Characterization of immunodominant epitopes of gag and pol gene-encoded proteins of human T-cell lymphotropic virus type I.

A series of synthetic peptides derived from the corresponding regions of the gag, pol, and env proteins of human T-cell lymphotropic virus types I (HTLV-I) and II (HTLV-II) were used in an enzyme immunoassay to map the immunodominant epitopes of HTLV. Serum specimens from 79 of 87 (91%) HTLV-I-infected patients reacted with the synthetic peptide Gag-1a (amino acids [a.a.] 102 to 117) derived from the C terminus of the p19gag protein of HTLV-I. Minimal cross-reactivity (11%) was observed with serum specimens from HTLV-II-infected patients. Peptide Pol-3, encoded by the pol region of HTLV-I (a.a. 487 to 502), reacted with serum specimens from both HTLV-I- and HTLV-II-infected patients (94 and 86%, respectively). The antibody levels to Pol-3 were significantly higher (P less than 0.01) in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis than in either adult T-cell leukemia patients or HTLV-I-positive asymptomatic carriers. None of the other peptides studied demonstrated significant binding to serum specimens obtained from HTLV-I- or HTLV-II-infected individuals. While Gag-1a did not react with serum specimens from normal controls, Pol-3 demonstrated some reaction with specimens from seronegative individuals (11.4%). The antibodies to Gag-1a and Pol-3 in serum specimens from HTLV-I-infected patients could be specifically inhibited by the corresponding synthetic peptides and by a crude HTLV-I antigen preparation, indicating that these peptides mimic native epitopes present in HTLV-I proteins that are recognized by serum antibodies from HTLV-I- and -II-infected individuals.     

Sequence rearrangement in JC virus DNAs molecularly cloned from immunosuppressed renal transplant patients.

From nonimmunocompromised individuals, we have recently identified a possible archetypal JC virus DNA sequence from which various regulatory sequences of JC virus isolates derived from patients with progressive multifocal leukoencephalopathy (PML) could have evolved. In this study, we analyzed the regulatory sequences of JCV DNAs cloned from urine samples of a PML risk group (renal transplant patients on immunosuppressive therapy). A number of JC virus DNAs were molecularly cloned from virions excreted in the urine of eight patients. Furthermore, fragments containing the regulatory region were amplified by the polymerase chain reaction and subsequently molecularly cloned from cell-associated JC virus excreted in the urine of two patients. The regulatory regions in all clones were analyzed with restriction enzymes, and those in representative clones were sequenced. We found that clones with the archetypal regulatory sequence were predominant in all urine samples, but a few clones carried regulatory sequences that diverged from the archetypal sequence by deletion or duplication. The finding that sequence rearrangement in the archetypal regulatory region occurs in the course of infection in immunosuppressed hosts is consistent with the adaptation hypothesis which has been put forward to explain the divergence of the regulatory regions in PML-derived JC virus isolates.     

Sensitive detection of viral antigens with a new method, "laser magnet immunoassay".

A new method, "laser magnet immunoassay" (LMIA), has been developed for sensitive detection of viral antigens. Target viruses captured on microbeads were made to react with antibodies labeled with magnetite particles. In a magnetic field, magnetically labeled antigens dispersed in water were attracted to and concentrated at one point on the surface, resulting in the lifting up of a small surface area. A laser beam which was incident on the point reflected, making an interference fringe. The intensity of the fringe indicates the amount of the magnetite conjugated with antigen. A very low concentration of antigens, such as 5 particles of influenza virus and 0.1 pg/ml of human immunodeficiency virus (HIV) p24 antigen in human serum, could be detected by this method. Application of this method to diagnoses of viral diseases in early stages is discussed.     

Three Japanese families with members carrying C7 silent allele (C7*Q0). Possibility for an association between C7*Q0 and C6*B

Three Japanese families with members carrying C7 silent allele(s) (C7*Q0) are presented. C6 types in the family members were also examined, and it was found that C7*Q0 was transmitted from a parent to offsprings as a haplotype, C7*Q0-C6*B. In another study of C6 types in sera from 3 volunteer blood donors with homozygous C7 deficiencies, the C6 phenotypes were found to be C6 B (homozygote). It seems remarkable that C7*Q0 can be associated with C6*B.     

Fast and easy detection of mouse sex chimeras using electrophoretic polymorphism of phosphoglycerate kinase-1, an X chromosome-linked enzyme

About half of the chimeras produced by aggregation of two mouse embryos are sex chimeras composed of both XX and XY cells. We developed a fast and easy method to identify sex chimeras by using electrophoretic bimorphism of an X-linked enzyme, phosphoglycerate kinase-1 (PGK-1), as a marker. When embryos resulting from the crossing of a Pgk-1b/Pgk-1b female and a Pgk-1a/Y male are aggregated, the genotype of sex chimeras is Pgk-1b/Pgk-1a----Pgk-1b/Y. Most of these were identifiable from the PGK-1 electrophoretic pattern of blood cells (i.e., AB type) and the appearance of genitalia (male type or apparently abnormal). Genotypes of functional sperm in the testes of the male-type sex chimeras were also identifiable from the PGK-1 electrophoretic pattern of progenies. Examination of gonads of the sex chimeras revealed that a considerable proportion was hermaphorditic. With this method, reasonable numbers of male-type sex chimeras and hermaphrodites may be selected and used as material for investigating sexual differentiation.     

Clonal nature of murine hemopoietic blast cell colonies

Murine hemopoietic blast cell colonies obtained from spleen cells of 5-fluorouracil (5-FU)-treated mice give rise to many multilineage colonies including granulocyte - erythrocyte - macrophage - megakaryocyte (GEMM) colonies in secondary cultures. Progenitor cells for blast cell colonies are considered to be more primitive than colony forming units (CFU)-GEMM. To determine whether they are clonal, we examined the phosphoglycerate kinase-1 (PGK-1) isozyme type of colonies originally grown from spleen cells of 5-FU-treated mice which had PGK-1 isozyme mosaicism. PGK assays of whole secondary colonies derived from one blast cell colony showed that they were either of type A or type B but not both. These results suggest that murine hemopoietic blast cell colonies are clonal.     

Proliferative response of seminal vesicle cells to androgen in mice castrated neonatally and pretreated with estrogen or androgen at adulthood

Seminal vesicle cells of neonatally castrated adult mice show poor response to androgen, compared to those of mice castrated at adulthood; effects of pretreatment with androgen or estrogen at adulthood on androgen-induced proliferation of the seminal vesicle cells were examined in neonatally castrated mice. Male mice castrated at day 0 after birth were pretreated with daily injections of testosterone propionate (TP, 100 micrograms/mouse), 17 beta-estradiol (E2, 5 micrograms/mouse) or vehicle for 20 days starting from day 60; daily TP injections (100 micrograms/mouse) for 30 days were started again from day 110 in all the pretreated mice to examine androgen-induced proliferation by incorporation of 5-[125I]iodo-2'-deoxyuridine into the whole seminal vesicles. Both TP and E2 pretreatments significantly increased the seminal vesicle weight found before TP treatment. However, androgen-induced proliferation of the seminal vesicle found in neonatally castrated mice (poor response; long duration with a low peak on day 3) was changed at least in part to that found in mice castrated at adulthood (good response; short duration with a high peak on day 3) only following the TP pretreatment but not at all following the E2 pretreatment. The E2 pretreatment induced poor androgen-induced proliferation with a low peak on day 7.     

N-hydroxy-2-acetylaminofluorene as effective inhibitor of aldehyde oxidase

N-Hydroxy-2-acetylaminofluorene has been found to be an effective inhibitor of aldehyde oxidase. At concentrations of 1 X 10(-6) M and 1 X 10(-5) M, 38% and 88% inhibition was observed on the oxidase activity towards N1-methylnicotinamide. The inhibition was of noncompetitive type and had a Ki value of 4.4 X 10(-6) M. In contrast, little inhibition of the enzyme was observed with 2-aminofluorene, 2-acetylaminofluorene and acetohydroxamic acid even at a concentration of 1 X 10(-4) M.     

Synergistic antitumor effect of interferon and anti-idiotype monoclonal antibody in murine lymphoma

Both IFN-alpha and anti-idiotype monoclonal antibody therapy have significant antitumor activity in vivo in a murine B cell lymphoma model. Combination therapy with syngeneic anti-idiotype antibody of the IgG2a or IgG2b isotype (a single i.p. injection of 100 micrograms) and recombinant human hybrid interferon-alpha A/D (10(4) to 10(6) U three times weekly for 3 wk) synergistically increased median survival time in mice challenged with a lethal dose of tumor cells compared with the sum of the median survival times of the two individual treatments. IFN-alpha has direct antiproliferative activity against 38C13 in vitro and enhances in vitro macrophage anti-idiotype antibody-specific cytolysis for IgG2a, IgG2b, and IgG1 isotypes.