Allorecognition and T cell repertoire selection in severe combined immunodeficiency lacking HLA class II antigens.

In order to elucidate the role of HLA class II molecules in generation of self-nonself discrimination of human T cells, we have analyzed T cell functions in an HLA class II-negative severe combined immunodeficiency patient. Patient PBL expressed no HLA-DR, -DQ, and -DP antigens as judged by immunofluorescence using mAb, and failed to elicit MLR responses from unrelated controls. Patient PBL contained mature T cells (CD3+ TCR alpha beta+) of the CD4 and CD8 subset, showing an apparently normal TCR diversity, as judged by use of anti-V beta 5, -V beta 6, -V beta 8, -V beta 12, and -V alpha 2 mAb. Patient PBL proliferated in response to anti-TCR/CD3 mAb and PHA, but not against recall Ag, despite immunization, and mounted proliferative, but not cytotoxic, responses against allogeneic cells. To find out whether the MLR responses were a consequence of self-nonself discrimination, the patient HLA-DR and -DQ genotype was determined using sequence specific oligonucleotide probes, revealing DRB1*0401 DQB1*0301 alleles, and MLR were set up against a panel of HLA-DR4 DQw3 stimulators matched or mismatched for DRB1*0401 DQB1*0301. Results showed no MLR against DRB1*0401 DQB1*0301 stimulators, but significant responses against stimulators expressing DRB1*0408 and/or DQB1*0302 alleles. Moreover, the DRB1*0401 DQB1*0301 APC reconstituted proliferation of patient PBL against PPD; this response was completely blocked by an anti-IL-2R (p55) mAb and partially also by anti-HLA-DR and -DQ mAb, indicating recognition of these molecules as restriction element presenting Ag--i.e., as self--by patient T cells. In conclusion, the novel demonstration of self-nonself discrimination by T cells from an HLA class II-negative SCID patient suggests that it may not be absolutely dependent on regular HLA class II expression within the differentiation environment in humans.

HLA-DR beta chain residues that are predicted to be located in the floor of the peptide-binding groove contribute to antibody-binding epitopes.

The purpose of this study was to identify polymorphic residues in HLA-DR beta chains that are involved in the epitopes recognized by DRw52-like mAbs. Flow cytometric analysis of antibody binding to mouse fibroblast transfectants expressing wild-type or mutant DRw52-associated DR beta chains demonstrated that the amino acid at position 77, which is predicted to be on a solvent-accessible surface of the alpha-helix, contributes to the epitopes recognized by the 7.3.19.1 and 21r5 mAbs. In contrast, residues that are not predicted to occupy accessible positions on the alpha-helix contribute to binding of the I-LR2 and UL-52 mAbs. Substitutions at positions 10, 12, and 51, but not 9, 11, and 13, affect these epitopes. It is interesting that antibody binding is influenced by amino acid substitutions at DR beta positions 10 and 12 because the class II model predicts that residues at these positions are located in the middle of the floor of the peptide-binding site, with their side chains pointing down. These findings emphasize the functional importance of polymorphic residues whose side chains are not predicted to point into or up from the antigen-binding site and they raise the possibility that peptides contribute to the generation of these epitopes.

Alloreactive T-cell clones raised in an HLA-B/D crossing-over family dissect HLA-DR5 and HLA-DQw3 subtypes.

This study was undertaken to resolve a positive mixed lymphocyte reaction between HLA-ABC identical, HLA-D different siblings. Three CD3+ CD4+ CD8- alloreactive T-lymphocyte clones, called 2/6, 7/1, and 7/2, were generated and extensively studied. Proliferation of 2/6 cells and 7/2 cells was blocked by anti-DQ monoclonal antibodies (mAbs), whereas anti-DR and DP were not effective. Stimulation of 7/1 cells was inhibited by anti-DR, but not by anti-DQ and DP mAbs. Testing on a well-characterized panel of reference B-lymphoblastoid cell lines showed that the DQ-specific clones 2/6 and 7/2 were able to proliferate upon stimulation by cells carrying the DQw7 and DQw8 but not the DQw9 subtype of DQw3. Clone 7/1 was proliferative towards cells expressing DRw11.1 but not towards DRw11.2- or DRw12-positive cells. Moreover, this clone detected determinants present on some DRw8 cells. Correlation of the reactivity of clone 7/1 with available sequence data suggests that amino acids 67, 71, and 86 of DR beta 1 molecules played a crucial role in forming the epitope recognized by this clone. In contrast, sharing of T-cell epitopes between DQw7 and DQw8 subtypes was not inferable from specific amino acid residues. The implication of these findings for T-cell allorecognition is discussed.

Diverse locations of amino acids in HLA-DR beta chains involved in polymorphic antibody binding epitopes on DR(alpha, beta 1*0101), DR(alpha, beta 1*1101), and DR(alpha,beta 3*0202) molecules.

In a previous study, we used transfectants expressing hybrid HLA-DR(beta 1*0403)/DR(beta 1*0701) chains to map sequences involved in polymorphic antibody binding epitopes on DR(alpha, beta 1*0403) or DR(alpha, beta 1*0701) molecules. Amino acids 1-40 of the beta 1 domain were found to make the major contributions to most of the antibody binding epitopes studied. To begin to localize sequences that contribute to polymorphic antibody epitopes on DR(alpha,beta 1*0101), DR(alpha,beta 1*1101) and DR(alpha,beta 3*0202) molecules, we used indirect immunofluorescence and flow cytometry to assess the binding of mAb to transfectants expressing hybrid DR(beta 1*0101)/DR(beta 1*1101) or DR(beta 1*1101)/DR(beta 3*0202) chains that divide the DR beta chain into three segments: amino acids 1-40, 41-97, and the beta 2 domain. The results indicate that amino acids 41-97 of the beta 1 domain on DR(beta 1*0101), DR(beta 1*1101), or DR(beta 3*0202) are critical in most of the epitopes, including those recognized by human antibodies MP4 and MP12, and mouse mAb GS88.2, I-LR1, 21r5, and 7.3.19.1, whereas amino acids 1-40 of DR(beta 1*1101) are critical in the epitope recognized by the MCS-7 mAb, and both segments 1-40 and 41-97 of DR(beta 1*1101) are important in the epitopes recognized by the I-LR2 and UL-52 mAbs. Based on these data and comparison of DR beta allelic protein sequences, the residues that may play critical roles in these antibody binding epitopes are predicted.

Noninvasive cardiac evaluation in chronic alcoholic patients with alcohol withdrawal syndrome.

Cardiac function was evaluated by noninvasive techniques in 50 patients hospitalized during acute alcohol withdrawal treatment. All patients had ingested large amounts of alcohol for at least five years, but discontinued alcohol intake 24 to 72 hours prior to admission. There was no clinical history of heart disease in any of the patients. Our study employed 12-lead electrocardiograms (daily) and 24-hour Holter monitoring. M-mode echocardiography and systolic time intervals were evaluated in 24 patients. The results indicate that marked electrical irritability of a depressed myocardium during the acute phase of alcohol withdrawal indicates the need for close cardiac observation of such patients in order to avoid potential life-threatening arrhythmias.

The significance of alpha-fetoprotein in the serum of patients with malignant teratomas and related gonadal neoplasms.

Sera from eleven patients with gonadal germ cell tumors were tested for alpha-fetoprotein (AFP) by counterelectrophoresis. Ovarian neoplasms containing either the endodermal sinus tumor (EST) or dysgerminoma and half of the testicular embryonal carcinomas elaborated the protein into the serum. The presence of AFP in the serum seemed to correlate with early metastasis or a fatal outcome. That the highest levels of AFP were found in cases of EST was in keeping with the theory that the tumor originates in cells of the yolk sac, a principal embryonic site of AFP synthesis.

Huge left atrial thrombus and valve degeneration in a patient with a bioprosthetic, porcine, mitral valve.

A 43 year old man with a Hancock porcine bioprosthetic valve in the mitral position developed a huge thrombus filling the entire left atrium and chronic degeneration of the bioprosthetic mitral valve. The effective valve orifice was less than 2 mm. These severe findings leading to the patient's death remained undetected while he was alive. This case illustrates the great need for a serial phonoechocardiographic studies in all patients with prosthetic valves.

Monoclonal antibody to a supertypic determinant associated with HLA-DRw52.

A cytotoxic mouse monoclonal antibody UL-52 (IgG2b) was obtained after immunization of a BALB/c mouse with the lymphoblastoid cell line STA homozygous for HLA-A3, B8, Cw7, DR3, DRw52, DQw2, DPw2. Fluorescence analysis on a panel of B lymphoblastoid cell lines from the 10th International Histocompatibility Workshop 1987 showed almost exact concordance of UL-52 reactivity with the presence of the HLA-DRw52 antigen. Cytotoxicity testing of UL-52 on mononuclear cells of HLA-typed individuals revealed a pattern of reactivity closely associated with the HLA-DRw52 specificity as defined by conventional alloantisera (R = 0.77). UL-52 precipitated appropriate 29,000- and 33,000-dalton bands on SDS- polyacrylamide gels under reducing conditions from an HLA-DRw52 positive B lymphoblastoid cell line. Thus, by serological and biochemical criteria UL-52 defines a supertypic determinant associated with HLA-DRw52. In contrast to most DRw52- like monoclonal antibodies, UL-52 binds to DRw8 positive cells.

Common overuse running injuries: diagnosis and management.

Running injuries are primarily caused by overuse due to training errors (i.e., running too far, too fast, too soon). A stress fracture should always be considered in a runner with pain, because long-term morbidity may occur if this injury is not recognized. The history and physical examination are usually sufficient to diagnose an overuse injury. Runners should be instructed to increase their mileage gradually in increments of 10 percent or less each week, to wear proper running shoes and to perform stretching and strengthening exercises for the lower extremities on a regular basis. In addition, they should not attempt to "run through pain." Treatment of overuse running injuries includes relative rest, nonsteroidal anti-inflammatory drugs, cross-training and stretching exercises, with a return to running as tolerated. Correction of biomechanical problems with the use of orthotics may be an adjunctive treatment measure.

Commonly missed orthopedic problems.

When not diagnosed early and managed appropriately, common musculoskeletal injuries may result in long-term disabling conditions. Anterior cruciate ligament tears are some of the most common knee ligament injuries. Slipped capital femoral epiphysis may present with little or no hip pain, and subtle or absent physical and radiographic findings. Femoral neck stress fractures, if left untreated, may result in avascular necrosis, refractures and pseudoarthrosis. A delay in diagnosis of scaphoid fractures may cause early wrist arthrosis if nonunion results. Ulnar collateral ligament tears are a frequently overlooked injury in skiers. The diagnosis of Achilles tendon rupture is missed as often as 25 percent of the time. Posterior tibial tendon tears may result in fixed bony planus if diagnosis is delayed, necessitating hindfoot fusion rather than simple soft tissue repair. Family physicians should be familiar with the initial assessment of these conditions and, when appropriate, refer patients promptly to an orthopedic surgeon.

Generation and characterization of three new monoclonal antibodies detecting the allospecificities HLA-A2,w69, HLA-A3 and HLA-B13.

Immunization of balb/c mice with peripheral blood mononuclear cells of a leukemic patient possessing the antigens HLA-A2,3; B7,35 resulted in the polymorphic monoclonal antibody (moab) UL-101/68 (IgM) defining HLA-A3. Immunization of a second group of balb/c mice with the lymphoblastoid cell line BER homozygous for HLA-A2; B13 revealed two polymorphic moabs UL-39/10 (IgG3) specific for HLA-B13 and UL-39/24 (IgG2b) defining HLA-A2,w69. Immunoprecipitation and polyacrylamide gel electrophoresis with the two moabs of the IgG isotype confirmed the class I structure of the recognized antigens.

Molecular analysis of the HLA-DR5 haplotype.

A panel of eleven HLA-DR5 homozygous lymphoblastoid cell lines was investigated for structural heterogeneity on the product level. HLA class II antigens were isolated by immunoprecipitation with different anti-class II monoclonal antibodies and separated by two-dimensional (2-D) gel electrophoresis. As a result, three distinct DRB1, one commonly expressed DRB3, and two distinct DQ gene products could be identified that combined to four different haplotypes associated with HLA-DR5. A hitherto serologically undetected split of HLA-DRw11 was presented by three cell lines. HLA-DRw11 and HLA-DRw12 were found to be related allospecifities that differ only in their DRB1 locus products, but are closely associated with the supertypic DRB3 allele HLA-DRw52b and with HLA-DQw7. The DRB3 alleles HLA-DRw52a and DRw52c were not detected in our cell line panel, indicating that these supertypic determinants are in negative linkage disequilibrium with HLA-DR5. Our data suggest that intra HLA-DR/DQ crossing-over events contribute to the development of the HLA class II polymorphism. Evidence is presented that the T cell defined HLA-D allospecifities are commonly determined by DRB1 and DQ gene products.

Characterization of the novel allele HLA-B*4417: implications for bone marrow transplantation.

The identification of the novel allele HLA-B*4417, which was found in a blood donor of Caucasian origin, is described. In the sequence analysis the new allele differs from B*4402 by three nucleotides in exon 3. At the protein level, the new allele has two residue differences compared to B*4402. Due to substantial differences with other B*44 variants a possible mismatch may impair clinical outcome of bone marrow transplantation.

Selection of unrelated bone marrow donors: does the current procedure warrant complete MHC class II identity?

Bone marrow transplantation from unrelated donors is being used increasingly for the treatment of patients with leukemia and several other hematologic disorders. Selection of unrelated bone marrow donors currently relies on serological HLA identity and negative mixed lymphocyte reactions between donor/recipient pairs. As serological HLA-DP typing is not feasible, we used the HLA-DPB1 oligonucleotide typing method to investigate whether the current selection procedure can guarantee complete MHC class II identity. In 40 consecutive patients, one third (62/193) serologically HLA-A, -B, -C, -DR and -DQ identical donors were found to be MLC negative with a relative response below 5%. HLA-DPB1 oligonucleotide typing of these MLC negative donors revealed that again only one third (20/62) was also identical for DP with their presumptive recipients. In the majority of pairs a disparity in graft-versus-host direction or in host-versus-graft direction of at least one allele was seen. These data indicate that in spite of the strict MLC criteria used, the current procedure did not warrant complete MHC class II identity. This implies that oligotyping for DPB1 can improve matching and should be introduced for typing of volunteers. We speculate that DP differences may contribute to the higher incidence of graft-versus-host disease or graft rejection in unrelated transplants.

HLA-DPB1 oligonucleotide typing of a southwest German Caucasian population.

HLA-DP genotyping with sequence-specific oligonucleotides can detected known sequence variations in the polymorphic segments of the DPB1 second exon. Since the allelic polymorphism of the 22 published alleles is based on recombination of sequence motifs from six variable regions, DPB1 typing depends on the reactivity pattern of many different probes rather than from typing with single allele-specific probes. By computer simulation, we have previously shown that the minimal set of probes to define the 22 different alleles and most of the heterozygous combinations is 18. Here we describe HLA-DPB1 typing results and allele frequencies in a panel of 200 unrelated Caucasians from Southwest Germany. The result confirmed the power of the new HLA-DPB1 typing method, but we failed to detect three of the previously described alleles in our panel. To accommodate with the observed 19 different alleles, the sequence and hybridization conditions of 17 oligonucleotide probes are given, which are able to differentiate all except two, resolved by group-specific amplification, of the 190 possible heterozygous phenotypes.

Selection of unrelated bone marrow donors: does the current procedure warrant complete MHC class II identity?

Bone marrow transplantation from unrelated donors is being used increasingly for the treatment of patients with leukemia and several other hematologic disorders. Selection of unrelated bone marrow donors currently relies on serological HLA identity and negative mixed lymphocyte reactions between donor/recipient pairs. As serological HLA-DP typing is not feasible, we used the HLA-DPB1 oligonucleotide typing method to investigate whether the current selection procedure can guarantee complete MHC class II identity. In 40 consecutive patients, one third (62/193) serologically HLA-A, -B, -C, -DR and -DQ identical donors were found to be MLC-negative with a relative response below 5%. HLA-DPB1 oligonucleotide typing of these MLC-negative donors revealed that again only one third (20/62) was also identical for DP with their presumptive recipients. In the majority of pairs a disparity in graft-versus-host direction or in host-versus-graft direction of at least one allele was seen. These data indicate that, in spite of the strict MLC criteria used, the current procedure did not warrant complete MHC class II identity. This implies that oligotyping for DPB1 can improve matching and should be introduced for typing of volunteers.