Mitsuaki Kakinuma

Demonstration of human Borna disease virus RNA in human peripheral blood mononuclear cells

BDV naturally infects horses and sheep, and causes sporadic neurological disease. Serological evidence suggests an association of BDV, or a related virus, with specific psychiatric diseases in humans. Here, by using a nested RT‐PCR technique, we demonstrate that human BDV RNA is present in the PBMC of psychiatric patients. In an examination of a total of 60 patients from 5 wards of a hospital in Japan, the detection rate differed within each ward, ranging from 8% to > 50% (37% on the average). Of particular note was the finding that the human derived BDV sequences, which included deleted forms in about 23% of the positive samples, were slightly different from those derived from horse BDV. These results suggest urgent consideration of the measures to be taken to cope with the effects of blood transfusion. In addition, the detection of a high level of BDV in the PBMC of patients will help our understanding of the pathogenesis in the disease.

Prevalence of Borna disease virus RNA in peripheral blood mononuclear cells from blood donors

The presence of Borna disease virus (BDV) in peripheral blood mononuclear cells (PBMC) of 100 blood donors from Sapporo and 72 blood donors from Tokyo was examined using nested reverse transcriptase/polymerase chain reaction amplification with specific-primers for BDV p24. Anti-BDV p24 antibodies in the plasma of the 100 blood donors from Sapporo also were studied by enzyme-linked immunosorbent assay and by Western blot. BDV RNA was detected in 3 (4.2%) of the 72 PBMC samples from Tokyo, and in 5 (5%) of the 100 PBMC samples from Sapporo. In contrast, anti-p24 antibodies were found in only 1 (1%) of the donors from Sapporo. These results suggest that BDV infection in humans may be more widespread than previously thought.

Cells surviving infection by human immunodeficiency virus type 1 : vif or vpu mutants produce non-infectious or markedly less cytopathic viruses

Under conditions in which a clonal cell line (M10) isolated from a human T cell lymphotrophic virus type I-transformed MT-4 cell line was completely killed by infection with wild-type human immunodeficiency virus type 1 (HIV-1), equivalent M10 cells survived infection with HIV-1 vif, vpr or vpu mutant virus after transient cytopathic effects. Several cell clones, which were isolated from the proliferating M10 cells after infection with vif and vpu mutant viruses (M10/vif- and M10/vpu-), had heterogeneous HIV-1 phenotypes in terms of HIV-1 antigen expression, their syncytium forming capacity, reverse transcriptase activity and the infectivity of HIV-1 particles produced. When the replication kinetics of the HIV-1 particles produced were assayed in M10 cells, the clones could be classified into three types, i.e. type I producing non-infectious HIV-1, type II producing infectious HIV-1 with low replicative ability and type III producing infectious HIV-1 with a replicative ability similar to that of wild-type HIV-1. HIV-1 major viral cell proteins and virus particle fractions were almost typical in types II and III but not in type I. Electron microscopic examination of particles released by I, II and III clones revealed rare defective, predominantly defective and essentially normal virions, respectively. Northern and Southern blot analyses revealed no apparent deletion in the proviral DNA and mRNA prepared from these clones, except in the case of type I and II clones isolated from M10/vpu- which contained large deletions in the mRNAs for gag and gag-pol proteins. Thus, M10 cells surviving infection with HIV-1 vif or vpu mutants are heterogeneous, persistently expressing HIV-1 antigens and producing non-infectious or less cytopathic virus.

Genetic Control of Granuloma Response to Oil-Associated BCG Cell Wall Vaccine in Mice

Intravenous injection of mice with BCG cell wall vaccine induces granuloma in the lung. The granuloma induced as measured by lung weight showed marked strain differences; C3H/HeMs bred in this Institute (C3H), C3HeB/FeJ, P/J and A/He were low responders, whereas C57BL/6 and AKR/He were high responded. When C57BL/6 and C3H were compared, the responsiveness appeared to be inherent in each strain of mice providing strong evidence for genetic control in this phenomenon. Breeding experiments suggested that multiple loci, including a MHC‐linked locus, were involved although the role of the MHC‐linked loci was marginal.

Heterogeneity of HLA-G Genes Identified by Polymerase Chain Reaction/Single Strand Conformational Polymorphism (PCR/SSCP)

A genomic HLA‐G clone named 7.0E was isolated from a Japanese placenta. The deduced amino acid sequence of the 7.0E was identical to two HLA‐G genomic clones and two cDNA clones previously described. The DNA sequences of α1 and α2 domains of the HLA‐G gene from 5 cell lines also encoded the same amino acids. However, a 14 bp insertion, ATTTGTTCATGCCT, was present in the 3′ untranslated region of 7.0E compared with the originally described HLA‐G clone (HLA 6.0). Polymerase chain reaction (PCR)/single strand conformational polymorphism (SSCP) analysis of exon 8 allowed the HLA‐G gene to be classified into two alternative types, G6.0 and 7.0 E, those correlated to the absence or the presence of the 14 bp stretch. Each group had minor sequence variant(s), and the alleles of the 7.0E‐type were more heterogeneous than those of the G6.0‐ type. The 14 bp deletion is present only in the G6.0‐type of HLA‐G alleles among HLA class I genes. Thus it was suggested that G6.0 alleles were generated after diversification of the HLA‐G.

Cell cycle dependent gene expressions and activities of protein phosphatases PP1 and PP2A in mouse NIH3T3 fibroblasts

We determined the mRNA levels and the activities in nuclear and non-nuclear fractions of protein phosphatase type 1 (PP1) and type 2A (PP2A) through the cell cycle in synchronized mouse NIH3T3 fibroblasts. The mRNA level for PP1 alpha was gradually elevated in late G1 phase, began to decrease in M phase, and reached the control level with entering into the next G1 phase. The mRNA level for PP2A was rapidly increased in early G1 phase, kept at the high level, and decreased after S phase. In nuclear fractions of cells, spontaneous activities of both PP1 and PP2A were gradually increased until M phase and rapidly decreased with entering the next G1 phase, while in non-nuclear fraction such dramatic alterations were not observed. Potential activities of PP1 in both fractions revealed by Co(2+)-trypsin treatment showed an oscillaion patterns similar to those of the spontaneous activities. These results strongly suggest that cell cycle dependent gene expressions and activities of PP1 and PP2A play roles in DNA synthesis and mitosis during the cell cycle.

Analyses of Ia Restriction Specificity of Helper T cells in H-2 Subregion Compatible Bone Marrow Chimera in Mice

Using irradiation bone marrow chimeras which had partial compatibility in H-2 subregions between donor and recipient mice, we found that H-2I matching was sufficient for the chimeras to generate anti-sheep erythrocyte plaque-forming cell (PFC) responses. In such chimeras, T cells appeared to encounter appropriate partner cells bearing the same Ia antigens as those which they had learned to recognize as self in the recipient micro-environment. Furthermore, the PFC number seen in I-A compatible chimeras was only about half of that seen in I-A, I-E compatible chimeras, suggesting the existence of two independent subpopulations of helper T cells. When incompatibility of donor and recipient mice existed on the left side of the H-2I region, the responses were very weak. However, even in such chimeras, marked responses were observed for both IgM and IgG type PFC following a sufficient period after immunization. This observation appears to indicate the existence of a minor subpopulation of helper T cells which can expand and interact effectively with antigen presenting cells of donor type.

Analyses of H-2 restriction specificity of helper T cells in fully allogeneic bone marrow chimera in mice

Using irradiation bone marrow chimeras to analyze restriction specificity of helper T cells, we found that recipient H-2 type dictated the H-2 type which the T cells recognize as self (adaptive differentiation). T cells from (H-2b----H-2k) chimeras cooperate with non-T cells bearing Iak to generate a vigorous PFC response to sheep erythrocytes (SRBC) in vitro, but not with genetically identical H-2b cells. However, when T cells from the chimeras and H-2b non-T cells were adoptively transferred into irradiated (donor X recipient) F1 mice with SRBC, marked responses were seen in recipient spleens where radio-resistant F1 macrophages might exist and act as antigen presenting cells (APC). From these in vitro and in vivo observations, we considered that in the primary antibody response to a T dependent antigen such as SRBC, only T cell-macrophage (APC) matching is required. In contrast, when T cells from H-2 incompatible chimeras which had been primed with SRBC in vivo were analyzed in vitro, these cells cooperated also with H-2b non-T cells. These findings indicate that there may be two separate stages of T cell differentiation during which the self restriction specificity is acquired: one appears to be responsive to intrathymic influences and is not associated with antigenic stimuli, and the other shows signs of being responsive to post-thymic stimuli and of involving antigenic presentation. Moreover, the latter appears to utilize the influence of donor type macrophages.

Specific elimination of the T lineage cells: Effect of in vitro treatment with anti-Thy 1 serum without complement on the adoptive cell transfer system☆

Thymocytes from C57BL/6(B6) mice treated with anti-Thy 1 antiserum without complement in vitro were transferred to lethally irradiated AKR mice. Five days following transfer, the proportion of Thy 1.2(+) cells recovered from the recipient spleen was significantly lower (7%) than that from the control mice which had received untreated cells (64%). the B6 spleen cells were treated in the same manner and transferred with SRBC (T-dependent antigen) or DNP-Ficoll (T-independent antigen) to irradiated syngeneic recipients. The recipients developed a response to SRBC which was significantly lower than that observed in control mice, but showed the same number of plaque-forming cell (PFC) against TNP-SRBC as the control group of mice which had received untreated B6 spleen cells. These results clearly show that in vitro pretreatment of lymphocytes with anti-Thy 1 serum without complement specifically resulted in elimination or inactivation of the T lineage cells in the host environment. The mechanisms of the elimination are discussed in this study.

STRUCTURE AND EXPRESSION OF THE MOUSE S10 GENE

We previously reported the cloning of a human S10 cDNA which encodes a small GTP-binding protein belonging to the Rab subfamily. Here we describe a mouse S10 cDNA and its genomic structure. Mouse S10 is 92.3% homologous at the nucleotide level and 98.3% identical at the amino acid level compared to human S10. The mouse S10 gene is comprised of two exons and a single intron. Northern blotting of tissue RNAs indicates that the S10 gene is predominantly expressed in brain.