Multidrug resistance protein 4 (MRP4) polymorphisms impact the 6-mercaptopurine dose tolerance during maintenance therapy in Japanese childhood acute lymphoblastic leukemia.

Multidrug resistance protein 4 (MRP4) is involved in the efflux of nucleoside derivatives and has a role in the determination of drug sensitivity. We investigated the relationship between MRP4 genetic polymorphisms and doses of the 6-mercaptopurine (6-MP) and methotrexate. Further, we evaluated the frequency of therapeutic interruption during maintenance therapy in Japanese children with acute lymphoblastic leukemia (ALL). Ninety-four patients received an initial 6-MP dose in the range of 30-50 mg m(-2) in this analysis. Patients with homozygous variant allele in any of MRP4 G2269A, C912A and G559T required high frequency of 6-MP dose reduction compared with non-homozygous individuals. Average 6-MP dose for patients with homozygous variant allele on either MRP4 or inosine triphosphate pyrophosphatase was significantly lower than that for patients with non-homozygous variant allele during maintenance therapy (30.5 versus 40.0 mg m(-2), P=0.024). Therefore, MRP4 genotyping may be useful for personalizing the therapeutic dose of 6-MP during the ALL maintenance therapy in Japanese.

Expression profiling of genes related to asthma exacerbations.

BACKGROUND: Asthma is a chronic airway inflammatory disease; however, the molecular mechanisms that underlie asthma exacerbation are only partially understood. OBJECTIVE: To identify gene expression signatures that reflect the acute exacerbation of asthma, we examined the differential expression of genes during asthma exacerbation and stable condition by using microarray analysis. METHODS: The subjects were mite-sensitive asthmatic children and non-asthmatic control children. The children were divided into four groups (AE: asthma exacerbation, n=12; SA: stable asthma, n=11; IC: infected control, n=6; and NC: non-infected control, n=5). Total RNA was extracted from peripheral blood mononuclear cells and subjected to microarray analysis with Illumina Human Ref8 BeadChip arrays. Welch's t-test was performed to identify genes whose expression was altered during asthma exacerbation. Quantitative real-time RT-PCR was performed on samples collected from 43 asthmatic children and 11 control children to verify the microarray results. RESULTS: The expression of 137/16 genes was significantly up/down-regulated during asthma exacerbation assessed by microarray analysis. Of the genes, 62 were also differentially expressed during upper respiratory infection. Many of the asthma exacerbation related genes were involved in defence responses and responses to external stimuli, but these associations disappeared after excluding the infection-related genes. Quantitative real-time RT-PCR confirmed that the genes related (S100A8 and GAS6) and unrelated to infections (CD200 and RBP7) were differentially expressed during asthma exacerbation (P<0.01). CONCLUSIONS: Previously unidentified immune responses during asthma exacerbation may provide further clarification of the molecular mechanisms underlying asthma.

Circulating antibodies to common food antigens in Japanese children with IDDM.

OBJECTIVE: To investigate the humoral immune response to common food antigens in Japanese children with IDDM. RESEARCH DESIGN AND METHODS: IgG antibodies to cow's milk, beta-lactoglobulin, bovine serum albumin (BSA), alpha-lactalbumin, and hens egg ovalbumin were examined by enzyme-linked immunosorbent assay in the sera of 33 patients with IDDM, ages 11.8 +/- 3.4 years. The data were compared with that of 50 normal subjects, ages 10.3 +/- 5.1 years, who acted as control subjects. A positive antibody to a food antigen was defined as an antibody titer greater than the 95th percentile value in normal subjects. RESULTS: Children with IDDM had significantly higher median titers of IgG antibodies to beta-lactoglobulin and ovalbumin (P = 0.03 and P = 0.0005 respectively). More children with IDDM than control subjects had positive IgG antibody to ovalbumin (21 vs. 6%, P = 0.04). Titers, as well as the number of positive antibodies to other food antigens, including BSA, did not differ between the two groups. CONCLUSIONS: Japanese children with IDDM show an enhanced humoral immune response to beta-lactoglobulin and ovalbumin, a phenomenon that may be related to the pathogenesis of the disease.

A case of fatal infectious mononucleosis presenting with fulminant hepatic failure associated with an extensive CD8-positive lymphocyte infiltration in the liver.

We describe a fatal case of infectious mononucleosis presenting with fulminant hepatic failure associated with extensive CD8-positive lymphocyte infiltration and diffuse karyorrhexis in the liver. Immunohistochemical analysis of mononuclear cells showed that Leu-2a (CD8)-positive lymphocytes were heavily distributed in the portal areas and the sinusoidal spaces, but Leu-3a (CD4)-, Leu-14 (CD22)-, or My 4 (CD14)-positive cells were undetectable in sections of the liver. Southern blot hybridization studies disclosed the presence of Epstein-Barr virus DNA fragments in the liver tissue. The unusual pathologic and immunologic responses observed in this case could not simply be explained by severe Epstein-Barr virus infection. Some superimposed factors should be considered.

Chronic active Epstein-Barr virus infection in an adult.

We report a rare adult case of chronic active Epstein-Barr virus (EBV) infection. A 54-year-old woman was admitted to our hospital with intermittent fever, weight loss, hepatosplenomegaly, pancytopenia and liver disturbance. In serological tests for EBV, anti-virus capsid antigen (VCA)-IgG antibody and anti-early antigen (EA)-IgG antibody were markedly elevated and anti-EBV nuclear antigen (EBNA) antibody was negative. EBV genome was detected in the bone marrow nucleated cells and peripheral lymphocytes by Southern blot hybridization. The patient developed left facial edema, bilateral breast tumor and pneumonia. She died one year after admission in spite of the administration of prednisolone, interferon and acyclovir.

[A case report of surgically treated tracheobrachiocephalic artery fistula following tracheostomy].

The patient was a 17 year-old-boy with mental retardation and cerebral palsy, who underwent tracheostomy because of tracheobronchomalacia and tongue swallowing three months ago. After minimal tracheostomy bleeding was noted for a few days, massive hemorrhage occurred suddenly. The patient was severely hypotensive. The cuff on the tracheostomy tube was hyperinflated, which stopped the bleeding temporarily. Through a median sternotomy and collar incision, a pin hole-sized defect in the medial surface of the brachiocephalic artery at the tracheal balloon was repaired by suture. The tracheal defect allowed visualization of the tracheal cannula balloon. The pectoralis major muscle flap was interposed between the tracheal defect and the artery. The tracheal defect was packed with muscle. Seven days after the operation, the second operation was done for recurrent bleeding. The defective segment of brachiocephalic artery was resected and the ends were oversewn with monofilament sutures. Continuous irrigation with povidone-iodine solution was continued for purulent mediastinitis. We believe that interruption of blood flow of the brachiocephalic artery, interposition and packing of muscle flap over the tracheal defect and continuous irrigation for infection were effective treatment of choice.

Potentiation of IgE production to common environmental allergens by storage house dust mite Dermatophagoides farinae.

Total serum IgE and IgE antibodies against ten common antigens, including Dermatophagoides farinae (D.f.), moth, ragweed, orchard grass, cryptomeria, alternaria, aspergillus, dog dander, cat dander and tetanus toxoid, were determined using the PRIST and the Sepharose-RAST, respectively, in 100 clinically non-allergic Japanese subjects and interrelations of IgE responses to these antigens were investigated. We obtained following results. The number of positive RAST antigens, to which the subjects responded, increased in parallel to their total serum IgE levels. Among ten antigens, D.f. and moth antigens contributed a great deal to the elevation of total serum IgE level as compared with other antigens. When the relationship between IgE response to D.f. and similar responses to the other nine antigens was investigated, positive RAST responses to various antigens occurred selectively in the subjects with positive RAST for D.f., and the number of positive RAST antigens to which the subjects responded increased depending upon their D.f. specific RAST levels. Such an association was not found between IgE response to moth and the other nine antigens. These findings suggest that non-specific activation of IgE-producing B cells occurs as a result of continuing stimulation by D.f.

Hypersensitive reactions to mosquito bites in congenital agammaglobulinemia.

A 6-year-old girl with congenital agammaglobulinemia exhibited severe delayed onset cutaneous reactions to mosquito bites with scar formation. The bites were not associated with immediate reactions accompanied by itching. The patient had non-detectable IgE levels (less than 0.2 IU/mL) in addition to absence of three other major classes of immunoglobulins, but showed normal T cell functions. Immunologic and histopathologic examination of the skin lesions revealed that this unusual reaction may be caused by a delayed type hypersensitivity response.

Mother-to-infant transmission of hepatitis C virus: a prospective study.

UNLABELLED: To investigate the risk of mother-to-infant transmission of hepatitis C virus (HCV) and the natural course of HCV-infected infants, we prospectively studied 31 offspring of pregnant women who were anti-HCV positive and anti-HIV negative. Sera were serially tested for anti-HCV by the second-generation ELISA-test (ELISA-2) and for HCV-RNA by the polymerase chain reaction procedure. The mean period of follow up was 19 months (range 6-41 months). The presence of HCV-RNA in the mothers was associated with a high titre of anti-HCV by ELISA-2 or a positivity of the second generation recombinant immunoblot assay. At birth, 26 babies were positive for anti-HCV. Passively transferred maternal antibodies became undetectable within 2-15 months. HCV-RNA was detected in only 3 infants (9.7%) within 1-4 weeks after birth and persisted thereafter. The genotype of HCV-RNA in each of the infants was consistent with that of their mother. These 3 showed chronic transaminase elevation during the follow up that started at 1-2 months of age, although they revealed no clinical symptoms. Reelevation of anti-HCV titre was observed in the HCV-infected infants within 10 months of age, suggesting an endogenous production of anti-HCV. The mean titre of HCV-RNA in three mothers of infected infants was higher than that in the mothers of uninfected infants (10(5.3 +/- 0.3) vs 10(4.4 +/- 0.2)/ml). CONCLUSION: Our findings indicate that HCV was most likely to have been transmitted from mothers to infants at the time of delivery and that it was capable of evoking the chronic carrier state.

Crossreactivity of IgE antibody against Dermatophagoides farinae with Limulus polyphemus agglutinin.

Crossreactivity of IgE antibody against Dermatophagoides farinae (Der f) with Limulus polyphemus agglutinin (LPA) was examined using RAST and immunoblot analysis. Of 40 Der f-sensitive asthmatic patients, 28 revealed a positive RAST reaction to LPA, while none of 20 Der f-insensitive hay fever patients showed this reaction. LPA-specific RAST levels of the 40 asthmatic patients correlated with their Der f-specific levels. The RAST reactivity to LPA was competitively inhibited by the addition of either soluble Der f or LPA, but not by the specific inhibitory sugar of sialic acid. LPA could also induce histamine release from leucocytes of Der f-sensitive asthmatic patients. IgE immunoblot analyses showed that the positive RAST sera for LPA had a strong IgE binding activity to the 30 kDa and 80 kDa components of Der f body extract, whereas gel filtration studies showed that the high molecular weight fractions above 150 kDa retained antigenic constituents associated with IgE reactivity to LPA. These results suggest that the antigenic materials of Dermatophagoides mites share some determinants with the haemagglutinin of horseshoe crabs.

Interaction of lectins with human IgE: IgE-binding property and histamine-releasing activity of twelve plant lectins.

We examined the IgE-binding reaction and the histamine-releasing response of basophils to a panel of 12 lectins: concanavalin A (Con A), Lens culinaris hemagglutinin (LcH), Pisum sativum agglutinin (PSA), wheat germ agglutinin (WGA), soybean agglutinin (SBA), Bauhinia purpurea agglutinin (BPA), peanut agglutinin (PNA), Ricinus communis agglutinin I (RCA-I), Lotus tetragonolobus agglutinin (Lotus A), Ulex europeus agglutinin I (UEA-I), phytohemagglutinin E (PHA-E) and phytohemagglutinin L (PHA-L), IgE from allergic patients bound with high affinity to Con A, LcH, PSA, RCA-I and PHA-E, and with lower affinity to WGA, BPA, Lotus A and UEA-I, but they did not bind to SBA, PNA or PHA-L. There was no apparent individual difference in the reactivity of IgE to these lectins between 10 IgE preparations from allergic patients. The binding to these lectins, except Lotus A and UEA-I, were competitively inhibited by the lectin-specific sugars or glycopeptide. Upon stimulation by Con A, LcH, PSA, WGA, RCA-1 and PHA-E, leukocytes from allergic patients showed a significant release of histamine, but cells from IgE-deficient subjects did not respond to these lectins. The histamine-releasing responses by these lectins were also inhibited by specific sugars or glycopeptides.

Relation between frequency of asthma and IgE antibody levels against Dermatophagoides farinae and total serum IgE levels in schoolchildren.

The relation between the frequency of wheezing illness and IgE antibody levels against Dermatophagoides farinae (Df) and total IgE levels was examined in 457 randomly selected schoolchildren. From the response to the ATS-DLD-78-C respiratory symptoms questionnaire, 14 subjects (3.1%) were found to have asthma syndrome (recurrent episodes of attacks of shortness of breath with wheezing) and 17 subjects (3.7%), wheezing syndrome (only wheezing). The percentage of the asthma syndrome increased with increasing levels of Df-specific IgE, and there was an intimate correlation between the percentage of asthma syndrome and Df-specific IgE levels (r = 0.97; p less than 0.001), whereas such association was not found between the two (r = -0.19; p greater than 0.5). Similar relations were found between the frequencies of the specific syndromes and total IgE levels. There was a significant correlation between total IgE levels and Df-specific IgE levels in the total population (r = 0.7; p less than 0.001). These results suggest that allergic reaction greatly contributes to the development of asthma in children.

Monoclonal antibody against bacterial lipopolysaccharide cross-reacts with DNA-histone.

Monoclonal antibodies to bacterial lipopolysaccharide (LPS) were prepared by fusing spleen cells from BALB/c mice immunized with Salmonella Minnesota Re 595 LPS to the mouse myeloma cell line P3U1. One of them, designated RS01, revealed a strong positive antinuclear activity and reacted with DNA-histone. RS01 also bound specifically to Salmonella Minnesota Re 595 LPS and eliminated the biological activity of LPS. The Salmonella completely inhibited the ANA activity of RS01 and DNA-histone blocked the reactivity of RS01 with LPS. Thus, it is clear that an anti-LPS monoclonal antibody, RS01 cross-reacts with DNA-histone.

Premenstrual asthma with seasonal variation.

A 19-year-old woman had premenstrual asthma (PMA) usually from April through October each year with normal and regular menstrual cycles. When the monthly variation in the patient's PMA between 1984 and 1990 was compared with the monthly admissions of children for acute asthma in a hospital in this region, there was a great similarity in pattern between the two. Although she had high sensitivity to house-dust mites, the monthly pattern of her PMA did not coincide with monthly variations in the number of mites in house-dust in her home.

Immune response to food antigens: kinetics of food-specific antibodies in the normal population.

The role of food-specific antibodies in the pathogenesis of food allergy is controversial. The first step in solving this controversy may be the assessment of antibody response to food antigens in the normal population. Most of the existing data in this field come from studies that used assays of different standards. This study investigated food-specific antibodies in the normal population using standardized assays. Normal levels of antibody titers were also derived for use as reference. Two hundred and eight individuals from different age groups participated. Immunoglobulin G (IgG) antibodies to cow's milk and its component proteins, and to hen's egg ovalbumin, IgA and IgM antibodies to beta-lactoglobulin and ovalbumin were measured by enzyme-linked immunosorbent assay. The sepharose-radioallergosorbent test was used to measure IgE antibodies to cow's milk and ovalbumin. Titers of IgG antibodies to cow's milk and its component proteins revealed an age-related trend, peaking in the 5 months-1 year age group and then decreased to negligible values in adults. A similar trend was observed with IgG anti-ovalbumin antibodies. Temporal association was less evident for antibodies of other classes. Only six subjects had positive IgE antibodies to cow's milk, while none had positive IgE anti-ovalbumin antibody. The prevalences of IgG antibodies to cow's milk, its component proteins, and ovalbumin are influenced by age and feeding habits. Cross-reactivity to related food antigens is common. The presence of IgE antibodies to food antigens is not a physiological phenomenon.

Rapidly progressive hepatitis C in a patient with common variable immunodeficiency.

UNLABELLED: A 15-year-old girl with common variable immunodeficiency contracted hepatitis C, which progressed to liver cirrhosis and finally to hepatic failure 5 years later. Since she was agammaglobulinaemic, hepatitis C virus (HCV) infection was diagnosed on the basis of HCV-RNA detection. Quantification of her sera showed high levels of HCV-RNA (more than 10(7) copies of RNA/mL), which implied active viral replication. There were no other hepatotoxic factors except HCV infection. The initial liver biopsy at 16 years of age and the autopsy confirmed a rapid progression in liver histopathological change over 4 years. CONCLUSION: Contrary to the widely held view of a benign short-term prognosis in paediatric hepatitis C, progressive fatal liver disease can develop in some patients with HCV infection. Such a rapid progression of liver injury provides the rationale for antiviral therapy in at least certain high risk groups of these children. Hepatitis C may progress rapidly in an immune deficiency condition.

Genotyping of hepatitis C virus: coinfection by multiple genotypes detected in children with chronic posttransfusion hepatitis C.

The distribution of genotypes of the hepatitis C virus (HCV) was studied in 24 children with chronic posttransfusion hepatitis C. Genotypes were determined by reverse transcription-nested polymerase chain reaction with type-specific primers. Twenty (83%) were infected by a single genotype: 14 by type II [1b], 3 by III [2a], and 3 IV [2b]. Four (17%) were coinfected by two genotypes. The amount of blood transfusion given to the patients infected by multiple genotypes was significantly larger than to those infected by a single genotype (mean +/- SD, 15.2 +/- 14.4 vs 78.6 +/- 42.0 U). Three of the four patients infected by multiple genotypes were considered to be immunocompromised by anticancer therapy for malignant disease. Eighteen patients showed a raised level of alanine aminotransferase throughout the follow-up, while the remaining six patients (three of type II [1b], two of IV [2b], and one of III [2a] + IV [2b]) achieved biochemical remission. Liver biopsy was performed on 19 patients. Compared to those with type III [2a] or IV [2b], those with type II [1b] appeared to show more severe histological changes with higher histological activity index scores, although there was no significant difference. The positive rates of antibody to C100-3 or 5-1-1 in patients with type III [2a] or IV [2b] were lower than in those with type II [1b] (33 and 33 vs 43 and 50%), whereas the antibody to C33C or C22-3 was detected in nearly all patients regardless of their genotypes. In the present study, we found a high incidence of multiple-genotype infection among children with chronic posttransfusion hepatitis C.

Persistence and clinical outcome of hepatitis G virus infection in pediatric bone marrow transplant recipients and children treated for hematological malignancy.

The natural course and the clinical significance of hepatitis G virus (HGV) infection were investigated in 106 pediatric patients who received chemotherapy for hematological malignancy or underwent bone marrow transplantation (BMT) using HGV-RNA and antibodies to the HGV-E2 protein (anti-E2). HGV markers were detected in 21 patients (19.8%; HGV-RNA in 19 and anti-E2 in 2). Longitudinal analysis of these HGV-infected patients showed that 1 had anti-E2 before the initial blood transfusion, 14 had persistent viremia, and 6 became clear of circulating HGV-RNA after completion of therapy, although 5 of the 6 HGV-cleared patients never developed anti-E2. Reactivation of HGV infection during chemotherapy was observed in two anti-E2-positive, HGV-RNA-negative patients; the reappearance of the same HGV strain was confirmed by phylogenetic analysis. Among BMT survivors without other known causes of liver dysfunction, HGV-RNA-positive patients had a higher peak serum alanine amino transferase (ALT) value than negative patients. Contrary to previous reports, immunosuppressed patients can apparently recover from HGV infection without detectable anti-E2 and some patients who supposedly recovered from HGV infection can nonetheless suffer exacerbation when subsequently immunosuppressed.

Natural course of HGV infection in haemophiliacs.

The natural course of hepatitis G virus (HGV) infection was clarified in 70 haemophiliacs by testing for HGV RNA and antibodies against HGV envelope protein (anti-E2). None of 12 patients treated with only virus-inactivated coagulation factors were infected with HGV. Of 58 patients who received non-inactivated products, 28 (48%) were positive for HGV RNA and/or anti-E2. Of 16 patients with anti-E2, 14 were negative for the viral RNA, and had recovered from HGV infections. HCV antibodies were detected in 59 patients, and eight patients were successively negative for HCV RNA. Thus, the recovery rate of HGV infection (14/28, 50%) was higher than that of HCV (8/59, 14%) (P<0.001). Longitudinal study revealed that anti-E2 developed either during viraemia or some years after seronegativity for HGV RNA. Hence the antibody response itself seemed not to play a major role in the clearance of HGV though anti-E2 was associated with the clearance of HGV RNA. In conclusion, HGV and HCV are prevalent in patients treated with unsterilized coagulation factor concentrates. However, in contrast to HCV, spontaneous recovery is frequently observed in HGV infections.

Multicolor flow-cytometric, morphologic, and clonogenic analysis of marrow CD10-positive cells in children with leukemia in remission or nonmalignant diseases.

PURPOSE: To characterize nonleukemic CD10-positive cells in the marrows of children with leukemia in remission or benign conditions. PATIENTS AND METHODS: Seventeen children with acute leukemia in complete remission, 12 with solid tumors, and 17 with benign blood diseases were included in this study. Bone marrow cells were analyzed by multicolor flow-cytometry and polymerase chain reaction (PCR) to detect immunoglobulin gene rearrangement. The CD10-positive cells were purely sorted and examined by light microscopy and single cell hemopoietic progenitor assay. RESULTS: In patients with acute leukemia, CD10-positive cells were present in higher proportion after completion of therapy than during chemotherapy. They were also higher in the patients of preschool age than in the older age group with benign blood diseases and solid tumors. These CD10-positive cells were morphologically compatible with immature lymphocytes but some blast-like cells also occurred in this population. Most CD10-positive cells coexpressed CD19 and HLA-DR, although only 10 to 30% coexpressed CD20 and CD34. Although some CD10-positive cells expressed CD34, they did not make any colonies. PCR analysis did not show monoclonal bands in CD10-positive bone marrow cells in any patients in remission. CONCLUSION: Marrow CD10-positive cells possess immature B-lymphocyte phenotype and are present in higher proportion in the marrows of children with acute leukemia in continuous complete remission after completion of therapy and children of preschool age than school-age children with benign diseases or solid tumors without marrow involvement. The clonality of these cells was excluded by PCR, which is a distinct point from CD10-positive ALL blasts.