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.

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.

Heterogeneity of the breakpoint in the ABL gene in cases with BCR/ABL transcript lacking ABL exon a2.

We report cases with a variant BCR/ABL mRNA expression lacking ABL exon a2 sequences. Two of these cases showed major breakpoint cluster region (BCR) exon 3 (b3) and ABL exon 3 (a3) junction (b3/a3), while the other case showed minor BCR exon 1 (e1) and a3 junction (e1/a3). One of the two cases with b3/a3 junction and the case with e1/a3 junction were diagnosed as acute lymphoblastic leukemia, and the remaining case with b3/a3 junction was chronic myeloid leukemia. Two of these cases, however, were found to have a breakpoint in the ABL gene outside of the intron between exons a2 and a3, probably 5' upstream of exon a2, suggesting that the BCR exon was spliced to ABL exon a3. These findings differ from those previously reported, in which the breakpoints in the ABL gene were between exons a2 and a3, and indicate a novel mechanism for the deletion of ABL exon a2 sequences in the formation of a variant BCR/ABL fusion transcript. The significance of the finding that a part of the SH3 region of ABL protein is missing in some Philadelphia chromosome-positive leukemias is discussed in reference to the cases reported previously.

Fluorescence intensity analysis through simplex optimization in flow cytometry.

Fluorescence intensity analysis in flow cytometric surface immunophenotyping has recently been appreciated in clinical applications. A curve fitting method to estimate the mean and SD values of fluorescence intensity is described in this report. A Gaussian distribution is aimed to be adapted for a specified distribution in logarithmically scaled histogram data through the simplex optimization, one of the non-linear least squares methods. In comparison with the conventional methods which include the detection of peak point and the direct calculation, this fitting method has demonstrated exceeding precisions in the estimation of both parameters with limited involved cell counts in typical lymphocytic phenotyping. The actual estimation for a precise SD value will develop the quality control approaches based on the fluorescence intensity analysis. While this method is not suitable for distributions that involve extremely small cell counts or that deviate markedly from a symmetric Gaussian, it has additional advantages of loose requirements, namely, narrow fitting regions, ordinarily small cell counts, practical computational periods and a simple programming.

Cell cycle-related expression of surface antigens on myelomonocytic leukemia cells.

We investigated the relationship between the expression of surface antigens and the cell cycle phase in leukemic cells from cell lines and one patient using two-color flow cytometry, in order to determine the reason for the uneven expression of some markers which frequently leads to equivocal results as to leukemic phenotyping. As a result, it was demonstrated that monocyte-related differentiation markers, including I2, My4, Mo1 and Mo2, on monocytoid leukemic cells are preferentially expressed at the G0/G1 phase. Consequently, it is expected that the positivities for such markers vary with the proliferation status of the leukemic cells.

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.

Two sisters with clinical diagnosis of Wiskott-Aldrich syndrome: is the condition in the family autosomal recessive?

We report two sisters in a family representing manifestations of Wiskott-Aldrich syndrome (WAS), an X-linked immunodeficiency disorder. An elder sister had suffered from recurrent infections, small thrombocytopenic petechiae, purpura, and eczema for 7 years. The younger sister had the same manifestations as the elder sister's for a 2-year period, and died of intracranial bleeding at age 2 years. All the laboratory data of the two patients were compatible with WAS, although they were females. Sialophorin analysis with the selective radioactive labeling method of this protein revealed that in the elder sister a 115-KD band that should be specific for sialophorin was reduced in quantity, and instead an additional 135-KD fragment was present as a main band. Polymerase chain reaction (PCR) analysis of the sialophorin gene and single-strand conformation polymorphism (SSCP) analysis of the PCR product demonstrated that there were no detectable size-change nor electrophoretic mobility change in the DNA from both patients. The results indicated that their sialophorin gene structure might be normal. Studies on the mother-daughter transmission of X chromosome using a pERT84-MaeIII polymorphic marker mapped at Xp21 and HPRT gene polymorphism at Xq26 suggested that each sister had inherited a different X chromosome from the mother. Two explanations are plausible for the occurrence of the WAS in our patients: the WAS in the patients is attributable to an autosomal gene mutation which may regulate the sialophorin gene expression through the WAS gene, or, alternatively, the condition in this family is an autosomal recessive disorder separated etiologically from the X-linked WAS.

T cell reconstitution by haploidentical BMT does not restore the diversification of the Ig heavy chain gene in patients with X-linked SCID.

We previously examined the Ig heavy (H) chain gene of pretransplant patients with X-linked SCID (XSCID), having defects in the gene of the IL-2 receptor (R) gamma chain. In the present study, we analyzed two post-transplant XSCID patients, in whom T cell-depleted haploidentical BMT resulted in lymphoid split chimeras, i.e., donor functional T cells coexisting with recipient B cells. Although the recipient B cells produced IgM, no isohemagglutinin or Ag-specific Ab was detected. To investigate the cause of failure to produce Ab in the patients, we sequenced the complementarity determining region 3 (CDR3) and adjacent region of Ig H chain gene, which govern Ab specificity. Among the 64 post-transplant CDR3 junctional sequences, combinatorial and junctional diversity were normal compared with those in age-matched controls. All of the post-transplant joining regions except one clone were equal to germline and the frequency of somatic mutation was significantly lower than that in age-matched controls. The results indicated that T cell reconstitution by BMT does not restore diversification of the Ig gene in the IL-2R gamma chain-deficient B cells, which might be associated with the defect in the Ag-specific Ab production.

Pulmonary miliary tuberculosis and T-cell abnormalities in a severe combined immunodeficient patient reconstituted with haploidentical bone marrow transplantation.

We report the development of miliary tuberculosis in a 7-year-old boy with severe combined immunodeficiency (SCID), whose immune system had been only partially reconstituted by haploidentical bone marrow transplantation. Although alpha beta and gamma delta T cells were of donor origin, alpha beta T cells in this patient showed defective interleukin-2 (IL-2) production, impaired IL-2 responsiveness and decreased cytolytic activity. However, gamma delta T cells could exhibit enough cytolytic activity after incubation with IL-2. Despite the presence of disseminated infection, C-reactive protein (CRP) remained negative. IL-2 therapy aggravated the disseminated tuberculosis though gamma delta T cells were supposed to be activated, and concurrently CRP became positive. These findings suggest that gamma delta T cells have no more than limited immunological roles in mycobacterium tuberculosis infection.

Peroxidase-negative and myelomonocytic antigen-positive acute leukemia.

Between 1983-1988 bone marrow samples obtained from 195 peroxidase-negative leukemia patients were analyzed for their surface antigens. Thirteen of these patients (6.7%) had myelomonocytic-positive and lymphoid-negative antigens. These leukemic cells reacted with CD13 in eight patients, CD33 in seven, CD11 in six and CDw41 in two. In none of these patients did the leukemic cells react with CD1, CD2, CD3, CD4, CD5, CD8, CD10, CD19 or CD20. Leukemic cells from two patients were reactive with CD7. These leukemic cells demonstrated L2 morphology in 11 patients and L1 morphology in one patient. The leukemic cells from the final patient were diagnosed as those of leukemic transformation of myelodysplastic syndrome. Chromosomal abnormality was observed in approximately half of the patients examined (6/10). Cytochemical analysis revealed that the leukemic cells were negative for periodic acid Schiff stain but positive for acid phosphatase. The prognosis of these patients was markedly poor as compared to acute lymphocytic leukemia or typical peroxidase-positive nonlymphocytic leukemia. Complete remission was induced in only 30% of patients and duration of survival was short (4.7 months). This suggests that myelomonocytic antigen-positive peroxidase-negative acute leukemia is a distinct type of leukemia and may require more aggressive therapy to improve survival.

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.

Defect of suppressor cell induction in patients with juvenile rheumatoid arthritis.

We studied the suppressor cell activity induced by concanavalin A (Con A) in 9 patients with acute febrile juvenile rheumatoid arthritis (JRA). The suppressor activity of JRA patients was higher than that of normal controls. However, the activity was significantly reduced by treating Con A-activated cells with mitomycin C (MMC) (P less than 0.05). On the other hand, the suppressor activity of normal controls and systemic lupus erythematosus (SLE) patients was not affected by MMC treatment. Two of 5 SLE patients showed low activity even before MMC treatment. The addition of the culture supernatant of Con A-stimulated peripheral blood mononuclear cells from a normal donor restored the induction of suppressor activity of JRA which was decreased by MMC treatment. The results indicated that patients with acute febrile type of JRA had reduced MMC resistant suppressor cell activity and that this was due to a defect in the ability of the cells to produce soluble factors needed to induce MMC resistant suppressor cells.

[In vitro detection of ovalbumin-specific IgA producing cells from lymphocytes of patients with hen-egg allergy].

We studied antigen-specific IgA production from the peripheral lymphocytes of children with hen-egg allergy by using the method of indirect plaque forming cell (PFC) assay. The number of OVA-specific IgA-PFC generated from the patient lymphocytes was low (6 +/- 3/7 X 10(4) non T cells) in comparison with age-matched normal children (112 +/- 18/7 X 10(4) non T cells). The numbers of OVA-specific IgG-PFC and IgM-PFC generated from the patient lymphocytes were not so different from those of the age-matched normal children. This indicates that the activity of OVA-specific IgA-antibody production was reduced in patients with hen-egg allergy.

[Thyroglobulin (Tg)-specific induction of IL2 responsiveness in lymphocytes from patients with Hashimoto thyroiditis and its dependency on HLA-DQ antigen].

The peripheral blood mononuclear cells from Hashimoto thyroiditis patients acquired IL2 responsiveness in response to thyroglobulin stimulation, when they were partially depleted of adherent cells. Non-adherent cells reconstituted with Tg-pulsed autologous adherent cells at the ratio of 9:1 also acquired IL2 responsiveness, but those cultured with OVA (ovalbumin)-pulse adherent cells did not. This indicates Tg-induced IL2 responsiveness was specific for the antigen. Although there was no clear correlation between the intensity of induced IL2 responsiveness and the serum titer of anti-Tg antibody, Tg-induced IL2 responsiveness was significant in the seropositive patients. Pretreatment of the mononuclear cells with the monoclonal antibody to the HLA-DQ framework (Leu 10), but not the HLA-DR framework (OKIal), blocked the induction of IL2 responsiveness. This indicated that DQ-bearing adherent cells play a key role in presenting Tg antigen to the responder cells.

[Skin reaction(s) to culture supernatant(s) from Dermatophagoides farinae (Df)-stimulated lymphocytes, and their correlation with Df-induced interleukin 2 responsiveness by lymphocytes from patients with bronchial asthma].

To investigate the inflammatory function of Dermatophagoides farinae (Df)-activated lymphocytes, supernatants from cultures of the lymphocytes were intracutaneously injected into donors' forearms. Injection of the supernatants of Df-stimulated lymphocytes from mite-sensitized atopic individuals with such diseases as bronchial asthma induced a profound inflammatory reaction in the autologous skin, characterized by erythema extending more than 15 mm in mean diameter and edema. The inflammation was at its peak after approximately 20 min, which was followed by gradually re-growing erythema lasting for as long as 24 hours after injection. Supernatants of unstimulated lymphocytes were also capable of inducing the same reaction, indicating the presence of in vivo activated lymphocytes, although the extent of the response was always smaller. The induced response in normal individuals was erythema of less than 15 mm in mean diameter. The supernatants obtained from cultures at 4 degrees C failed to induce such inflammation. The culture supernatants of ovalbumin-restimulated lymphocytes were also incapable of augmenting the response. The combined data show that the production of inflammatory response-inducing factor(s) from Df-stimulated lymphocytes was antigen specific. Significant skin reactions was correlated with the IL2 responsiveness of Df-stimulated lymphocytes. The skin reaction induced by factor(s) derived from Df-stimulated lymphocytes, which might be similar to the bronchial hyperreactivity of patients with bronchial asthma. The in vitro assay for Df-induced IL2 responsiveness by lymphocytes might reflect the in vitro immediate, late and/or delayed type hypersensitivity.

[Studies on the effect of lentinan on human immune system. II. In vivo effect on NK activity, MLR induced killer activity and PHA induced blastic response of lymphocytes in cancer patients].

Immunological activities of lymphocytes were studied in patients with cancer of digestive organs and mamma after administration of lentinan. When 11 cancer patients received lentinan i.v. 2 mg, NK activity of lymphocytes was increased in 4 patients on the following day. With the same condition, MLR induced killer activity against Raji cell of lymphocytes was increased in 3/7 patients and PHA response was also increased in 3/7 patients. These activities were returned to values before the administration of lentinan. Then each activity was examined after administration of lentinan twice a week. NK activity, MLR induced killer activity and PHA response were increased in 4/15, 8/12 and 5/11 patients, respectively.

[Anti-CD45R antibody-treated normal lymphocytes respond to interleukin 2 (IL2) after stimulation by allergens].

Interleukin 2 (IL2) responsiveness was specifically induced by Dermatophagoides farinae (Df) antigen in Df-sensitized lymphocytes from asthmatic children, but not in normal lymphocytes. Df-induced IL2 responsiveness was also observed in normal lymphocytes pretreated (Day 0) with anti-CD45R antibody, which recognize suppressor inducer subset among CD4+ T cells. However anti-CD45R antibody was no longer effective when the lymphocytes were cultured for more than one day with the antigen, suggesting its effect in the initial phase of the reaction. The intensity of the response induced in normal lymphocytes by the anti-CD45R was comparable to that of the patients sensitized to the nominal antigen. The response of the patients was no longer augmented by the anti-CD45R antibody. Taken together, these data suggest that even normal lymphocytes have potentiality to elicit Df-induced IL2 responsiveness and it is probably derepressed by inhibiting suppressor inducer subset with the anti-CD45R antibody. Also suggested is a defective suppressor inducer activity in the lymphocytes which may lead to hyperreactivity to allergens in asthmatic children.

[Correlation of interleukin-2 (IL-2) responsiveness by egg white-stimulated lymphocytes with hen egg oral provocation test in atopic children].

One hundred and twenty five cases of atopic children such as atopic dermatitis and bronchial asthma were orally provocated with rare hen egg every 20 minutes one by one upto the whole amount. In one week observation 75 cases showed any symptoms of allergy including eruption and exacerbation of atopic eczema in an immediate, late, and/or delayed responses. Frequency of positive egg white-induce IL-2 responsiveness test in patients with positive oral provocation was 90.7% (68 out of 75 cases; sensitivity). That of negative test in patients with negative provocation was 84.0% (42 out of 50 cases; specificity). In contrast, specificity of IgE RAST for egg white were 88.0% comparable to the value of antigen-specific IL-2 responsiveness (AIR) test, but the specificity was lower value (37.3%) for screening the etiological antigens as compared to that of AIR test. High frequency of positive egg white-induced IL-2 responsiveness test was observed over an immediate, late and delayed responses, while low frequency of positive IgE RAST for hen egg was observed largely in patients showing delayed but not immediate response. The results indicate that IgE RAST in this study reflects IgE-mediated immediate type hypersensitivity, whereas AIR test reflects, in addition to immediate responses, late and delayed type hypersensitivity. The combined results suggest that AIR test in hen egg allergy is a useful method in vitro for both screening and determining etiological allergens, and might be able to substitute for provocation test in vivo for which many times, labours, expenses, and patients' risks are required, and to cover IgE RAST which fails to determine etiological allergens in 62.7% of patients with positive oral provocation.

[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.

[Cytokine therapy of immunodeficiency].

The cellular immunodeficiency diseases especially those with impaired IL-2 production are successfully treated by every day injection of rhIL-2. IL-2 is also effective on some patients with antibody deficiency probably caused by the lack of T cell help for B cells. Prolonged infection of EB-virus, human immunodeficiency virus, fungi and mycobacteria can be ameliorated by IL-2 treatment. Superoxide production and bacteriocidal activity of the leukocytes from some cases of chronic granulomatous disease are improved by injection of interferon gamma. Succeeding injection of G-CSF is effective to maintain the leukocyte count of congenital neutropenia to the level competent to protect bacterial infections.