Null alleles of human complement C4. Evidence for pseudogenes at the C4A locus and for gene conversion at the C4B locus.

The two genes for the C4A and C4B isotypes of the fourth component of human complement are located in the MHC class III region. Previous studies have demonstrated the unusual expression of C4 genes in the form of aberrant or duplicated haplotypes. Null alleles of C4A or C4B (AQ0 or BQ0) have been defined by the absence of gene products and occur at frequencies of 0.1-0.3. However, only some C4 null alleles are due to gene deletions, the remainder were thought to be nonexpressed genes. We have analyzed the C4 gene structure of 26 individuals lacking either C4A or C4B protein. The DNA of individuals with apparently nonexpressed C4 genes was tested for the presence of C4A- and C4B-specific sequences using restriction fragment analysis and isotype-specific oligonucleotide hybridization of DNA amplified by polymerase chain reaction. All nondeleted AQ0 allels had C4A-specific sequences and may thus be described as pseudogenes, whereas the nondeleted BQ0 alleles had C4A-instead of C4B-specific sequences. Gene conversion is the probable mechanism by which a C4A gene is found at the second C4 locus normally occupied by C4B genes.

Elevated levels of circulating adhesion molecules in IDDM patients and in subjects at risk for IDDM.

Serum levels of recently discovered circulating forms of adhesion molecules, ICAM-1 and L-selectin, were found to be elevated in IDDM patients and in subjects at risk for developing IDDM compared with 100 normal, nondiabetic blood donors. Both adhesion molecules were determined by sandwich ELISA. Serum concentrations of either clCAM-1 or cL-selectin were > 2SD of normal mean in 10 of 14 recent-onset IDDM patients (P < 0.05). Serum levels of clCAM-1 and cL-selectin did not correlate. In first-degree relatives, elevated adhesion molecule levels were observed in the 6 ICA+ individuals and in the ICA- individuals all (n = 14) with a genetic risk of IDDM (sharing HLA-DR3 and/or-DR4 with the diabetic relative) but not in the HLA-DR3- and/or -DR4- relatives (n = 13). We conclude that elevated clCAM-1 and cL-selectin levels occur independently of ICA status and probably reflect ongoing immune processes in recent-onset IDDM patients and first-degree relatives at risk for IDDM.

Systemic bias of cytokine production toward cell-mediated immune regulation in IDDM and toward humoral immunity in Graves' disease.

Disturbed immune regulation has been postulated to be crucial in the pathogenesis of IDDM and other autoimmune or allergic diseases. We therefore tested the hypothesis of a general bias in the peripheral immune system in patients with recent-onset IDDM or Graves' disease in comparison to healthy control subjects by studying whole blood cultures stimulated with phytohemagglutinin. Cells from IDDM patients (n = 53) produced significantly higher amounts of Th1 cytokines gamma-interferon (IFN-gamma) (P = 0.028) and tumor necrosis factor alpha (TNF-alpha) (P = 0.007) than normal control subjects (n = 56), while Th2 cytokine levels (interleukin [IL]-4, IL-10) were similar. Low levels of islet cell antibodies (ICAs) in IDDM patients were associated with high levels of Th1 and Th2 cytokines. Antibodies to GAD, ICA512, or insulin did not correlate with individual cytokine profiles. Also, HLA-DQ types did not significantly correlate with either Th1 or Th2 cytokine production. Conversely, whole blood cultures from patients with Graves' disease (n = 18) produced significantly less TNF-alpha and IL-4 than normal subjects (P = 0.001-0.006). However, when the balance between Th1 and Th2 cytokine production was analyzed in individuals, the ratio between IFN-gamma or TNF-alpha and IL-4 or IL-10 was clearly biased toward Th1 reactivity in patients with IDDM (P = 0.0001), while a dominance of Th2 cytokine production was seen in Graves' disease (P = 0.0001). The ratio of counterregulatory cytokines appeared to be the most reliable marker of the individual disease process. This study provides first evidence of a systemic bias in the immune regulation of humans, which might be either toward cell-mediated immunity (Th1) in IDDM or humoral immunity (Th2) in Graves' disease.

Linear loss of insulin secretory capacity during the last six months preceding IDDM. No effect of antiedematous therapy with ketotifen.

OBJECTIVE: To investigate the effect of an antiedematous therapy with the histamine antagonist ketotifen on beta-cell function in late prediabetes. RESEARCH DESIGN AND METHODS: In a randomized double-blind placebo-controlled study, ketotifen was administered for 3 months to 9 islet cell antibody positive (ICA+) prediabetic patients with a first-phase insulin response (FPIR) below the 2.5th percentile to preserve residual beta-cell function. Patients were followed by intravenous glucose tolerance tests (IVGTTs) every 4-6 weeks for determination of FPIR, HbA1, ICAs, and insulin autoantibodies. In 5 patients, the immune activation state was followed by determination of serum levels of tumor necrosis factor-alpha (TNF-alpha), beta 2-microglobulin, and C-reactive protein (CRP). RESULTS: Seven of nine patients developed diabetes within one year of follow-up. Irrespective of treatment with ketotifen, a slow and linear decline (P < 0.05) of 1 + 3-min insulin values was observed in sequential IVGTTs in those 7 patients who developed insulin-dependent diabetes mellitus (IDDM) during follow-up. The 2 other patients showed wide fluctuations of the insulin response with a threefold increase of initial insulin levels. HbA1 did not correlate with FPIR. Fasting blood glucose increased significantly during the study (P < 0.05). Individual levels of serum TNF-alpha, CRP, and beta 2-microglobulin did not change during the study. CONCLUSIONS: The study could not demonstrate preservation of beta-cell function by ketotifen in the late stage before manifestation of clinical diabetes. Manifestation is preceded in the last 6 months by a steady loss of the FPIR without rapid deterioration immediately before diagnosis and without signs of increased immune activity.

The influence of common variables on T cell subset analysis by monoclonal antibodies.

Normal values for T cell subsets as defined by the most commonly used monoclonal antibodies of the OKT series were determined in a group of 142 unrelated normal individuals. In most age classes females had significantly greater portions of OKT3 and OKT4 antigen bearing lymphocytes and accordingly a higher T4/T8 index. The range of individual normal values within the control population was remarkably wide. Intra-individual differences between the T subset composition remained rather constant over a period of 2 months. Time of day, food absorption and physical exercise had no influence on the results of subset analyses. Storage of the blood sample and freezing of isolated lymphocytes can result in significantly reduced OKT3 and OKT4 positive cell numbers. If lymphocytes were isolated soon after the blood was taken and resuspended in a stabilization medium they exhibited only minimal change.

Normal distribution of factor B (Bf) allotypes in multiple sclerosis.

Bf allotypes were determined in 200 multiple sclerosis (MS) patients and compared to analogous control frequencies. No significant deviation of any Bf phenotype was found. Also in HLA-B7- as well as Dw2-positive patients normal Bf frequencies existed.

Missing evidence for HLA antigen association with Eales' disease, chorioretinitis, central serous retinopathy, and malignant choroidal melanoma.

Patients with Eales' disease, chorioretinitis, central serous retinopathy, or malignant choroidal melanoma were tested for HLA antigen deviation. When corrected p values (pc) are used, the first three disorders did not show any significant deviation, whereas a significant increase of HLA-Aw32 (pc = 0.026) was found in the malignant melanoma group. For conclusive evidence the latter finding needs confirmation by analysis of a greater number of patients with this disorder.

Homogeneity of HLA-DQ,DR phenotypes in lymphocyte subsets of normal individuals.

The study was performed to evaluate the association of HLA-DQ and DR antigens with the composition of peripheral blood lymphocytes. B cells, T cells, and T subsets were enumerated in 200 normal individuals by indirect immunofluorescence microscopy using the monoclonal antibodies OKT3, OKT4, OKT8, and OKIa1. Results were related to the HLA-DQ,DR phenotypes of the investigated population. No significant correlation between any of the lymphocyte subsets and of the HLA-DQ,DR antigens was found.

Gene and haplotype frequencies of the fourth component of complement (C4) in type 1 diabetics and normal controls.

C4 gene and haplotype frequencies were calculated from phenotype data of 380 unrelated Caucasian patients with insulin dependent (type 1) diabetes mellitus and were compared with analogous frequencies of 382 unrelated healthy Caucasian individuals. In diabetics, a significantly increased frequency of the rare allele C4B 3 (p less than 10(-7] and of the silent alleles C4A Q0 (p less than 10(-7] and B Q0 (p less than 0.002) was observed. Accordingly, insulin dependent diabetes is associated with partial C4 deficiency, which may contribute to the pathogenesis of the disease.

Effective treatment after failure of omeprazole plus amoxycillin to eradicate Helicobacter pylori infection in peptic ulcer disease.

METHODS: Fifty patients with relapsing or complicated Helicobacter pylori positive duodenal (n = 41) or gastric ulcer disease (n = 9) and failure of a combined treatment with omeprazole plus amoxycillin to eradicate H. pylori infection were re-treated with either oral triple therapy (bismuth subsalicylate, metronidazole, tetracycline) plus ranitidine [group I: n = 22] or high-dose omeprazole (40 mg b.d. to t.d.s.) plus amoxycillin (1 g t.d.s.) [group II: n = 28]. RESULTS: Patients of group I and II had similar demographic and clinical characteristics. The overall proportion of eradication of H. pylori infection was 81.8% in group I and 78.6% in group II (P = N.S.) as judged from negative bacterial findings by means of an urease test, specific culture and histology after modified Giemsa stain. Ulcer healing was observed in all patients after a maximum duration of 10 weeks. Ten patients on triple therapy and only one patient on omeprazole plus amoxycillin (45.5% vs. 3.6%; P < 0.001) complained of side effects without necessity of discontinuation of the study medication in either group. Twenty patients (group I: n = 10; group II: n = 10) with relapsing duodenal ulcer disease and successful cure were prospectively followed for one year without any evidence of ulcer relapse or H. pylori re-infection. CONCLUSION: Oral triple therapy plus ranitidine or high-dose omeprazole plus amoxycillin remain highly effective in eradicating H. pylori infection in patients with peptic ulcer disease and treatment failure of omeprazole/amoxycillin, but the omeprazole enhanced antibiotic monotherapy seems to be superior with regard to side effects. Thus, high-dose omeprazole/amoxycillin is recommended as the treatment of first choice in these selected patients. Triple therapy should be reserved for patients intolerant of amoxycillin.

Biostatistical basis of individualization and segregation analysis using the multilocus DNA probe MZ 1.3: results of a collaborative study.

A collaborative study using the multilocus minisatellite DNA probe MZ 1.3 was carried out to investigate segregation information, mutation rate, DNA fragment frequencies as well as band sharing characteristics. The fingerprint patterns of 393 children as well as 694 unrelated individuals were analysed after digestion of DNA with the restriction enzyme HinfI. A mutation rate of 1% per meiosis or 0.04% per band was found with a mean number of 26 bands/individual. It was shown that maternal and paternal fragments are inherited in equal proportions. Population frequencies of restriction fragments demonstrated a distribution with increasing frequencies in the small fragment size range below 10 kb as well as the absence of very common or very rare fragments. Our data can be used to calculate simple exclusion probabilities based on the number of non-maternal bands in the child.

Crohn's disease in four members of a family, two of whom are dizygotic twins.

Familial occurrence of Crohn's disease (CD) is well known, but the disease is rarely reported to occur in dizygotic twins. We present an additional case of dizygotic twins, both of whom developed CD, from a family in which two other members are affected. The 16-year-old son contracted the disease 2 years before his 50-year-old father, and 13 years before his twin sister. Another sister was affected 6 years after the onset of the disease in the propositus. HLA haplotyping of the three children matched the Mendelian ratio. The multiple occurrence of CD in blood relatives, especially in siblings, emphasizes the importance of genetic factors in the development of this disease. This family history, however, could point to psychic influences promoting the occurrence of CD on the basis of a polygenic disease susceptibility.

Clinical features of inflammatory joint and spine manifestations in Crohn's disease.

167 patients with Crohn's disease were investigated for joint and spine inflammation. Arthritis was observed in 23 patients (14%), sacroiliitis in 24 (14%), and sacroiliitis in combination with arthritis in 11 patients (7%). 15 patients (9%) had ankylosing spondylitis; 9 of them were HLA-B27 positive (60%). A parallel pattern in the course of bowel disease and joint inflammation was observed in 22 out of 34 patients with arthritis (59%). An association between the localization of Crohn's disease and the type of spondylarthritis could not be demonstrated. Patients with arthritis alone developed erythema nodosum (35%) or aphthous stomatitis more often (21%) than patients without spondylarthritis+ (6% and 12%, respectively). Other extra-intestinal manifestations of Crohn's disease did not reveal any association with the development of spondyloarthritis.

[Pathogenesis and immunotherapy of insulin-dependent (type I) diabetes mellitus]. / Zur Pathogenese und Immuntherapie des insulinbedürftigen (Typ I) Diabetes mellitus.

The association of insulin-dependent (type I) diabetes mellitus with HLA-DR3 and DR4 and with several epidemiological, virological, immunological, and clinical data suggests a heterogenous pathogenesis. the initial lesion in most cases is a virologically induced autoimmune process. It is only rarely that insulin-dependent diabetes results from a pure viral infection or as part of polyendocrine autoimmune deficiencies. The knowledge of the genetical risk factors and of disease-specific humoral and cellular immune deviations exhibits possibilities of successful intervention by means of immunotherapy.