高橋 清

A simian cell line, Si-IIA, harboring Epstein-Barr-virus (EBV) -related herpesvirus (Si-IIA-EBV), produces malignant lymphoma in rabbits when administered by intravenous inoculation. In this study, we analyzed the Si-IIA-EBV genome and compared it with human EBV and herpesvirus macaca fascicularis 1 (HVMF 1 ), which is associated with B-cell lymphoma developing in SIV-infected immunosuppressed monkeys. DNA from Si-IIA-EBV was amplified by the polymerase chain reaction using three different primer pairs complementary to human EBV (B95-8) DNA; two of the primer pairs covered part of the long internal repeat 1 region (IR 1) and the third covered part of the BRRF 1 region. Direct sequencing of the three PCR products revealed that Si-IIA-EBV DNA had about 82% nucleotide homology to the human EBV DNA in all three regions and 92.4% homology to HVMF1 in the IR1 region. The blotting pattern by Southern blot analysis was different between Si-IIA-EBV and human EBV.

<P>Reed-Sternberg cells (RS cells) of Hodgkin's disease (HD) are frequently infected with Epstein-Barr virus (EBV) and express EBV-encoded nonpolyadenylated RNA transcripts (EBER)-1. EBV latency has been classified into three distinct forms: Latency I, expressing only one of the latent proteins, EBV nuclear antigen (EBNA)-1, latency II, coexpressing EBNA-1 and LMPs, and latency III, expressing all latent viral proteins. RS cells express LMP-1 in addition to EBNA-1 and are considered to be EBV latency II frequently encountered in nasopharyngeal carcinoma. We examined 13 cases of EBV-infected HD by combined EBER-1 in situ hybridization and immunostaining for LMP-1. All of the RS cells expressed EBER-1, but a substantial number of EBER-1+ RS, cells were negative for LMP-1. The percentage of LMP-1+ RS cells out of EBER-1+ RS cells varied from 7% to 100% (average 69%). In this study, we showed that all EBV-infected RS cells were not restricted to latency II, and some belonged to latency I.</P>

Circulating hepatitis C virus (HCV) particles can be fractionated by means of differential flotation centrifugation. It is reported that in the bottom fraction HCV is in the form immune complexes, whereas in the top, it is free of antibodies. We evaluated the significance of circulating complex and free HCV in chronic hepatitis C, and assessed the relationship in terms of the response to interferon (IFN) therapy. We examined sera before, just after, and 1 year after administering IFN to 18 patients with chronic hepatitis C, 10 of whom responded (group CR), and 8 did not (group NR). The amounts of virus were similar between both groups before therapy. After differential flotation centrifugation with 1.063 g/ml of NaCl, the top and bottom fractions were assayed for HCV RNA. Before therapy, HCV RNA was detected in the top fraction in 1 of 10 in group CR, and in 6 of 8 in group NR (P < 0.05, chi-square test). HCV RNA was positive in the bottom fraction of all samples. In a follow-up study of group NR, HCV RNA was detected in the top fraction in 3 of 8 just after IFN therapy, and in 7 of 8 after 1 year. This study suggests that the presence of HCV in the top fraction can predict a poor response to IFN therapy.

To elucidate the latent state and reactivation of Epstein-Barr virus (EBV) in non-neoplastic lymphoid lesions, we investigated 144 non-neoplastic lymphoid lesions by in situ hybridization (ISH) to detect the expression of EBV-encoded small RNAs (EBER)-1 and BCRF-1 and by immunostaining for latent membrane protein (LMP)-1 and ZEBRA. ISH for EBER-1 detected EBER-1-positive cells (EPC) in 31 of the 144 examined lesions (22%). EPC were detected in 4 of 49 cases of nonspecific lymphoid hyperplasia, in 16 of 20 abscess-forming granulomatous lymphadenitis (AFGL), 5 of 25 Kikuchi's disease, and in 3 of 3 infectious mononucleosis. LMP-1 was expressed in 6 of 124 non-neoplastic lymphoid lesions (4.8%). LMP-1-positive cells were observed in 6 of the 31 EBER-1-positive cases (19%). EPC were detected significantly more frequently in LMP-1- and ZEBRA-positive specimens than in the LMP-1- and ZEBRA-negative specimens. BCRF-1 was expressed in 4 of 11 cases examined: 2 of 3 AFGL, 1 of 2 Kikuchi's disease, and in the 1 case of atypical lymphoid hyperplasia. This study suggests that Epstein-Barr virus is prevalent and can be reactivated in the lymph nodes effaced by destructive inflammation, such as AFGL. Such inflammation may provide a local milieu that is conducive for EBV to enter the lytic cycle.

The sensitivity and specificity of single cell polymerase chain reaction (PCR) were studied. Its high sensitivity enabled detection of a single-copy gene, such as human T-lymphotropic virus type I genome in paraffin sections. The rate of obtaining positive signals with this method was affected by the number of copies of the gene in the target cell. Specificity was satisfactory if the procedure was properly and carefully followed. Since the single cell PCR is a time-consuming method which requires skill and experience to pick up the target cells accurately, the applicability of this method is limited. It works best when it is used to analyze a single or a few copy genes in histologically identified cells.

Previously, we reported that interleukin-2 (IL-2)-stimulated helper T cells produced an unknown soluble factor which induced dendritic cell-like differentiation in primary cultures of monocytic leukemia cells and we referred to this factor as dendritic cell differentiation factor (DCDF). In this study, we attempted to purify and characterize DCDF and investigated its biological effect on normal human monocytes. Gel filtration chromatography indicated that the molecular weight of DCDF is approximately 30-35 kDa. Chromatofocusing indicated that the isoelectric point of DCDF is approximately 5.0. DCDF, partially purified by subsequent gel filtration, chromatofocusing, and hydrophobic chromatography, significantly enhanced the HLA-DR expression of normal human monocytes and a human monocytic leukemia cell line, THP-1. This biological activity was not neutralized by any known antibodies to human cytokines. DCDF significantly amplified the T-cell stimulatory activity of monocytes in the allogeneic mixed leukocyte reaction (MLR). Moreover, DCDF significantly enhanced IL-1 beta and IL-6 production by monocytes in a dose-dependent manner. These results suggest that DCDF is a novel human cytokine which stimulates the accessory cell function of monocytes.

To confirm the formation of Charcot-Leyden crystals (CLC) in basophils, we observed basophils in sputum and peripheral blood. Sealed slide and suspension culture methods were used to observe the process of CLC formation in peripheral blood basophils and eosinophils under electron microscopy. CLC formation was observed in basophils and eosinophils, and was found to be augmented by sealed slide method. A temperature of 4 degrees C was better than 37 degrees C for promoting the formation of crystals. There was no correlation between the degranulation of these cells and the formation of CLC after stimulation with anti-IgE or anti-IgG antibodies. CLC were initially detected in the cytoplasmic granules of basophils where they continued to enlarge. No CLC were identified in mast cells under any conditions studied. These findings confirm that CLC in sputum are not exclusive to eosinophils and that CLC appear to be present in basophil-rich sites under the cell damage.

Southern blot hybridization was used to detect the rearrangement and amplification of five proto-oncogenes (bcl-2, bcl-1, c-myc, c-myb and c-Ha-ras) and one tumor suppressor gene (RB-1) in 55 Japanese patients with non-Hodgkin's lymphoma; 16 with T-cell lymphomas and 39 with B-cell lymphomas (7 follicular and 32 diffuse lymphomas). Genetic abnormalities of the proto-oncogenes were detected in 7 of the 55 (13%). Genetic abnormalities of bcl-2 plus other genes were detected in 5 of 7 cases of follicular lymphoma (71%), rearrangements of bcl-2 and c-myc, rearrangement of bcl-2 and amplification of c-myb. Genetic abnormalities were observed in only three cases of diffuse lymphoma. In each of 3 cases of B-cell lymphoma, one of the genes, blc-2 mbr, bcl-2 mcr and c-myc, was rearranged respectively. The incidence of genetic abnormalities in diffuse lymphomas (6.3%) was lower than that in follicular lymphomas. None of diffuse lymphomas had double oncogene abnormality. No abnormalities were found in RB-1, bcl-1, and Ha-ras. These findings suggest that follicular lymphomas are associated with some abnormalities of oncogenes not restricted to bcl-2 that facilitate growth which may be associated with their clinical features.

Serum levels of total IgE, specific IgE, IgG and IgG4 against house dust mite were measured in mite-sensitive asthma patients receiving immunotherapy with house dust. Serum levels of total IgE, mite specific IgE and IgG did not significantly change during the course of hyposensitization. Increased levels of mite specific IgG4 were observed in patients during immunotherapy. The increase in specific IgG4 was dependent on the total dose of house dust administered in both children (r = 0.636, p less than 0.001) and adults (r = 0.629, p less than 0.01). However, the increase of specific IgG4 in adults was not as apparent as in children. These results might suggest that mite specific IgG4 is a useful immunological marker in the immunotherapy for allergic asthma, and that IgG4 antibody acts as a blocking antibody in atopic bronchial asthma.

We have attempted to clarify the characteristics of monoclonal antibodies (MAbs) detecting lymphocyte subsets in fixed materials. We examined by means of flow cytometric technique influences of fixatives and reactivity with malignant lymphomas (MLs). Specific markers for T-cells were UCHL1 and OPD4, which reacted especially with helper/inducer T-cells. MT1 recognized almost all of T-cells from peripheral blood and tonsils, but reacted with a part of B-MLs. As for B-cell markers, L26 was the most reliable marker for B-MLs. L26 and MB1 antigens could not be detected on living cells flow cytometrically. LN1 reacted with a part of T-cells as well as B-cells, but fluorescent intensity of the former was apparently stronger than that of the latter. Although LN2 antigen was located mainly in the cytoplasm close to the nuclear membrane immunohistochemically, it could be detected on living cells flow cytometrically. LN2 positive cells belonged to B-cells in peripheral blood and tonsils. When fixed for relatively short time, B5 and buffered formalin were better for examining MAbs than non-buffered formalin and ethanol.