Yasuko Sagara

Human T-cell leukemia virus type I (HTLV-1) proviral load and disease progression in asymptomatic HTLV-1 carriers: a nationwide prospective study in Japan

Definitive risk factors for the development of adult T-cell leukemia (ATL) among asymptomatic human T-cell leukemia virus type I (HTLV-1) carriers remain unclear. Recently, HTLV-1 proviral loads have been evaluated as important predictors of ATL, but a few small prospective studies have been conducted. We prospectively evaluated 1218 asymptomatic HTLV-1 carriers (426 males and 792 females) who were enrolled during 2002 to 2008. The proviral load at enrollment was significantly higher in males than females (median, 2.10 vs 1.39 copies/100 peripheral blood mononuclear cells [PBMCs]; P < .001), in those 40 to 49 and 50 to 59 years of age than that of those 40 years of age and younger (P = .02 and .007, respectively), and in those with a family history of ATL than those without the history (median, 2.32 vs 1.33 copies/100 PBMCs; P = .005). During follow-up, 14 participants progressed to overt ATL. Their baseline proviral load was high (range, 4.17-28.58 copies/100 PBMCs). None developed ATL among those with a baseline proviral load lower than approximately 4 copies. Multivariate Cox analyses indicated that not only a higher proviral load, advanced age, family history of ATL, and first opportunity for HTLV-1 testing during treatment for other diseases were independent risk factors for progression of ATL.

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.

Incidence of human T-lymphotropic virus 1 infection in adolescent and adult blood donors in Japan: a nationwide retrospective cohort analysis

BACKGROUND Human T-lymphotropic virus 1 (HTLV-1) infection has an especially high prevalence in Japan. Transmission has been confirmed in infancy through breastfeeding; however, little is known about the epidemiological aspects of new HTLV-1 infections later in life. We aimed to estimate the nationwide annual number of new HTLV-1 infections among adolescents and adults in Japan. METHODS In this retrospective cohort analysis, we assessed new HTLV-1 infections of repeat blood donors aged 16-69 years between Jan 1, 2005, and Dec 31, 2006, in the Japanese Red Cross Blood Centres database. We used results of antibody tests done in repeat blood samples collected until Dec 31, 2011, to assess the number who seroconverted to HTLV-1. We calculated the incidence density by dividing the number of seroconverters by the number of person-years of follow-up, and then extrapolated densities to regional populations to estimate the annual number of new HTLV-1 infections. FINDINGS We included 3 375 821 HTLV-1-seronegative blood donors (2 100 915 men and 1 274 906 women). Within a median follow-up of 4·5 years (IQR 2·3-5·8), 532 people (204 men and 328 women) had seroconverted. The incidence density was significantly higher in women (6·88 per 100 000 person-years; 95% CI 6·17-7·66) than in men (2·29 per 100 000 person-years; 95% CI 1·99-2·62; p<0·0001). The estimated annual number of new HTLV-1 infections was 4190 (95% CI 4064-4318) with 975 (914-1038) infections in men and 3215 (3104-3328) in women. INTERPRETATION New HTLV-1 infections in adolescents and adults are an important public health concern in Japan and preventive strategies are needed to reduce new transmission. FUNDING Ministry of Health, Labour, and Welfare of Japan; Japan Agency for Medical Research and Development.

Phosphatidylglycerol Participates in Syncytium Formation Induced by HTLV Type 1-Bearing Cells

We previously reported that 71-kDa heat shock cognate protein (HSC70) was expressed on the cell surface of human T cell lymphotropic virus type 1 (HTLV-1)-susceptible cells and that HSC70, beta-actin, and a lipid-like component on the target cell membrane participated in syncytium formation by HTLV-1. We have now identified this lipid-like component to be palmitoyl (16:0)-oleoyl (18:1)-phosphatidylglycerol (POPG), using preparative thin-layer chromatographic fractionation and tandem mass spectrometric analysis. In the syncytium formation assay, exogenously added PG inhibited cell-to-cell transmission of HTLV-1 in a dose-dependent manner. Other phospholipids showed less (PE) or no effect (PC, PS, PI, PA, lysoPC, lysoPE, and CL). Binding experiments showed that PG interacted with three synthetic peptides, gp46--111, gp46--197, and gp21--400, which correspond to regions Lys111--Asp138 and Asp197--Leu216 on the gp46 surface glycoprotein, and to region Cys400--Leu429 on the gp21 transmembrane glycoprotein, respectively, as well as with intact gp46 and gp21 proteins of HTLV-1. On the other hand, HSC70 and beta-actin interacted with gp46--197 and gp46, not with gp46--111. However, the eluate from an affinity column coupled with gp46--111 contained not only PG but also HSC70 and beta-actin, despite the lack of direct interaction between gp46--111 and these proteins. In the in vitro binding assay, HSC70 showed interaction with both PG and beta-actin, while there was no evidence of any interaction between PG and beta-actin. These results suggest that HSC70 molecules on target cell surface interact with both PG in lipid bilayers and intracellular beta-actin and that these three cellular components form a receptor complex that plays a critical role in syncytium formation induced by HTLV-1-bearing cells.

Involvement of molecular mimicry between human T-cell leukemia virus type 1 gp46 and osteoprotegerin in induction of hypercalcemia.

Human T‐cell leukemia virus type‐1 (HTLV‐1) causes adult T‐cell leukemia/lymphoma (ATL), frequently associated with hypercalcemia and bone destruction. A positive correlation between the appearance of an antibody recognizing the central region (Asp197 to Leu216) on Gp46, gp46‐197, and the severity of ATL has been demonstrated. In this study, five male Nihon Hakusyoku rabbits were immunized with a synthetic peptide corresponding to the gp46‐197 region to clarify its action and mechanism. Two of the rabbits showed piloerection, anorexia, and somnolence, and died soon after booster administration. The serum calcium level of the dead rabbits was significantly high, compared to those of surviving rabbits. Interestingly, amino acid sequences homologous with gp46‐197 were found in the carboxyl‐terminal half of osteoprotegerin (OPG), an osteoclast inhibitory factor. To confirm the effect of the gp46‐197 region on osteogenesis in vivo, the peptide was intraperitoneally administered to male Sprague‐Dawley rats. The administration of the gp46‐197 peptide resulted in a decrease of bone mineral density (BMD), a significant increase of serum calcium level, and inhibition of normal bone growth in both short‐ and long‐term experiments. In rats, femoral growth inhibition by the gp46‐197 peptide was restored by the coadministration of recombinant human OPG. Improvement by OPG in the adverse effect indicates that the central region of HTLV‐1 Gp46 acts as an antagonist for OPG and leads to hypercalcemia. (Cancer Sci 2009; 100: 490–496)

Novel biomarker of HTLV-1-associated disease: specific appearance of antibody recognizing the receptor-binding site on HTLV-1 envelope protein.

We previously showed that 71‐kDa heat shock cognate protein (HSC70) functions as a cellular receptor for gp46 protein via the gp46–197 region, corresponding to Asp197 to Leu216 of human T‐cell lymphotropic virus type 1 (HTLV‐1), leading to cell‐to‐cell transmission of HTLV‐1. We found that HSC70 protein was contained in goat serum and casein used as blocking agents in the usual ELISA method. Here, it was demonstrated that HSC70 contamination in the blocking agents causes a false‐negative result in the detection of anti‐gp46–197 antibody in serum samples from HTLV‐1‐infected individuals. By using ELISA without the blocking agents, we detected antibodies recognizing the HSC70‐binding site of gp46, and the anti‐gp46–197 antibody specifically appeared in sera from patients with HTLV‐1‐associated diseases. The frequency of serum anti‐gp46–197 antibody‐positive individuals was 98% and 100% among ATLL and HAM/TSP patients, respectively, but only 6% among asymptomatic HTLV‐1‐infected carriers (ACs). The antibody titer in ATLL and HAM/TSP patients was higher than that in ACs (P>0.002 for ATLL; P>0.0001 for HAM/ TSP). These findings suggest that appearance of the anti‐gp46–197 antibody is a predictive marker for the onset of HTLV‐1‐associated disease.

Standardization of Quantitative PCR for Human T-cell Leukemia Virus Type 1 in Japan: A Collaborative Study

ABSTRACT Quantitative PCR (qPCR) analysis of human T-cell leukemia virus type 1 (HTLV-1) was used to assess the amount of HTLV-1 provirus DNA integrated into the genomic DNA of host blood cells. Accumulating evidence indicates that a high proviral load is one of the risk factors for the development of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis. However, interlaboratory variability in qPCR results makes it difficult to assess the differences in reported proviral loads between laboratories. To remedy this situation, we attempted to minimize discrepancies between laboratories through standardization of HTLV-1 qPCR in a collaborative study. TL-Om1 cells that harbor the HTLV-1 provirus were serially diluted with peripheral blood mononuclear cells to prepare a candidate standard. By statistically evaluating the proviral loads of the standard and those determined using in-house qPCR methods at each laboratory, we determined the relative ratios of the measured values in the laboratories to the theoretical values of the TL-Om1 standard. The relative ratios of the laboratories ranged from 0.84 to 4.45. Next, we corrected the proviral loads of the clinical samples from HTLV-1 carriers using the relative ratio. As expected, the overall differences between the laboratories were reduced by half, from 7.4-fold to 3.8-fold on average, after applying the correction. HTLV-1 qPCR can be standardized using TL-Om1 cells as a standard and by determining the relative ratio of the measured to the theoretical standard values in each laboratory.

Analysis of HTLV-1 proviral load (PVL) and antibody detected with various kinds of tests in Japanese blood donors to understand the relationship between PVL and antibody level and to gain insights toward better antibody testing

Adult T‐cell leukemia/lymphoma (ATL) occurs in approximately 5% of individuals infected with human T‐cell leukemia virus type 1 (HTLV‐1). A high proviral load (PVL; more than four copies per 100 peripheral blood mononuclear cells (PBMCs) or 1.6 copies per 100 blood leukocytes) and being male are risk factors for ATL development. Whether anti‐HTLV‐1 antibody level is related to such risk is unknown. Here, PVL and antibody levels were examined using real‐time PCR and other tests in 600 HTLV‐1 positive screened Japanese blood donors to understand the relationship between PVL and antibody level in asymptomatic carriers and to gain insights toward better antibody testing for HTLV‐1 infection. The 430 donors in whom proviral DNA was detected were considered as true positives for HTLV‐1 infection. Among donors aged 40 years or older, more males than females had a PVL corresponding to more than 1.6% infected leukocytes, and an antibody titer below the median (P = 0.0018). In antibody tests using an HTLV‐1 positive cell line or Env antigens there was a large discrepancy in antibody titer among 13 provirus‐positive samples, probably suggesting that antibody‐based screening tests should incorporate multiple HTLV‐1 antigens, such as Gag and Env antigens.

Frequent expression of gp46 in adult T‐cell leukemia/lymphoma: An immunohistochemical study of 40 cases

Adult T‐cell leukemia/lymphoma (ATLL) is a lymphoproliferative disease caused by human T‐cell lymphotropic virus type 1 (HTLV‐1) infection. HTLV‐1 is spread by cell‐to‐cell transmission via the gp46‐197 region, from Asp197 to Leu216, in the envelope protein gp46. A correlation exists between the prevalence and titer of the antibody recognizing the gp46‐197 region (anti‐gp46‐197 antibody) and the severity of ATLL. In the present study, immunohistochemical staining was performed on samples of paraffin embedded lymph nodes of three different histological types of ATLL (anaplastic large cell type, n = 10; pleomorphic type, n = 10; and Hodgkins‐like type, n = 10) from 30 cases and 10 cases of HTLV‐associated lymphadenitis. Of the three ATLL subtypes, gp46 expression was highest in the anaplastic large cell type, followed by the pleomorphic type and Hodgkins‐like type (mean: 53.4%, 34.9% and 16.0%, respectively; P= 0.0003). In HTLV‐1 associated lymphadenitis cases, gp46 positive cells were rarely seen (4.0%). These results suggest that gp46‐197 immunohistochemical staining can be a useful histological indicator for prediction of the aggressiveness of ATLL and prognosis for ATLL patients.