Cellular immunity in vitro. Clonal proliferation of antigen-stimulated lymphocytes.

When sensitive lymphocytes are cultured with the appropriate antigen, lymphoblasts appear after 24-48 hr of incubation and the number of these increases steadily from the 2nd to the 6th or 7th day. Our problem was to discover, at a cellular level, how this increase takes place; whether it is a massive response of many cells, stepwise recruitment of cells into the lymphoblast class, or simply repeated division of a few cells to form clones. In these experiments lymphocytes were incubated with antigen in culture tubes for 2-4 days and then a few cells, usually less than 200, were transferred to special microchambers for further culture. In these microchambers the cells could be viewed continually with a microscope and their fate recorded over the next 3-5 days by time-lapse cinemicrography. Examination of the film produced in this way showed that lymphoblasts divided and redivided to produce clones of 64 cells or more. It was possible to measure generation times from the film for 301 cells; the majority were between 8 and 13 hr but the range was 7.5-38.0 hr. There was no clear difference between generation times of human lymphocytes stimulated with tuberculin, streptokinase-streptodrnase, extract of the American pokeweed, or in the mixed leukocyte reaction. Similar times were also found for rat cells in the mixed leukocyte reaction. While these observations show that clonal proliferation does occur and could reasonably account for all the increase of lymphoblasts in lymphocyte cultures, the experiments, because of their design, do not exclude the possibility that other mechanisms such as recruitment may play a role as well, particularly during the first 48 hr after contact between sensitive cells and antigens.

Coordinate defects in human histocompatibility leukocyte antigen class II expression and antigen presentation in bare lymphocyte syndrome.

The human immunodeficiency, type II bare lymphocyte syndrome (BLS), has been attributed to a defect in the transcription of class II histocompatibility genes. Immunocompetence, as assessed by functional exogenous antigen presentation, was not restored in immortalized B cells, derived from a BLS patient, after transfection with HLA-DR class II structural genes. Incubation of protein antigens, as well as infectious virus, with DR-transfected BLS cells failed to induce activation of antigen-specific helper T lymphocytes. Peptide antigens were presented by class II molecules displayed on BLS cells, although the conformation of these class II proteins was altered as indicated by epitope mapping. This defect in antigen presentation was independent of the specific class II DR allele transfected into BLS cells. Genetic complementation analysis has been used with BLS cells to demonstrate that the defect in class II gene transcription is linked to the absence of a trans-acting factor. Similarly, functional class II dimers were restored after in vitro fusion of cells derived from two distinct BLS complementation groups, implying that specific transcriptional control elements are shared by a gene critical for antigen presentation and genes encoding HLA class II antigens. Thus, two important functionally linked pathways of class II molecules, structural gene expression and antigen presentation, share a common regulatory pathway defective in BLS.

A study of human leukocyte D locus related antigens in Graves' disease.

An association between Graves' disease and the human leukocyte antigen (HLA) system has previously been reported. The disease was more strongly associated with the HLA D locus antigen Dw3 than with HLA B8. Products of the HLA D locus are determined by the interaction of test cells with standard typing lymphocytes, a technically difficult procedure. Recently, it has been possible to type serologically for D locus related (DRw) specificities on peripheral bone marrow-derived (B) lymphocytes. Blood B lymphocytes from 50 unrelated controls and 41 patients with Graves' disease were typed for seven HLA DRw specificities. 28 patients with Graves' disease (68%) were positive for DRw3, in contrast to 14 controls (28%); whereas only 21 patients (50%) were HLA B8 positive, compared with 13 (26%) controls. Thus, positivity for DRw3 afforded a relative risk for Graves' disease of 5.5, whereas that for HLA B8 amounted to 3.0. Additionally, a family with multiple cases of Graves' disease in which the disease was previously shown to be inherited with the haplotype, was linked to DRw2, which suggests that the susceptibility to the disease was inherited in association with that antigen. Two HLA B/glyoxalase recombination events were observed in this family; in both instances HLA DRw followed HLA B. This study thus demonstrates that the disease susceptibility gene for Graves' disease is in strong linkage disequilibrium with DRw3; however, it may be associated with other DRw specificities and inherited within family units in association with them.

Thyroid autoimmune disease in a large Newfoundland family: the influence of HLA.

Ninety-eight members of a large Newfoundland family, seven of whose members over three generations suffered from Graves' disease, were studied with respect to the mode of transmission of the disease and its association with HLA. Compared to Newfoundland communities of similar size and geographical location, very little consanguinity was documented in this family. The susceptibility to Graves' disease appeared to be inherited as a dominant with a variable degree of expressivity; the degree of expressivity being determined by the female sex. In part of the pedigree, the susceptibility to Graves' disease entered the family with a wife. Three of her offspring who subsequently developed Graves' disease shared with her the haplotype A9, Bw16. Of the three remaining affected family members, two shared the haplotype A1, B8, whereas the third carried the haplotypes Aw32,b8; a9,bw16. Graves' disease could be associated with either of these two haplotypes in the last individual. This study shows that the susceptibility to Graves' disease is inherited associated with HLA and that whereas the disease susceptibility gene for Graves' disease is in linkage disequilibrium with HLA-B8 in Caucasians, it can be randomly associated with other HLA-B antigens.

Cerebral infarction and migraine: clinical and radiologic correlations.

Cerebral infarction was documented by arteriography and serial computed cranial tomography (CT) in four young adults (ages 16 to 32 years) with migraine. In one case, posterior cerebral artery occlusion produced a deep parietotemporal infarct. The other three cases all had frontotemporal infarcts (one hemorrhagic) in the territory of the middle cerebral artery, without major arterial occlusion. Two infarcts produced lasting neurologic deficits; one was associated with mild, transitory symptoms, and one was asymtomatic. Laboratory investigations in two cases revealed no hematologic or cardiovascular predisposition to cerebrovascular disease. Cerebral infarction, as revealed by CT, may be more prevalent in "complicated" migraine than is generally appreciated. Such lesions may or may not develop in chronologic and anatomic relationship to the headache, and may involve either large or small arteries. The prognosis for functional recovery, based on this limited sample, seems favorable.

Glutaraldehyde fixation of target cells to plastic for ELISA assays of monoclonal anti-HLA antibodies produces artefacts.

We found that an ELISA method for screening mouse monoclonal antibodies raised against antigens of the HLA system, that involves glutaraldehyde fixation of the target cells, produces both false negatives and false positives. The false negative results are due to destruction and/or modification of the antigenicity of some HLA-D region coded molecules; the false positives are mostly caused by non-specific adhesion of IgM, even at 1.625 micrograms/ml, to glutaraldehyde fixed cells. To a lesser extent there is nonspecific binding of IgG2a and IgG2b monoclonal antibodies particularly at concentrations above 12.5 micrograms/ml. These findings are unfortunate because the fixation of cells to the plates appeared to be a major technical advance as it removed the need for multiple centrifugation steps in the ELISA assay.

A plaque assay for cells making antibody against HLA antigens.

A plaque assay has been devised, for detecting cells making antibodies to HLA antigens, using intact nucleated cells as targets. The principle is to use a green fluorescent dye to stain living cells and a red fluorescent dye to stain the cells in a plaque that have been lysed by antibody and complement. Plaques are counted under a fluorescence microscope and the preparations can be made permanent and virtually non-quenchable by drying.

Toxoplasmosis presenting as brain abscesses. Diagnosis by computerized tomography and cytology of aspirated purulent material.

A patient with brain abscesses caused by Toxoplasma gondii is described. Presence of brain abscesses was confirmed by computerized tomography, and T. gondii was identified as the etiologic agent in cytologic preparations of aspirated purulent material from one of the abscesses.

Blood coagulation and fibrinolysis in thyroid disease.

Fibrinolytic activity and factor VIII concentration were studied in 30 patients with moderate to minimal hypothyroidism and in 7 patients with hyperthyroidism. In the hypothyroid group, the results were related to serum thyroxine levels, HL-A phenotypes and thyroid autoantibody titres. As serum thyroxine decreased so did factor VIII concentration, however, euglobulin lysis time was correspondingly prolonged. Factor VIII appears to be the most sensitive among coagulation factors to the deterioration of thyroid function tests. There was a significant correlation between the reciprocal of thyroid antibody titres and fibrinolysis; however, there was no relationship between factor VIII concentration or fibrinolysis and a specific HL-A phenotype although the incidence of HL-A8 was increased in the group as a whole. Euglobulin lysis time was prolonged in 6 out of 7 patients with Graves' hyperthyroidism. Factor VIII was elevated in only 3 of these patients.

Transfection of HLA genes using genomic DNA.

Mouse L cells expressing HLA genes are potentially useful for producing and analyzing monoclonal antibodies (mAb) to HLA molecules. This paper describes the preparation of transfectants using uncloned human DNA and three methods to isolate the HLA-expressing transfectants. Transfectant libraries were made by cotransfecting mouse thymidine kinase (tk)-deficient L cells with a calcium phosphate precipitate containing genomic DNA and tk plasmid DNA. Transfectants expressing HLA genes were isolated using these methods: immunomagnetism, replicate-plating combined with cellular enzyme-linked immunoassay, and sorting using a fluorescence-activated cell sorter. Two HLA-A2 transfectant were isolated using immunomagnetism, two HLA-A24 transfectants by replicate plating, and one HLA-Bw60 transfectant by FACS. However, no transfectants were isolated that stably expressed class II genes. The class I transfectants have been useful in characterizing several locally prepared mAbs which bind to monomorphic determinants on class I HLA molecules. Two of the transfectant lines, one expressing HLA-A2 (8001) and the other HLA-A24 (8008), have been included in the collection of lines distributed for use in the Eleventh International Histocompatibility Workshop.

Analysis of monoclonal antibodies specific for unique and shared determinants on HLA-DR4 molecules.

The specificities for seven mAbs to HLA-DR4 were determined initially using homozygous BCLs and L-cell transfectants expressing wild-type DR molecules. Three antibodies (NFLD.D1, NFLD.M1, and NFLD.D7) bound all DR4 molecules, but only one was specific for DR4. Four antibodies (NFLD.D2, NFLD.D3, NFLD.D8, and NFLD.D10) reacted with some but not all DR4 subtypes and had extra reactions, particularly with DR gene products associated with susceptibility to RA. To localize the antibody-binding epitopes on DR4 molecules, the antibodies were then analyzed on transfectants expressing hybrid genes, which were generated by exon shuffling of DRB1*0403 and DRB1*0701. Two of the pan-DR4 antibodies bound epitopes that require the beta 2 domain while the third mapped primarily to the HVR-I region. One antibody NFLD.D10 to subtypes of DR4 mapped to residues 40-97 on DR beta 1*0403 chains. Comparison of reaction patterns with amino acid sequences suggest that the antibodies against subtypes of DR4 are specific primarily for a region containing sequences postulated to determine susceptibility to RA.

HLA-DR residues accessible under the peptide-binding groove contribute to polymorphic antibody epitopes.

Many residues involved in polymorphic antibody-binding epitopes on class II molecules are located on the alpha-helix of DR beta chains. Although they have received less attention, residues in the peptide-binding groove and second domain of the DR beta chain may also be critical for polymorphic anti-DR antibody epitopes. In this study, we used transfectants expressing site-directed mutations at positions in the HLA-DR beta 1 and beta 2 domains and flow cytometry to define the epitopes of several polymorphic anti-DR antibodies. Both DR(beta 1*0403) residues 14 and 25 were shown to be involved in the epitopes of mAbs DA6. 164, HU-20, Q5/6, and 50D6, and DR(beta 1*0701) residue 14 was shown to be critical for the epitopes of two DR7-specific mAbs, SFR 16-DR7M and TAL13.1. Unlike most other residues shown to be important in antibody-binding epitopes, residue 14 is located in the floor of the peptide-binding groove and residue 25 is in an outer loop, each with their side chains pointing down, such that antibodies may directly contact these residues from below the binding groove. Two residues in the beta 2 domain, beta 180 and beta 181, were also shown to be involved in the epitopes of three polymorphic anti-DR mAbs, NFLD.D1, NFLD.M1, and LY9. Although these two residues are close to the transmembrane domain in the linear sequence, their solvent accessibility in the DR1 structures is quite impressive. Our data provide new evidence that residues accessible under the peptide-binding groove contribute to polymorphic antibody-binding epitopes.

HLA-DP epitope typing using monoclonal antibodies.

We have made a panel of murine anti-DP monoclonal antibodies for serological typing of HLA-DP polymorphisms; they can be used in microcytotoxicity (for 7 epitopes) and binding assays (for 8 epitopes). The antibodies detect polymorphic differences in both alpha and beta chains. As immunogens we sometimes used B-lymphoblastoid lines or purified DP molecules but mostly used mouse fibroblast transfectants expressing DP molecules. The DP beta genes were made from a cloned DPB1*0201 gene by replacing its major area of polymorphism with matching stretches of DNA amplified from other alleles; cloned DPA1*01 and DPA1*02 genes were used for transfection along with the beta chain genes. The monoclonal antibodies showed reaction patterns that correlated with the presence of particular amino-acid sequence motifs; thus none of the antibodies is allele-specific. They bind instead to epitopes which are found on a number of different HLA-DP types. We have constructed frequency tables so that the epitope (motif) data can be interpreted as the most likely genotype in each case. The basic assumption to justify this work is that HLA-DP matching or mismatching will likely influence transplant outcome, particularly in bone marrow transplantation. The present challenge is to define permissive and nonpermissive combinations of HLA-DP; it may be that matching for epitopes, rather than for full alleles, will help to resolve this issue.

Influence on antibody recognition of amino acid substitutions in the cleft of HLA-DQ2 molecules. Suggestive evidence of peptide-dependent epitopes.

The purpose of this study was to identify polymorphic residues of DQ2 beta chains which are involved in the epitopes recognized by DQ2-reactive mAbs. Binding studies were performed on a panel of DQ2 mutant cells made by transfection of a DQ (alpha 1*0501, beta 1*0301)-positive B-LCL with DQB1 genes encoding either wild-type or mutated DQ beta 1 *0202. Several aa substitutions were found to influence the binding of DQ2 mAbs. All but one of the mAbs were clearly sensitive to the introduced aa substitutions, but individual mAbs were differentially influenced, suggesting that they recognized different epitopes on the DQ2 molecule. Substitutions in positions 30 and 37 of the peptide-binding cleft were also found to influence the binding of some mAbs. Second, two mAbs, NFLD.M71 and NFLD.M102, which bound to DQ(alpha 1*0501, beta 1*0202) expressed in human cells and were sensitive to the introduction of aa substitutions in the cleft, bound weakly to the DQ(alpha 1*0501, beta 1*0202) heterodimer when expressed in a transfected murine NIH 3T3 cell line. Together this indicates that some DQ2-reactive mAbs may recognize peptide-dependent epitopes.

Inbreeding in outport Newfoundland.

We investigated inbreeding in 3 outport Newfoundland study areas in which persistent genetic isolation was demonstrated previously. The inbreeding coefficient of every person born in each area was calculated from reconstructed pedigree data. The average inbreeding coefficient for persons born between 1960 and 1979 is 0.0032 in Trepassey Parish (southern Avalon Peninsula), 0.0171 for a group of communities on the west coast of the Great Northern Peninsula, and 0.0007 for southeastern Labrador. Average inbreeding of these populations was higher earlier in this century. Averages are high compared to those reported for other populations, consistent with genetic isolation. Coefficients of kinship were calculated for large samples of pairs of births in each study area, and frequency distributions of inbreeding coefficients compared with frequency distributions of kinship coefficients, to evaluate random and nonrandom mating with respect to consanguinity. These comparisons show that matings between unrelated individuals are more frequent than expected, that matings between first cousins are not strongly avoided in 2 of the 3 study areas, that matings between first cousins once removed are favored, and that matings between distantly related individuals are becoming more frequent. To gain an impression of the potential contribution of inbreeding to the prevalence of recessive disease, we calculated indirect estimates of the expected excess of prereproductive mortality due to inbreeding. These estimates are 15% for Trepassey Parish, 49% for the West Coast study area, and 2% for southeastern Labrador. It is unlikely that genetic isolation of outport Newfoundland populations will decrease. Elevated prevalences of recessive disease, due primarily to matings between persons unaware of their distant consanguinity, therefore require consideration in health care planning in Newfoundland.

Persistent genetic isolation in outport Newfoundland.

The historical development of genetic isolation has been evaluated for three outport Newfoundland study areas. An attempt was made to ascertain all livebirths in each study area, and determine the parentage of each. Data from records of baptism and marriage were used for this, supplemented with other historical and ethnographic information. Parent-offspring migration was used as a measure of genetic exchange between subpopulations within study areas, and gene flow into the study areas. Currently, 1-8% of parents originate outside the study areas; these rates are low compared to earlier periods, and compared to present-day rates for European isolate populations. Average kinship was estimated, to measure genetic relatedness within and between subpopulations of each area and the potential for random inbreeding; these values, which are minimum estimates, are now at historically high levels. Increased migration into the study areas, which would decrease average kinship, is not likely. Thus, any regionally or locally elevated frequencies of deleterious alleles will persist, and must be taken into account in providing genetic counseling and evaluating the utility of local screening programs.

Parentage testing implications of male fertility after allogeneic bone marrow transplantation.

Fertility is expected to be reduced after the extensive chemotherapy and/or radiotherapy that is needed for conditioning prior to bone marrow transplantation. However, a male patient can be fertile, and in very rare situations such as reported here, this may confuse subsequent paternity testing. The patient, initially excluded as the biological father by red cell types but not by HLA, was subsequently included after the history of his previous marrow transplant was revealed, a review of the HLA results and further RFLP testing on buccal mucosal cells. This case points to the need for good history taking before performing paternity testing.

Left-right temporal region asymmetry in infants and children.

Thirty-two consecutive cranial computed tomographic (CT) scans in normal infants ranging from 1 day to 3 years of age were evaluated for asymmetry of the temporal lobes as evidenced by differences in the size of the sylvian fissures. The left sylvian fissure was larger than the right in 23 of the 32 cases, which was statistically significant (p less than 0.0001). In five other cases the two sides were equal; in the four remaining cases, the right side was larger than the left. The results show that asymmetry of the temporal lobes can be demonstrated in vivo even at birth and that this asymmetry is a normal developmental difference between the two hemispheres and not secondary to an acquired abnormality.

CT of intracranial metastases with skull and scalp involvement.

Twenty-eight persons with contiguous intracranial skull, and often extracranial metastatic disease are reported. These lesions comprised 7.6% of a series of 250 consecutive patients with intracranial metastatic disease. Only three of 28 patients had other intracranial lesions and only seven of 28 patients has other skull lesions demonstrable on computed tomography (CT). Carcinoma of the prostate and breast, multiple myeloma, and neuroblastoma are especially likely to appear in this manner. All metastases enhanced. The bone destruction was so pervasive that in 19 of the patients it was obvious at routine CT settings. In the nine other patients, it could be clearly seen only at bone settings (high window and level). The CT demonstration of an enhancing intracranial mass involving the skull and often the scalp is highly suggestive but not diagnostic of a metastatic lesion.

Intraoperative sonography through a burr hole: guide for brain biopsy.

Sonographic imaging of the brain was accomplished through a burr hole using a small, real-time, 5 MHz transducer in five patients. Brain tumors were accurately imaged, corresponding closely in morphology to the computed tomographic scan. Intraoperative sonography through a burr hole allows flexibility in technique and greatly facilitates brain biopsy procedures.