Establishment of a novel human T-cell leukemia virus type 1 infection model using cell-free virus.

The primary mode of infection by human T-cell leukemia virus type 1 (HTLV-1) is cell-to-cell transmission during contact between infected cells and target cells. Cell-free HTLV-1 infections are known to be less efficient than infections with other retroviruses, and transmission of free HTLV-1 is considered not to occur in vivo. However, it has been demonstrated that cell-free HTLV-1 virions can infect primary lymphocytes and dendritic cells in vitro, and that virions embedded in biofilms on cell membranes can contribute to transmission. The establishment of an efficient cell-free HTLV-1 infection model would be a useful tool for analyzing the replication process of HTLV-1 and the clonal expansion of infected cells. We first succeeded in obtaining supernatants with high-titer cell-free HTLV-1 using a highly efficient virus-producing cell line. The HTLV-1 virions retained the structural characteristics of retroviruses. Using this cell-free infection model, we confirmed that a variety of cell lines and primary cultured cells can be infected with HTLV-1 and demonstrated that the provirus was randomly integrated into all chromosomes in the target cells. The provirus-integrated cell lines were HTLV-1-productive. Furthermore, we demonstrated for the first time that cell-free HTLV-1 is infectious in vivo using a humanized mouse model. These results indicate that this cell-free infection model recapitulates the HTLV-1 life cycle, including entry, reverse transcription, integration into the host genome, viral replication, and secondary infection. The new cell-free HTLV-1 infection model is promising as a practical resource for studying HTLV-1 infection.IMPORTANCECo-culture of infected and target cells is frequently used for studying HTLV-1 infection. Although this method efficiently infects HTLV-1, the cell mixture is complex, and it is extremely difficult to distinguish donor infected cells from target cells. In contrast, cell-free HTLV-1 infection models allow for more strict experimental conditions. In this study, we established a novel and efficient cell-free HTLV-1 infection model. Using this model, we successfully evaluated the infectivity titers of cell-free HTLV-1 as proviral loads (copies per 100 cells) in various cell lines, primary cultured cells, and a humanized mouse model. Interestingly, the HTLV-1-associated viral biofilms played an important role in enhancing the infectivity of the cell-free infection model. This cell-free HTLV-1 infection model reproduces the replication cycle of HTLV-1 and provides a simple, powerful, and alternative tool for researching HTLV-1 infection.

Establishment of a novel diagnostic test algorithm for human T-cell leukemia virus type 1 infection with line immunoassay replacement of western blotting: a collaborative study for performance evaluation of diagnostic assays in Japan.

BACKGROUND: The reliable diagnosis of human T-cell leukemia virus type 1 (HTLV-1) infection is important, particularly as it can be vertically transmitted by breast feeding mothers to their infants. However, current diagnosis in Japan requires a confirmatory western blot (WB) test after screening/primary testing for HTLV-1 antibodies, but this test often gives indeterminate results. Thus, this collaborative study evaluated the reliability of diagnostic assays for HTLV-1 infection, including a WB-based one, along with line immunoassay (LIA) as an alternative to WB for confirmatory testing. RESULTS: Using peripheral blood samples from blood donors and pregnant women previously serologically screened and subjected to WB analysis, we analyzed the performances of 10 HTLV-1 antibody assay kits commercially available in Japan. No marked differences in the performances of eight of the screening kits were apparent. However, LIA determined most of the WB-indeterminate samples to be conclusively positive or negative (an 88.0% detection rate). When we also compared the sensitivity to HTLV-1 envelope gp21 with that of other antigens by LIA, the sensitivity to gp21 was the strongest. When we also compared the sensitivity to envelope gp46 by LIA with that of WB, LIA showed stronger sensitivity to gp46 than WB did. These findings indicate that LIA is an alternative confirmatory test to WB analysis without gp21. Therefore, we established a novel diagnostic test algorithm for HTLV-1 infection in Japan, including both the performance of a confirmatory test where LIA replaced WB on primary test-reactive samples and an additional decision based on a standardized nucleic acid detection step (polymerase chain reaction, PCR) on the confirmatory test-indeterminate samples. The final assessment of the clinical usefulness of this algorithm involved performing WB analysis, LIA, and/or PCR in parallel for confirmatory testing of known reactive samples serologically screened at clinical laboratories. Consequently, LIA followed by PCR (LIA/PCR), but neither WB/PCR nor PCR/LIA, was found to be the most reliable diagnostic algorithm. CONCLUSIONS: Because the above results show that our novel algorithm is clinically useful, we propose that it is recommended for solving the aforementioned WB-associated reliability issues and for providing a more rapid and precise diagnosis of HTLV-1 infection.

Natural Course of Human T-Cell Leukemia Virus Type 1 Proviral DNA Levels in Carriers During Pregnancy.

The measurement of human T-cell leukemia virus type 1 (HTLV-1) proviral DNA levels by using polymerase chain reaction has been beneficial for confirming HTLV-1 infection during pregnancy. However, the influence of pregnancy on HTLV-1 infection and proviral DNA levels among pregnant women with HTLV-1 has not been clarified. We prospectively gathered blood samples from 36 pregnant women in whom HTLV-1 carriage was previously diagnosed and sequentially measured their proviral DNA levels. The HTLV-1 proviral DNA levels remained at a plateau during pregnancy but were elevated after delivery. Moreover, flow cytometry and serological analyses revealed that the regulatory T-cell population and soluble interleukin 2 receptor levels were similarly elevated after birth in comparison with those in control pregnant women. This study is the first to provide data on sequential changes in HTLV-1 proviral DNA levels during and after pregnancy. These findings will guide the establishment of a better program to prevent mother-to-child transmission of HTLV-1.

A significant association between rs8067378 at 17q12 and invasive cervical cancer originally identified by a genome-wide association study in Han Chinese is replicated in a Japanese population.

In this study, associations between invasive cervical cancer and four cervical cancer susceptibility loci (rs13117307 at 4q12, rs8067378 at 17q12, and rs4282438 and rs9277952 at 6p21.32) in the Han Chinese population were investigated in a Japanese population. Human leukocyte antigen (HLA)-DPB1 alleles were also investigated for their association with cervical cancer risk in the Japanese population. After receiving written informed consent, 214 unrelated Japanese women with invasive cervical cancer and 288 cancer-free Japanese women were recruited, and DNA samples were obtained (study protocol approved by Institutional Review Board of Nagasaki University). Of the four single-nucleotide polymorphisms, rs8067378 showed a significant association with invasive cervical cancer (P=0.0071). Under a recessive model, the minor allele G of rs8067378 contributed to the risk of invasive cervical cancer (odds ratio=2.92, 95% confidence interval=1.40-6.36; P=0.0021). No association was detected between HLA-DPB1 alleles and cervical cancer risk in the Japanese population. In conclusion, we show for the first time, to the best of our knowledge, that an association between increased risk of invasive cervical cancer and rs8067378 in the Han Chinese population is replicated in a Japanese population. In addition, Japanese women with the GG genotype of rs8067378 are a candidate high-risk group for invasive cervical carcinoma.

Adult T-cell leukemia-lymphoma in a pregnant woman diagnosed as a human T-cell lymphotropic virus type 1 carrier.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia-lymphoma (ATL), which is difficult to cure. In Japan, a nationwide HTLV-1 screening test in pregnant women has been recommended since 2011. A 30-year-old woman was diagnosed as being an HTLV-1 carrier in her previous pregnancy. During the current pregnancy, she had persistent fever and cough. Although she had treatment with antibiotics, peripheral white blood cell count remained high, with an abnormal lymphocyte count. Given that she was an HTLV-1 carrier, she was diagnosed with unfavorable chronic ATL (aggressive ATL) at 12 weeks gestation. After pregnancy termination, her ATL status became favorable chronic ATL (indolent ATL). Therefore, watchful waiting was performed until disease progression. This is the first case report of chronic ATL in early pregnancy, in a woman already diagnosed as an HTLV-1 carrier on screening test.

Circulating chromosome 19 miRNA cluster microRNAs in pregnant women with severe pre-eclampsia.

AIM: To clarify the association between circulating chromosome 19 miRNA cluster (C19MC) microRNAs in maternal plasma and severe pre-eclampsia. METHOD: Maternal blood samples (7 mL) at 27-34 weeks of gestation were obtained from 20 pregnant women with severe pre-eclampsia (sPE group) and from 20 uncomplicated pregnant women (NP group). Twenty cases of severe pre-eclampsia were classified into late onset (sPELO group; n = 14) and early onset (sPEEO group; n = 6). Plasma concentration of C19MC microRNAs (miR-518b, -1323, -516b, -516a-5p, -525-5p, -515-5p, -520 h, -520a-5p, -519d and -526b) was measured on quantitative real-time reverse transcription-polymerase chain reaction. RESULTS: The circulating levels of all 10 C19MC microRNAs in maternal plasma were significantly increased in the sPE group compared with the NP group. Plasma concentration of all 10 C19MC microRNAs tested was significantly increased in the sPEEO group compared with the NP group, while plasma concentration of nine miRNAs, except for miR-519d, was significantly increased in the sPELO group compared with the NP group. Of the 10 C19MC microRNAs measured, plasma concentration of eight miRNAs, except for miR-518b and miR-519d, was significantly increased in the sPEEO group compared with the sPELO group. CONCLUSIONS: Increased levels of C19MC microRNAs in maternal plasma are a characteristic phenomenon of established severe pre-eclampsia, and it has been shown for the first time that the upregulation of C19MC miRNAs occurred as a consequence of, not in advance of, the onset of pre-eclampsia.

Frequency of HTLV-1 seroconversion between pregnancies in Nagasaki, Japan, 2011-2018.

Background: Human T-cell leukemia virus type-1 (HTLV-1) is transmitted vertically from an infected mother to her child via breastfeeding during infancy or horizontally via sexual contact. However, little information is available on the HTLV-1 seroconversion rate in pregnant mothers and the impact of new HTLV-1 infection on mothers and babies during the perinatal period. Methods: From the database of a prefecture-wide antenatal adult T-cell leukemia prevention program in Nagasaki, Japan, we extracted data on 57,323 pregnant women who were screened for anti-HTLV-1 antibody during 2011-2018. Data on the 16,863 subjects whose HTLV-1 proviral load (PVL) was measured more than twice were included in our analyses. Results: In total, 133 (0.79%) pregnant women were HTLV-1-positive during their first pregnancy and nine (0.05%) seroconverted before or during subsequent pregnancies (between pregnancies). The median PVL (per 100 peripheral blood mononuclear cells) was significantly lower in the seroconverted mothers (0.10%) than in the initially seropositive mothers (0.15%). A repeated measures correlation analysis for the individual PVLs of the HTLV-1-positive pregnant women showed that PVL increased with parity number (rrm = 0.25) with no perinatal problems. Conclusion: The HTLV-1 seroconversion rate between pregnancies was 0.05%, and their HTLV-1 PVL increased annually but no perinatal problems were noted.

Mesenchymal stem cell-derived extracellular vesicles as probable triggers of radiation-induced heart disease.

BACKGROUND: Radiation-induced heart disease has been reported, but the underlying mechanisms remain unclear. Mesenchymal stem cells (MSCs), also residing in the heart, are highly susceptible to radiation. We examined the hypothesis that the altered secretion of extracellular vesicles (EVs) from MSCs is the trigger of radiation-induced heart disease. METHODS: By exposing human placental tissue-derived MSCs to 5 Gy γ-rays, we then isolated EVs from the culture medium 48 h later and evaluated the changes in quantity and quality of EVs from MSCs after radiation exposure. The biological effects of EVs from irradiated MSCs on HUVECs and H9c2 cells were also examined. RESULTS: Although the amount and size distribution of EVs did not differ between the nonirradiated and irradiated MSCs, miRNA sequences indicated many upregulated or downregulated miRNAs in irradiated MSCs EVs. In vitro experiments using HUVEC and H9c2 cells showed that irradiated MSC-EVs decreased cell proliferation (P < 0.01), but increased cell apoptosis and DNA damage. Moreover, irradiated MSC-EVs impaired the HUVEC tube formation and induced calcium overload in H9c2 cells. CONCLUSIONS: EVs released from irradiated MSCs show altered miRNA profiles and harmful effects on heart cells, which provides new insight into the mechanism of radiation-related heart disease risks.

HTLV-1 targets human placental trophoblasts in seropositive pregnant women.

Human T cell leukemia virus type 1 (HTLV-1) is mainly transmitted vertically through breast milk. The rate of mother-to-child transmission (MTCT) through formula feeding, although significantly lower than through breastfeeding, is approximately 2.4%-3.6%, suggesting the possibility of alternative transmission routes. MTCT of HTLV-1 might occur through the uterus, birth canal, or placental tissues; the latter is known as transplacental transmission. Here, we found that HTLV-1 proviral DNA was present in the placental villous tissues of the fetuses of nearly half of pregnant carriers and in a small number of cord blood samples. An RNA ISH assay showed that HTLV-1-expressing cells were present in nearly all subjects with HTLV-1-positive placental villous tissues, and their frequency was significantly higher in subjects with HTLV-1-positive cord blood samples. Furthermore, placental villous trophoblasts expressed HTLV-1 receptors and showed increased susceptibility to HTLV-1 infection. In addition, HTLV-1-infected trophoblasts expressed high levels of viral antigens and promoted the de novo infection of target T cells in a humanized mouse model. In summary, during pregnancy of HTLV-1 carriers, HTLV-1 was highly expressed in placental villous tissues, and villous trophoblasts showed high HTLV-1 sensitivity, suggesting that MTCT of HTLV-1 occurs through the placenta.

Corrigendum: Feasibility of placenta-derived mesenchymal stem cells as a tool for studying pregnancy-related disorders.

This corrects the article DOI: 10.1038/srep46220.

Feasibility of placenta-derived mesenchymal stem cells as a tool for studying pregnancy-related disorders.

The cellular and molecular mechanisms responsible for pregnancy-related disorders remain unclear. We investigated the feasibility of using placenta-derived mesenchymal stem cells (MSCs) as a tool to study such pregnancy-related disorders. We isolated and expanded adequate numbers of cells with characteristic features of MSCs from the chorionic plate (CP-MSCs), chorionic villi (CV-MSCs), and decidua basalis (DB-MSCs) of human term placental tissues. All placenta-derived MSCs expressed pregnancy-associated C14MC microRNA (miRNA) (miR-323-3p). Interestingly, the placenta-specific C19MC miRNAs (miR-518b and miR517a) were clearly expressed in CP-MSCs and CV-MSCs of foetal origin, but were barely expressed in DB-MSCs of maternal origin. Furthermore, expression levels of placenta-specific C19MC miRNAs in CV-MSCs remained stable during the ex vivo expansion process and across different pregnancy phases (first trimester versus third trimester). High-efficiency siRNA transfection was confirmed in twice-passaged CV-MSCs with little toxicity, and microarray analysis was used to screen for miR-518b target genes. Placenta-derived MSCs, especially CV-MSCs, are a potential tool for investigating the role of placental miRNAs in pregnancy-related disorders.

Carbon dioxide angiography and arterial embolization could successfully control postpartum uterine hemorrhage for the patient with hypersensitivity to iodine compound.

Transcatheter arterial embolization has become a common management option for intractable postpartum uterine hemorrhage. Iodinated contrast agents are commonly used for embolotherapy but cannot be used in patients with renal dysfunction or iodine allergy. A 37-year-old woman spontaneously delivered a healthy girl but developed severe postpartum uterine hemorrhage, probably due to placenta accrete. Contrast-enhanced computed tomography revealed extravasation of the iodinated contrast agent into the uterine cavity. The patient subsequently developed allergic reaction to this agent. After recovery from the allergic reaction with appropriate treatment, we performed carbon dioxide angiography and found that the extravasation originated from the right uterine artery. Embolization of the right internal iliac artery was performed, and hemostasis was verified. Based on this experience, we suggest carbon dioxide angiography and arterial embolization can be used for treating intractable postpartum hemorrhage in patients with iodinated contrast media allergy or renal dysfunction.

Circulating Levels of Pregnancy-Associated, Placenta-Specific microRNAs in Pregnant Women With Placental Abruption.

The aim of this study was to clarify the association between circulating pregnancy-associated, placenta-specific microRNAs (miRNAs) in maternal plasma and placental abruption. All samples were obtained after receiving written informed consent, and the study protocol was approved by the institutional review board. Maternal blood samples (7 mL) were obtained at 25 to 40 weeks of gestation from 15 cases of placental abruption (placental abruption group) and from 24 cases of uncomplicated pregnancies (uncomplicated pregnancy group). The plasma concentrations of pregnancy-associated, placenta-specific miRNAs (miR-515-3p, -517a, -517c, and -518b) were measured by quantitative real-time reverse transcription-polymerase chain reaction. There were no significant differences in clinical characteristics between the 2 groups. The median concentration of plasma cell-free miR-517c in the placental abruption group was 21 672.2 copies/mL, whereas that in the uncomplicated pregnancy group was 13 452.0 copies/mL (Mann-Whitney U test, P = .047). Receiver operating characteristic curve analysis revealed that plasma cell-free miR-517c levels discriminated placental abruption from uncomplicated pregnancy with an area under the curve of 0.692. When a cutoff negative/positive value of 15 669.6 copies/mL was selected, the sensitivity and specificity were 73.3% and 62.5%, respectively. In addition, the positive and negative predictive values were 55.0% and 78.9%, respectively. Plasma cell-free miR-517a and miR-517c levels in the large abruption (degree of abruption ≥50% of placenta) group were significantly higher than in the small abruption (<50%) group ( P = .03 for both miRNAs). In conclusion, the circulating level of cell-free miR-517c in maternal plasma was increased as a consequence of placental abruption and may be a potential biomedical marker for placental abruption.