Quantifiable analysis of human immunoglobulin heavy chain class-switch recombination to all isotypes

Somatic recombinational events, including the immunoglobulin heavy chain class-switch, are a normal feature of B-cell maturation. To enable comprehensive and sensitive class-switch analysis in ex vivo human B cells, we have developed multiple digestion-circularization PCR (DC-PCR) techniques for quantifiable detection of switching to all immunoglobulin isotypes. This technology was validated by extensive sequencing of PCR products, tests with control non-lymphoid cells and B-cell lines of known isotypic specificities, and by demonstrating DC-PCR selectivity in a model system. With tonsillar B-cell DNA, switching to gamma 3, gamma 1, alpha1, gamma 2, gamma 4 and alpha2 isotypes was reproducibly detectable among different individuals. Levels of epsilon switching were relatively low and usually required higher total amounts of template DNAs for detection. Quantitation of alpha1 class switching in a panel of human tonsillar whole B cells was performed by the internal-competitor approach, and showed a pattern consistent with previous studies on IgA+ tonsillar cells. We demonstrate that these assays can rapidly show germline status or specific switch rearrangements in B lymphoid cell lines.     

Human genetic defects in class-switch recombination (hyper-IgM syndromes).

Several genetic defects in class-switch recombination, leading to hyper-IgM syndromes, have been recently described in humans. Besides the well-known role of interaction between CD40-ligand and CD40, these pathological conditions definitively demonstrate the requirement of CD40-mediated NF-kappaB activation and the essential role of a newly described molecule, activation-induced cytidine deaminase (AID), in B cell terminal differentiation.     

Anti-D immunoglobulin treatment for thrombocytopenia associated with primary antibody deficiency

AIMS: To review our experience of anti-D immunoglobulin for immune thrombocytopenia (ITP) in patients with primary antibody deficiency. METHODS/PATIENTS: A retrospective case notes review of four Rhesus positive patients with ITP and primary antibody deficiency, treated with anti-D. Patients were refractory to steroids and high dose intravenous immunoglobulin (IVIG). Two patients were previously splenectomised. RESULTS: All patients responded to anti-D immunoglobulin. Improved platelet counts were sustained for at least three months. Side effects included a fall in haemoglobin in all cases; one patient required red blood cell transfusion. Two patients had transient neutropenia (< 1 x 10(9)/litre). CONCLUSION: Anti-D immunoglobulin may be an effective treatment for antibody deficiency associated thrombocytopenia, even after splenectomy. Anti-D immunoglobulin may have considerable clinical advantages in this group of patients, where treatments resulting in further immunosuppression are relatively contraindicated.     

Plasma ascorbate deficiency is associated with impaired reduction of sulfamethoxazole-nitroso in HIV infection

OBJECTIVE: The objective of these studies was to determine the role of ascorbate deficiency in HIV infection in the defective detoxification of sulfamethoxazole-nitroso, the metabolite thought to mediate sulfonamide hypersensitivity reactions. METHODS: Fifty-one HIV-infected patients and 26 healthy volunteers were evaluated. Vitamin supplementation histories were obtained, and blood samples were collected for determination of plasma ascorbate, dehydroascorbate, and cysteine concentrations, erythrocyte glutathione concentrations, and plasma reduction of sulfamethoxazole-nitroso in vitro. RESULTS: Plasma ascorbate concentrations were significantly lower in HIV-positive patients not taking vitamin supplements (29.5 +/- 22.3 microM) than in healthy subjects (54.8 +/- 22.3 microM; P = 0.0005) and patients taking 500-1000 mg of ascorbate daily (82.5 +/- 26.3 microM; P < 0.0001). Plasma ascorbate deficiency was strongly correlated with impaired reduction of sulfamethoxazole-nitroso to its hydroxylamine (r = 0.60, P < 0.0001), and during in vitro reduction, the loss of plasma ascorbate was strongly associated with the amount of nitroso reduced (r = 0.70, P < 0.0001). Ascorbate added ex vivo normalized this reduction pathway. Erythrocyte glutathione concentrations were significantly lower in HIV-positive patients (0.98+/-0.32 mM) than in healthy subjects (1.45+/-0.49 mM; P = 0.001), but this finding was unrelated to ascorbate supplementation. There was trend toward lower plasma cysteine concentrations in patients (8.4+/-3.9 microM) than in controls (10.3+/-4.3 microM), but this trend was similarly unrelated to ascorbate supplementation. Dehydroascorbate concentrations were not significantly higher in HIV-positive patients (7.4+/-10.5%) than in healthy controls (4.0+/-6.2%), even in the subset of patients taking ascorbate (8.4+/-9.4%). CONCLUSIONS: Ascorbate deficiency is common in HIV-positive patients and is associated with impaired detoxification of sulfamethoxazole-nitroso, the suspected proximate toxin in sulfonamide hypersensitivity. Patients taking daily ascorbate supplements (500-1000 mg) achieved high plasma ascorbate concentrations and did not show this detoxification defect. Ascorbate deficiency (or supplementation) was not associated with changes in glutathione or cysteine concentrations. These data suggest that ascorbate deficiency, independent of thiol status, may be an important determinant of impaired drug detoxification in HIV infection.     

Myopathic carnitine deficiency associated with lymphocytic malignant non-hodgkin lymphoma and monoclonal immunoglobulin G-K

Summary A 47-year-old male patient suffered from recurrent myalgia, induced by fasting or physical exercise. Later, he developed progressive muscular weakness. Serum levels of creatine phosphokinase (CPK) were elevated to approx. 400 U/l. Muscle biopsies showed lipid storage myopathy and signs of acute fiber necrosis, muscle carnitine was decreased to below 20% of controls, carnitine palmitoyl transferase (CPT) activity was normal. Carnitine was also moderately decreased in a liver biopsy and in plasma. Urine excretion of carnitine was low, no elevation of short-chain dicarboxylic acids could be found. The patient was also found to suffer from a lymphocytic malignant non-Hodgkin lymphoma, a monoclonal immunoglobulin (Ig) G-k in plasma and lymphocytic infiltration of bone marrow were demonstrated. At that time no evidence had been obtained to indicate that these two diseases could be related to each other. Autoantibodies against skeletal muscle could not be demonstrated. Absorption of L-carnitine p.o. was normal, however, plasma levels of carnitine fell again rapidly. Administration of 3 × 2 g L-carnitine per day normalized the patient's plasma carnitine levels and led to an increase of plasma short-chain acylcarnitine and ketone bodies, particularly-hydroxybutyrate (B-HOB). However, no significant clinical improvement could be seen. Additional application of prednisone led to normalization of CPK serum levels.Abbreviations AST alanine aminotransferase - ALT aspartate aminotransferase - CAT carnitine acetyl transferase - CPT carnitine palmitoyl transferase - CPK creatine phosphokinase - -HOB -hydroxybutyrate - LDH lactate dehydrogenase - NCP non-collagen-protein This work has been supported by the Deutsche Forschungsgemeinschaft and the Friedrich-Baur-Stiftung     

Inherited 7S immunoglobulin deficiency of chickens is associated with bursal degeneration anomalies

Partially inbred line UCD 140 chickens develop an age dependent inherited 7S immunoglobulin deficiency with features similar to acquired human agammaglobulinemia. Serial and developmental observations in line UCD 140 and control lines 440 and 444 reveal a significant progressive premature involution of the bursa of Fabricius. These bursal changes are characterized by epithelial and medullary degeneration, reduced follicular bursacyte mitosis, and decreased follicular plasma cells. These abnormalities have not been previously described in other avian systems and suggest that this immune deficiency is due to a primary bursal disease.     

Human CD36 deficiency is associated with elevation in low-density lipoprotein-cholesterol

To find out whether CD36 plays a role in the human lipoprotein metabolism, we studied lipoprotein profiles in subjects with CD36 deficiency. Apparently healthy Japanese volunteers (n = 790) were classified by flow cytometry into three groups of normal (platelet and monocyte CD36+, n = 741, 93.8%), type-II deficiency (platelet CD36- and monocyte CD36+, n = 45, 5.7%), and type-I deficiency (platelet and monocyte CD36-, n = 4, 0.5%). At least one of reported mutations in the CD36 gene was found in all four subjects with type-I deficiency and in 23 of the 45 subjects with type II. Among 779 subjects (731 normals, 44 type II, and four type I) with serum triglyceride levels of <400 mg/dL, serum total cholesterol and low-density lipoprotein (LDL) cholesterol were significantly elevated in type-II deficiency (P = 0.0095 and 0.0382 versus normal, respectively, Scheffe's F-test), while differences were not significant in triglyceride and high-density lipoprotein-cholesterol. Similar tendency was observed in type-I deficiency, although the differences were not statistically significant because of small sample size. We conclude that CD36 deficiency elevates LDL cholesterol, indicating a contribution of CD36 to LDL metabolism.     

Human and animal models of V(D)J recombination deficiency

V(D)J recombination not only comprises the molecular mechanism that insures diversity of the immune system but also constitutes a critical checkpoint in the developmental program of B and T lymphocytes. The analysis of human patients with severe combined immune deficiency (SCID) has enabled (and will enable in the future) the discovery of important factors involved in this reaction. The finding that the V(D)J recombinase apparatus includes components of the general DNA repair machinery of the cells has provided some new and interesting insights into the role of V(D)J recombination deficiency in the development of lymphoid malignancies, a hypothesis that has been tackled and proven in several animal models.     

Systemic antibody deficiency in patients without serum immunoglobulin deficiency or with selective IgA deficiency.

Serum IgG tetanus toxoid antibody (IgGTTab) concentrations were measured in patients with chronic chest infections or recurrent acute chest infections following immunization and compared with results obtained in a group of 43 controls. Apart from selective IgA deficiency in some patients, all had normal or high serum immunoglobulins. Using an enzyme linked immunosorbent assay (ELISA) for IgG TTab, antibody was present following one immunization in all controls who had previously been immunized and following two immunizations in those not previously immunized. Ninety-seven and a half per cent of controls had a serum antibody concentration of greater than 4 micrograms/ml. Following the same immunization schedule, eight of 45 (18%) patients with chronic chest infections and three of 11 (27%) patients with recurrent acute infections had a serum IgG TTab of less than 4 micrograms/ml. The three patients with a low IgG TTab concentration and recurrent acute infections all had selective IgA deficiency. Two of these patients have benefited from injections of normal human immunoglobulin. It is suggested that systemic antibody deficiency as a cause of chronic or recurrent respiratory tract infections cannot be excluded by measuring serum immunoglobulin concentrations alone and that it is of value to measure antibody responses following immunization.     

Human T-cell response to meningococcal immunoglobulin A1 protease associated alpha-proteins

A unique feature of the immunoglobulin A1 (IgA1) protease from pathogenic Neisseriae, i.e. N. meningitidis and N. gonorrhoeae, is its co-secretion with an amphipathic a-protein. Polymerase chain reaction (PCR) analysis of the respective iga(alpha). gene region in 48 meningococcal strains revealed that this protein domain is conserved throughout all isolates in four different principal variants. Despite strain-dependent size and sequence variations, sequence analysis showed common structural characteristics. More than 80% of the amino acid sequence of all a-proteins is dependent on the five amino acids Q, E, A, K and R, resulting in a pI> 10. The sequences are highly conserved at the N-terminus and the C-terminus and contain long amphipathic alpha-helical stretches. These stretches have a strong probability of forming coiled coil conformations and comprise short repetitive sequence modules with pronounced similarities to T-cell epitopes. We therefore analyzed the T-cell response of 20 volunteer blood donors to four peptides, representing such predicted epitopes, and a recombinant meningococcal alpha-protein. Sixteen donors reacted against at least one peptide after culture of peripheral blood mononuclear cells in interleukin (IL)-2-rich medium, while two individuals showed a positive reaction only against an IgA1 protease-derived control peptide. From one donor, we established and maintained T-cell clones specific for purified alpha-protein. Characterization of the T-cell clones revealed a CD3- and a CD4-positive phenotype and the secretion of IL-2 and interferon-gamma (IFN-gamma),     

Vitamin D deficiency in children with recurrent respiratory infections,with or without immunoglobulin deficiency

Purpose

The objective of this study was to evaluate thevitamin D concentration in patients with recurrent respiratory infections with or without immunoglobulin G, A or M (IgG, IgA, IgM) deficiency, and to find a correlation between the vitamin D concentration and the response to hepatitis B vaccination.

Methylation status of immunoglobulin x e segments correlates with their recombination potential

We have previously shown that unlike endogenous ? genes, unrearranged ? transgenes undergo V?-J? recombination in T as well as B cells of transgenic mice. To determine whether the difference in recombination specificity of the transgenic and endogenous ? genes is associated with differences in DNA structure, the methylation status of the endogenous genes and three unrearranged ? transgenes was compared. The J?-C? locus of the transgenes was found to be hypomethylated in all tissues of the transgenic mice. In contrast, methylation of the endogenous ? genes was tissue and developmentally regulated. Hypomethylation of the endogenous J?-C? region occurs only in cells of the B lineage undergoing, or having completed ? gene recombination. Transfection of fibroblasts from transgenic and control mice with the recombination activating genes, Rag1 and Rag2, led to a high level of rearrangement of the hypomethylated transgenic, but not the endogenous ? genes. These results suggest that hypomethylation defines an accessible state of the ? locus and that methylation/demethylation could be involved in the control of ? gene rearrangement during lymphocyte differentiation.