Bodil K. Jakobsen

HLA-D and -DR antigens in genetic analysis of insulin dependent diabetes mellitus

SummaryThree groups of patients with insulin-dependent diabetes mellitus, ascertained by different procedures, were investigated for HLA-A, B, C and D antigens (n=164), and a subset (n=93) for HLA-DR. Both HLA-D/DR3 and D/DR4 were strongly positively associated and D/DR2 was negatively associated with insulin-dependent diabetes. HLA-DR4 was found to be a better marker for insulin-dependent diabetes than Dw4. The HLA-B associations (B8, B15 and B18) were clearly secondary to the increases of HLA-D/DR3 and D/DR 4. The HLA associations did not differ between familial and isolated cases indicating that these two groups may well have a common genetic background. Based on analysis of HLA-haplotype sharing in affected sibling pairs, a simple dominant model of inheritance could be ruled out, and a simple recessive model was found unlikely. The relative risks for the HLA-Dw3,4 and HLA-DR3,4 phenotype were 21.2 and 44.4 respectively and exceeded those of both the HLA-Dw3 and HLA-DR3 (5.6 and 4.3) as well as the HLA-Dw4 and DR4 (10.1 and 10.5) phenotypes. This argues against an intermediate genetic model but further studies are needed to clarify whether there is more than one susceptibility gene for insulin-dependent diabetes mellitus within the HLA-system.

Association between HLA-DR2 and Production of Tumour Necrosis Factor α and Interleukin 1 by Mononuclear Cells Activated by Lipopolysaccharide

The production of tumour necrosis factor (TNF) and interleukin 1 (IL‐1) by lipopolysaccharide‐activated mononuclear cells from 39 healthy donors was studied in vitro by bioassay and ELISA. The donors were typed for HLA‐A, ‐B, ‐C, ‐DR, and ‐DP antigens. There was no detectable production of TNFβ (lymphotoxin). The intracellular levels of bioactive TNFα were minimal or undetectable in all cases. Cells from HLA‐DR2+ individuals secreted significantly lower amounts of TNFα than cells from HLA‐DR2− donors [2 ng/ml (1.5–4.4) and 7.5 ng/ml (3.9–8.3) respectively (medians 25–75%); P<0.01]. The difference disappeared if the cells were preactivated for 2 days with 1000 U/ml of recombinant gamma interferon (rIFN‐γ). In some individuals, the TNFα response increased considerably after IFN‐γ priming, in particular in those possessing the HLA‐DR2 antigen. In contrast, there was no detectable difference in the production of IL‐1β between the donors, and the IL‐1β response decreased significantly after rIFN‐γ priming in HLA‐DR2+ individuals [2.3 ng/ml (1.1–8.4) versus 7.2 ng/ml (5–7.9); P<0.05] and in HLA‐DR2− individuals [3 ng/ml (1.1–5.3) versus 5.7 ng/ml (3.9–7.5); P<0.01]. There was no correlation between the TNFα and IL‐1 responses and any of the other HLA‐DR, ‐DP, or ‐B antigens. There was a significant positive correlation between the levels of TNFα measured by ELISA and by cytotoxicity assay. However, the TNFα‐containing supernatants from 9 out of 37 individuals appeared to contain inhibitor(s) of the biological activity of TNFα. The presence of inhibitor(s) was not associated with any HLA antigens.

NcoI restriction fragment length polymorphism (RFLP) of the tumour necrosis factor (TNF alpha) region in primary biliary cirrhosis and in healthy Danes

The restriction fragment length polymorphism of the human tumour necrosis factor (TNFα) region was investigated by means of 20 different restriction enzymes and a human TNFα cDNA probe. Only one of the enzymes, NcoI, revealed a polymorphic pattern consisting of fragments of 10.5 and 5.5 kb, which behaved as alleles. In a panel of 108 random, healthy, unrelated Danes, the phenotype frequencies of the 10.5 and 5.5 kb bands were 0.94 and 0.53 and the gene frequencies were 0.71 and 0.29, respectively. The test for Hardy‐Weinberg equilibrium showed no significant deviation from the expected values. The 5.5 kb band was strongly positively associated with HLA‐DR3, HLA‐B8, and HLA‐A1. In 22 patients with primary biliary cirrhosis a significantly (corrected P= 0.024) decreased frequency of the 10.5 kb fragment was found. Additional studies are in progress to substantiate this association.

DNA polymorphism of HLA class II genes in primary biliary cirrhosis

We investigated the DNA restriction fragment length polymorphism of the major histocompatibility complex class II genes: HLA-DRB,-DQA,-DQB, DPA,-DPB, the serologically defined HLA-A, B, C, DR antigens, and the primed lymphocyte typing defined HLA-DP antigens in 23 Danish patients with primary biliary cirrhosis (PBC) and in healthy Danes. The following genetic markers were found with increased frequencies in PBC: HLA-B8 (relative risk, RR=2.4, P<0.05, ‘corrected’ P>0.05), HLA-DR3 (RR=3.4, P<0.01, ‘corrected’ P<0.05), the DRB3*01/02/03 (DRw52) associated DRB Bgl II 9.1 kilobase (kb) fragment (RR= 2.9; P<0.05, ‘corrected’ P>0.05), the DQA1*0501 associated DQA Taq I4.8 kb fragment (RR=3.1; P<0.05, ‘corrected’ P>0.05), the DQB1*0201 (DQw2) associated DQB Hin dIII 11.5 kb fragment (RR=3.1; P<0.05, ‘corrected’ P>0.05). No DNA fragments specific for DRB1*0301 (DR3) could be identified. The frequencies in PBC of other genetic markers including DRw8, DRB1*08, HLA-DP antigens, DPA, and DPB genes did not differ significantly from those in controls. The associations between PBC and B8, DR3, DQA1*0501, and DQB*0201, which are frequently found together on the same haplotype, are at variance with recent reports on associations between PBC and Drw8. The discrepancy suggests that PBC is genetically heterogenous.

Haematopoietic stem cell transplantation with non‐myeloablative conditioning in the outpatient setting: results, complications and admission requirements in a single institution

Thirty patients with haematological malignancies received peripheral blood stem cells from human leucocyte antigen (HLA)‐identical sibling donors after non‐myeloablative conditioning with fludarabine and total body irradiation. Twenty‐seven patients received the transplant as an outpatient procedure. All patients engrafted. The probability of acute graft‐versus‐host disease (GVHD) grades II–IV and extensive chronic GVHD was 57% and 80%, respectively. Patients alive on day +365 experienced a median of 44 d (range 4–151) of hospitalization during the first year. In the entire cohort, GVHD accounted for 22%, infections for 18%, thrombotic thrombocytopenic purpura (TTP) for 16% and engraftment syndrome for 14% of the time in hospital. The 1‐year risk of TTP was 26%. Acute GVHD was a risk factor for the development of TTP (P = 0·008). With a median follow‐up of 602 d, the 2‐year estimates for overall survival, progression‐free survival, non‐relapse mortality and relapse related mortality were 68%, 43%, 22% and 13%, respectively. This transplantation regimen is feasible and induces long‐term remissions in heavily pretreated patients. The procedure can be performed in the outpatient setting, but complications could result in a substantial number of admissions during the first year.

Investigation of immunosuppressive properties of inactivated human immunodeficiency virus and possible neutralization of this effect by some patient sera

Retroviral infections are accompanied by immunosuppression in a variety of species. For feline leukemia virus, the immunosuppression has been ascribed to the transmembrane envelope protein, p15E, which suppresses the proliferative responses of cat, mouse, and human lymphocytes. A similar suppressive effect has been shown for a lysate of human immunodeficiency virus (HIV), strain HTLV-IIIB. Here we determined that detergent-disrupted HTLV-IIIB lystate exerted a strong suppressive effect on PHA-stimulated lymphocytes. Preparations of whole virions, a lysate of a local HIV isolate grown on MP-6 cells, and a commercially obtained UV and psoralene-inactivated lysate were examined and demonstrated to have a similar suppressive effect. The HIV lysate was not directly cytotoxic to lymphocytes and did not contain tumor necrosis factor or lymphotoxin. The HIV lysate specifically suppressed the proliferation of a range of hemopoietic cell lines from man and mouse including three EBV transformed CD4- and IL-2 receptor-negative B-cell lines. The lysate also suppressed the formation of human bone marrow colonies, whereas the lysate had only a slight or no effect on fibroblasts. The suppression of lymphocyte proliferation was not abrogated by addition of IL-2 or IL-1 and the HIV lysate inhibited the expression of IL-2 receptors on suboptimal PHA-stimulated mononuclear cells. The suppressive factor(s) has not been characterized in molecular terms, but suppressive activity was recovered in fractions with a molecular weight of about 67,000 and in both the glycoprotein fraction and in the glycoprotein-depleted fraction of the HIV lysate. Sera from one-third of a small series (N = 13) of individuals with antibodies to HIV seem to be able to neutralize the suppressive properties of HIV lysate in cultures.

Immunological studies in the acquired immunodeficiency syndrome. II. Active suppression or intrinsic defect--investigated by mixing AIDS cells with HLA-DR identical normal cells.

The lymphocyte transformation responses to mitogens (phytohaemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM)), allogeneic cells, and the antigen‐purified protein derivative (PPD) were studied in six acquired immunodeficiency syndrome (AIDS) patients and in six healthy controls, each of whom was HLA‐DR‐ and mixed lymphocyte culture (MLC)‐identical with one of the AIDS patients. No evidence of suppression was observed when irradiated or non‐irradiated AIDS peripheral blood mononuclear cells (PBMC) were added to cultures of HLA‐DR‐identical PMBC from healthy controls stimulated with the strong mitogens PHA and Con A or with allogeneic cells, but suppression may be involved in the decreased responses in cultures stimulated with PWM or PPD. Addition of supernatants from macrocultures of AIDS cells did not suppress responses of control PBMC. Thus, suppression by any lymphocyte subset or soluble factor alone cannot explain the generally severely depressed transformation responses in AIDS. Addition of heavily irradiated HLA‐DR‐identical PBMC from healthy controls or supernatants from these cultures led to increased responses in cultures of mitogen‐stimulated AIDS PBMC and in some cultures of antigen or allogeneic cell‐stimulated AIDS PBMC, which were of the same magnitude as seen after the addition of commercially obtained T‐cell growth factor (TCGF). This indicates that AIDS cells are deficient in producing TCGF.

Prognostic value of immunologic abnormalities and HIV antigenemia in asymptomatic HIV-infected individuals: proposal of immunologic staging

The prognostic value of various immunologic tests was investigated in 150 HIV-seropositive homosexual men, who were initially without HIV-related symptoms or AIDS and who were followed for a median of 12 months (range 3-28 months). The laboratory investigations included HIV antigen in serum, total lymphocyte count, T-helper (CD4) and T-cytotoxic/suppressor (CD8) counts, and lymphocyte transformation responses to the mitogens phytohemagglutinin (PHA) and pokeweed mitogen (PWM), and to antigenic extracts from Candida albicans and cytomegalovirus. 24 individuals developed HIV-related symptoms or AIDS (11 cases). All parameters except the CD8 count were of prognostic value, but a multivariate analysis of symptom-free survival showed that HIV antigenemia, a CD4 count less than 0.5 x 10(9)/l, and relative response to PWM below 25% of controls contained all the prognostic information. Individuals abnormal at entry for these 3 variables had a theoretical 36 times as high hazard of developing symptoms within the observation period as had individuals with normal parameters. There was no significant covariation between HIV antigenemia on the one hand and CD4 count and response to PWM on the other. Although, the latter 2 variables covaried, each of them provided independent information, and both were used to classify the degree of the immunodeficiency in 3 stages: Im-0 with normal values, Im-1 with one, and Im-2 with both tests abnormal. Individuals in stage Im-2 had a 10 times increased risk of developing symptoms. The immunologic staging correlated significantly with the clinical grouping (CDC criteria). This staging improved in only 1, but deteriorated in half of 36 individuals observed for at least 18 months. Thus, the staging is likely to prove useful when attempts to arrest the immunodeficiency of HIV-infected individuals has to be monitored.

Immunogenetic Heterogeneity in Single-System and Multisystem Langerhans Cell Histiocytosis

Langerhans cell histiocytosis is a rare disease with an unknown etiology and poorly understood pathogenesis. Immunologic, viral, and proliferative clonality causes have all been considered. To determine whether Langerhans cell histiocytosis and its two main subgroups, single-system and multisystem disease, are associated with HLA-A, -B, -Cw, or -DR alleles, a total of 84 patients <15 y of age at the time of diagnosis and of Nordic origin were analyzed, 82 for HLA class I and 76 for HLA class II. Stratification of the patients into two subgroups, singlesystem disease (skin only, and monostotic and polyostotic disease) and multisystem disease with or without organ dysfunction, showed that patients with single-system disease (17 of 45, 38%) more often (p = 0.00018 and, after correction, p = 0.029) had the phenotype HLA-DRB1*03 compared with patients with multisystem disease (1 of 31, 3%). In the patients with multisystem disease a nonsignificant reduction of the frequency of this phenotype was seen compared with controls (p = 0.02, uncorrected). In 14 of the patients with single-system disease, but none with multisystem disease, the deduced haplotype HLA-A*01, B*08, DRB1*03 was found. High-resolution typing, performed in nine patients, revealed that all had the HLA-A*0101, B*0801, DRB1*0301, DQB1*0201 alleles. Our findings suggest an immunogenetic heterogeneity in the two clinical entities of Langerhans cell histiocytosis and indicate that HLA-DRB1*03 may play a protective role against developing multisystem disease. Further studies to confirm these findings are desired.

HLA Antigen Frequencies in Juvenile Chronic Arthritis

HLA-A, B, C, D, and DR typing was performed in 104 patients with Juvenile Chronic Arthritis (JCA). The majority of these (88 patients) participated in a follow-up study of a series of consecutive patients including patients in remission. The study confirmed that JCA is positively associated with B27, Dw8, and possibly Dw5, and negatively associated with Dw2 and Dw7. In JCA patients in remission, the frequency of Dw4 was significantly decreased to 5.0%, compared with 25.0% in healthy Danes and 23.7% in JCA patients with active disease. In pauciarticular onset JCA, the frequency of Dw4 was significantly decreased to 8.1% compared with 25.0% both in controls and in polyarticular onset JCA. These data indicate that Dw4 may be a risk factor of chronicity and multiple joint involvement in JCA. Chronic iritis was present in 18.2% of Dw8-positive patients, compared with 7.0% in Dw8-negative patients, and the frequency of Dw8 was 50.0% in JCA patients with chronic iritis. Thus, Dw8 may be a risk factor of chronic iritis in JCA. Genetically, three distinct subgroups seem to exist: (i) a B27-associated group; (ii) a D/DR5- and D/DRw8-associated group, and (iii) a D/DR4-associated group.