Diabetes and tolerance in transgenic mice expressing class II MHC molecules in pancreatic beta cells

Insulin-dependent diabetes is caused by the loss of insulin-producing beta cells in pancreatic islets. It has been proposed that aberrant expression of Class II Major Histocompatibility Complex (MHC) molecules on beta cells stimulates an autoimmune attack against beta cell antigens. To test this hypothesis, we generated transgenic mice that express Class II MHC molecules (E alpha d/E beta b, or I-Eb) on beta cells. Diabetes was found in 100% of transgenic progeny from three expressing transgenic mouse lines, but without evidence for lymphocytic infiltrates. Furthermore, T lymphocytes appeared to be tolerant to the transgene I-Eb molecule, despite the absence of expression of I-Eb in the thymus or any other lymphoid tissue. The results suggest that novel expression of Class II MHC molecules on nonlymphoid cells is by itself insufficient to initiate autoimmune responses against tissue-specific antigens.     

Restriction site polymorphisms in the pig beta-globin gene cluster

A restriction fragment length polymorphism was detected in pig DNA digested with Hind III restriction endonuclease and probed with rabbit beta 1-globin gene. Eight different phenotypes were observed and for six of them family data demonstrated that they are determined by three alleles. As this polymorphism is not found with four other restriction endonucleases (Bam HI, Eco RI, Kpn I, and Pst I), single point mutations are proposed to explain the observed differences.     

Restriction fragment length polymorphisms detected in N-ras-related sequences of rats and their linkage analyses

Novel restriction fragment length polymorphisms (RFLPs) in inbred rats were revealed with the human N-ras gene as probe. Three fragments hybridizing to the probe were detected by Southern blot hybridization under highly stringent conditions, and one of the fragments showed variation in inbred rat strains. Furthermore, on hybridization under low-stringency conditions, an additional fragment hybridizing to the probe was observed, and this fragment also showed interstrain variation. These two variant fragments showed different distributions in 27 inbred rat strains and segregated in backcross progeny as codominant alleles of independent single autosomal loci. Therefore, the loci for these RFLPs were named Nras-1 and Nras-2, respectively. Analyses of linkages between the RFLPs and 11 other loci revealed that the Nras-2 locus was closely linked to the c locus (3.7 +/- 2.6%), which belongs to rat linkage group I.     

Restriction fragment length polymorphisms of horse class II MHC genes observed using various human alpha-and beta-chain cDNA probes

Genomic DNA isolated from 20 horses was digested with up to six restriction endonucleases and subjected to southern blot hybridization analysis using various human class II alpha- and beta-chain cDNA probes. A high degree of restriction fragment length polymorphism (RFLP) was found for the DQ alpha, DP beta, DQ beta and DR beta probes, about 20 polymorphic bands being detected for each. DR alpha showed 2-4 polymorphic bands, whereas no evidence for DP alpha-like genes was found. A number of correlations of RFLPs with individual alloantisera were apparent.     

Sequence variability analysis of human class I and class II MHC molecules: functional and structural correlates of amino acid polymorphisms

Major histocompatibility complex class I (MHCI) and class II (MHCII) molecules display peptides on antigen-presenting cell surfaces for subsequent T-cell recognition. Within the human population, allelic variation among the classical MHCI and II gene products is the basis for differential peptide binding, thymic repertoire bias and allograft rejection. While available 3D structural analysis suggests that polymorphisms are found primarily within the peptide-binding site, a broader informatic approach pinpointing functional polymorphisms relevant for immune recognition is currently lacking. To this end, we have now analyzed known human class I (774) and class II (485) alleles at each amino acid position using a variability metric (V). Polymorphisms (V>1) have been identified in residues that contact the peptide and/or T-cell receptor (TCR). Using sequence logos to investigate TCR contact sites on HLA molecules, we have identified conserved MHCI residues distinct from those of conserved MHCII residues. In addition, specific class II (HLA-DP, -DQ, -DR) and class I (HLA-A, -B, -C) contacts for TCR binding are revealed. We discuss these findings in the context of TCR restriction and alloreactivity.     

Selective expression of class II E alpha d gene in transgenic mice

Class II genes of the MHC must be expressed by APC for activation of CD4+ T cells and efficient delivery of T cell help to B lymphocytes. Class II genes have restricted tissue expression and are under complex regulation. By using various deletion constructs of the class II E alpha d gene in transgenic mice we have mapped different 5' flanking regions which control E alpha d gene expression in distinct cell types. We demonstrate dissociate expression of E alpha d within the macrophage lineage as well as within the B cell lineage, and present evidence for a repressive element operative in B cells and macrophages. We describe the generation of novel transgenic lines with limited constitutive and inducible E alpha mRNA and I-E protein.     

Helicobacter-induced gastritis in mice not expressing metallothionein-I and II

Background. Helicobacter pylori a primary cause of gastritis and peptic ulcer disease, is associated with increased production of reactive oxygen species within the gastric mucosa. Metallothionein (MT), a low‐molecular‐weight, cysteine‐rich, metal‐binding ligand, has been shown to sequester reactive oxygen species and reduce tissue damage. This study investigates the role of MT in H. pylori‐induced gastritis in mice. Materials and Methods. Control (MT+/+) and MT‐null (MT–/–) mice were inoculated with either 1 × 10⁸H. pylori or H. felis, and were infected for 4, 8 and 16 weeks or 8 weeks, respectively. H. pylori load was determined by culture. Myloperoxidase activity and MT levels were also determined. Results. The stomachs of H. felis‐infected mice were more severely inflamed than those of H. pylori‐infected mice. H. felis‐induced gastritis was more severe (p = .003) in MT–/– than in MT+/+ mice. MT–/– mice also had higher (60%; p < .05) H. pylori loads than MT+/+ mice 4 weeks after infection but not 8 or 16 weeks after infection. Myloperoxidase activity with H. pylori was similar between MT+/+ and MT–/– mice. Thirty‐three per cent greater (p < .05) myloperoxidase activity was observed in MT–/– than in MT+/+ mice infected with H. felis. In MT+/+ mice infected with H. pylori, liver MT was increased by 33 and 39% (p < .05) at 8 and 16 weeks, respectively, whereas gastric MT increased by 46% (p < .05) at 4 weeks and declined to baseline levels at 8 and 16 weeks. Conclusions. Mice lacking MT are more susceptible to H. pylori colonization and gastric inflammation, indicating that MT may be protective against H. pylori‐induced gastritis.     

Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP

We generated transgenic mice in which red, green, yellow, or cyan fluorescent proteins (together termed XFPs) were selectively expressed in neurons. All four XFPs labeled neurons in their entirety, including axons, nerve terminals, dendrites, and dendritic spines. Remarkably, each of 25 independently generated transgenic lines expressed XFP in a unique pattern, even though all incorporated identical regulatory elements (from the thyl gene). For example, all retinal ganglion cells or many cortical neurons were XFP positive in some lines, whereas only a few ganglion cells or only layer 5 cortical pyramids were labeled in others. In some lines, intense labeling of small neuronal subsets provided a Golgi-like vital stain. In double transgenic mice expressing two different XFPs, it was possible to differentially label 3 neuronal subsets in a single animal.     

Restriction fragment length polymorphisms at the growth hormone gene in pigs

Pigs from four Danish and two Swedish populations were examined for restriction fragment length polymorphism (RFLP) at the growth hormone (GH) gene. Polymorphism was detected with the restriction enzymes DraI and TaqI. A comparison of the Danish populations showed significant differences among their allelic frequencies.     

Restriction fragment length polymorphisms in bovine major histocompatibility complex class II ß-chain genes using bovine exon-containing hybridization probes

Restriction fragment length polymorphisms (RFLPs) have been identified in the bovine MHC class II region using five hybridization probes constructed from two bovine genomic clones. Four probes were constructed from a bovine DR beta-like gene, BoLA-DRB2. These included a probe containing the complete beta 1 exon (R2-beta 1), a probe containing the last 129 base pairs of the beta 2 exon (R2-beta 2), a probe containing intron immediately 5' of the beta 2 exon (R2-5' beta 2), and a probe containing the complete transmembrane exon (R2-TM). A fifth probe was constructed from a novel bovine beta-chain gene, BoLA-DIB, and contained the entire TM exon (I1-TM). R2-beta 1 defined very little polymorphism. R2-beta 2 hybridized to several fragments but one or two fragments hybridized much stronger on all Southern blots and it was presumed these corresponded to BoLA-DRB2 fragments. By using R2-5' beta 2 as a probe, these BoLA-DRB2 fragments were confirmed: 6.4 and 2.7-kb Eco RI alleles, 1.7- and 1.5-kb Pvu II alleles, 5.9-, 5.4-, 3.7- and 1.9-kb TaqI alleles, and a non-polymorphic 22.5-kb BamHI fragment. I1-TM identified three alleles with TaqI. To investigate the linkage between the RFLP alleles, 166 offspring of five sires were tested. Complete linkage was found for all RFLPs identified with the BoLA-DRB2 probes. However, the RFLP patterns of 13 calves out of 58 indicated recombination between BoLA-DRB2 and BoLA-DIB.     

Association of MHC class I and class II gene polymorphisms with vaccine or challenge response to Salmonella enteritidis in young chicks

Salmonella enteritidis (SE) is a worldwide source of salmonellosis in humans, caused mainly by consumption of contaminated poultry products. The major histocompatibility complex (MHC) class I and class II molecules, which function as antigen-presentation structures, are involved in both macrophage and dendritic cell immune response to Salmonella and may, therefore, play an important role in host resistance to infections. The chicken MHC class I and class II were investigated as candidate genes for immune response to SE. We characterized the complete MHC class I cDNA sequences for an outbred broiler line and four diverse highly inbred lines: two MHC-congenic Leghorn (G-B2 and G-B1), one Egyptian Fayoumi (M15.2), and one Spanish (Sp21.1), to define the allelic sequences within these lines, so that we might study the association between particular MHC polymorphisms and response to SE. The F1 offspring of outbred broiler sires crossed with three inbred lines (G-B1, G-B2, and Fayoumi) were evaluated as young chicks for either bacterial load in spleen and cecum after pathogenic SE inoculation or antibody level after SE vaccination. Alleles defined by a Lys(148)-->Met(148) polymorphism in the MHC class I alpha(2) domain were associated with spleen bacterial load after SE challenge. These results suggest that particular MHC haplotypes may contribute to control of responses to SE, and that particular polymorphisms may serve as markers for genetic resistance to SE in the chicken.     

Restriction fragment length polymorphisms of a chloroplast photosystem II gene from poplar and their use for species identification.

In a total DNA library from the poplar clone Beaupré (Populus trichocarpa x P. deltoides) one DNA clone was found to identify restriction site polymorphisms in different poplar species. This clone represents a cpDNA gene that shows close homology to a photosystem II gene of pea and spinach coding for the D2 protein and the 44 kDa reaction centre. In Southern blot analysis this probe identified interspecific restriction site variation among the different poplar species; intraspecific variation was not detectable. As the chloroplast genome is maternally inherited in poplars this cpDNA probe was used for identification of P. nigra or P. deltoides as the seed parents of F1 hybrid trees in natural stands of western Germany.     

T cell development in mice expressing CD1d directed by a classical MHC class II promoter

CD1d and nonclassical MHC molecules differ markedly from classical MHC ligands in their ability to promote the selection and differentiation of developing T cells. Whereas classical MHC-restricted T cells have a predominantly naive phenotype and a broad TCR repertoire, most other T cells have a memory and/or NKT phenotype with a restricted repertoire. Because the nonclassical ligands selecting these memory-type cells are expressed by bone marrow-derived cells, it has been suggested that the development of large repertoires of naive-type cells was dependent on the classical MHC expression pattern in the thymus cortex, high on epithelial cells and low on cortical thymocytes. We redirected CD1d expression using the classical MHC II Ealpha promoter. pEalpha-CD1d mice lacked memory-type NKT cells, but, surprisingly, they did not acquire the reciprocal ability to select a diverse population of naive CD1d-restricted cells. These findings suggest that, whereas the development of NKT cells is dependent on the pattern of CD1d expression, the absence of a broad, naive CD1d-restricted T cell repertoire may reflect intrinsic limitations of the pool of TCR genes or lipid Ags.     

Transgenic wheat expressing a barley class II chitinase gene has enhanced resistance against Fusarium graminearum

Fusarium head blight (FHB; scab), primarily caused by Fusarium graminearum, is a devastating disease of wheat worldwide. FHB causes yield reductions and contamination of grains with trichothecene mycotoxins such as deoxynivalenol (DON). The genetic variation in existing wheat germplasm pools for FHB resistance is low and may not provide sufficient resistance to develop cultivars through traditional breeding approaches. Thus, genetic engineering provides an additional approach to enhance FHB resistance. The objectives of this study were to develop transgenic wheat expressing a barley class II chitinase and to test the transgenic lines against F. graminearum infection under greenhouse and field conditions. A barley class II chitinase gene was introduced into the spring wheat cultivar, Bobwhite, by biolistic bombardment. Seven transgenic lines were identified that expressed the chitinase transgene and exhibited enhanced Type II resistance in the greenhouse evaluations. These seven transgenic lines were tested under field conditions for percentage FHB severity, percentage visually scabby kernels (VSK), and DON accumulation. Two lines (C8 and C17) that exhibited high chitinase protein levels also showed reduced FHB severity and VSK compared to Bobwhite. One of the lines (C8) also exhibited reduced DON concentration compared with Bobwhite. These results showed that transgenic wheat expressing a barley class II chitinase exhibited enhanced resistance against F. graminearum in greenhouse and field conditions.     

Major histocompatibility complex class II polymorphisms in humans and chimpanzees

Allelic variation at the MhcPatr-DR and -DQ loci was studied by molecular biological techniques and compared to available HLA data. With regard to the number of allelic lineages, the chimpanzee shows a condensation of its major histocompatibility complex (MHC) class II repertoire as compared to humans. This does not have an impact on the overall degree of MHC class II polymorphism in the chimpanzee since a few lineages that are oligomorphic in humans display an extensive degree of polymorphism in the chimpanzee.     

Restriction site polymorphisms in the pig β-globin gene cluster

A restriction fragment length polymorphism was detected in pig DNA digested with Hind III restriction endo nuclease and probed with rabbit β₁-globin gene. Eight different phenotypes were observed and for six of them family data demonstrated that they are determined by three alleles. As this polymorphism is not found with four other restriction endo nucleases (Bam HI, Eco RI, Kpn I, and Pst I), single point mutations are proposed to explain the observed differences.     

Regulation of class II MHC gene expression by macrophages from Bcgr and Bcgs mice

The presence of class II mRNA was determined following stimulation of macrophages from Bcgr and Bcgs mice with rIFN-gamma. Despite the continuous expression of surface I-A glycoprotein by macrophages from Bcgr mice, class II mRNA was no longer present. The transient expression of I-A by macrophages from Bcgs mice, however, was accompanied by the disappearance of class II mRNA from the cells. Restimulation of macrophages from Bcgs mice, with rIFN-gamma resulted in the reappearance of class II mRNA and surface I-A expression. The reappearance of class II mRNA and the surface expression of I-A glycoprotein was inhibited by PGE2. These results indicate that differences in I-A expression by macrophages from Bcgr and Bcgs are not at the level of class II gene expression.     

T cells that recognize peptide sequences of self MHC class II molecules exist in syngeneic mice.

T cell reactivity toward self MHC class II molecules has been recognized in syngeneic MLR in a number of studies, where the T cells are believed to recognize the combination of self/nonself peptide and self MHC molecule. We investigated the stimulation of T cell proliferation by synthetic peptides of sequences corresponding to the first polymorphic amino terminal domain of alpha- and beta-chains of self I-A molecules. Both unprimed and primed T cells responded to a number of peptides of alpha 1 and beta 1 domains of self I-Ad molecules. The response was dependent on the presentation of I-Ad peptides by syngeneic APC and was blocked by anti-class II MHC mAb. Upon further investigation it was observed that I-Ad peptides could inhibit the stimulation of Ag-specific MHC class II-restricted T cell hybridoma due to self presentation of peptides rather than to direct binding of free peptides to the TCR, further supporting their affinity/interaction with intact self MHC class II molecules. The peptide I-A beta d 62-78 showed high affinity toward intact self MHC II molecule as determined by the inhibition of Ag-specific T cell stimulation and yet was nonstimulatory for syngeneic T cells, therefore representing an MHC determinant that may have induced self tolerance. Thus we have shown that strong T cell proliferative responses can be generated in normal mice against the peptides derived from self MHC class II molecules and these cells are part of the normal T cell repertoire. Therefore complete tolerance toward potentially powerful immunodominant but cryptic determinants of self Ag may not be necessary to prevent autoimmune diseases.     

Inhibition of class II MHC gene expression by anti-sense RNA in transgenic mice.

We have established transgenic mice carrying the anti-sense DNA to the gene encoding beta chain of the class II major histocompatibility complex (I-A) molecule. The amount of I-A molecule on splenic B lymphocytes from the mice was reduced in the presence of a large amount of the exogenous anti-sense RNA. The amount of I-A beta chain RNA was selectively reduced and inversely correlated with the amount of anti-sense RNA in the spleens. These results suggest that the I-A beta chain RNA is rapidly degraded by duplex formation with the anti-sense RNA in splenic B cells from the transgenic mice.