Expression of latent and replicative-infection genes of Epstein-Barr virus in macrophage.

Unlike other herpesviruses, Epstein-Barr virus (EBV) has not yet been shown to infect macrophages. Six macrophage cultures were isolated from normal and affected samples. Nested polymerase chain reaction revealed the existence of the EBV genome in all these macrophages. EBV latent genes expression in all cultures were detected by mRNA in situ hybridization and immunofluorescence staining. Some cultures also expressed EBV replicative-infection proteins, while in other cultures induction of these proteins was demonstrated. These findings are the first to show expression of several latent and replicative-infection genes of EBV in macrophages, indicating that EBV proliferates in macrophages.

Hepatitis C virus infection in children with hemophilia: characterization of antibody response to four different antigens and relationship of antibody response, viremia, and hepatic dysfunction.

We studied hepatitis C virus (HCV) infection in children with hemophilia by characterizing the antibody responses to four different HCV antigens and investigating the relationship of the antibody response to viremia and hepatic dysfunction. Three antigens (core, nonstructural (NS) 3, and NS5) were expressed in Escherichia coli transfected with plasmids that contained fragments of the putative core and of the NS3 and NS5 regions of the HCV genome, respectively. Antibody responses to these three antigens and the commercially available C100 antigen were detected by enzyme-linked immunosorbent assay. In 45 children with hemophilia, the percentage of children with seropositivity for C100, core, NS3, and NS5 protein in one or more specimens was 82%, 91%, 91%, and 89%, respectively. The time course of changes in the antibody response to the four antigens was determined by using sera obtained from 44 of the 45 patients at intervals of 1 to 4 years. Antibodies to the core and NS3 antigens appeared earlier and persisted longer than those to C100 and NS5 after HCV infection. The relationship of antibody response to viremia and hepatic dysfunction was investigated in 27 children by using the polymerase chain reaction assay. Five children whose tests results were negative for all four antigens did not have viremia or hepatic dysfunction; 13 of the 16 children with positive results for the four antigens had both viremia and hepatic dysfunction. Five of the six children whose serum had the core and NS3 antibodies but not either C100 or NS5, or both, had viremia, and three of them also had hepatic dysfunction. These results suggest that detection of antibodies to the core and NS3 antigens is useful for the serologic diagnosis of HCV infection and that both antibodies are more related to viremia than are the antibodies to C100 and NS5. In addition, viremia is strongly associated with hepatic dysfunction.

Molecular basis of impaired pyruvate kinase isozyme conversion in erythroid cells: a single amino acid substitution near the active site and decreased mRNA content of the R-type PK.

Conversion of pyruvate kinase (PK) isozymes from M2- to R-PK has been observed during erythroid cell maturation. To understand this mechanism, we analyzed the PK gene of a R-PK deficient patient, in whose erythrocytes the M2-PK was persistently expressed. A point mutation, 1102 GTC-->TTC was identified in the R-PK cDNA, and it caused a single amino acid substitution from 368Val-->Phe. The residue is very close to the 372nd Gln, the putative binding site of the monovalent cation (K+). The impaired K+ binding would cause the decreased affinity for phosphoenolpyruvate, consequently the variant PK may be extremely unstable. Although the proband's other PK allele did not have any structural change, the R-PK mRNA level in reticulocytes was decreased. These findings suggested that both the structural mutation near the active site and the decreased mRNA level of the R-PK were responsible for the disorder.

[Flow cytometric analysis of platelets in patients with variant thrombasthenia].

Three cases of suspected variant thrombasthenia patients (out of 10 cases of Glanzmann's thrombasthenia), who had significant amounts of platelet GPIIbIIIa, underwent flow cytometry to analyse the binding capacity of monoclonal antibodies against GPIIbIIIa to platelets. The monoclonal antibodies used in this study were as follows: PLT-1 and AP-2 recognizing the IIbIIIa complex; TP 80, P2 and AP-4 recognizing IIb:;AP-5 recognizing IIIa;OP-G2, which binds an epitope near the RGD binding site and 3F11. OP-G2 also recognizes conformational changes of activated platelets by increased binding. Case 1 platelets showed a binding capacity of 28-63% of that of normal platelets for TP80, AP-2, AP-4, and 3F11, but no binding to OP-G2. Case 2 platelets also showed 16-44% binding with TP80, AP-2, AP-4, AP-5, and 3F11, but no binding to OP-G2. These findings indicated the presence of structural abnormalities of the functional site of platelet GPIIbIIIa in cases 1 and 2. Case 3 platelets bound with all monoclonal antibodies normally, but normal increase in the binding of OP-G2 to platelets activated by thrombin or ADP was not seen, indicating a lack of activation of the fibrinogen binding site of platelet GPIIbIIIa.

Effects of biological response modifiers on childhood ALL being in remission after chemotherapy.

Of 125 children with acute lymphoblastic leukemia (ALL), who had been in continuous remission for three years on chemotherapy, 108 patients received biological response modifiers (BRM) such as Bestatin, N-CWS, OK-432 and/or PSK in order to prevent relapse after treatment suspension. From 20 patients who were treated with PSK, 6 relapsed within 13 months. This relapse rate was quite similar to the rate observed with those children who were off therapy (4 relapses in 17 patients within 13 months). In contrast to these 37 patients, only 3 out of 31 patients who received Bestatin (p less than 0.05) and 8 out of 57 patients who received N-CWS or OK-432 relapsed. Based on these findings, BRMs used in the present study seems to be effective to prevent relapse of leukemia among childhood ALL who have electively stopped chemotherapy.

Long-term prognosis and residual abnormalities of idiopathic acquired aplastic anemia in children.

We evaluated the long-term prognosis and quality of cure of idiopathic acquired aplastic anemia in children. Of the 244 patients registered from 1965 to 1985, those registered in 1965-1975 and 1976-1985 had a survival rate of 50.1% and 62.0%. The percentage of cure, undertreatment and death was 30, 30 and 40%, respectively. About 40% of the patients with moderate cases, died dead or required frequent blood transfusions. In the case of pediatric patients, as the success rate of bone marrow transplantation was high. This modality should be considered for patients with moderate severity who require blood transfusion 3 months after the diagnosis and an HLA identical donor is available. Physical development was almost normal but 35% of the patients showed residual abnormalities such as bleeding tendency, and hepatic disorders due to treatment. Thrombocytopenia and ineffective hematopoiesis were observed in one-third of the patients and all of the patients showed abnormal committed stem cell assay. The CD 4/8 ratio was reduced in 50% of the patients and 15% exhibited psychological problems. These residual abnormalities last for years, and sometimes a lifetime.

Long-term follow-up study of children with chronic ITP.

A total of 126 children with chronic idiopathic thrombocytopenic purpura, including 35 splenectomized cases, were investigated in a long-term follow-up study, with regard to residual hematologic and immunologic abnormalities, complications and physical growth. Such hemorrhagic symptoms as petechiae, ecchymosis and epistaxis were still observed in about 22%-28% of the patients with a period of morbidity ranging from 3 to 15 years after onset. Residual thrombocytopenia below 150,000/microliters was found in 62% of patients within 5 years, 59% within 5 to 9 years and 57% within 10-14 years after onset. Other abnormalities were mild anemia, low serum level of IgA or IgM, positive antinuclear antibody, rheumatoid factor, and positive Coombs test in a small number of patients. Increased platelet-associated IgG was still obtained in patients with subnormal platelet counts whose morbid periods were 6 to 27 years after onset. Investigation of the patients by questionnaire revealed such complications as obesity, striae atrophicae, abdominal pain, headache, cataract, Perthes' disease, and cardiac complication in some patients. No apparent disturbances except for obesity were observed in their physical growth.

Characterization of polyethylene glycol-modified L-asparaginase from Escherichia coli and its application to therapy of leukemia.

For the purpose of clinical application to the therapy of human leukemia and lymphosarcoma, L-asparaginase from Escherichia coli was modified with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine (activated PEG2) by an improved method, which involves a purification step of activated PEG2 by gel filtration. The PEG2-modified asparaginase retained approximately 30% (73 IU/mg of protein) of the enzymic activity of the native enzyme, while it had almost wholly lost the immunoreactivity towards anti-asparaginase antibodies. The modified enzyme retained the characteristics of the native enzyme in terms of the pH- and temperature-dependencies of activity and stability, and the Km value for L-asparagine. Administration of the modified enzyme to a dog with spontaneous lymphosarcoma induced complete remission without any toxic side effects. Seven children with multiple relapses of acute leukemia were treated with a regimen of cycles of methotrexate and native asparaginase. Three of these children developed anaphylactic shock. In contrast to the native enzyme, the successive administration of PEG2-modified asparaginase to those three patients was therapeutically effective without causing any allergic reaction.

Testicular histology and function following long-term chemotherapy of acute leukemia in children and outcome of the patients who received testicular biopsy.

Wedge biopsy of the testis was performed in 46 children who had received long-term chemotherapy for acute lymphoblastic leukemia. Occult testicular infiltration was noted in three children (6.5%). Two of three children with biopsy-proven infiltration died of systemic disease in spite of local irradiation and reinduction chemotherapy. Six of 43 children shown to be negative by testicular biopsy relapsed 11 months to 15 years later, and 3 of 6 patients died of systemic disease, but none of the cases developed testicular disease. Chemotherapy-induced gonadal damage was observed in 30 of 46 children, and tubular damage was occasionally still seen 4 years after cessation of treatment. Although gonadal damage usually depends on the cumulative dosage of cyclophosphamide, intact tubular fertility index was found in several children who had received a greater dose of cyclophosphamide intermittently. Induction and maintenance chemotherapy for acute lymphoblastic leukemia had little influence on hormonal function. Testicular biopsy at the time of cessation of chemotherapy seems to be worthwhile for the subsequent strategy of treatment, and long-term surveillance for gonadal damage of long-term survivors will be required.

[Therapeutic effect of cefotaxime in the field of pediatrics].

Cefotaxime (CTX) was administered to 130 children with various bacterial infections of 41 to 400 mg/kg/day for 2 to 21 days. The clinical effect of CTX was very satisfactory in respiratory tract infection, urinary tract infection and meningitis. The overall clinical effect was excellent in 59, good in 39, fair in 17 and failure in 8 with effective rate of 79.7%. During this therapy, side effects were seen in 3 cases, diarrhea in 1 and rash in 2. Abnormal laboratory findings were seen in 5 cases, elevation of GOT in 1, GOT, GPT and A1-P in 1, GOT, GPT and T. Bil. in 1, elevation of BUN, increase of number of basophils and albuminuria in 1 and observation of albuminuria in 1. The above results demonstrate that CTX is a clinically useful antibiotic for the therapy of pediatric infections.

Varicella infection in a children's hospital: prevention by vaccine and an episode of airborne transmission.

Varicella vaccine was used safely and effectively for preventing ward infection with varicella. Ward infection was experienced 34 times in 5 years between 1977 and 1982. During these ward infections, varicella developed in 4 of 142 patients who received vaccine, 21 of 47 patients who did not receive vaccine and 1 of 9 who received transfusion with vaccine-boostered blood. Of the 142 vaccinated patients, the four in whom varicella developed showed symptoms 3 to 10 days after vaccination, indicating that vaccination had been too late. Details of a ward infection with varicella by airborne transmission and its prevention by vaccination are presented.

[The clinical effects of a new antiviral 9-(2-hydroxyethoxymethyl) guanine (aciclovir) against herpes virus infections].

The clinical effects of a new anti-viral 9-(2-hydroxymethoxymethyl) guanine (Aciclovir) against Herpes virus infections have been investigated. The patients had malignant tumours or auto-immune disease complicated by shingles and chicken pox due to Vaicella zoster virus (VZV) (43 cases), Herpes simplex virus (HSV) (10 cases) and 9 cases which were clinically diagnosed as Herpes, though the virus was not confirmed as the causative agent. As a general principle the dosage of Aciclovir was 5 mg/kg, t. i. d. for 5 days by slow intravenous infusion. The clinically effective rate against VZV was 93%, being excellent in 42% and against HSV it was 80%, being excellent in 40% and when the results of the cases of unknown origin were included it was excellent in 40% and the cumulative effective rate was 88%. Concerning the efficacy in reduction of pain, swelling, disappearance of vesicles and new scab formation, the effect was most noticeable after the third day of treatment. Treatment given early in the disease is likely to provide better results. Concerning side effects, one of 62 patients had proteinuria and the other had a drug rash and an abnormal liver function test. It is likely that the combination of treatment and the primary disease had some influence, but the cause/effect relationship to Aciclovir treatment is not clear.

Prevention of varicella in urgent cases by passive transfer of vaccine-induced immunity.

For the purpose of preventing spread of infection to high risk children whose immunities were severely impaired by intensive chemotherapy or for some other reason, when cases of varicella occurred in a children's ward or in a family, healthy adults (mothers and a doctor) were immediately given live varicella vaccine, blood was collected from these adults 5 to 7 days after vaccination and the whole blood or plasma including the buffy coat was transferred in the high risk children. Subsequently the children showed little or no clinical reaction, and follow-up studies by the neutralizing test and skin test with varicella antigen indicated that their inapparent or subclinical varicella infection occurred in them and that their immunity to varicella was lasting. Skin tests with varicella antigen showed that booster reaction occurred in adults with a previous history of varicella as early as 5 to 7 days after vaccination. The cellular immunity thus induced in the donors may have played a role in preventing a clinical reaction in the high risk children. Thus passive transfer of vaccine-induced immunity seems a convenient and effective method for preventing infection in subjects whose immune capacities are severely impaired.