains a superantigen. M. tuberculosis induced strong T cell proliferative responses in tuberculin-negative healthy donors in vitro, with preferential expansion of V beta 8+ T cells, independent of the CDR3 region. T cell stimulation was MHC class II-dependent and did not require antigen processing by the antigen-presenting cells. These findings are consistent with the presence of a superantigen in M. tuberculosis, aspects of which may contribute to the immunopathology of tuberculosis and to the adjuvant properties of M. tuberculosis.

T cells bearing V beta 6 T cell receptors in the cell-mediated immune response to Mycobacterium leprae.

The skin lesions of leprosy provide a window into the immunoregulatory events involved in the human immune response to infection. T cells are thought to play a vital role in the pathogenesis of different forms of the disease. To identify predominant specific T cell subpopulations in leprosy lesions, the TCR-beta chain repertoire was simultaneously studied in skin biopsy specimens and PBMC from both immunologically resistant tuberculoid leprosy and susceptible lepromatous leprosy patients. This was accomplished by obtaining RNA from lesions and PBMC, synthesizing cDNA, and performing the polymerase chain reaction. We found that TCR gene subfamilies V beta 6.1 through V beta 6.4 (V beta 6.1-4) were strikingly overrepresented in lesions vs PBMC of seven of nine tuberculoid patients but only one of nine lepromatous patients. Similarly, V beta 6.5/6.8/6.9 subfamilies were predominant in four of nine tuberculoid patients, but none of the nine lepromatous patients. To explore the influence of the complementarity-determining region 3 (CDR3) in selection of T cells expressing V beta 6 TCR, we sequenced the V beta 6.1-4-C beta polymerase chain reaction products derived from the lesions and PBMC of two tuberculoid patients. From the analysis of deduced amino acid sequences, we found conserved amino acid residues and amino acid motifs in the CDR3 region of the lesion-derived sequences from each patient. Our data suggest that the nominal Ag select T cells bearing V beta 6 TCR in the cell-mediated immune response to Mycobacterium leprae.

Local expression of antiinflammatory cytokines in cancer.

To characterize the nature of the local cytokine response to cancer, we chose to investigate cytokine patterns in biopsy specimens of basal cell carcinoma (BCC). We hypothesized that a distinct pattern of local cytokine production may be characteristic of BCC, a neoplasia of epidermis, in comparison to the pattern of seborrheic keratosis (SK), a benign growth of epidermis. We analyzed cytokine mRNAs in BCC versus SK by performing polymerase chain reaction on mRNA derived from biopsy specimens. The mRNAs encoding cytokines for IL-4, IL-5, IL-10, and granulocyte macrophage colony-stimulating factor were strongly expressed in BCC lesions and weakly expressed in SK lesions. Conversely, IL-2, IFN-gamma, and lymphotoxin mRNAs were strongly expressed in SK lesions and weakly expressed in BCC lesions. The response to malignancy, BCC, was typified by cytokines characteristic of murine TH2 cells. This cytokine pattern favors humoral immunity with concomitant immunosuppression of cell-mediated immune responses. In comparison, the response to the benign growth, SK, was typified by cytokines characteristic of murine TH1 cells that favor cell-mediated immune reactions. The findings of a distinct cytokine pattern in skin cancer provide a framework to develop strategies for immunologic intervention.

Selection of T lymphocytes bearing limited T-cell receptor beta chains in the response to a human pathogen.

Delayed-type hypersensitivity (DTH) is a classic measure of T-cell responsiveness to foreign antigen. To estimate the extent of the T-cell repertoire in the DTH response to a human pathogen, we measured T-cell receptor (TCR) beta-chain variable-region (V beta) gene usage in reversal reactions in leprosy. Reversal reactions represent naturally occurring DTH responses in leprosy, in which augmentation of T-cell responses to Mycobacterium leprae is concomitant with clearance of bacilli from lesions. T cells using the V beta 6-, V beta 12-, V beta 14-, and V beta 19-encoded TCRs were strikingly overrepresented in the lesions of patients as compared to blood and pre-DTH lesions from the same individuals. Furthermore, these data indicate a possible association between the predominant expression of a V beta gene segment in lesions and the major histocompatibility complex class II haplotype of the individual. V beta 6 was prominent in the lesions of four patients who were DR15, a marker of resistance in leprosy infection. Sequence analysis of V beta 6 TCRs showed frequent use of V beta 6.1 and J beta 2.7 gene segments and a conserved amino acid motif in the V-J junction in a reversal-reaction lesion, but not in blood from the same patient. The limited TCR repertoire expressed by the infiltrating T cells suggests that a limited set of antigens is recognized in the DTH response to a human pathogen. We suggest that the mechanism by which major histocompatibility complex haplotype influences DTH in this disease involves the presentation of specific peptides, with subsequent selection of specific TCRs followed by local oligoclonal expansion.

Selective expansion of V delta 1 + T cells from leprosy skin lesions.

T cells bearing gamma delta T-cell receptors (TCRs) are prominent residents of murine epidermis and appear to be important participants in the immune response to infection in human skin. The Mitsuda reaction in leprosy, induced by intradermal challenge with Mycobacterium leprae, provides an opportunity to study the cellular events that mediate a form of delayed-type hypersensitivity (DTH) in skin. T cells bearing gamma delta TCRs comprise a significant proportion of the T-cell population in these DTH reactions. Presently we have generated T-cell lines from Mitsuda reactions in vitro and compared their TCR repertoire to that found in situ. gamma delta T cells comprised 20-40% of lines derived from these skin lesions, but < 10% of lines derived from the peripheral blood of the same individuals. Flow-cytometric analysis of variable (V) chain usage in T-cell lines derived from skin lesions indicated that V delta 1 was predominant. Evaluation of the TCR repertoire using PCR indicated that V delta 1-J delta 1 and V gamma 2-J gamma P gene rearrangements were prevalent. In comparison, V delta 2-J delta 1 gene rearrangements predominated in situ. Furthermore, nucleotide sequence analysis of the V-J junction of one T-cell line revealed limited genetic diversity of the gamma delta TCR. These findings suggest that the V delta 1 subpopulation of gamma delta T cells in Mitsuda skin reactions selectively outgrows from leprosy skin lesions in vitro. Such V delta 1 + T-cell lines should be useful for determining the relevant antigens and restriction elements in this response to a pathogen in skin.

Limited T-cell receptor beta-chain diversity of a T-helper cell type 1-like response to Mycobacterium leprae.

Delayed-type hypersensitivity (DTH) is the standard measure of T-cell responsiveness to infectious organisms. For leprosy, the Mitsuda reaction, a local immune response to cutaneous challenge with Mycobacterium leprae, is considered to represent a measure of DTH against the pathogen. We analyzed the diversity of the T-cell receptor beta-chain repertoire in Mitsuda reactions to determine the breadth of the mycobacterial antigens involved. The polymerase chain reaction was used to compare V beta usage in the Mitsuda reaction T-cell lines established and unstimulated peripheral blood. These molecular analyses revealed a skewed T-cell receptor V beta gene usage in the Mitsuda reaction and in T-cell lines from lesions. To examine the reactivity of T cells from these lesions, T-cell lines were tested against the available native and recombinant antigens of M. leprae. T-cell lines derived from Mitsuda reactions responded more strongly to the 10-kDa M. leprae antigen, a homolog of GroES in Escherichia coli, than to other M. leprae proteins. T-cell lines were also shown to proliferate strongly in response to the 17- and 3-kDa proteins. The pattern of the lymphokine mRNA of these cells was reminiscent of the pattern of murine TH1 cells, positive for interleukin-2 and gamma interferon and weakly positive for interleukin-4. These data indicate that a limited array of T cells, perhaps recognizing stress proteins, secrete a type 1 lymphokine profile in the DTH response to mycobacteria.

Cytokine patterns of immunologically mediated tissue damage.

Reactional states in leprosy are produced by different immunologic mechanisms and are responsible for a major component of tissue damage of the disease. Reversal reactions exhibit increased CD4 T cell infiltration in lesions and augmented cell-mediated immune reactivity to Ag of Mycobacterium leprae that can rapidly produce nerve damage. Erythema nodosum leprosum (ENL) reactions also have CD4 T cell infiltration but appear to be associated with the formation of immune complexes that are responsible for panniculitis, arthritis, vasculitis, and nerve injury. Because these reactional states may serve as paradigms for other types of human immunologically mediated tissue damage, this study sought to characterize the dynamic changes in cytokines associated with these reactions. Expression of cytokine mRNA in lesions of leprosy reactional states were measured by PCR. In reversal reactions, IL-1 beta, TNF-alpha, IL-2, and IFN-gamma mRNA were prominent and found to increase during the reaction, concomitant with decreases in expression of mRNA for IL-4, IL-5, and IL-10. In ENL, selective increases in the expression of IL-6, IL-8, and IL-10 mRNA was observed, with persistent expression of IL-4 and IL-5 mRNA. Reversal reactions represent naturally occurring delayed-type hypersensitivity reactions that favor macrophage activation and protective immunity, but which can engender concomitant cell injury. In contrast, ENL lesions represent immediate-type hypersensitivity reactions reflecting the selective stimulation of cytokines that attract neutrophils, stimulate antibody production, and down-regulate macrophage activation. The analysis of cytokine dynamics within different inflammatory responses can provide insights into immune mechanisms of tissue damage, and provide a useful framework for developing strategies for therapeutic intervention.