Evidence for a primary association of celiac disease to a particular HLA-DQ alpha/beta heterodimer.

Typing of DNA from 94 unrelated children with celiac disease (CD) with HLA-DQA1 and -DQB1 allele-specific oligonucleotide probes revealed that all but one (i.e., 98.9%) may share a particular combination of a DQA1 and a DQB1 gene. These genes are arranged in cis position on the DR3DQw2 haplotype and in trans position in DR5DQw7/DR7DQw2 heterozygous individuals. Thus, most CD patients may share the same cis- or trans-encoded HLA-DQ alpha/beta heterodimer.

MS and clinically isolated syndromes: shared specificity but diverging clonal patterns of virus-specific IgG antibodies produced in vivo and by CSF B cells in vitro.

BACKGROUND: Intrathecal synthesis of oligoclonal IgG antibodies against measles virus (MeV), varicella zoster virus (VZV) and herpes simplex virus type-1 (HSV-1) is a characteristic feature multiple sclerosis (MS). METHODS: We have used isoelectric focusing-immunoblot to define the clonal patterns of IgG and of IgG antibodies to MeV, VZV and HSV-1 in supernatants of in vitro cultures of peripheral blood lymphocytes (PBL) and cerebrospinal fluid (CSF) cells and in sera and CSF from three patients with MS and three patients with clinically isolated syndromes (CIS) suspective of demyelinating disease. RESULTS: In vitro synthesis of IgG by PBL was not detected in any patient. In contrast, in vitro synthesis by CSF cells of oligoclonal IgG and oligoclonal IgG antibodies to one or two of the three viruses tested was observed in all six patients. The clonal patterns of the in vitro synthesized IgG and virus specific IgG differed to varying extent from those synthesized intrathecally in vivo. However, in each patient, the in vitro and in vivo intrathecally produced antibodies displayed specificity for the same viruses. The addition of B cell activating factor (BAFF) had no effect on the amounts or clonal patterns of either total IgG or virus-specific IgG produced by CSF cells in vitro. CONCLUSION: Virus specific B cells capable of spontaneous IgG synthesis are clonally expanded in the CSF of patients with MS. The B-cell repertoire in CSF samples is only partially representative of the intrathecal B-cell repertoire.

GAD65 IgG autoantibodies in stiff person syndrome: clonality, avidity and persistence.

BACKGROUND AND PURPOSE: Persistent intrathecal production of IgG autoantibodies against glutamic acid decarboxylase 65 (GAD65 IgG) and oligoclonal IgG of undetermined specificity has been reported in stiff person syndrome (SPS). METHODS: To chart the avidity and clonal patterns of GAD65 IgG, we performed scatchard plot of binding characteristics and isoelectric focusing-immunoblot of cerebrospinal fluid (CSF) and serum from five SPS patients. RESULTS: Oligoclonal GAD65 IgG bands, predominantly restricted to the IgG1 subclass, were detected in CSF and serum in all patients. The distribution of GAD65-specific IgG bands in serum and CSF revealed intrathecal synthesis of oligoclonal GAD65 IgG in all five patients, whilst radioimmunoassay demonstrated intrathecal synthesis of GAD65 IgG in four. The binding avidity of GAD65 IgG from CSF was more than 10 times higher than in serum in two of the patients but did not differ substantially in the remaining three. These differences were not related to symptom severity. The pattern of oligoclonal GAD65 IgG bands in CSF and serum in three patients examined remained unchanged for up to 7 years after symptom debut. CONCLUSION: This study confirms the persistent systemic and intrathecal production of GAD65-specific IgG in SPS, and further shows that this immune response is oligoclonal and mediated by a stable population of affinity maturated B cell clones.

T lymphocyte subset changes in human immunodeficiency virus infection.

The absolute number of CD4+ and CD8+ T cells was determined by an immunomagnetic technique directly in the blood of 75 healthy controls and 223 HIV-infected individuals. The HIV-seropositive individuals were also classified clinically according to the system recommended by the Centers for Disease Control (CDC), and the CDC classification was correlated with the patients' T cell subset counts. Compared to the control group, the HIV-infected individuals demonstrated an early and sustained increase in the number of CD8+ T cells with median values of CDC groups II, III, and IV C2 twice that observed in the control group. Patients with AIDS had CD8+ T cell counts comparable to those of the control group. The HIV-infected individuals showed a decrease in the number of CD4+ T cells correlating with clinical deterioration of the disease. T cell subset counts significantly distinguished the group of healthy seropositive individuals from those with HIV-related disease, and the group of patients with AIDS from those with other HIV-related opportunistic infections.

In vitro replication of HIV-1 in naturally infected CD4+ T cells is inhibited by rIFN alpha 2 and by a soluble factor secreted by activated CD8+ T cells, but not by rIFN beta, rIFN gamma, or recombinant tumor necrosis factor-alpha.

The effect of lymphokines on the replication of HIV-1 has previously been investigated using HIV-1-infected cell lines or PBMCs infected in vitro with HIV-1. We have examined the effect of rIFN alpha 2, rFIN beta, and rIFN gamma and recombinant tumor necrosis factor-alpha (rTNF alpha) on the replication of HIV-1 in vitro in naturally HIV-1-infected CD4+ T cells from asymptomatic HIV-1-seropositive individuals. rIFN alpha 2 inhibited the replication of HIV-1 effectively at concentrations that can be achieved in vivo. The inhibitory activity was most efficacious when rIFN alpha 2 was added as the CD4+ T cells were being activated, and less but still considerable when rIFN alpha 2 was added 4-96 h after CD4+ T-cell activation. rIFN alpha 2 exerted a suppressive effect on the proliferation of the CD4+ T cells, but this effect was small at concentrations that caused 90% inhibition of the replication of HIV-1. rIFN beta, rIFN gamma, and rTNF alpha had no effect on the replication of HIV-1, but rIFN beta and rTNF alpha had a costimulatory effect on CD4+ T-cell proliferation. Activated CD8+ T cells secrete a HIV-1-inhibitory soluble factor. Blocking experiments using an IFN alpha 2-neutralizing MAb showed that this HIV-1-inhibitory factor is not IFN alpha 2.

Imprint immunofixation of electrofocused immunoglobulins.

An imprint immunofixation (IIF) technique for the characterization of electrofocused immunoglobulins (Ig) is described. Electrofocused proteins are blotted (imprinted) from the separating polyacrylamide gel to agarose gels by gel-to-gel overlays. The protein imprints are chemically fixed in the agarose gels with a solution of 46% methanol, 8% acetic acid and 46% water. The imprinted Ig are then identified radioimmunologically, using an indirect system with monoclonal mouse anti-human Ig antibodies in the first layer and 125I-labelled rabbit anti-mouse Ig in the second, followed by autoradiography. The method is sensitive and permits characterization of Ig in unconcentrated cerebrospinal fluid. By sequential imprinting, each separated specimen can be characterized for up to 10 separate antigenic determinants without loss of sensitivity.

Immunofluorescence staining of agarose-embedded cells. A new technique developed for immunological characterization of markers on a small number of cells.

A simple new method for the immunofluorescence staining of small numbers of cells is described: cell suspensions are mixed with low-temperature-gelling agarose at 37 degrees C and 2 microliter samples of agarose containing cells are dispensed onto multitest microslides precoated with agarose. The cells are subsequently stained by immunofluorescence techniques. Alternatively, the cell slides can be stored in liquid nitrogen until immunofluorescence staining is carried out. Since cells are entrapped within the agarose matrix, cell loss is prevented during staining and washing procedures. The method permits staining of as few as 250 cells for each marker, thus enabling simultaneous characterization of several separate cell markers in cerebrospinal fluid or other body compartments from which comparatively few cells are obtainable.

Depletion of T lymphocytes from human bone marrow. Use of magnetic monosized polymer microspheres coated with T-lymphocyte-specific monoclonal antibodies.

A new technique for depletion of T cells from bone marrow is presented. Bone marrow cells (BMC) were rosetted with magnetic monosized polystyrene microspheres coated with monoclonal antibodies (MAbs) specific for T cell CD2 and CD3 antigens. Rosetted T cells were subsequently removed from non-T cells with the aid of a magnet. This immunomagnetic separation procedure was carried out in less than 40 min and reproducibly removed T cells, leaving a maximum of 0.025% sheep-red-blood-cell (SRBC) rosette-forming cells and less than 0.02% T cells as detected by a T cell limiting dilution assay. The efficacy of the depletion procedure was further shown by flow cytometry data, by effective removal of cells from a T cell line added to the BMC prior to immunomagnetic separation, and by abrogation of interleukin 2 (IL-2)-producing capacity in T-cell-depleted BMC (BMC-T). The T cell depletion procedure provided a 43-74% recovery of non-T cells present in the Isopaque-Ficoll-isolated bone marrow mononuclear cell fraction and did not disturb the growth potential of stem cells, as assayed by hematopoietic stem cell assays.

A strong impact of matching for a limited number of HLA-DR antigens on graft survival and rejection episodes: a single-center study of first cadaveric kidneys to nonsensitized recipients.

BACKGROUND: A single-center study of 655 nonsensitized recipients of primary cadaveric kidney grafts is presented. RESULTS: Graft survival in serologically HLA-DR 1-10 antigen-matched grafts to nonsensitized recipients at 1 year was 90%, compared with 82% (P=0.004) and 73% (P=0.001) in one and two DR antigen-mismatched grafts. The corresponding figures at 5 years were 76%, 62%, and 56%, respectively. Matching for the DR antigens 11-14, or for some DR alleles only detectable by genomic typing, further improved graft survival, but the differences did not reach statistical significance. Matching also for the serologically defined HLA-A and -B antigens did not significantly further improve overall graft survival, but some effects for grafts surviving at least 1 year were observed. Among recipients of grafts mismatched for zero, one, or two HLA-DR antigens, acute rejection episodes were experienced in 48%, 64% (P<0.001), and 82% (P<0.001), respectively, within the first 3 months. HLA-A and -B mismatches showed no significant correlation to acute rejection episodes. CONCLUSION: Matching for the DR antigens 1-10 significantly secures and prolongs the survival of first cadaveric renal grafts. Our results also show that DR 1-10 antigen-matched combinations can often be obtained even in rather small recipient pools, when actively sought for.

No linkage or association of the nitric oxide synthase genes to multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) of unknown etiology. Nitric oxide (NO) is a free radical that participates in a variety of biological processes. It is an important mediator in the immune response. Several studies indicate involvement of NO in the pathogenesis of MS. We studied five markers within the three NO synthase genes with regards to susceptibility and disease course in 156 affected sib-pairs and in 96 "benign" and 96 "severe" definite MS patients and 148 controls. We found no significant association or evidence for linkage in our data sets.

No association of multiple sclerosis to alleles at the TAP2 locus.

MS is associated with genes in the HLA complex. We have previously proposed that the primary HLA-associated susceptibility may be conferred by particular DQ alleles. Furthermore, we have previously found that there is no association of MS to DP alleles. This study shows that MS is not associated to alleles of the TAP2 locus, which is located close to DQ on its centromeric side. This observation provides further support to the notion that HLA-associated susceptibility to MS maps telomeric to the TAP2 locus.

HLA-DQA1 and HLA-DQB1 genes may jointly determine susceptibility to develop multiple sclerosis.

Serologic DR typing and genomic DRB1, DQA1, DQB1, DPA1, and DPB1 typing using sequence-specific oligonucleotides were performed in 69 multiple sclerosis (MS) patients and 181 healthy controls on in vitro amplified DNA. The frequencies of DR2 as well as the DR2-associated DQA1*0102 and DQB1*0602 alleles were increased whereas DR7 was decreased among MS patients. The distribution of DR4 subtypes as well as DP alleles were similar in patients and healthy controls. All but one of 23 DR4-positive MS patients carried the DQB1*0302 allele, whereas five of five DR7-positive MS patients carried the DQB1*0303 allele. Of the MS patients, 99% compared to 79% of the controls carried DQA1 alleles encoding glutamine at residue 34, while 97% of the MS patients compared to 72% of the controls carried DQB1 alleles encoding DQ beta chains sharing long polymorphic stretches. A combination of such DQA1 and DQB1 alleles was carried by 96% of the MS patients and 60% of the controls, suggesting an association between MS and a combination of particular DQA1 alleles and DQB1 alleles. The corresponding DQ alpha beta heterodimers may have in common an ability to bind a particular peptide.

Patients with multiple sclerosis carry DQB1 genes which encode shared polymorphic amino acid sequences.

Of 61 Norwegian multiple sclerosis patients tested, 59, i.e., 97%, were positive for at least one of the HLA specificities DR2, DR4, or DRw6. Typing with sequence-specific oligonucleotide probes revealed that the same 59 patients carried DR2-, DR4-, or DRw6-associated HLA-DQB1 genes which encode shared polymorphic amino acid sequences in the membrane-distal part of their HLA-DQ beta chains. This shared DQ beta polymorphism may explain previously reported DR associations and could thus be the primary HLA association in MS.

HLA-DR and -DQ genotypes of celiac disease patients serologically typed to be non-DR3 or non-DR5/7.

The susceptibility to develop celiac disease (CD) seems to be primarily associated to a particular HLA-DQ alpha/beta heterodimer encoded by the DQA1*0501 and DQB1*0201 alleles, in cis position on the DR3-DQ2 haplotype or in trans position by DR5-DQ7/DR7-DQ2 heterozygotes. However, exceptional patients exist who are neither DR3 nor DR5/DR7, particularly among Southern European populations. We therefore examined the DRB1, DQA1, and DQB1 alleles of 13 Spanish CD patients who were serologically typed to be neither DR3 nor DR5/DR7. Five patients were found to carry the DQA1*0501 and DQB1*0201 alleles either in cis or in trans position, three of them had previously been serologically mistyped. However, two of these patients carried DQA1*0501 and DQB1*0201 on haplotypes other than DR3 or DR5 in combination with DR7. One of the latter patients carried an unusual DR4-DQ2 haplotype, while another had an unusual DR8-DQ2 haplotype. Four of the remaining eight patients carried DR4-DQ8 haplotypes. Taken together, our findings provide further evidence that the DQ alpha/beta heterodimer encoded by the DQA1*0501 and the DQB1*0201 alleles confers the primary HLA-associated susceptibility to develop CD. However, our studies also corroborate that a second (and "weaker") HLA-associated CD susceptibility gene may be present on some DR4-carrying haplotypes.

Distribution of HLA class II alleles among Norwegian Caucasians.

We report genomic HLA class II typing of 181 randomly selected Norwegian controls. Seventeen DRB1, 7 DQA1, 10 DQB1, 2 DPA1, and 16 DPB1 alleles were found in the tested population. HLA class II antigen and allele frequencies are given, as well as the distribution of DRB1, DQA1, DQB1 haplotypes. Linkage disequilibrium between some DPB1 alleles and DRB1 and/or DQB1 alleles are also reported.

Heterogeneity of T cells specific for a particular peptide/HLA-DQ complex.

Whether T cells specific for a particular peptide/HLA-DQ complex are restricted with respect to TCR usage has not been fully established. TCR usage of T cells specific for a peptide presented by a given HLA-DQ molecule has not been studied before. We therefore sequenced the TCR genes of five different TCCs derived from the same donor, which were specific for a p21 ras-derived synthetic peptide presented by the HLA-DQ(alpha 1*0102,beta*0602) (DQ6) molecule. We found that these T cells which recognized the same peptide/HLA-DQ complex used highly diverse TCRs. However, dose-response experiments using various truncations of the p21 ras-derived peptide revealed that the peptide fine specificities of the five TCCs were not completely identical. This may explain the heterogeneity in TCR usage.

Susceptibility to develop celiac disease is primarily associated with HLA-DQ alleles.

We have recently reported that the susceptibility to develop celiac disease (CD) seems to be primarily associated to a particular combination of an HLA-DQA1 (DQA1*0501) and an HLA-DQB1 (DQB1*0201) allele: i.e., a particular DQ alpha/beta heterodimer. To investigate whether certain DP alleles might also contribute to the genetic susceptibility, DPA1 and DPB1 genes of 94 CD patients and 132 healthy controls were examined by probing in vitro amplified DNA with sequence-specific oligonucleotide probes corresponding to all hitherto known DPA1 and DPB1 alleles. The frequencies of the DPA1*0201 and of the DPB1*0101 alleles were increased in CD patients compared to healthy controls (0.31 versus 0.14 and 0.25 versus 0.08, respectively). However, these DP alleles were in linkage disequilibrium with CD-associated DQ alleles in the normal population, and the difference in frequency of these DP alleles was no longer significant when CD patients and healthy controls carrying the CD-associated DQA1*0501 and DQB1*0201 alleles were compared. DQB1*0201 homozygous individuals were overrepresented among DQB1*0201-positive patients compared to controls. When DQB1*0201 heterozygous patients and controls were compared, nearly identical frequencies of the DPA1*0201 and the DPB1*0101 alleles were found. Thus, the observed increase of the DPA1*0201 and DPB1*0101 alleles among CD patients seems mainly to be caused by linkage disequilibrium to the CD-associated DQ alleles.

Reliable isolation of human immunodeficiency virus from cultures of naturally infected CD4+ T cells.

A procedure for reliable and reproducible isolation of human immunodeficiency virus (HIV) from cultures of CD4+ T cells from healthy HIV seropositive individuals is described. Using immunomagnetic cell separation techniques, CD4+ T cells were positively selected from blood or peripheral blood mononuclear cells from 34 HIV infected individuals in CDC group II. The cells were stimulated with beads coated with a monoclonal antibody specific for the T cell receptor alpha/beta dimer and cultured in medium containing recombinant IL 2. HIV was isolated from 100% of the 41 cultures from 34 individuals. As this culture system allows reproducible isolation of HIV from cultures of naturally infected CD4+ T cells in the absence of other autologous or allogeneic cells, it may provide a good test system for the study of factors affecting the replication of HIV at low multiplicities of infection.

Herpes simplex virus encephalitis: intrathecal synthesis of oligoclonal virus-specific IgG, IgA and IgM antibodies.

Paired specimens of serum and CSF from seven patients with acute herpes simplex virus encephalitis were examined during the acute illness or the convalescent stage or during both stages. Imprint immunofixation analyses of viral antibodies separated by agarose electrophoresis and by electrofocusing disclosed intrathecal production of herpes simplex virus IgG antibodies in all seven patients, and of IgA and IgM antibodies in six and three of six patients, respectively. Intrathecal production of herpes simplex virus-specific IgG and IgA was observed in two patients from whom samples were collected after 1 year, while intrathecal production of virus-specific IgM was not demonstrated later than 5 weeks after onset. The intrathecally synthesized IgG and IgM, and to a lesser extent IgA antibodies displayed oligoclonal characteristics. Oligoclonal bands of IgG were observed in the CSF of all patients. Evidence is presented to show that the bulk of the oligoclonal CSF IgG represents herpes simplex virus-specific antibodies. Intrathecally synthesized populations of herpes simplex virus antibodies cross-reacting with varicella-zoster virus were identified in three of the patients.

Multiple sclerosis. Electrofocused "bands" of oligoclonal CSF IgG do not carry antibody activity against measles, varicella-zoster or rotaviruses.

Electrofocused serum and cerebrospinal fluid (CSF) specimens from patients with multiple sclerosis (MS) were analysed for immunoglobulins (Ig) and for antibodies to measles, varicella-zoster and rotaviruses by an imprint immunofixation method. Patterns of intrathecally synthesized antibodies to the 3 viruses differed from patterns of oligoclonal IgG in the CSF. A variable proportion of virus antibody bands (average 19% for measles antibodies, 8% for varicella-zoster antibodies, 31% for rotavirus antibodies) displayed isoelectric points identical to bands of IgG, but absorption with measles, varicella-zoster and rotavirus antigens produced no change in the bands of IgG and no quantifiable decrease of the CSF IgG. The results confirm previous evidence that the intrathecally synthesized viral antibodies so far demonstrated in MS are not carried by the oligoclonal bands of CSF IgG and account for only a minor fraction of the CSF IgG.