Mutations in TNFRSF13B encoding TACI are associated with common variable immunodeficiency in humans.

The functional interaction of BAFF and APRIL with TNF receptor superfamily members BAFFR, TACI and BCMA is crucial for development and maintenance of humoral immunity in mice and humans. Using a candidate gene approach, we identified homozygous and heterozygous mutations in TNFRSF13B, encoding TACI, in 13 individuals with common variable immunodeficiency. Homozygosity with respect to mutations causing the amino acid substitutions S144X and C104R abrogated APRIL binding and resulted in loss of TACI function, as evidenced by impaired proliferative response to IgM-APRIL costimulation and defective class switch recombination induced by IL-10 and APRIL or BAFF. Family members heterozygous with respect to the C104R mutation and individuals with sporadic common variable immunodeficiency who were heterozygous with respect to the amino acid substitutions A181E, S194X and R202H had humoral immunodeficiency. Although signs of autoimmunity and lymphoproliferation are evident, the human phenotype differs from that of the Tnfrsf13b-/- mouse model.

Clustering of missense mutations in the ataxia-telangiectasia gene in a sporadic T-cell leukaemia.

Ataxia-telangiectasia (A-T) is a recessive multi-system disorder caused by mutations in the ATM gene at 11q22-q23 (ref. 3). The risk of cancer, especially lymphoid neoplasias, is substantially elevated in A-T patients and has long been associated with chromosomal instability. By analysing tumour DNA from patients with sporadic T-cell prolymphocytic leukaemia (T-PLL), a rare clonal malignancy with similarities to a mature T-cell leukaemia seen in A-T, we demonstrate a high frequency of ATM mutations in T-PLL. In marked contrast to the ATM mutation pattern in A-T, the most frequent nucleotide changes in this leukaemia were missense mutations. These clustered in the region corresponding to the kinase domain, which is highly conserved in ATM-related proteins in mouse, yeast and Drosophila. The resulting amino-acid substitutions are predicted to interfere with ATP binding or substrate recognition. Two of seventeen mutated T-PLL samples had a previously reported A-T allele. In contrast, no mutations were detected in the p53 gene, suggesting that this tumour suppressor is not frequently altered in this leukaemia. Occasional missense mutations in ATM were also found in tumour DNA from patients with B-cell non-Hodgkin's lymphomas (B-NHL) and a B-NHL cell line. The evidence of a significant proportion of loss-of-function mutations and a complete absence of the normal copy of ATM in the majority of mutated tumours establishes somatic inactivation of this gene in the pathogenesis of sporadic T-PLL and suggests that ATM acts as a tumour suppressor. As constitutional DNA was not available, a putative hereditary predisposition to T-PLL will require further investigation.

The molecular basis of hereditary complement factor I deficiency.

The molecular basis of hereditary complement factor I deficiency is described in two pedigrees. In one pedigree, there were two factor I-deficient siblings, one of whom was asymptomatic and the other suffered from recurrent pyogenic infections. Their factor I mRNA was analyzed by reverse transcription of fibroblast RNA followed by amplification using the polymerase chain reaction. Both siblings were homozygous for the same transversion (adenine to thymine) at nucleotide 1282 in the cDNA. This mutation causes histidine-400 to be replaced by leucine. The altered histidine is a semi-conserved residue within the serine proteinase family, although no function has been ascribed to it. The proband of the second pedigree studied was found to be a compound heterozygote. One allele had the same mutation as the first family, the second allele had a donor splice site mutation that resulted in the deletion of the mRNA encoded in the fifth exon (a low-density lipoprotein receptor domain) from its transcript.

ATM mutations in cancer families.

Ataxia-telangiectasia (A-T) is a multisystem recessive disease characterized clinically by cerebellar ataxia, oculocutaneous telangiectasias, immunodeficiency, sensitivity to radiomimetic agents, and cancer predisposition. This pleiotropic disorder is caused by mutations in the ATM (mutated in A-T) gene, which is located in the human chromosomal region 11q22-q23. The ATM gene product is a member of a novel family of large proteins implicated in the regulation of the cell cycle and response to DNA damage. Heterozygosity for A-T was previously suggested to be associated with an increased risk of tumors, particularly female breast cancer. Because of loss of constitutional heterozygosity at 11q22-q23 is a frequent event in breast and other tumors, suggesting the presence of a tumor suppressor gene(s) in this region, we screened blood DNA samples from 88 unrelated breast cancer patients of Swedish cancer families for ATM mutations using single-strand conformation polymorphism analysis. All patients had a family history of tumors previously associated with A-T heterozygosity or homozygosity. We demonstrate the first three germ-line mutations in ATM identified by screening of breast cancer patients. Two mutations were previously found in A-T homozygotes and one mutation was a 1-bp insertion. All mutations were found in families with a large number of tumors, however, they did not cosegregate with malignancies. Although the proportion of A-T carriers in this sample seems to be higher than expected by chance, larger studies and pooled data sets will be required to establish that an A-T allele confers cancer susceptibility in heterozygotes.

The ATM gene and susceptibility to breast cancer: analysis of 38 breast tumors reveals no evidence for mutation.

Heterozygosity for ataxia-telangiectasia (A-T), a cancer-prone recessive syndrome, has been associated with an increased risk of breast cancer. The gene for A-T (ATM) is located at chromosomal region 11q22-q23, a region of frequent loss of constitutional heterozygosity in breast and other tumors. Loss of constitutional heterozygosity at 1lq22-q23 was found in 47% of informative cases in the series of primary tumors analyzed in this study. To investigate the role of ATM in breast cancer, we have determined the complete genomic organization of the gene, developed an exon-scanning PCR single-strand conformation polymorphism (PCR-SSCP) assay for mutation detection of ATM, and screened 38 consecutive breast tumors for mutations using both genomic DNA- and cDNA-based assays. In addition to common ATM polymorphisms detected both in the coding sequence and in flanking introns, seven unique SSCP alleles were identified in six tumor DNAs. Sequence analysis of these alleles revealed rive nucleotide substitutions that were predicted to change the encoded amino acid. However, PCR-SSCP and nucleotide sequencing analysis of the paired blood samples and of an extended sample size of a total of 224 chromosomes indicated that these SSCP patterns represent constitutional rare polymorphisms with a frequency between 0.005 and 0.023. Because the majority of A-T mutations are null mutations and none of the ATM alleles found in breast cancer samples would lead to the truncation of the translation product, we conclude that, in this initial sample of sporadic breast cancer patients, there was no evidence for an increased number of A-T carriers. In addition, because no somatic mutations were found, our study rules out the ATM gene as the frequently altered tumor suppressor gene at 11q23.

Ataxia-telangiectasia and T-cell leukemias: no evidence for somatic ATM mutation in sporadic T-ALL or for hypermethylation of the ATM-NPAT/E14 bidirectional promoter in T-PLL.

The ATM gene deficient in ataxia-telangiectasia, a recessive multisystem disease associated with a high risk of lymphomas and leukemias, was found previously to be inactivated in a rare sporadic malignancy, T-cell prolymphocytic leukemia (T-PLL), which is often associated with cytogenetic aberrations of chromosome 14. The ATM gene was shown to sustain frequent loss-of-function mutations in T-PLL tumor cells, consistent with functioning as a tumor suppressor gene in this leukemia. To investigate the possibility of nonmutational or nonrecombinational mechanisms of T-PLL development, we have used bisulfite genomic sequencing to analyze DNA methylation in the putative bidirectional promoter region of the closely linked ATM and NPAT/E14 genes within the CpG island at 11q22-q23. We show that this region is completely demethylated in lymphocytes expressing ATM; however, no extensive hypermethylation was found in 9 T-PLL tumor DNA samples without evidence of ATM/p53 mutations. Because acute T-cell lymphoblastic leukemias (T-ALL) were also observed in ataxia-telangiectasia patients and T-ALL tumor cells contain chromosome 14 abnormalities, 19 presentation samples of T-ALL patients were analyzed for ATM mutations. Although T-ALL patients exhibited rare nucleotide substitutions not previously found in ATM, all were identified in the germ-line, indicating constitutional polymorphisms, potentially confined to ethnic subpopulations. The absence of somatic nucleotide changes in ATM in T-ALL as compared with T-PLL suggests a distinct pattern of genetic events in the development of the two leukemias.

Cutaneous basophil hypersensitivity to inhalant allergens in atopic dermatitis patients: elicitation of delayed responses containing basophils following local transfer of immune serum but not IgE antibody.

Inhalant allergens applied to the skin of sensitive atopic dermatitis patients by means of a modified patch test technique, induce acute eczematous lesions. These lesions contain basophils, eosinophils, mononuclear cells, and neutrophils and represent an example of human cutaneous basophil hypersensitivity. The role of IgE antibody in this eczematous reaction was studied by systemic and local passive transfer experiments. Plasma with high IgE antibody when infused into patients with hypogammaglobulinemia as part of their replacement treatment resulted, post infusion, in cutaneous mast cell and blood basophil sensitization as measured by quantitative skin testing and leukocyte histamine release. Subsequent patch tests on these patients using the house dust mite antigen, antigen P1, produced macroscopic erythematous responses containing mononuclear cells, and eosinophils but not basophils. Local transfer of atopic dermatitis serum with high IgE antibody produced weak macroscopic responses and in these lesions mononuclear cells and both basophils and eosinophils were present. The serum activity which allowed transfer of basophil and eosinophil recruitment was heat labile. Specifically purified antibody to the mite antigen P1 (containing IgE and IgG antibody), when transferred, allowed eosinophil but not basophil recruitment to patch test sites. These results suggest that while the allergen-induced patch test response may involve IgE antibodies, as well as the cells normally involved in delayed responses, another serum activity is also involved.

Exon-scanning mutation analysis of the ATM gene in patients with ataxia-telangiectasia.

Using a polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) assay, which amplifies individually all coding exons of the ATM gene deficient ataxia-telangiectasia (A-T), we have analyzed 10 patients with A-T for ATM mutations. Mutation were detected in 9 patients. We describe the first ATM mutation in the splice junction found in the 5' splice site of intron 17, leading to exon skipping. However, most mutations were small deletions or insertions resulting in premature termination of the translation product. The development of DNA-based methods for detection of unknown mutations and further characterization of ATM mutation pattern will facilitate identification of A-T carriers and assessment of their cancer risk.

Short tandem repeat (STR) haplotypes in HLA: an integrated 50-kb STR/linkage disequilibrium/gene map between the RING3 and HLA-B genes and identification of STR haplotype diversification in the class III region.

We present a dense STR/linkage disequilibrium(LD)/gene map between the RING3 and HLA-B loci, reference allelic sizes on the most prevalent HLA haplotypes and their allelic frequencies in pedigree founders. This resource will facilitate LD, evolution and gene mapping studies, including comparisons of HLA and STR haplotypes and identification of HLA recombinants. The map was constructed by testing novel and previously reported STRs using a panel of 885 individuals in 211 families and 60 DNA samples from cell lines and bone marrow donors homozygous in the HLA-A, -B and -DR loci selected from over 15 000 entries into the registry of Swedish bone marrow donors. We have also analysed the variability of STR alleles/haplotypes on the most prevalent HLA haplotypes to identify STRs useful for fine mapping of disease genes in the region previously implicated in susceptibility to many disorders. The analysis of 40 HLA-A*01, B*0801, DRB1*03011, DQB1*0201 haplotypes in homozygous donors showed a surprising stability in 23 STRs between the class II recombination hot spot and HLA-B, with the average of 1.9% (16/838) variant alleles. However, 40% variant alleles were found at the D6S2670 locus in intron 19 of the tenascin-X gene both in the families and homozygous donors. The nucleotide sequence analysis of this STR showed a complex polymorphism consisting of tetra- (CTTT)(8-18) and penta-nucleotide (CTTTT)(1-2) repeats, separated by an intervening non-polymorphic sequence of 42 bp. The HLA-A1, B*0801, DRB1*03011, DQB1*0201 haplotypes had five (CTTT)(14-18)/(CTTTT)(2) variants with a predominant (CTTT)(16) allele, implicating the tetranucleotide component as the source of this ancestral haplotype diversification, which may be due to the location of D6S2670 in the region of the highest GC content in the human MHC.

Humoral immunity before and after thymectomy in myasthenia gravis.

Humoral immunity was studied in 10 patients with myasthenia gravis before thymectomy, in 15 different patients over 10 years after thymectomy, and in normal controls. Antibody titers to acetylcholine receptor were significantly (p less than 0.01) lower in the post-thymectomy group. However, other antibody titers to common viruses, and to Escherichia coli, and isohemagglutinins showed no significant change. Levels of IgM and IgE (with atopic subjects excluded) decreased following thymectomy (p less than 0.05). Autoantibodies persisted, apart from those directed against the acetylcholine receptor. The absence of any significant changes in humoral immunity after thymectomy for myasthenia gravis suggests that there is no generalized loss of helper T-cell function.

A radioimmunoassay for antibody to pneumococcal polysaccharides.

A radioimmunoassay for type specific pneumococcal polysaccharides is described. This involves obtaining specific polysaccharides from culture supernatants by precipitation with ethanol saturated with potassium acetate, and then after further purification labelling the tyraminated polysaccharide with 125 iodine. The radioimmunoassay itself is a standard ammonium sulphate precipitation technique (Farr assay) using IgG myeloma protein as a carrier. We have found that measuring antibodies after test immunization with a polyvalent pneumococcal vaccine is a useful aid in diagnosing functional antibody deficiency.

Determination of intracellular cytokines by flow-cytometry following whole-blood culture.

Various methods have been reported for measuring intracellular cytokines in peripheral blood mononuclear cells isolated by density-gradient centrifugation. In this report, we describe a whole-blood method for the determination of intracellular cytokines (IFN-gamma, TNF-alpha and IL-2) that uses small-volume (500 microl) blood samples. Directly conjugated anti-cytokine antibodies and commercial cell membrane fixation and permeabilisation reagents were used. Blood was cultured in a 1:3 dilution with a combination of PMA and ionomycin to reveal the cytokine synthetic potential of each cell, together with monensin to increase the sensitivity by retaining cytokines within the cell to detectable levels. The optimum concentrations of PMA (10 ng/ml (16.2 nmol/l)), ionomycin (2 micromol/l) and monensin (3 micromol/l) were determined. Kinetic studies showed maximal cytokine expression after 2 h of culture for TNF-alpha and IFN-gamma and 4 h for IL-2. Assessment of TNF-alpha and IFN-gamma production within the CD4 and CD8 lymphocytes from 10 normal volunteers showed that considerably more CD8 + than CD4 + cells produced IFN-gamma. This technique could be used by routine immunology laboratories and will be of use in studies to determine whether cytokine assays are of value in the investigation of immune disorders.

Serum immunoglobulin concentrations in preschool children measured by laser nephelometry: reference ranges for IgG, IgA, IgM.

Serum immunoglobulin concentrations were determined on sera from 298 healthy children aged six months to six years using the Hyland laser nephelometer PDQ system. Age-specific 95% reference ranges for serum IgG, IgA and IgM are presented; considerable care has been taken to ensure statistical validity of the reference ranges. The wide range of values in children under two years suggest that measuring immunoglobulin concentrations in this age group is of little value in diagnosing immunodeficiency.

Microbial and metabolic profile of achlorhydric stomach: comparison of pernicious anaemia and hypogammaglobulinaemia.

The microbial flora and some of its metabolites and enzymes in the stomach were compared in patients with achlorhydria, pernicious anaemia, and primary hypogammaglobulinaemia and in patients with dyspepsia with normal gastric acidity. Detailed analysis of the flora of the gastric juice and of the mucosa from the antrum, body, and fundus in six patients with hypogammaglobulinaemia (mean pH 8.2), seven patients with pernicious anaemia (mean pH 7.3), and five patients with dyspepsia (mean pH 1.9) yielded 22 different genera of bacteria, mainly from the patients with achlorhydria, the most common being streptococci, micrococci, staphylococci, veillonella, and lactobacilli. A similar flora was found associated with the mucosa at all three sites. Various metabolites were also looked for. beta Glucoronidase and C14 lipase were found in patients with hypogammaglobulinaemia but not in those with pernicious anaemia or dyspepsia. Volatile fatty acids were not found. Relatively high concentrations of ethanol were found in the patients with hypogammaglobulinaemia compared with those with pernicious anaemia (p = 0.02). Similar concentrations of dimethylamine were found in all three groups, but the concentrations of trimethylamine were much higher in patients with pernicious anaemia and hypogammaglobulinaemia. The high concentrations of some microbial enzymes and ethanol differentiated the group with hypogammaglobulinaemia from the rest, and these may bear some relation to the high incidence of gastric cancer in patients with hypogammaglobulinaemia.

CD4+ lymphocytopenia due to common variable immunodeficiency mimicking AIDS.

There are an increasing number of published reports of patients with acquired immunodeficiency without evidence of HIV infection, who have been labelled as having "idiopathic CD4+ lymphocytopenia". The case is reported here of a young man who presented with Pneumocystis carinii pneumonia (PCP), CD4+ lymphopenia, and hypogammaglobulinaemia attributable to common variable immunodeficiency (CVID). The presentation of this condition, with many of the clinical and laboratory features of AIDS, highlights CVID as a diagnosis to be considered in the differential diagnosis of CD4+ lymphocytopenia.

Serum factors for opsonisation of non-typable Haemophilus influenzae.

Neutrophil chemiluminescence was used to assess the opsonins required for phagocytosis of non-typable Haemophilus influenzae isolated from sputum samples of patients with hypogammaglobulinaemia. Immunoglobulin was the major opsonin, whereas complement was relatively unimportant. Evidence was found for a heat-labile opsonin other than complement that enhanced phagocytosis of these organisms. Tuftsin was shown to aid phagocytosis of H. influenzae without triggering chemiluminesence.

Nitrate- and nitrite-reducing bacteria in the achlorhydric stomach.

The microbial composition of samples of gastric juice from eight achlorhydric patients was determined by aerobic and rigorously anaerobic culture techniques. Bacteria from 16 genera were commonly isolated, but representatives of only three genera, (streptococci, neisseriae and haemophili) were isolated from every patient. Nitrate and nitrite were both reduced by veillonellae, haemophili, staphylococci, corynebacteria, lactobacilli, flavobacteria and fusobacteria, but the potential rate of nitrate reduction by suspensions of veillonellae, Haemophilus parainfluenzae and members of the Enterobacteriaceae were up to ten times more rapid than the rate of nitrite reduction. Conversely, although all Neisseria spp. reduced nitrite only some strains reduced nitrate. Streptococci did not reduce nitrate. Streptococcus sanguis reduced nitrite when grown with haematin; other streptococci did not reduce nitrite. Bacterial nitrate and nitrite reduction were active over the pH range 6-8, similar to the pH range of the achlorhydric stomach. From a knowledge of the composition of the bacterial flora and their potential rates of nitrate and nitrite reduction under prevailing conditions, predictions were made about the tendency of nitrite to accumulate during nitrate reduction. Studies of the transient accumulation of nitrite by mixed cultures of H. parainfluenzae and N. subflava were consistent with these predictions. Haemophili and veillonellae could be responsible for the accumulation of nitrite in the gastric juice of some patients, whereas streptococci and neisseriae would tend to remove nitrite from the stomach as rapidly as it formed.

Bactericidal activity of serum for Klebsiella rhinoscleromatis: studies on serum from a patient with rhinoscleroma and sera deficient in antibody or complement.

The in-vitro bactericidal effect of serum for Klebsiella rhinoscleromatis was tested. Experiments with C2-deficient and hypogammaglobulinaemic human sera suggested that killing depended on activation of the classical complement pathway, although the alternative pathway probably amplified the effect. Serum from a patient with active rhinoscleroma, and another cured of the disease, showed normal killing.

DNA probes bind non-specifically to eosinophils during in situ hybridization: carbol chromotrope blocks binding to eosinophils but does not inhibit hybridization to specific nucleotide sequences.

Eosinophils were found to bind radiolabelled DNA probes non-specifically during in situ hybridization. Pretreatment of cells with carbol chromotrope blocked non-specific binding without interfering with the recognition of specific nucleotide sequences.

Quantitation of HIV-1 activity in tissue culture supernatants: effects of culture condition on syncytial assays and virus production.

We have compared the infectivity titres, reverse transcriptase levels and antigen titres in the supernatants from persistently infected cell lines that produce a variety of HIV-1 isolates. We found a poor correlation between the different assays, and a variability in viral activity dependent on culture medium.