IL-2 and IL-7 induce heterodimerization of STAT5 isoforms in human peripheral blood T lymphoblasts

The CD40 ligand expressed on activated T cells plays a pivotal role in B cell proliferation and differentiation. Mutations in the CD40 ligand gene, which alter its expression on the surface of activated T cells, are associated with the X-linked form of Hyper-IgM syndrome (XHIM). A rapid and simple, three-color whole blood flow cytometry procedure was developed for maximal expression and detection of the CD40L on the surface of in vitro activated CD4+ T cells. Approximately 90% of in vitro activated CD4+ T cells obtained from healthy controls expressed the CD40L compared to only 5% of in vitro activated CD4+ T cells obtained from the XHIM patients. The CD40L was expressed on approximately 50% of the in vitro activated CD4+ T cells obtained from the mothers of XHIM patients, consistent with a diagnosis of their carrier status. This is the first report of a whole blood procedure adapted for routine clinical use which is able to detect abnormal CD40L expression in XHIM patients and carriers.     

Mutations of the CD40 ligand gene in 13 Japanese patients with X-linked hyper-IgM syndrome

X-linked hyper-IgM syndrome (XHIM) is a rare primary immunodeficiency caused by a defective CD40 ligand. We identified mutations of the CD40 ligand gene in 13 unrelated Japanese XHIM patients. Of the four patients with missense mutations, one had a mutation within the transmembrane domain, and the three others had mutations affecting the TNF homology region of the extracellular domain. Two of the missense mutations resulted in the substitution of amino acids that are highly conserved in TNF family proteins. Three patients had nonsense mutations, all of which resulted in the truncation of the TNF homology domain of the CD40 ligand. Three patients had genomic DNA deletions of 2, 3 or 4 nucleotides, respectively. All of the deletions were flanked by direct repeat sequences, suggesting that these deletions were caused by slipped mispairing. Three patients had mutations within introns resulting in altered splicing, and multiple splicing products were found in one patient. Thus, each of the 13 Japanese patients had different mutations, 9 of them being novel mutations. These results indicate that mutations in XHIM are highly heterogeneous, although codon 140 seems to be a hot spot of the CD40 ligand gene since two additional point mutations were located at Trp 140, bringing the total numbers of mutations affecting codon 140 to six. In one XHIM family with a missense mutation, prenatal diagnosis was performed by single-strand conformation polymorphism analysis of genomic DNA of a male fetus.     

Parvovirus B19 infection-related complications in renal transplant recipients: treatment with intravenous immunoglobulin

BACKGROUND: Chronic red cell aplasia can develop in immunocompromised patients including transplant recipients infected with parvovirus B19 (PV B19). Renal involvement with PV B19 infection is not well-recognized. METHODS: We diagnosed erythroid hypoplasia associated with PV B19 infection in three renal transplant recipients; one of them developed de novo collapsing glomerulopathy. These patients were treated with intravenous immunoglobulin (IVIG). RESULTS: In two patients, anemia responded promptly to IVIG therapy. One of them had recurrence of anemia that responded to a second course of IVIG. Despite IVIG treatment, persistent infection with PV B19, recurrent anemia, and de novo collapsing glomerulopathy leading to allograft failure developed in the third patient, who had received the most intense immunosuppression. CONCLUSIONS: These findings indicate that PV B19 infection in transplant recipients can cause chronic red cell aplasia that generally responds to IVIG therapy. In some patients, particularly those who are heavily immunosuppressed, infection may persist despite treatment. As the cellular receptor for PV B19 is expressed in the kidney, persistent infection may result in development of glomerulopathies in these patients.     

Leukocyte transfusion-associated granulocyte responses in a patient with X-linked hyper-IgM syndrome

X-linked hyper-IgM syndrome (XHIM) is a severe congenital immunodeficiency caused by mutations in CD154 (CD40 ligand, gp39), the T cell ligand for CD40 on B cells. Chronic or cyclic neutropenia is a frequent complicating feature that heightens susceptibility to severe infections. We describe a patient with a variant of XHIM who produced elevated levels of serum IgA as well as IgM and suffered from chronic severe neutropenia. Eight of ten leukocyte transfusions with cells from a maternal aunt, performed because of mucosal infections, resulted in similar episodes of endogenous granulocyte production. Transfection studies with the mutant CD154 protein indicate that the protein is expressed at the cell surface and forms an aberrant trimer that does not interact with CD40. The data suggest that allogeneic cells from the patient's aunt, probably activated T cells bearing functional CD154, may interact with CD40+ recipient cells to produce maturation of myeloid precursors in the bone marrow.     

CD40 cross-linking inhibits specific antibody production by human B cells

Ligation of CD40 on B cells is a co-stimulatory signal for proliferation, antibody secretion, heavy chain switching and rescue from apoptosis after somatic mutation in the germinal centre. The importance of these manifold responses to CD40 activation for humoral immunity is exemplified by the inability of boys with X-linked hyper IgM syndrome to make IgG, IgE or IgA due to a mutation in in the gene coding for CD40 ligand (CD40L). In the present study, we have investigated the effect of CD40 ligation on specific antibody production by human B cells to influenza virus. The antibody response was T cell dependent and specific for the strain of influenza virus used as antigen. Addition of either CD40 mAb or recombinant trimeric CD40L profoundly inhibited specific antibody production. Antibody production by unseparated tonsillar mononuclear cells and by T-depleted B cells stimulated with antigen in the presence of T cell replacing factor were equally inhibited with CD40 antibody showing that the effect was due to ligation of CD40 on B cells rather than blocking of T cell help. The specific antibody detected in these experiments was mostly IgG with little or no IgM and was obtained from surface IgM B cells consistent with activation of a secondary (memory) response. Co-stimulation of tonsillar B cells with CD40 antibody and anti-IgG induced proliferation of IgG+ B cells. These results suggest that CD40 ligation can inhibit specific antibody responses and stimulate proliferation in the same IgG+ (memory) B cell subpopulation. Addition of CD40 antibody during the first 24-48 h of the response was required for inhibition, suggesting that the effect was on early B cell activation and/or proliferation required for antibody production. There was no correlation, however, between the ability of CD40 mAb to stimulate proliferation and inhibit antibody production. We suggest that early activation of CD40 in the specific antibody response inhibits the formation of plasma cells and promotes instead the generation of memory cells.     

CD40 ligand gene defects responsible for X-linked hyper-IgM syndrome

The ligand for CD40 (CD40L) is a membrane glycoprotein on activated T cells that induces B cell proliferation and immunoglobulin secretion. Abnormalities in the CD40L gene were associated with an X-linked immunodeficiency in humans [hyper-IgM (immunoglobulin M) syndrome]. This disease is characterized by elevated concentrations of serum IgM and decreased amounts of all other isotypes. CD40L complementary DNAs from three of four patients with this syndrome contained distinct point mutations. Recombinant expression of two of the mutant CD40L complementary DNAs resulted in proteins incapable of binding to CD40 and unable to induce proliferation or IgE secretion from normal B cells. Activated T cells from the four affected patients failed to express wild-type CD40L, although their B cells responded normally to wild-type CD40L. Thus, these CD40L defects lead to a T cell abnormality that results in the failure of patient B cells to undergo immunoglobulin class switching.     

Characterization of nine novel mutations in the CD40 ligand gene in patients with X-linked hyper IgM syndrome of various ancestry

X-linked immunodeficiency with hyper-IgM (HIGMX-1) is a rare disorder caused by defective expression of the CD40 ligand (CD40L) by activated T lymphocytes, resulting in inefficient T-B cell cooperation and failure of B cells to undergo immunoglobulin isotype switch. In the present work, we describe nine patients of various ancestry who bear different mutations in the X chromosome-specific CD40L gene. Two of the mutations were nonsense mutations, one each resulting in premature stop codons at amino acid residues 39 and 140. Three patients had single point missense mutations, one each at codons 126, 140, and 144. Another patient had a 4-bp genomic deletion in exon 2, resulting in a frameshift and premature termination. Three patients showed insertions, one each of 1, 2, and 4 nt, probably because of polymerase slippage, resulting in frameshift mutation and premature termination. Overall, these observations confirm the heterogeneity of mutations in HIGMX-1. However, the identification of two patients whose mutation involves codon 140 (previously shown to be altered in two other unrelated subjects) suggests that this may be a hotspot of mutation in HIGMX-1. In two additional patients with clinical and immunological features indistinguishable from canonical HIGMX-1, no mutation was detected in the coding sequence, in the 5' flanking region, or in the 3' UTR.     

Chronic lymphocytic leukemia B cells can express CD40 ligand and demonstrate T-cell type costimulatory capacity

Chronic lymphocytic leukemia (CLL) is characterized by a clonal expansion of CD5(+) B cells in the peripheral blood. Associated immune aberrations include abnormal Th-cell function and pathogenic autoantibodies. Under most circumstances, CLL B cells do not proliferate in culture and express a limited repertoire of surface antigens, including CD19, CD20, CD23, CD27, CD40, and CD70. In this report, we demonstrate that freshly isolated B cells from a subset of CLL cases constitutively express CD40 ligand (CD40L, CD154), a member of the tumor necrosis factor family which is normally expressed by activated CD4(+) T cells and mediates T-cell-dependent B-cell proliferation and antibody production. The degree of CD40L expression varied considerably among the CLL cases examined. CD40L was detected in purified CLL B cells by immunofluorescence flow cytometry, by RT-PCR, and by immunoprecipitation. To demonstrate that CD40L in the CLL B cells is functional, we used irradiated CLL cells to stimulate IgG production by target, nonmalignant B cells in coculture. The CLL B cells induced IgG production by normal B cells to a similar degree as did purified T cells in a process which was partially inhibited by monoclonal antibody to CD40L. This is one of the first reports of CD40L expression in a B-cell tumor. The data suggest that CD40L in the tumor cells may be a factor in the generation of pathologic antibodies by normal B cells in some patients with CLL.     

Psychological adjustment of children with mild and moderately severe asthma

In chronic hepatitis B virus (HBV) infection seroconversion from hepatitis B e antigen (HBeAg) to hepatitis B e antibody (HBeAb) may be followed either by remission of the disease with low-level viraemia, or by continuing inflammation with high-level viraemia. In both situations the virus may acquire a mutation in the precore sequence which prevents it from encoding HBeAg. We now show that the number of amino acid substitutions in the HBV core is low in viral sequences from patients with HBeAg positive chronic liver disease and HBeAg negative HBeAb positive patients in remission, but the frequency of substitutions is high in HBeAg, negative HBeAb positive patients with active liver disease. Furthermore we show that these substitutions cluster in the promiscuous CD4+ T-helper-cell epitope and in HBV core/e antibody binding determinants, but are not found in regions recognized by major histocompatability complex (MHC) restricted cytotoxic T lymphocytes. Sequential viral sequences from patients before and after HBeAg/HbeAb seroconversion shows that core mutations arise either at the same time or after the precore stop mutation which prevents the virus from encoding HBeAg. These results are consistent with the hypothesis that after clearance of HBeAg, mutations in regions of the virus recognized by CD4+ helper T cells and B cells allow persistence of the HBe negative virus in HBeAb positive patients with viraemia and active hepatitis.     

Correction of neutropenia and hypogammaglobulinemia in X-linked hyper-IgM syndrome by allogeneic bone marrow transplantation

X-linked hyper-IgM (X-HIM) syndrome is a primary immunodeficiency disease characterized by defects in both cellular and humoral immunity. X-HIM is caused by mutations in the gene for CD40 ligand (CD40L), a T cell membrane protein that mediates T cell-dependent immune functions. We report the case of a 6-year-old male with X-HIM due to an intronic mutation resulting in aberrant CD40L RNA splicing and absence of detectable CD40L protein. The patient had a history of multiple infectious complications and chronic neutropenia requiring treatment with recombinant granulocyte colony-stimulating factor, and underwent allogeneic bone marrow transplantation from an HLA-matched sibling donor. Following successful engraftment, T cell CD40L expression and immunoglobulin isotype switching were reconstituted and neutropenia resolved. Allogeneic bone marrow transplantation can correct neutropenia and reconstitute immune function in X-HIM.     

Clinical relevance of parvovirus B19 as a cause of anemia in patients with human immunodeficiency virus infection

Parvovirus B19 (B19) DNA was detected by dot blot hybridization in sera from 5 (17%) of 30 human immunodeficiency virus (HIV)-infected patients with hematocrits (HCT) of < or =24 and 4 (31%) of 13 HRV-infected patients with HCT of < or =20, suggesting that B19 is a reasonably common cause of severe anemia in HIV infection. The anemia promptly remitted after immunoglobulin therapy in 3 of 4 treated patients. The presence of IgM to B19, the clinical circumstance in which anemia developed, and the marrow morphology were poor predictors of chronic B19 infection. DNA hybridization studies of sera from 191 HIV-infected and 117 HIV-seronegative homosexual males attending a clinic in the Seattle area revealed that 1 (0.5%) and 2 (2%) samples, respectively, from the 2 groups contained B19. However, when assayed by polymerase chain reaction (PCR), 5% of the serum samples from HIV-infected persons and 9% from uninfected persons contained B19, although each had an HCT of > or =40. The data argue that anemia results from chronic high-titer B19 infection. Although a negative PCR assay excludes this diagnosis, DNA hybridization may be the more specific serum test.     

Psychometric properties and clinical utility of the scale for suicide ideation with inpatient children

The aim of this study was to identify mutations in the lipoprotein lipase (LPL) gene in 20 unrelated patients with familial lipoprotein deficiency (FLLD) and to investigate the genotype/phenotype relationship. The previously reported G188E mutation (Monsalve et al., J Clin Invest 86:728-734, 1990) was screened for and found to be present in seven individuals (12/40 alleles). In addition, three patients were heterozygous for the 2.0 kb insertion (Langlois et al., Proc Nalt Acad Sci US 86:948-952, 1989). Two approaches were taken for new mutation detection; single-strand conformation polymorphism and sequencing to identify micro-mutations in the proximal promoter and exons 1-9 of the LPL gene and Southern blotting to identify gross mutations. Ten different point mutations were found (W86G, A158T, H183Q, G188E, S193R, P207L, L252X, N291S, M301T, L303P). Additionally, a two nucleotide deletion in exon 6 (delta1006-1007), a six nucleotide deletion in exon 8 (delta1441-1447), and a silent substitution in the wobble position of codon E118 were identified. In vitro mutagenesis and expression in COS-B cells suggested that the A158T and S193R substitutions virtually abolished enzyme activity. In analysing the genotype/phenotype relationship, there was no strong association between age at diagnosis, severity of symptoms, lipid levels, and the nature/position of the mutation. Triglyceride levels, however, were higher in compound heterozygotes compared to true homozygotes, possibly reflecting increased instability of heterodimers. Overall, 29 of 40 (72.5%) mutant alleles were identified. Failure to identify the mutation in 11 alleles might reflect the inadequacy of the method or the possibility that mutations lie within regions of the gene not screened in the study because of lack of availability of sequence.     

The superantigen toxic shock syndrome toxin-1 induces CD40 ligand expression and modulates IgE isotype switching

Isotype switching to IgE requires two signals. The first signal is provided by the cytokines IL-4 or IL-13, and the second signal is delivered by the interaction between the B cell antigen CD40 and its ligand (CD40L) which is expressed on activated T cells. Since superantigens have been shown to activate T cells, we examined the effect of the superantigen toxic shock syndrome toxin-1 (TSST-1) on CD40L expression on T cells. TSST-1 induced expression of CD40L in both freshly isolated T cells and in T cell lines expanded by re-stimulation with TSST-1. CD40L was preferentially expressed in the V beta 2 subset of T cells expanded by TSST-1. We next examined the effect of TSST-1 on IgE synthesis by human peripheral blood mononuclear cells (PBMC). Addition of TSST-1 to PBMC inhibited IL-4-induced IgE synthesis in a dose-dependent manner. This inhibition was reversed partly by adding a neutralizing antibody to IFN-gamma. In contrast, TSST-1 induced high amounts of IgE synthesis in the presence of IL-4 at low T:B cell ratios (0.5:10 to 4:10), a condition which circumvents the inhibitory effect of IFN-gamma. TSST-1 induction of IgE synthesis was inhibited by a mAb to CD40L. These results indicate that superantigens induce CD40L expression in T cells and cause isotype switching in B cells which is mediated by CD40L-CD40 interaction.     

Sequential changes in full-length genomes of hepatitis B virus accompanying acute exacerbation of chronic hepatitis B

BACKGROUND/AIM: During the course of persistent hepatitis B virus infection, viral replication markedly decreases after acute exacerbation of liver inflammation accompanied by emergence of antihepatitis B e antibody (anti-HBe) and/or anti-hepatitis B surface antibody (anti-HBs). In some cases, however, persistent viral replication continues even after such exacerbation with or without HBeAg/anti-HBe seroconversion. The aim of the present study was to investigate the extent of genetic variations of HBV in this phenomenon. METHODS: Full-length HBV genomes were amplified by polymerase chain reaction from sera of three patients before and after acute exacerbation and were directly sequenced. RESULTS: In the whole genomes of 3215 nucleotides, only six nucleotide mutations for six amino acid substitutions (2 in the surface gene, 2 in the X gene, 1 in the core gene and 1 in the polymerase gene) were observed in patient 1, 15 mutations for 14 amino acid substitutions (1 in the pre-core codon 28, 4 in the surface gene, 4 in the core gene and 5 in the polymerase gene) were observed in patient 2, and 5 mutations for 6 amino acid substitutions (2 in the surface gene, 2 in the X gene, pre-core stop codon mutation and 1 in the polymerase gene) were observed in patient 3. Substitution in the a determinant of the surface gene, which encodes target epitopes for neutralizing antibodies, as well as those in the pre-core/core gene, which encodes epitopes for cytotoxic T cells, were mainly found. CONCLUSION: HBV that remained after the emergence of anti-HBe and anti-HBs are considered to possess mutations in epitopes for both humoral and cellular immunity. These mutant HBV may be involved in the pathogenesis of persistent hepatic injury after acute exacerbation.     

Clinical variability of Fanconi anemia (type C) results from expression of an amino terminal truncated Fanconi anemia complementation group C polypeptide with partial activity

Fanconi anemia (FA) is an autosomal recessive disease characterized by congenital anomalies, aplastic anemia, and cancer susceptibility. Mutations within the FA complementation group C (FAC) gene account for approximately 14% of diagnosed FA cases. Two mutations, one in exon 1 (delG322) and one in exon 4 (IVS4 + 4 A to T), account for 90% of known FAC mutations. The delG322 mutation results in a mild FA phenotype, while the IVS4 + 4 A to T mutation results in severe FA phenotype. To determine the molecular basis for this clinical variability, we analyzed patient-derived cell lines for the expression of characteristic mutant FAC polypeptides. All cell lines with the delG322 mutation expressed a 50-kD FAC polypeptides, FRP-50 (FAC-related protein), shown to be an amino terminal truncated isoform of FAC reinitiated at methionine 55. All cell lines with the IVS4 + 4 A to T mutation lacked FRP-50. Overexpression of a cDNA encoding FRP-50 in an FA(C) cell line resulted in partial correction of mitomycin C sensitivity. In conclusion, expression of an amino terminal truncated FAC protein accounts, at least in part, for the clinical heterogeneity among FA(C) patients.     

High incidence of the CD8/9 (+G) beta 0-thalassemia mutation in Spain

BACKGROUND AND OBJECTIVE: In Spain, as in other Mediterranean regions the most common beta-thalassemia mutations are due to point mutations in gene regions that are critical for production of mRNA, such as [IVS-I-nt1 (G-->A), IVS-I-nt6 (T-->C), IVS-I-nt110 (G-->A)] which interrupt normal RNA processing or nonsense mutations [CD39 (C-->T)] which interrupt the translation of mRNA. The frameshift mutation CD8/9 (+G) is a very common allele in Asian Indians but is rare in the Mediterranean regions in which isolated alleles with this mutation have been found in Israel, Greece, Portugal and Turkey. DESIGN AND METHODS: We performed a molecular analysis of 175 chromosomes corresponding to 233 beta-thalassemia patients (221 heterozygous, 10 homozygous and 2 compound heterozygous) who belong to 169 Spanish families. The study of beta-thalassemia was made by PCR-ARMS, the alpha genes by Southern blot, the phenotype of Hb Lepore by enzymatic amplification and the presence of -158 gamma G C-->T mutation by PCR and digestion with the restriction enzyme XmnL. RESULTS: Twenty of these 233 patients showed the beta-thalassemia mutation CD8/9 (+G) (17 were heterozygous, 2 homozygous and in one patient the mutation was associated with a structural variant Hb Lepore Boston). INTERPRETATION AND CONCLUSIONS: These data reveal the heterogeneity of beta-thalassemia in Spain and the relatively high frequency (8.6%) of the frameshift mutation CD8/9 (+G). It is surprising that homozygotes for beta zero-thalassemia due to this mutation with very high Hb F values (around 90%) present a phenotype of intermediate thalassemia.     

Prevalence of mutations in core promoter/precore region in Japanese patients with chronic hepatitis B virus infection

We examined the frequency and significance of mutations in the core promoter and precore region in 103 Japanese patients with chronic hepatitis B virus (HBV) infection. HBV DNAs from the patients' sera were amplified by polymerase chain reaction and were directly sequenced. A double mutation (T1762 A1764) in the core promoter was frequently observed in the patients regardless of HBeAg status except for asymptomatic carriers with HBeAg. Furthermore, a mutation at nucleotide 1753 from T to C or G was frequently found in anti-HBe positive patients and was often accompanied by the double mutation. The A1896 mutation was found in only about one fourth of the patients with anti-HBe. These data suggest that the patients with chronic liver diseases frequently had a double mutation regardless of HBeAg status and a mutation at nucleotide 1753 might be associated with HBeAg-negative chronic hepatitis B virus infection.     

Deficiency of the Fas apoptosis pathway without Fas gene mutations in pediatric patients with autoimmunity/lymphoproliferation

Fas (CD95) is a transmembrane molecule that induces programmed cell death (PCD) of lymphocytes. We examined its function in children with chronic thrombocytopenia, serum autoantibodies, and lymphadenopathy and/or splenomegaly. We found that T-cell lines from six of seven patients with this autoimmune/lymphoproliferative disease (ALD) were relatively resistant to PCD induced by monoclonal antibodies to Fas. By contrast, Fas function was normal in control patients with typical chronic idiopathic thrombocytopenic purpura (ITP) without lymphadenopathy. The defect was not due to decreased Fas expression, nor to over-production of soluble forms of Fas. Moreover, it specifically involved the Fas system because PCD was induced in the normal way by methylprednisolone. Complementary DNA sequencing of the Fas gene did not identify any causal mutation in patients with ALD. This distinguished them from patients with the human autoimmune lymphoproliferative syndrome (ALPS), who carry mutations of the Fas gene. Moreover, patients with ALD did not show the peripheral expansion of CD4/CD8 double-negative T cells that characterizes the ALPS phenotype. Fas signaling involves activation of a sphingomyelinase-catalyzing production of ceramide. We found that ceramide-induced PCD was defective in patients with ALD and not in patients with typical chronic ITP. These data suggest that the ALD patient defect involves the Fas signaling pathway downstream from the sphingomyelinase and that Fas gene mutations and double-negative T-cell expansion are not the only signs of a defective Fas system.