RFLP is an abbreviation of restriction fragment length polymorphism. It is a commonly employed tool to check the small but specific variations in a sequence of double-stranded DNA. It is based on the specificity of restriction endonucleases, which recognize a set of nucleotides called restriction site and cleave the DNA at those sites. A specific RFLP pattern emerges on electrophoretic separation of digested DNA, producing variable lengths of cleavage fragments which are characteristic of a sequence of DNA. Individuals, species, or organisms can be distinguished on the basis of RFLP pattern. RFLP are widely used in genotyping, DNA fingerprinting, mapping of genes, and diagnosis of genetic disorders.
Three infertile subjects with no expression of CD46 (membrane cofactor protein of complement) on their spermatozoa were found when screening 542 idiopathic male infertile patients. The sperm CD46 isoform was reported to be associated with the sperm-egg interaction, yet a ubiquitous expression of CD46 confers resistance to complement-mediated injury on host cells. All three patients expressed normal CD46 isoforms on their lymphocytes and granulocytes. Thus, the loss of CD46 is sperm-specific, probably due to testicular germ cell-specific regulation of CD46 production. Recently, a mechanism of the gene regulation of human CD46 was elucidated in which the silencer element of the 3' UT and the promoter region of human CD46 gene partly participate. Here, we analyzed these regions of the CD46 gene in our 3 patients. We found no abnormality in 3' and 5' regions of the CD46 genome in the 3 patients. Thus, in these infertile patients sperm-specific depletion of CD46 is not governed by the so for identified regulators in the CD46 gene. Other unknown factors outside the known regulatory regions would play a role in the regulation of sperm-specific CD46 expression.
We isolated a CD59 cDNA from a HeLa cell library which encoded a mutated form of CD59, having a single base substitution (G to T) that changed Arg55 to Met. Since this mutation occurred in the vicinity of the putative active site of CD59, we expressed the aberrant form of the protein in Chinese hamster ovary cells in order to test for effects upon function. We found that the mutation did not influence complement inhibitory activity of CD59. However, the epitopes recognised by the function-blocking CD59 monoclonal antibodies BRIC229 and YTH 53.1 were significantly affected. The G to T substitution caused loss of an Mnl I restriction site which permitted PCR-RFLP analysis. All of 52 human subjects studied, and our in-house HeLa cells, were homozygous for the normal CD59 sequence, indicating that the altered sequence was not due to normal variation in the general population. Therefore this mutation probably arose spontaneously in the HeLa cell line used to generate the commercially obtained cDNA library.
To obtain human deoxyribonuclease I (DNase I) as an immunogen, we have developed a procedure that is more useful and effective than the conventional procedure, which uses human urine as a starting material. In the new procedure, we culture COS-7 cells transfected with expression vector carrying human DNase I cDNA, and then purify the enzyme from the culture medium. The enzyme can be easily isolated to apparent homogeneity by passage through only three chromatography columns. The rabbit antiserum that we used against the recombinant DNase I was not inferior to that used against DNase I from human urine, in terms of both its ability to discriminate DNase I phenotypes and its ability to neutralize enzyme activity. Therefore, our procedure may be useful for producing an antibody specific for human DNase I.
The suppressors of cytokine signalling (SOCS) proteins comprise a newly identified family of negative feedback regulators of cytokine signalling. SOCS expression is differentially induced upon cytokine stimulation in different cell types. Here we show that interferon-α (IFNα) is a potent inducer of SOCS expression in human T cells, as high expression of CIS, SOCS-1, SOCS-2, and SOCS-3 was detectable after IFNα stimulation. After 4 h of stimulation, CIS, SOCS-1, and SOCS-3 expression had returned to baseline levels, whereas SOCS-2 expression had not declined. In contrast, after IL-2 induction neither CIS, SOCS-1, nor SOCS-2 expression levels declined after 6 h. In conclusion, we provide the first evidence that IFNα induces SOCS expression in human T cells. Moreover, we show that IFNα and IL-2 induce distinct patterns of expression kinetics, suggesting that dynamic changes in cytokine sensitivity might be mediated via induction of SOCS expression with different kinetics in T cells.
‘Nomenclature of the Human Immunoglobulin Kappa (IGK) Genes’, the 17th report of the ‘IMGT Locus in Focus’ section, provides the first complete list of all the human IGK genes. In the most frequent haplotypes, the human IGK locus spans 1,820 kb and the IGKV genes are organized in two clusters separated by 800 kb. In those haplotypes where both the proximal and distal IGKV clusters are present, the total number of human IGK genes per haploid genome is 82 (107 genes if the orphons are included) of which 37–41 are functional. If only the proximal IGKV cluster is present, which is found in a rare haplotype, the total number of genes per haploid genome is 46 (71 genes if the orphons are included) of which 23–25 genes are functional. IMGT/HUGO gene names and definitions of the human IGK genes on chromosome 2p11.2, and IGK orphons on chromosomes 1, 2, 15, and 22 are provided with the gene functionality and the number of alleles according to the rules of the IMGT Scientific chart, with the accession numbers of the IMGT reference sequences, and the accession ID of the Genome Database GDB and NCBI LocusLink databases in which all the IMGT human IGK genes have been entered. The tables are available at the IMGT Marie-Paule page from IMGT, the international ImMunoGeneTics database (http://imgt.cines.fr:8104) created by Marie-Paule Lefranc, Université Montpellier II, CNRS, France.
Objectives: Salivary gland organogenesis was evaluated in NOD mice, an animal model for autoimmune exocrinopathy, to determine when disease onset is first present in the target tissues. Methods: Submandibular glands were removed for histological, immunohistochemical and biochemical evaluation from neonatal NOD and congenic strains as well as healthy control C57BL/6 mice. Results: Histomorphological analyses of neonatal submandibular glands, the primary target for autoimmune exocrinopathy at 1 day postpartum, revealed delayed morphological differentiation during organogenesis in autoimmune-susceptible NOD mice when compared to nonsusceptible C57BL/6 mice. Acinar cell proliferation was reduced, while expression of Fas, FasL and bcl-2 were increased. Acinar cell proliferation was reduced, while expression, of Fas, FasL and bcl-2 were increased. Throughout the preweaning period (21 days) submandibular glands from NOD and NOD congenic strains aberrantly expressed an increased matrix metalloproteinase (MMP)-2 and MMP-9 activity. Substitution of two susceptibility alleles (Idd3 and Idd5) in NOD mice resulted in an hierarchical and additive reversal of delayed organogenesis, elevated MMP-9 activity, and aberrant expression of parotid secretory protein. Discussion: NOD-derived mice whose submandibular glands showed normal organogenesis did not progress to develop autoimmune exocrinopathy. Altered organogenesis of target tissue may therefore provide a cellular microenvironment capable of activating autoimmunity.
The class II transactivator is a major transcriptional factor acting on the promoters of MHC class II genes. Transcription of the CIITA gene is driven by four alternative promoters, which exhibit cell-type-specific activity. The CIITA promoter III (PIII) is constitutively active in B cells, whereas promoter IV (PIV) becomes activated upon interferon-gamma activation. The aim of this study was to investigate whether these two promoters exhibit a sequence variability like the MHC class II promoters do. We isolated PIII and PIV fragments from healthy individuals and rheumatoid arthritis patients and screened them for sequence polymorphisms. Single base pair substitutions within the CIITA PIV were found in 9% of the individuals analyzed. The majority of the substitutions were located upstream of the known cis-acting elements of the promoter. PIII was non-polymorphic. To evaluate the functional relevance of the detected polymorphism we cloned variable PIV upstream of the luciferase reporter gene. Such prepared constructs were transfected into monocytes, melanoma and HeLa cells, which were subsequently stimulated with interferon-gamma. The analysis of promoter activities did not reveal significant differences in all three cell types. We conclude that the level of CIITA expression does not vary within the population. Thus the differences in the level of MHC class II expression, which are observed between individuals, stem for the polymorphisms of the MHC class II promoters themselves.
A simple bidirectional allele-specific PCR method is described for determining the -1082 A and G alleles in the interleukin-10 (IL-10) promoter region. This polymorphism is associated with IL-10 production capacity, and it is thus interesting to see whether different infectious and autoimmune conditions are associated with it. With our method, the A and G alleles may be studied simultaneously in a single PCR reaction, as amplification of the different alleles is performed by using 3′-mismatched and partly overlapping allele-specific upstream and downstream primers around the -1082 site. The fast and simple method described here is especially suitable for large-scale association studies.
Objective: The aim of this study was to investigate the relationship between HLA molecules and the positive or negative response of atopic patients to specific immunotherapy (SIT). Methods: We studied 42 atopic multisensitive patients undergoing grass pollen immunotherapy, 42 parents of patients (30 mothers and 12 fathers) and 173 control individuals. HLA class I and class II antigens were typed by a microlymphocytotoxicity test. The typing of DRB1* alleles for atopic patients and their parents was based on the reverse hybridization principle, while for the control group, DNA-RFLP and PCR-SSP methods were used. Results: The frequency of B14 and DRB1*1101-4 antigens/alleles, as well as the A2B5DR11 haplotype, showed a statistically significant difference in those patients who responded to immunotherapy. On the other hand, HLA-A28, B8 and DRB1*0301 antigens/alleles, as well as the frequency of the A1B8 and A1B8DR3 haplotypes, were found to be significantly higher in patients who responded poorly to SIT. Discussion: Our findings support the hypothesis that treatment responsiveness may show an association to HLA molecules, which could thus play a role in the immunological selection and monitoring of atopic patient candidacy for SIT.
Four patients with mental illness were found to be deficient in plasma α1,3-fucosyltransferase for the first time in Japan [Exp Clin Immunogenet 1999;16:125–130]. Complete sequencing of FUT6 genes in these individuals revealed the presence of two point mutations, i.e., G739 to A (Glu→247 to Lys) and C945 to A (Tyr→315 to stop). In addition to two reported alleles having G739 to A (pf1) and G739 to A and C945 to A (pf3), a new mutated allele having C945 to A (pf2) was found to be present and all the individuals who lack α1,3-fucosyltransferase activity in plasma were found to possess pf genes homozygously (pf/pf). In order to detect such lethal mutations in FUT6 genes easily, PCR-RFLP methods have also been developed and applied for the screening of FUT6 deficiency in a large number of samples which resulted in the demonstration of three additional FUT6-deficient individuals. The absence of α1,3-fucosylated molecules on α1-acid glycoprotein in plasma from all the 7 individuals was confirmed to result from the plasma α1,3-fucosyltransferase deficiency.
Objective: The monoclonal IgG anti-double-stranded (ds) DNA antibody 32B9, obtained from a patient with systemic lupus erythematosus, was found to be encoded by somatically mutated immunoglobulin genes. We examined the input of several somatic mutations into antibody specificity and affinity. Methods: Five single-chain (sc) Fv fragments [variable domain of the heavy chain (VH)-linker-variable domain of the light chain (VL)] derived from 32B9 were constructed and expressed in Escherichia coli. These scFv fragments contained VH or VL fragments, differing in the somatic mutation pattern. The antigen binding features of the 32B9 IgG were compared with the corresponding scFv fragments, and the binding to DNA of all fragments was analyzed by ELISA. Binding constants to dsDNA were determined by surface plasmon resonance and ELISA. Results: The scFv 32B9 reflected the binding features of the 32B9 IgG. Independently of the somatic mutations, all scFv fragments bound to dsDNA in ELISA. The affinity data indicated that the mutations studied had only a marginal effect on affinity maturation of the 32B9. Discussion: We discuss the approach to constructing scFv fragments as a tool to study autoantibody maturation.
Objective: To determine whether the immunoglobulin heavy chain genes contribute to the occurrence of bullous pemphigoid (BP), polymorphisms of both the immunoglobulin constant IGHC and variable IGHV groups were studied in 100 Caucasian BP patients and 143 ethnically matched healthy individuals. Methods: To analyze the restriction fragment length polymorphism (RFLP) of the IGHG constant locus, a genomic immunoglobulin gamma 3 probe which detects polymorphisms in the gamma constant genes was hybridized to BstEII- or BamHI/SacI-digested germline DNA, while IGHV3 subgroup polymorphism was analyzed by hybridizing a cloned VH3 probe to EcoRI-digested DNA. Results: No difference in the frequencies of the genotypes defined by the constant probe was observed between patients and controls. Analysis of the RFLP obtained with the VH3 probe showed that within the range of 4.5 and 1.5 kb, the observed band pattern was composed of 8 monomorphic and 7 polymorphic bands. Among the latter, 4 allowed to define 10 different restriction patterns. One pattern was shown to be significantly less frequent in patients than in controls. Conclusion: IGHV3 polymorphism might be a factor conferring susceptibility to BP.
Genetic variation in immunoglobulin γ (GM) and ĸ (KM) chains was associated with systemic lupus erythematosus (SLE) in some studies. However, the data are conflicting, and only one study examined associations in African-Americans. We examined GM and KM allotypes, by race, in a population-based case-control study of SLE. Sera from patients (n = 222) and controls (n = 273) were typed for GM and KM allotypes by a hemagglutination inhibition method. GM phenotypes were not significantly associated with SLE in African-Americans or Caucasians. However, the frequency of KM phenotypes in Caucasian patients was significantly different from that in controls (p = 0.032). KM3,3 was associated with an increased risk, whereas KM1,3 was associated with a lower relative risk of SLE. In African-Americans, however, the pattern of associations with KM phenotypes differed from that in Caucasians, and the overall difference between patients and controls was not statistically significant.
GM and KM allotypes--genetic markers of immunoglobulin (Ig) gamma and kappa chains, respectively--have been shown to play an important role in genetic predisposition to some autoimmune diseases. To determine their role in susceptibility to systemic sclerosis (SSc; scleroderma) and in the generation of anti-fibrillin-1 antibodies, 148 SSc patients and 191 controls were typed for several GM and KM allotypes by a standard hemagglutination inhibition method. IgG and IgM antibodies to fibrillin-1 were measured by radioimmunoassay. GM and KM phenotypes were not significantly associated with SSc. However, these determinants significantly influenced the production of anti-fibrillin-1 antibodies in SSc patients. In Caucasians, GM1,3,17 23 5,13,21 and GM3 23 5,13 phenotypes were associated with the presence and absence of IgG autoantibodies, respectively. The production of these autoantibodies was also associated with KM allotypes, KM1,3 heterozygosity being associated with response and homozygosity for the KM3 allele with nonresponse to fibrillin-1. In African-Americans, the KM1 homozygotes were associated with the absence of anti-fibrillin-1 antibodies and the KM3 homozygotes with the presence of autoantibodies. In this ethnic group, the GM1,17 5,13 phenotype was associated with the absence of IgM autoantibodies. This represents the first description of genetic control of autoimmunity to fibrillin-1 in scleroderma.
Objective: The balance between mucosal CD4+ T cells producing IFN-γ or IL-10 is essential in the maintenance of intestinal homeostasis. We aimed to investigate how in situ activated T cells were expanded in vitro according to a new cell culture protocol and if it selected for continuous clonal CD4+ T cells capable of producing mainly IFN-γ or IL-10. Methods: T cell cultures were established from colonic biopsy specimens of 27 patients with Crohn’s disease and from 10 healthy controls in a medium supplemented with IL-2 and IL-4 but without addition of exogenous antigen or mitogen. Cytokine production was measured after stimulation (IL-12, super antigen) and inhibition (ciclosporin and methylprednisolone). Results: Cytokine production was increased in cultures from patients with Crohn’s disease compared to controls (IFN-γ, p = 0.005; IL-10, p = 0.003; TNF-α, p = 0.03). Early cultures were highly responsive to IL-12 stimulation. We established CD4+ T-cell clones escaping cellular senescence with an inflammatory or regulatory cytokine profile. Discussion: The data indicate that cultures of in situ activated inflammatory and regulatory subpopulations of intestinal T lymphocytes with pathogenic importance in Crohn’s disease can be established preserving their functional properties during growth.
Signal transducer and activator of transcription 6 (STAT6) is essential for the biological activities of interleukin-4 (IL-4) and the development of allergic responses in mice. Here we report on a sensitive and specific assay for STAT6 activation in response to IL-4. We took advantage of double-stranded oligonucleotide probes containing a STAT6-binding gene-sequence from the promotor of the immunoglobulin heavy chain germline Ε transcript to study the IL-4-induced DNA binding of STAT6. Using these probes, we show that repeated adjacent STAT6-binding sites result in enhanced STAT6-DNA binding. Moreover, the distance between the binding sites is critical for STAT-DNA binding, i.e. STAT6 binding is decreased at distances above 20 nucleotides between neighbouring binding sites. Using this assay to study cross-talk between IL-4 and chemokines, we provide evidence that MIP-1β and MIG inhibit IL-4-induced STAT6 activation, whereas other chemokines and cytokines do not. In conclusion, our data show that oligonucleotide fishing is a supplementary tool for studying cytokine cross-talk at a genomic level.
