Hematopoietic stem cell transplantation for 30 patients with primary immunodeficiency diseases: 20 years experience of a single team.

We retrospectively analyzed our results of 30 patients with three distinctive primary immunodeficiency diseases (PIDs)--severe combined immunodeficiency (SCID, n = 11), Wiskott-Aldrich syndrome (WAS, n = 11) and X-linked hyper-immunoglobulin M (IgM) syndrome (XHIM, n = 8)--who underwent hematopoietic SCT (HSCT) during the past 20 years. Until 1995, all donors were HLA-haploidentical relatives with T-cell depletion (TCD) (n = 8). Since 1996, the donors have been HLA-matched related donors (MRD) (n = 8), unrelated BM (UR-BM) (n = 7) and unrelated cord blood (UR-CB) (n = 7). Twenty-seven of 30 patients had various pre-existing infections with or without organ damages before HSCT. Conditioning regimen and GVHD prophylaxis were determined according to disease, donor and pretransplant status. Although one of eight patients transplanted with TCD is alive with full engraftment, the other seven died. On the other hand, 18 of 22 patients transplanted without TCD are alive and well, including six of eight transplanted from MRD, seven of seven from UR-BM and five of seven from UR-CB. All 19 survivors did not require Ig supplementation after HSCT. These results indicate that UR-CBT as well as UR-BMT provides good results for PID comparable to MRD-SCT, and that early diagnosis, HSCT at early stage, careful supportive therapy and monitoring for various pathogens are important for the successful HSCT.

Mutations of the WASP gene in 10 Japanese patients with Wiskott-Aldrich syndrome and X-linked thrombocytopenia.

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, immunodeficiency, and eczema. X-linked thrombocytopenia (XLT) is a mild form of WAS with isolated thrombocytopenia. Both phenotypes are caused by mutation of the Wiskott-Aldrich syndrome protein (WASP) gene. In this study, we identified mutations of the WASP gene in 10 Japanese patients from 9 unrelated families with WAS/XLT. All XLT patients (n = 3) and one WAS patient had a missense mutation at the PH domain of WASP. Two WAS patients had nonsense mutations. One WAS patient had exon 8 skipping caused by one nucleotide deletion at the acceptor site of intron 7. Three WAS patients had genomic deletions; one of the three had a large genomic deletion involving exons 3 to 7. Codons 45 and 86 seem to be the hot spots of the WASP mutation, because missense mutations in these codons have been reported previously in several WAS/XLT patients in addition to the patients in this report, and patients with the same mutation show a similar clinical phenotype. All other mutations are novel, indicating that the mutations of WASP are heterogeneous. EB virus-transformed cell lines from XLT patients expressed nearly normal amounts of WASP, whereas those from typical WAS patients expressed almost undetectable amounts of WASP. We conclude that the analysis of gene mutation and protein expression of WASP are useful together in assessing the severity of WAS.

WASP is involved in proliferation and differentiation of human haemopoietic progenitors in vitro.

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, immunodeficiency and eczema. X-linked thrombocytopenia (XLT) is a mild form of WAS with isolated thrombocytopenia. Both phenotypes are caused by mutation of the Wiskott-Aldrich syndrome protein (WASP) gene. In this study we investigated the role of WASP in the differentiation of CD34-positive (CD34+) cells isolated from the bone marrow of patients with WAS (n = 5) or with XLT (n = 4). Megakaryocyte colony formation was significantly decreased in patients with WAS when compared with normal controls. The formation of granulocyte-macrophage colonies and erythroid bursts were also decreased in WAS patinets. In contrast, in XLT patients, formation of all these colonies was normal. However, in vitro proplatelet formation of megakaryocytes induced by thrombopoietin was markedly decreased in both XLT and WAS. Electron microscopic examination revealed that megakaryocytes obtained from WAS or XLT patients grown in vitro had abnormal morphologic features, which seemed to be caused by defective actin cytoskeletal organization, including labyrinth-like structures of the demarcation membrane system and deviated distribution of the alpha-granules and demarcation membrane system. These observations indicate that WASP is involved in the proliferation and differentiation of CD34+ haemopoietic progenitor cells probably by its participation in signal transduction and in the regulation of the cytoskeleton.

EBV infection induced transformation of benign T lymphoproliferative state in patient with chronic active EBV infection into malignant lymphoma: implication of EBV infection as additive oncogenic factor in tumorigenesis.

An 11-year-old girl with chronic EBV (Epstein-Barr virus) infection, who later developed malignant lymphoma in the lung, is reported. She had an increased number of V alpha2, V beta8, CD3, CD4, and HLADR positive activated lymphocytes (20-30% of total lymphocytes) in peripheral blood. One year later, she developed lymphoma in the lung, which was V alpha2, V beta8, CD3, CD4, HLADR and IL2Rbeta positive. At that time, the population in the peripheral blood increased up to 40%, but there was no evidence of lymphoma in the bone marrow. In situ hybridization revealed lymphoma cells were EBER-1 positive but gp350/220 and LMP mRNA negative. The EBV genome was detected in the tumor, but not in the peripheral T cells. Clonal analysis of the lymphoma cells revealed monoclonal rearrangement of the TcRbeta and gamma gene, however, investigation of the terminal repeat of EBV gene did not show the monoclonal pattern. These results indicate that infection of EBV into clonally activated T cells was related with transformation from benign lymphoproliferative disease to malignant lymphoma in this patient.

Ku in the cytoplasm associates with CD40 in human B cells and translocates into the nucleus following incubation with IL-4 and anti-CD40 mAb.

CD40 plays a critical role in survival, growth, differentiation, and class switching of B lymphocytes. Although Ku is required for immunoglobulin class switching, how CD40 signal transduction is coupled to Ku is still unknown. Here, we show that CD40 directly interacts with Ku through the membrane-proximal region of cytoplasmic CD40. Ku was confined to the cytoplasm in human primary B cells, and the engagement of CD40 on the B cells cultured in the presence of IL-4 resulted in the dissociation of Ku from CD40, translocation of Ku into the nucleus, and increase in the activity of DNA-dependent protein kinase. These findings indicate that Ku is involved in the CD40 signal transduction pathway and may play an important role in the CD40-mediated events.

The pleckstrin homology domain of the Wiskott-Aldrich syndrome protein is involved in the organization of actin cytoskeleton.

In this study, we investigated the role of the pleckstrin homology (PH) domain of the Wiskott-Aldrich syndrome protein (WASP) in the regulation of actin cytoskeleton, which is defective in patients with Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT). Overexpression of the WASP in COS-7 cells cultured in the presence of fetal calf serum (FCS) resulted in large cluster formation of polymerized actin and WASP in the cytoplasm. In contrast, when the WASP transfected cells were cultured in the absence of FCS, activation with PMA or EGF was required to induce cluster formation. Overexpression of WASP with a missense mutation in the N-terminus of the PH domain failed to induce the large cluster formation in COS-7 cells even in the presence of FCS. We also found that phosphatidylinositol 4,5-bisphosphate (PIP(2)), which is known to regulate the actin cytoskeleton, binds to the PH domain of WASP, and the binding was abolished by the introduction of a missense mutation into the N-terminus but not the C-terminus of the PH domain. Together with the observations that most of the missense mutations observed in patients with WAS and XLT are located within the PH domain, these results indicate that the PH domain of WASP plays important roles in the regulation of actin cytoskeleton and suggested that the binding of PIP(2) to the PH domain is necessary for WASP to function properly.

Involvement of wiskott-aldrich syndrome protein in B-cell cytoplasmic tyrosine kinase pathway.

Bruton's tyrosine kinase (Btk) has been shown to play a role in normal B-lymphocyte development. Defective expression of Btk leads to human and murine immunodeficiencies. However, the exact role of Btk in the cytoplasmic signal transduction in B cells is still unclear. This study represents a search for the substrate for Btk in vivo. We identified one of the major phosphoproteins associated with Btk in the preB cell line NALM6 as the Wiskott-Aldrich syndrome protein (WASP), the gene product responsible for Wiskott-Aldrich syndrome, which is another hereditary immunodeficiency with distinct abnormalities in hematopoietic cells. We demonstrated that WASP was transiently tyrosine-phosphorylated after B-cell antigen receptor cross-linking on B cells, suggesting that WASP is located downstream of cytoplasmic tyrosine kinases. An in vivo reconstitution system demonstrated that WASP is physically associated with Btk and can serve as the substrate for Btk. A protein binding assay suggested that the tyrosine-phosphorylation of WASP alters the association between WASP and a cellular protein. Furthermore, identification of the phosphorylation site of WASP in reconstituted cells allowed us to evaluate the catalytic specificity of Btk, the exact nature of which is still unknown.

Anti-tumor activity of antizyme which targets the ornithine decarboxylase (ODC) required for cell growth and transformation.

Anti-tumor activity of antizyme which targets the ornithine decarboxylase (ODC) required for cell growth and transformation Cell proliferation and transformation induced by growth factor stimulation or by carcinogens, viruses, or oncogenes are characterized by an associated increase in polyamine levels, which is mediated by increased polyamine biosynthesis and enhanced uptake of polyamines. Polyamine biosynthesis is catalyzed particularly, in the level of ornithine decarboxylase (ODC). The elevation of cellular polyamine levels on the other hand accelerates the induction of ornithine decarboxylase antizyme (antizyme), which is involved not only in ODC-degradation, but in the negative regulation of polyamine transport. Taking advantage of these characteristics of antizyme, the potential of antizyme as a factor having anti-cell growth and anti-tumor activity was investigated. We show that antizyme can induce cell death associated with a rapid decline of intracellular polyamine contents. The possible anti-tumor activities of ectopically expressed antizyme were tested in p21H-ras (Val 12)-transformed NIH3T3 cells and several human malignant cell lines including a line with loss of p53 expression, and they were shown to be as sensitive as nontransformed NIH3T3 cells in vitro. The in vivo anti-tumor activity was also tested using nude mice inoculated with H-ras transformed NIH3T3 cells that had been transfected with inducible antizyme expression vector and the results showed that antizyme expression in vivo blocks tumor formation in these mice. These results suggest that ectopic antizyme expression is of possible therapeutic benefit in the treatment of cancer, which is mediated by ODC inactivation and intracellular polyamine depletion.

Human mast cells produce IL-13 by high-affinity IgE receptor cross-linking: enhanced IL-13 production by IL-4-primed human mast cells.

BACKGROUND: Mast cells play a central role not only in the early phase of the allergic reaction, but also participate in the late phase of the allergic reaction through the allergen and IgE-dependent release of multifunctional cytokines. OBJECTIVE: Using the recently established culture system for human mast cells, we examined the expression of a variety of cytokines in cord blood-derived human cultured mast cells (HCMCs) in response to different stimuli. METHODS: HCMCs were grown from cord blood mononuclear cells in the presence of stem cell factor and IL-6 for 10 weeks. Cytokine mRNA expression in HCMCs by the different stimuli was examined by RT-PCR. Then taking 2 important cytokines, IL-13 and IL4, that share several functional properties and play important roles in allergic diseases, we examined protein as well as mRNA expression of both cytokines in HCMCs. RESULTS: HCMCs did not express either IL-13 or IL-4 spontaneously. Stimulation with PMA + A23187 induced the expression of IL4 protein, as well as IL-13 protein, in their cytoplasm, although IL-4 secreted in the supernatant was below detectable levels in contrast to a significant amount of IL-13. Stimulation of HCMCs by cross-linking of the high-affinity IgE receptor (Fc(epsilon)RI) induced the expression of IL-13 mRNA and protein, but not IL4. Although we previously found that IL-4 upregulates Fc(epsilon)RI expression on HCMCs, when HCMCs were first cultured in the presence of IL4 and then activated through FC(epsilon)RI cross-linking, remarkable increase was found in IL-13 production. Furthermore, although IL-4 was still undetectable at protein level, IL-4 mRNA expression was induced in the IL-4-primed HCMCs stimulating Fc(epsilon)RI cross-linking. In addition, we examined the effects of these cytokines on the surface molecule expression in HCMCs. Although IL4 remarkably upregulated lymphocyte function-associated antigen-1, intercellular adhesion molecule-1, and Fc(epsilon)RI expression and downregulated c-kit expression in HCMCs, IL-13 did not. CONCLUSIONS: Our observation that HCMCs produce IL-13 on cross-linking of Fc(epsilon)RI, which was enhanced by IL-4 priming, supports an important role of mast cells in amplification of allergic reaction and further suggests one of the mechanisms enhancing mast cell function in the microenvironment.

Correlation between antigen-specific IL-2 response test and provocation test for egg allergy in atopic dermatitis.

BACKGROUND: The antigen-specific interleukin-2 response (AIR) test using lymphocytes is effective in searching for the antigen which causes allergic diseases and understanding their disease activity. OBJECTIVE AND METHODS: The correlation between the raw egg oral provocation test and egg white antigen-specific interleukin-2 (IL-2) response test was investigated in 123 children with infantile atopic dermatitis and 13 children with bronchial asthma. RESULTS: Among the 83 who showed positive reactions to provocation, 75 also reacted positively to the AIR test (sensitivity, 90.4%), while among the 53 children who showed negative responses to antigen provocation, 45 produced negative responses to the AIR test (specificity, 84.9%). The specificity of egg white IgE RAST score and skin-prick test are 88.7 and 81.3% which are comparable to that of the AIR test. However, their sensitivity was low (38.6 and 66.7%). In the patterns of symptom developed in the provocation AIR displayed late and delayed type allergic responses in addition to the immediate type which RAST reflected. The RAST-negative group composed of 98 patients included 51 (52.0%) who exhibited positive reactions to the provocation test. Among these 44 responded positively to the AIR test (86.3%). CONCLUSION: The AIR test is effective for screening egg white antigen as part of the tests for antigens responsible for allergic diseases and as a test to ascertain the relevant antigens, and that the conditions that could not be diagnosed by RAST can be detected by the AIR test.

Functional analysis of peripheral blood B cells in patients with X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disease caused by mutations of Bruton tyrosine kinase (Btk); Btk plays an essential role in the development of mature B cells. However, small numbers of B cells ("leaky B cells") are present in the peripheral blood of most XLA patients. In this study, we analyzed the function of these leaky B cells obtained from XLA patients. Enough numbers of B cells were available for analysis from five of nine XLA patients originally screened. Sequence analysis revealed missense mutations of Btk in four of the five XLA patients. No mutation was found in the coding region of Btk in one patient. Western blotting and/or flow cytometric analysis failed to detect Btk protein in all five patients. B cells isolated from peripheral blood of these XLA patients were CD5-, CD20+, CD19+, and CD21-. If stimulated with anti-CD40 and IL-4, XLA B cells proliferated normally and produced significant amounts of IgE. Anti-CD40 stimulation of XLA B cells resulted in normal expression of CD23. In addition, three of the five XLA patients studied were immunized with bacteriophage phiX174 and produced low but detectable levels of antiphage-specific Ab. Similarly, X-linked immunodeficiency mice, which carry a missense mutation in Btk, produced substantial amounts of antiphage Ab. These results indicate that CD40 signaling is intact in B cells lacking demonstrable Btk, and that leaky B cells in XLA patients can proliferate, undergo isotype switching, and differentiate into specific Ab-producing cells.

[Intravenous immunoglobulin (GB-0998) for prophylaxis of recurrent acute otitis media and lower respiratory tract infection in infancy with IgG 2 deficiency].

The prophylactic effect of intravenous immunoglobulin (GB-0998) on the recurrent acute otitis media, bronchitis and bronchopneumonia in IgG 2 deficient infants was investigated in a multicenter trial. GB-0998 was administered 6 times every 4 weeks. The doses were 300 mg/kg wt. during the first treatment, followed by 5 doses of 200 mg/kg wt. The results indicated that GB-0998 was effective in the prophylaxis of the recurrent infection of acute otitis media, bronchitis and bronchopneumonia in infancy with IgG 2 deficiency and/or IgG 2 antibody deficiency specific for Streptococcus pneumoniae.

Diminished expression of CD40 ligand may contribute to the defective humoral immunity in patients with MHC class II deficiency.

Major histocompatibility complex (MHC) class II deficiency (bare lymphocyte syndrome, BLS) is a rare primary immunodeficiency classified as a subgroup of severe combined immunodeficiency. We studied T and B lymphocyte function by examining the CD40 ligand/CD40 system in three BLS patients from two unrelated families. CD40 ligand expression by maximally activated BLS T cells was diminished. This abnormality may represent immunological naïveté rather than a general T cell defect, since expression of activation marker CD69 and proliferative responses to PHA or anti-CD3 were normal, and BLS T cells primed and restimulated in vitro expressed normal amounts of CD40 ligand. BLS B cells proliferated and produced IgE if stimulated with anti-CD40 or soluble CD40 ligand and IL-4. Activation of BLS B cells with soluble CD40 ligand and IL-4 induced normal expression of activation markers, although MHC class II expression remained absent. Depressed antibody titers, lack of amplification and failure to undergo isotype switching in response to immunization with bacteriophage phi x 174 demonstrated defective T cell help. We conclude that BLS B cells are functionally normal if appropriately stimulated, and that the defective humoral immunity observed may be related to diminished expression of CD40 ligand on BLS T cells.

Interleukin-4 promotes the development of tryptase and chymase double-positive human mast cells accompanied by cell maturation.

Human cultured mast cells (HCMCs) grown from cord blood mononuclear cells in the presence of stem cell factor (SCF) and interleukin-6 (IL-6) expressed tryptase but no or low chymase in their cytoplasm. The addition of IL-4 to these cells strikingly increased chymase expression. Consequently, the activity of chymase was significantly higher in IL-4-treated mast cells than that in IL-4-nontreated mast cells, whereas the activity of tryptase and histamine content were comparable in both cells. Electron microscopic immunocytochemistry also showed that secretary granules containing chymase increased in IL-4-treated mast cells. Interestingly, the IL-4-induced increase of chymase expression in HCMCs was accompanied by morphological maturation of the cells. Cytoplasmic projections were few in IL-4-nontreated HCMCs, and a small number of secretary granules were observed, most of which were empty or partially filled with discrete scrolls with rough particles showing immaturity. In contrast, IL-4-treated HCMCs had extremely abundant cytoplasmic projections and had many secretary granules filled with electron-dense crystal materials. Taken together, immature HCMCs grown only with SCF and IL-6 expressed tryptase with no or a low amount of chymase, and addition of IL-4 promoted cell maturation together with the expression of both tryptase and a high amount of chymase. Our findings will raise a possibility of a linear pathway of human mast cell development from tryptase single positive mast cells into tryptase and chymase double positive mast cells as the cells mature and will suggest that this maturation process is promoted by IL-4.

Interleukin-4 induces homotypic aggregation of human mast cells by promoting LFA-1/ICAM-1 adhesion molecules.

We report here that interleukin-4 (IL-4) induces homotypic aggregation of cultured human mast cells, grown from cord blood mononuclear cells in the presence of stem cell factor and IL-6. This aggregation was specifically induced by IL-4, because other cytokines including IL-1alpha, IL-1beta, IL-2, IL-3, IL-5, IL-9, IL-10, interferon-gamma, IL-12, granulocyte-macrophage colony-stimulating factor, NGF-beta, and tumor necrosis factor-alpha failed to show such effect. Flow cytometric analysis of the cultured mast cells showed that IL-4 increases the expression of lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1), but not of very late antigen (VLA) family adhesion molecules or vascular cell adhesion molecule-1 (VCAM-1). Neutralizing monoclonal antibodies specific for LFA-1alpha, LFA-1beta, or ICAM-1 inhibited the IL-4-induced homotypic aggregation of the mast cells, indicating that the aggregation was mediated mainly by LFA-1/ICAM-1 interaction. In addition, IL-4-treated but not untreated mast cells bound to immobilized ICAM-1. This binding was also inhibited by anti-LFA-1 or anti-ICAM-1. These results show that IL-4 promotes expression of ICAM-1 and LFA-1 molecules on mast cells, and suggest that IL-4 may contribute to the migration of mast cells into the inflamed tissue and to the cellular interaction with other inflammatory cells by upregulating adhesion molecules.

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.