Kazuo Okochi

Spontaneous proliferation of peripheral blood lymphocytes increased in patients with HTLV‐I‐associated myelopathy

We found unstimulated (spontaneous) peripheral blood lymphocyte (PBL) proliferation significantly increased in 14 patients with human T-lymphotropic virus (HTLV)-I-associated myelopathy (HAM) compared with findings in HTLV-I seropositive non-HAM carriers (N = 8) or HTLV-I seronegative controls (N = 16). The proliferative response to phytohemagglutinin, concanavalin A, or pokeweed mitogen was decreased in the HAM patients. Cell clusters were frequent in cultures of unstimulated PBL from the HAM patients, but much less common in the controls or carriers. This spontaneous PBL proliferation was depressed when adherent-cell populations were depleted from the cultures. IL-2 activity increased in the supernatant of 3-day cultured cells from HAM patients, but not in cultured cells from the controls. Since IL-2 receptor positive cells increased in HAM, this spontaneous PBL proliferation is probably a response to IL-2 through the expression of IL-2 receptors.

Infection of the erythroid cell line, KU812Ep6 with human parvovirus B19 and its application to titration of B19 infectivity.

A human parvovirus B19 (B19) infectivity assay was developed using the erythroid cell line, KU812Ep6. KU812Ep6 was cloned for high efficiency infection with B19 in vitro, in the presence of erythropoietin by a limiting dilution method from the parent cell line, KU812. B19 was effectively propagated in KU812Ep6 and was detected for B19 antigens, VP1 and VP2. The titers of B19 positive sera measured with KU812Ep6 cells were in the range of 10(6) to 10(8) TCID50 ml. This KU812Ep6 infectivity assay had a 10(3)-10(4.5) higher sensitivity than the colony forming unit-erythroid (CFU-e) injury assay. It was calculated that one TCID50 needed 10(3) B19 genome copies, judging from the infectivity assay and semi-quantitative PCR. The KU812Ep6 infectivity assay was also used to determine infectivity of B19 in vitro, and to evaluate inactivation, as well as clearance of the virus. The inactivation of B19 by heating was carried out and infectivity declined from 10(4) TCID50 ml to < 10 TCID50 ml (lower limit of detection) at 60 degrees C for 3 h or at 70 degrees C for 30 min, but only decreased to 10(2.5) TCID50 ml at 50 degrees C for 8 h.

Altered subsets of peripheral blood lymphocytes in patients with HTLV‐I associated myelopathy (HAM)

We studied subsets of peripheral blood lymphocytes (PBL) from 12 patients with human T-lymphotropic virus type I (HTLV-I) associated myelopathy (HAM). The percent of OKT8+ cells was significantly decreased, and the percentages of OKDR+ cells and IL-2R+ cells and the ratio of OKT4+ cells to OKT8+ cells were significantly increased compared with either HTLV-I seronegative healthy adults (controls) or HTLV-I seropositive non-HAM subjects (carriers). The subsets of PBL from eight carriers showed no differences from those in controls. The patients with HAM showed significantly higher antibody titers to HTLV-I compared with carriers. These altered PBL subsets and high HTLV-I antibody titers in the patients suggest that immune functions are in an activated state in HAM.

Screening of Blood Donors for Human Parvovirus B19 and Characterization of the Results

Background and Objectives: Human parvovirus B19 (B19 virus) can be transmitted through blood transfusion and plasma–derived products. In a previous report, we utilized the simple hemagglutination method based on the interaction between the B19 virus and P antigen on human erythrocytes in order to screen the blood donors. We called this method receptor–mediated hemagglutination (RHA) [Lancet 1995;346:1237–1238]. In this paper, we report on a large–scale screening of the B19 virus by RHA and discuss the results. Materials and Methods: Donor sera from September 1995 to March 1997 and seroconversion panels were enrolled. Donor sera were examined by RHA for large–scale screening. The positive sera in the first screening were then further investigated by the RHA inhibition test, countercurrent immunoelectrophoresis (CIE), an enzyme–linked immunosorbent assay, and polymerase chain reaction (PCR). We also evaluated the infectivity and neutralizing activity of various kinds of sera by the erythroid colony forming unit (CFU–e) assay. To examine the detection limits of the B19 virus by RHA, B19–viremic sera were purified by sucrose gradient ultracentrifugation. Results: Among 257,710 sera specimens, 293 sera (0.11%) gave a positive reaction in the first screening using RHA. Out of these 293 sera specimens, 31 were positive for PCR, of which 28 were also RHA inhibition–positive, and 25 of the 28 CIE–positive. In the CFU–e injury assay, all the RHA inhibition (+) sera showed a decrease in the number of erythroid colonies. The RHA inhibition (–) PCR (+) B19 antibody (+) sera did not affect the erythroid colony formation and protected CFU–e from injury by the B19 virus. By measuring the amount of purified B19 protein and its RHA titer, the detection limit of the B19 virus by RHA was calculated to the 0.37±0.03 ng/ml. Conclusion: These results suggest that the RHA(+) RHA inhibition (+) sera were infectious in vitro. The combination of RHA and the RHA inhibition test is considered to be useful for the large–scale screening of infectious B19 virus in blood donors with high specificity.

Detection of Serum Thermolabile β-2 Macroglycoprotein (Hakata Antigen) by Enzyme-Linked Immunosorbent Assay Using Polysaccharide Produced by Aerococcus viridans

ABSTRACT Although a serum thermolabile β-2 macroglycoprotein (TMG) may play a role in host defense as a lectin, little is known of its related physiological functions, mainly due to a lack of appropriate methods for tracing the functions of TMG. We identified a polysaccharide fromAerococcus viridans, PSA, which reacts with TMG, and based on this finding, we developed an enzyme-linked immunosorbent assay to trace the functions of TMG. Using ethanol precipitation and DEAE-Sepharose and Sephacryl S-400 column chromatographies, we isolated PSA from cultured medium of A. viridans, and it exhibited specific binding against TMG in blood samples. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the isolated PSA showed ladder bands that implied the existence of repeating units composed of d-glucose,N-acetyl-d-glucosamine, d-mannose, and d-xylose, as confirmed by gas chromatography-mass spectrometry. SDS-PAGE and immunochemical analysis, using rabbit anti-TMG antibody, showed that PSA specifically binds solely to intact serum TMG but not to TMG heated at 56°C for 30 min, a condition under which antigenicity is lost. TMG in serum samples bound to PSA in a dose-dependent manner, and this binding was clearly suppressed by addition of PSA. These observations indicate that PSA is a useful adsorbent to TMG and can be used to develop appropriate methods for tracing the functions of TMG.

Serum concentration of Hakata antigen, a member of the ficolins, is linked with inhibition of Aerococcus viridans growth

BACKGROUND Hakata antigen (Hakata) is a novel serum glycoprotein that consists of collagen- and fibrinogen-like domains, similar to ficolin/p35. Our research suggested that serum Hakata may be a target of a polysaccharide (PSA) produced by Aerococcus viridans. METHODS A. viridans was incubated with human plasma and Hakata-depleted plasma to examine Hakata binding and growth inhibition of A. viridans through binding with PSA. RESULTS When A. viridans was mixed with human acid citrate dextrose-one (ACD-A) plasma, it pulled down Hakata complexed with mannose-binding lectin (MBL)-associated serine proteases 1 and 2 (MASP-1 and MASP-2). This complex had the potential for C4 deposition. Serum Hakata circulates as Hakata-MASPs complex in the blood and is proteolytically active. By mixing A. viridans with human plasma, we prepared a Hakata-depleted plasma, deficient in Hakata-MASPs complex. This plasma failed to inhibit A. viridans growth plasma, but does not inhibit Staphylococcus aureus, Yersinia enterocolitica and Escherichia coli. However, a decrease of growth inhibition of A. viridans in Hakata-depleted plasma could be restored by adding a Hakata-MASPs complex preparation in a dose-dependent manner. On the other hand, the Hakata-MASPs complex exhibited strong binding to A. viridans, but not to S. aureus, Y. enterocolitica and E. coli. CONCLUSIONS The serum concentration of Hakata is linked with growth inhibition of A. viridans upon binding of Hakata via PSA.

Human Parvovirus B19 Infection in Blood Donors

Using an immunodiffusion assay, we tested all of the blood units donated at the Fukuoka Red Cross Blood Center from June 1991 to July 1994 for B19 antigen. Over this 3‐year trial period, we detected 16 viremic cases out of approximately 560,000 blood donors. Interestingly, most of the viremic donors (15 out of 16) were detected between February 1992 and January 1993, which coincided with a local erythemia infectiosum epidemic in the Fukuoka area (December 1991 to August 1992). In particular, we detected 4 cases of viremia in March 1992, which was the peak of the erythema infectiosum epidemic. The incidence of B19 viremia in this peak period was approximately 1/4,000. The viremic donors ranged in age from 17 to 45 years, and most (11/16) were between 31 and 39 years old. By ELISA, using virus particles purified from viremic donor plasma as antigen, we analyzed the prevalence of B19‐specific antibody among blood donors. The antibody‐positive rate was approximately 40% in donors 16–30 years old, gradually increased in middle age, and reached a peak of 92% in donors more than 61 years old.

Incidence of antibody to HTLV-I is not increased in Japanese MS patients.

To examine the association between MS and anti-human T-cell lymphotropic virus-I (HTLV-I) antibody, we studied serum and CSF antibody to HTLV-I in 27 Japanese MS patients by an indirect immunofluorescence method sensitive and specific enough to detect carriers of HTLV-I. The antibody was detected in 3 of 27 MS patients (11.1%), in 4 of 48 patients (8.3%) with other neurologic diseases, and in 8.3% of 2,500 healthy blood donors. There was no significant difference in the incidence between the three groups. The titer of the antibody was low in CSF when compared with that in serum in all seropositive MS patients. Fluctuations in the CSF antibody titer were not observed in any of 14 MS patients sampled repeatedly.

Four putative subtypes of human parvovirus B19 based on amino acid polymorphism in the C-terminal region of non-structural protein.

The nucleotide sequence of 10 isolates of human parvovirus B19 (B19) were determined and compared throughout 96.3% of the open reading frames (4145 nucleotides from nt. 509–4653). In the 4145 nucleotides, 122 mutation sites were found, of which 24 were accompanied by amino acid displacement. Furthermore, the polymorphism of the amino acids was seen in about 110 bases near the carboxy terminal of the non‐structural protein, ranging from nt. 2011 to 2123, where four amino acid mutation points were found to exist. Based on the amino acid polymorphism of these four mutation sites in this area, 10 isolates of the B19 parvovirus could be divided into 4 subtypes (subtypes A, B, C, and D). The frequency of isolation of the subtypes depended on the time and location of collection of the B19 viremic blood specimens. J. Med. Virol. 62:60–69, 2000.

Elevated serum antibody titers to Epstein‐Barr virus in HTLV‐I‐associated myelopathy (HAM)

We studied the serum antibody titers to Epstein-Barr virus (EBV) in 11 patients with HTLV-I-associated myelopathy (HAM). HAM patients showed significant increases in IgG antibodies to EBV-capsid antigen (VCA) and -early antigen (EA) and in VCA-specific IgA compared with 25 seronegative subjects. However, there were no significant differences in antibodies to EBV-nuclear antigen (EBNA) or to other viruses between the two groups. In HAM patients, altered antibody responses occurred specifically to EBV-associated antigens, not to other viruses.