Linkage disequilibrium mapping of a type 1 diabetes susceptibility gene (IDDM7) to chromosome 2q31-q33.

The role of human chromosome 2 in type 1 diabetes was evaluated by analysing linkage and linkage disequilibrium at 21 microsatellite marker loci, using 348 affected sibpair families and 107 simplex families. The microsatellite D2S152 was linked to, and associated with, disease in families from three different populations. Our evidence localizes a new diabetes susceptibility gene, IDDM7, to within two centiMorgans of D2S152. This places it in a region of chromosome 2q that shows conserved synteny with the region of mouse chromosome 1 containing the murine type 1 diabetes gene, Idd5. These results demonstrate the utility of polymorphic microsatellites for linkage disequilibrium mapping of genes for complex diseases.

Three Ia species with different structures and alloantigenic determinants in an HLA-homozygous cell line.

Three distinct molecular subsets with different structures and alloantigenic determinants were identified in human Ia antigens from cells of an HLA-Dw7 homozygous cell line. The subsets carried DR7 specificity, BR4X7 supertypic specificity and MB2 supertypic specificity, respectively, and were immunospecifically separated by the use of operationally monospecific alloantisera. These specificities showed HLA-linked segregation in families and they were distributed in the population according to different but partially overlapping patterns. On peptide mapping analysis, the three subsets showed marked differences in the beta-chains. The alpha-chains of DR7 and BR4X7 subsets were very similar to each other, whereas the alpha-chains of MB2 subset were distinctive from those of DR7 and BR4X7. These data indicate the presence of a minimum of three HLA-linked loci; DR locus, a locus that encodes BR4X7, and a locus that encodes MB2, and substantiate the three-loci concept for the genetic control of human I antigens.

Immunological dissection of human Ia molecules.

The immunochemical analysis of Daudi Ia molecules by a variety of alloantisera led to the recognition of at least three molecular species carrying different antigenic determinants: DRw6, DC-1, and DC-2. Genetic as well as structural evidence indicates that DRw6 and DC-1 molecules are controlled by separate, HLA-linked loci, rather than by alleles at the same locus. The alloantigenic determinants appear to be expressed on the small Ia subunit. DC-1 and DC-2 determinants discussed had not been defined by serological analysis at the population level, but were demonstrated to be present by immunochemical analysis at the molecular level.

Serological and immunochemical analysis of the products of a single HLA DR-alpha and DR-beta chain gene expressed in a mouse cell line after DNA-mediated cotransformation reveals that the beta chain carries a known supertypic specificity.

Using a mouse cell line transformed with and expressing a single HLA DR-alpha and DR-beta chain gene, we present evidence that the product of the DR-beta chain gene carries a supertypic determinant, BR3, previously defined by serology. The amino acid sequence of this beta chain gene is determined from the DNA sequence. Another DR-associated supertypic specificity defined by monoclonal antibody MCS7 was not encoded by this DR-beta chain gene. This provides formal proof that a supertypic specificity can be associated with a product of a distinct DR-beta locus. We propose that haplotypes sharing such specificities are evolutionarily related.

Electrophoretically homogeneous antibody synthesized by spleen foci of irradiated repopulated mice.

10(6) splenocytes from primed donors were injected, together with sheep erythrocytes (SRBC), into X-irradiated syngeneic mice. 8 days later the spleens were excised and cut into small fragments, keeping track of their location in the organ. Each fragment was cultured individually for 24 hr in the presence of (14)C amino acids and the culture fluids were assayed for antibody activity. The antibody-producing fragments were found to be clustered in few restricted areas (foci) surrounded by negative tissue. The anti-SRBC antibody from single foci was purified by absorption on stroma followed by acid elution. Thereafter, it was subjected to electrophoresis and immunoelectrophoresis. The radioautography of the runs showed a considerable degree of homogeneity. Distinct and sharp spikes were localized in the beta and gamma region. The pattern of each focus is unique from the point of view of the number of spikes and their mobility. Eluates obtained from many pooled fragments gave a broad radioactive smear in beta-gamma region. Many foci synthesized antibody migrating as a single band. This homogeneous protein is probably the product of a clone of cells homogeneously differentiated. However, some foci producing two and probably more antibody bands were also encountered. Two interpretations of the finding can be given. Either more than one precursor may participate in the formation of a focus or a differentiation switch may occur during the clonal expansion.

Simultaneous bilateral retinal detachment following coronary artery bypass graft: case report.

PURPOSE: To present an unusual case of simultaneous bilateral retinal detachment (RD) following a coronary artery bypass graft in a patient with acute myocardial infarction (AMI). METHODS: A 78-year-old man was first seen for bilateral sudden visual loss after surgical treatment of AMI. The patient underwent ultrasound biomicroscopy (UBM) and ocular B-scan echographic examination. RESULTS: The ocular assessment showed a bilateral seclusion of the pupil with bombe of the iris, an anterior chamber without cells or flare, and hypotonia. The evaluation of the visual acuity revealed no light perception in the right eye (RE) and uncertain light perception in the left eye (LE). The UBM analysis of the anterior segment confirmed the presence of bilateral pupillary block due to the seclusion of the pupil and a peripheral serous choroidal detachment involving the RE. The echographic B-scan analysis of the posterior segment showed a bilateral closed funnel-shaped RD and confirmed the presence of the peripheral flat serous choroidal detachment in RE. CONCLUSIONS: The cause for simultaneous bilateral RD remained unclear. It may have been a consequence of a persistent choroidal detachment with multiple swelling and 'kissing' of retinal surface. The increased venous pressure caused by congestive heart failure due to AMI could have caused a bilateral uveal effusion. Alternatively, the absence of retinal tears, the presence of a closed funnel-shaped morphology, and seclusion of the pupils allowed us to suspect an exudative pathogenetic mechanism due to a previous unrecognized ocular inflammatory state.

An explanation for the neutral effect of DR2 on IDDM susceptibility in central Italy.

Susceptibility to IDDM is strongly associated with HLA. Some HLA allelic combinations (haplotypes) can be found in most patients, whereas other haplotypes are encountered only rarely. It has been proposed that this difference in susceptibility depends on the absence (in the DR3 and DR4 haplotypes) or the presence (in the DR2 haplotype) of Asp57 in the DQ beta-chain. Data on southern European populations challenge this hypothesis because the DR2 haplotype has not been associated negatively with IDDM, as reported in northern European populations. This study on a selected panel of DR2-positive Italian IDDM patients shows that 19 of 21 (90.5%) DR2 haplotypes possess a non-Asp57 DQB allele. Moreover, the same non-Asp57 subtype has a comparatively high frequency (9/28, or 32.1%, DR2 haplotypes) also in the DR2-positive healthy Italian population. The difference between patients and control subjects is significant (P less than 0.0001). This is the largest series of DR2-positive patients analyzed so far. Comparison with cumulated data in various white populations shows a distinct northern European-to-southern European gradient. Toward southern Europe, the relative frequency of the non-Asp57 DR2 subtype increases. Concomitantly, the apparent protective effect of the DR2 haplotype disappears. Therefore, the observed differences in DR2-IDDM association in white populations can be explained adequately by the Asp57 hypothesis, which this study's data strongly support.

The HLA class I-CML association revisited taking into account the two forms of gene fusion in the Philadelphia chromosome. A multicenter study.

CML patients possess either a b3-a2 or a b2-a2 fusion between the BCR and ABL genes. Depending on the type of fusion, two different series of non-self potentially immunogenic peptides may be produced. If they are presented by HLA class I molecules and recognized by cytotoxic CD8 lymphocytes, individuals could be more susceptible or resistant to leukemic cells bearing one or the other form of fusion according to their HLA class I phenotype. To test this point, the frequencies of HLA-A and HLA-B alleles were compared between b3-a2 and the b2-a2 CML patients. In essence, no difference was found whose significance could withstand correction for multiple comparisons.

Two brc-abl junction peptides bind HLA-A3 molecules and allow specific induction of human cytotoxic T lymphocytes.

Intracellular processing of the products of the bcr-abl junction region in CML Philadelphia chromosomes would generate novel peptides which, if they are capable of binding to HLA-class I molecules, would be potential targets of a cytotoxic T cell response. The 18 nonamers corresponding to the b2-a2 and b3-a2 fusions and differing from the parental bcr and abl sequences for at least one amino acid have been synthesized and tested for binding with HLA class I alpha chain preparations from HLA-homozygous B lymphoblastoid cell lines. Two peptides derived from the b3-a2 junction bound to HLA-A3 and elicited detectable specific CTL responses in vitro. The binding affinity of one of the two peptides could be increased by appropriate substitutions of the anchor residues with those of the known HLA-A3 anchor motifs. More important, the modified peptide had increased capacity to prime a specific CTL response in vitro. The interaction with HLA-A3 of these two peptides and their substitution derivatives seems to be promising for target trials aimed at eliciting a specific CD8 T cell response against CML.

Influence of ionic strength on the antibody binding of MHC products.

Ionic strength has a strong influence on the binding of human Ia and HLA(A,B,C) molecules by the corresponding antibodies. A control system, BSA-anti-BSA, was influenced only minimally. The effect on the binding of anti-beta 2-microglobulin was very small when isolated chains were used, but more pronounced when using whole HLA(A,B,C) molecules. An inverse relationship exists between degree of inhibition and antibody concentration.

A spin label study of the ionic strength dependent conformational change in the human Ia molecule.

Purified human Ia molecules were labelled with maleimide or isothiocyanate spin labels or by reacting "TEMPAMINE" spin label with the neuraminic acid of their carbohydrate residues. It was found that increasing the ionic strength from 0.05 to 0.75 markedly increased the dipolar interaction between the maleimide-attached labels, but no effect was found of ionic strength or motion on dipolar interaction with the other two labels. The effect of increasing ionic strength could be blocked by the prior addition of Ia-specific antibody, but could not be reversed by the addition of antibody after ionic strength was increased. These findings complement an earlier finding that increasing ionic strength over the range 0.05-0.75 has an inhibitory effect on the combination of Ia with its antibody. Because the maleimide spin labels attach predominantly to SH groups it is suggested that increasing ionic strength causes conformational changes in the immunoglobulin loop region which alter the accessibility of the Ia antibody-binding site.

Microfingerprinting analysis of human Ia molecules favours a three loci model.

Alpha subunits from DC1 Ia molecules, when compared with DR alpha subunits, are shown to possess distinctive features revealed by differences in microfingerprinting patterns after peptic digestion. Alpha chains from BR4X7 molecules differ from DC1 alpha chains and are more similar to DR alpha chains. Since DC1 and BR4X7 beta chains (which carry the HLA-controlled alloantigenic determinants) associate with different alpha subunits, it is considered unlikely that they are controlled by alleles at the same locus. The proposed model implies the existence of three tightly linked HLA loci controlling the beta subunits of DR, DC and BR molecules respectively.

Linkage analysis of multiple sclerosis with candidate region markers in Sardinian and Continental Italian families.

Previous genome screens in multiple sclerosis have shown some evidence of linkage in scattered chromosomal regions. Although in no case the evidence of each single study was compelling and although in general the linkage 'peaks' of the different studies did not coincide, some regions can be considered likely candidates for the presence of MS risk genes because of the clustering of MLS scores and homology with eae loci. We performed a linkage analysis of markers in these regions and of intragenic markers of some individual candidate genes (HLA-DRB1, CTLA-4, IL9, APOE, BCL2, TNFR2). For the first time, Southern European populations were targeted, namely Continental Italians and Sardinians. A total of 69 multiplex families were typed for 67 markers by a semi-automatic fluorescence-based assay. Results were analysed for linkage by two non-parametric tests: GENEHUNTER and SimIBD. In general, the linkage scores obtained were low, confirming the conclusion that no gene is playing a major role in the disease. However, some markers, in 2p11, 3q21.1, 7p15.2 and 22q13.1 stood out as promising since they showed higher scores with one or the other test. This stimulates further association analysis of a large number of simplex families from the same populations.

Constancy of cross reactivity patterns during the anti-HL-A response.

Three immunizations yielding cross reacting anti-HL-A antibodies were followed for 1 year or more. The specificities of the antisera obtained at different times were determined from their reactivity against a selected cell panel. Each antiserum was titrated against cells presenting different degrees of cross reaction. An extensive absorption study of each antiserum, diluted to one cytotoxic unit, was also performed. The data obtained suggest that the cross reactivity pattern in each response is rather rigidly established from the beginning and that it has little chance to vary even after secondary stimulation from the same donor.

A safe blood transfusion procedure for immunization against major histocompatibility complex determinants in man.

A standardized procedure is proposed for deliberate immunizations against human major histocompatibility complex determinants. The data presented demonstrate its effectiveness and, by using a number of necessary precautions, this procedure has proven to be very safe. The following points are especially important: (1) exclusive utilization of regular blood donors as immunizers; (3) use of whole blood as an immunizing agent; and (3) use of small immunizing stimuli rather than large transfusions. This procedure can be recommended for the production of monospecific anti-HLA antisera and it may be useful if and when a deliberate transfusion policy for prospective kidney recipients is adopted.

HLA-DR typing by radioimmunoassay.

A radioimmunoassay procedure is described by which peripheral blood lymphocytes can be typed for HLA-DR specificities. The major advantages of this method are the following: simple and reproducible procedure, no need for B lymphocyte separation, no need for optimal viability, and no need for preabsorption of antisera with platelets. This method will find an application in the genetic and biochemical analysis of the HLA complex, and in the clinical tests of Ia antigens for diagnostic or prognostic purposes and in retrospective transplant studies.

Subgrouping of DR4 alleles by DNA heteroduplex analysis.

Amplified DNA molecules from six DR4 alleles at the DRB1 locus were denatured and cross-hybridized pairwise. Several of the DNA heteroduplexes thus generated were found to possess distinct mobilities in polyacrylamide gel electrophoresis. The degree of retardation as compared to homoduplexes depends strongly on the position of mismatched nucleotide pairs. In critical positions, the type of mispairing also influences the number of heteroduplex bands since a transversion-type substitution yields two reciprocal pairings (purine-purine or pyrimidine-pyrimidine) whereas a transition-type substitution generates symmetrical (purine-pyrimidine) pairings. Based on their heteroduplex pattern the DR4 alleles can be subdivided in four groups: group I = DRB*0401, group II = 02 and 06, group III = 03 and 04, and group IV = 05. Each group can be recognized by the heteroduplex bands generated by cross-hybridizing with group II reference DNA (either the 02 or 06). This subgrouping is obtained with a single electrophoretic run and without the use of probes. However, the alleles within groups II and III, and notably the alleles 03 and 04, which are both present in the Caucasoid population, can be distinguished only by oligonucleotide hybridization (dot blot) analysis. With this limitation, the method can be recommended either in conjunction with dot blot typing or independently, thus avoiding completely the use of probes in the cases where it is not essential to discriminate between 03 and 04. The data also show that distinguishable heteroduplexes may be generated by a single mismatch. This opens the possibility of applying the same technique to genetic systems of lower degree of polymorphism.

A current view of the human Ia system based on serological and immunochemical analysis.

We have approached the analysis of the human Ia system by localizing antigenic determinants on subsets of Class II molecules. For this purpose two classes of reagents have been used: alloantisera and mouse anti human monoclonal antibodies (moabs). We summarize here the most recent data obtained by these two approaches.

A new allodeterminant on HLA-DQ molecules carrying the DQw3 specificity.

The la subset that reacts with alloantiserum HON known to possess a strong anti-DRw53 activity was isolated from a 125I-labeled Ia preparation obtained from cells of RPMI 8057 cell line (DR1,4) and was found on peptide mapping to be lacking in the pattern characteristic of DR-like molecules carrying the DRw53 specificity and to display the structural features of DQ molecules, particularly those carrying the DQw3 specificity. Distribution analysis on a panel of selected la-positive cell lines indicated that the specificity involved is associated only with DR4 and DRw9, differing from the known DRw53 pattern (DR4, 7, and w9) and also from the known DQw3 pattern (DR4 and 5). Reciprocal sequential binding experiments demonstrated that the HON-defined specificity resides along with DQw3 specificity on the same molecules. Thus, HON alloantiserum possesses two different antibody activities; one directed to DRw53 specificity and another directed to a new DR4- and w9-associated DQ specificity.