Search

The antibody titer is known to wane within months after receiving two doses of the Pfizer-BioNTech BNT162b2 mRNA SARS-CoV-2 vaccine. However, knowledge of the cellular immune response dynamics following vaccination is limited. This study to aimed to determine antibody and cellular immune responses following vaccination, and the incidence and determinants of breakthrough infection.This prospective cohort study a 6-month follow-up period was conducted among Japanese healthcare workers. All participants received two doses of BNT162b2 vaccine. Anti-SARS-CoV-2 antibody titers and T-cell immune responses were measured in serum samples collected at several timepoints before and after vaccination.A total of 608 participants were included in the analysis. Antibody titers were elevated 3 weeks after vaccination and waned over the remainder of the study period. T-cell immune responses showed similar dynamics. Six participants without predisposing medical conditions seroconverted from negative to positive on the IgG assay for nucleocapsid proteins, indicating breakthrough SARS-CoV-2 infection. Five of the six breakthrough infections were asymptomatic.Both humoral and cellular immunity waned within 6 months after BNT162b2 vaccination. The incidence of asymptomatic breakthrough infection within 6 months after vaccination was approximately one percent.UMIN000043340.

Background SARS-CoV-2 vaccination has started worldwide, including Japan. Although high rates of vaccine response and adverse reactions of BNT162b2 vaccine have been reported, knowledge about the relationship between sex differences and antibody response is limited. Furthermore, it is uncertain whether adverse reactions are associated with the vaccine response. Methods This prospective observational study included 673 Japanese participants working in a medical school and its affiliated hospital in Tokyo, Japan (UMIN000043340). Serum samples were collected before the first dose and three weeks after the second dose of BNT162b2 vaccine, and antibody titers against the receptor-binding domain of the spike protein of SARS-CoV-2 were measured. Answers to questionnaires about background characteristics and adverse reactions were obtained at the time of sample collection, and the relationship between antibody titers was analyzed. Results After excluding participants who did not complete receiving two doses of vaccination or two series of serum sample collection, 646 participants were analyzed. Although all participants became sero-positive after vaccination, antibody titers were highly variable among individuals (260.9–57,399.7A U/mL), with a median titer of 13478.0AU/mL. Mean titer was higher in females than in males and higher in young (≤45 years old) participants than in aged (>45 years old) participants. Participants who experienced adverse reactions demonstrated a higher antibody titer after vaccination than those without adverse reactions. Multivariable analysis demonstrated that young age, female sex, and adverse reactions after the second dose were independently related to higher antibody titers after the second dose. Discussion A favorable antibody response was observed after two doses of BNT162b2 vaccination among mostly healthy Japanese participants, especially among female and young participants. Although further investigation is essential, our results imply that the systemic adverse reactions (i.e., fever and general fatigue) are associated with a higher antibody response that indicates the acquisition of humoral immunity.

Memory T cell responses have been analyzed only in small cohorts of COVID-19 vaccines. Herein, we aimed to assess anti-SARS-CoV-2 cellular immunity in a large cohort using QuantiFERON assays, which are IFN-γ release assays (IGRAs) based on short-term whole blood culture. The study included 571 individuals receiving the viral spike (S) protein-expressing BNT162b2 mRNA vaccine. QuantiFERON assays revealed antigen-specific IFN-γ production in most individuals 8 weeks after the second dose. Simultaneous flow cytometric assays to detect T cells expressing activation-induced markers (AIMs) performed for 28 randomly selected individuals provided data correlating with the QuantiFERON data. Simultaneous IFN-γ enzyme-linked immunospot and AIM assays for another subset of 31 individuals, based on short-term peripheral blood mononuclear cell culture, also indicated a correlation between IFN-γ production and AIM positivity. These observations indicated the acquisition of T cell memory responses and supported the usability of IGRAs to assess cellular immunity. The QuantiFERON results were weakly correlated with serum IgG titers against the receptor-binding domain of the S protein and were associated with pre-vaccination infection and adverse reactions after the second dose. The present study revealed cellular immunity after COVID-19 vaccination, providing insights into the effects and adverse reactions of vaccination.

While the prognosis and the quality of life of hemophilia A patients have been improved greatly, liver diseases due to hepatitis virus infection from blood products previously administered, which require liver transplantation, are increasing. How to optimize perioperative factor VIII (FVIII) replacement through hemostatic monitoring is critically important in hemophilia A patients undergoing liver transplantation. Clot waveform analysis (CWA) extends the interpretation of measurement curves for activated partial thromboplastin time (APTT) or prothrombin time to provide global information about coagulation abnormalities and disorders such as coagulation factor deficiencies. We performed CWA to monitor perioperative hemostasis in a hemophilia A patient on emicizumab undergoing liver transplantation using a brain-dead donor for liver cirrhosis due to hepatitis virus C infection. To gain accurate results of APTT-CWA as well as of APTT and FVIII activity measurement, plasma samples were treated with anti-idiotype monoclonal antibodies against emicizumab to cancel its interference with the coagulation assays due to binding human activated FIX (FIXa) and FX to functionally mimic FVIIIa. Kinetics of the maximum coagulation velocity and acceleration roughly mimicked that of the FVIII activity. These CWA parameters better correlated with FVIII activity than APTT. The plateaus of them were observed at FVIII activity of 100% or more, likely supporting the protocol for perioperative FVIII replacement. Thus, CWA may measure coagulation potential in hemophilia A patients undergoing liver transplantation, aiding in optimizing perioperative FVIII replacement for proper hemostatic management.

Persistence of leukemic stem cells (LSCs) results in the recurrence of chronic myeloid leukemia (CML) after the administration of tyrosine kinase inhibitors (TKIs). Thus, the detection of minimal residual disease (MRD) with LSC potential can improve prognosis. Here, we analyzed 115 CML patients and found that CD25 was preferentially expressed on the phenotypic stem and progenitor cells (SPCs), and TKI therapy decreased the number of CD25-positive cells in the SPC fraction. To detect MRD harboring BCR-ABL1 fusion DNA, we developed a highly-sensitive method using patient-specific primers and next-generation sequencing. By using this method, we identified that in patients who achieved molecular remission, almost all residual CD25-positive SPCs were BCR-ABL1-negative. Moreover, in some patients BCR-ABL1 was detectable in peripheral B cells but not in SPCs. We conclude that CD25 marks LSCs at diagnosis but does not mark MRD following TKI treatment and that analysis of peripheral B cells can allow sensitive detection of MRD.

Assessing the percentage of reticulocytes (%Retic) is useful for diagnosing and treating blood diseases that present with anaemia. The Celltac G+™ hematology analyzer (HA) uses a novel reticulocyte identification method that involves metachromatic nucleic acid staining with acridine orange and crossover analysis of emission light of DNA/RNA (determination of red cells, nucleic acid-containing cells, and platelets, RNP Determination™). The red and green fluorescence generated by stained single-stranded RNA and double-stranded DNA express immaturity and morphological abnormality of erythrocytes by detecting erythrocyte RNA and DNA content.The basic performance of the test automated analyzer (TAA) Celltac G+ was evaluated and compared with the flow cytometry reference method and the comparative automated analyzer (CAA) XN-1000/2000™. In addition, its precision, limit of quantity (LoQ), linearity, analytical measurement interval (AMI), accuracy, and comparability and the effects of interfering substances were evaluated.Evaluation of %Retic by the TAA demonstrated good precision and linearity. The AMI was confirmed from 0.02 to 8.23, and the LoQ in %Retic as the coefficient of variation within an 11% limit (SD, within a 0.01 limit) was 0.14. TAA correlated well with the reference method and routine HA (CAA). Some deviations were found between TAA and CAA in DNA measurements of erythrocytes from abnormal samples.Celltac G+ uses a novel measurement principle and can assess erythrocyte immaturity independent of DNA contents. It represents a new HA that provides novel, useful information on immaturity and morphological abnormality of erythrocytes.

Generation of antigen-specific memory T cells has been analyzed only for few coronavirus disease 2019 (COVID-19) vaccinees, whereas antibody titers have been serologically measured for a large number of individuals. Here, we assessed the anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular immune response in a large cohort using interferon (IFN)-γ release assays (IGRAs) based on short-term whole blood culture. The study included 571 individuals who received the viral spike (S) protein-expressing BNT162b2 mRNA SARS-CoV-2 vaccine. Serum IgG titers against the receptor-binding domain (RBD) of S protein were measured. Samples of 28 vaccinees were subjected to flow cytometry analysis of T cells derived from short-term whole blood culture. IFN-γ production triggered by S antigens was observed in most individuals 8 weeks after receiving the second dose of the vaccine, indicating acquisition of T cell memory responses. The frequencies of activated T cell subsets were strongly correlated with IFN-γ levels, supporting the usability of our approach. S antigen-stimulated IFN-γ levels were weakly correlated with anti-RBD IgG titers and associated with pre-vaccination infection and adverse reactions after the second dose. Our approach revealed cellular immunity acquired after COVID-19 vaccination, providing insights regarding the effects and adverse reactions of vaccination.

Osmunda regalis and O. japonica are geographically isolated independent species. Contrary to this morphologically based taxonomy, previous plastid DNA data have shown that O. regalis is paraphyletic, with the American O. regalis sister to O. japonica and O. lancea. It remains to be confirmed whether this paraphyletic topology results from hybridization, budding speciation, or classification issues. To clarify the evolutionary history of O. regalis and O. japonica, we performed multiple molecular phylogenetic analyses using 38 worldwide samples and sequences from 15 nuclear and six plastid DNA regions. The phylogenetic trees of the nuclear and plastid single-region analyses were incongruent. Phylogenetic networks were also discordant between the nuclear and plastid regions. Statistical analysis for detecting the introgressed sequences found hybridization signals between the American O. regalis and O. japonica lineage, particularly in plastid regions. Along with the paleobotanical data, the results suggest that the ancient introgression occurred at an estimated time of 12.8–4.1 Ma in East Asia, when and where both species coexisted. Post-hybridization geographical fluctuation led to the present amphi-Pacific distribution pattern. The phylogenetic network suggests that the New World O. regalis spread across continents in a relatively short time, while the Old World O. regalis experienced long-term geographical isolation between (sub)continents.

Premise of research. Osmunda regalis has a worldwide distribution, except for in eastern Asia, where Osmunda japonica and Osmunda lancea can be found instead. However, the present geographic separation does not necessarily mean geographical speciation. Molecular data indicated that American O. regalis is sister to O. japonica and O. lancea rather than to European O. regalis, but the relationship between the distribution and phylogeny remained unclear. An analysis of vegetative pinnule fossils, which were abundant in the Cenozoic time, would help elucidate the historical biogeography and evolution of these species if the taxonomy of the fossils would be clarified.Methodology. We measured interveinlet intervals in mature and juvenile plants of the extant species and fossils from Japan and the rest of the world, as well as the size of their pinnules, and compared them through geological time and during ontogeny.Pivotal results. The interveinlet interval differs between O. regalis and O. japonica, and part of...

The chromosome number and phylogenetic position of the monotypic eusporangiate genus Mankyua (Ophioglossaceae), which shares peculiar morphological characteristics with Ophioglossum s.l. and Helminthostachys, were determined. Meiotic and mitotic chromosome observations of Mankyua show n = 130 and 2n = ca. 260, suggesting that the base number of this genus is x = 130, which is a new finding and the highest base number reported so far for Ophioglossaceae. The molecular phylogenetic trees derived from nucleotide sequence variation of rbcL and matK genes differ in terms of the phylogenetic position of Mankyua. The molecular phylogenetic trees based on combined data sets of rbcL and matK suggest that Mankyua might be the earliest-diverging lineage of Ophioglossaceae.

The southern Chinese Osmunda ×mildei has been suggested to be an intersubgeneric hybrid, i.e., O. japonica (subgenus Osmunda) × O. angustifolia (subgenus Plenasium) or O. japonica × O. vachellii (subgenus Plenasium). These interpretations were based on morphological, cytological, and/or chloroplast DNA data, yet the parents of the hybrid remained unclear. Molecular phylogenetic relationships inferred here from chloroplast rbcL sequences and three nuclear DNA markers show that O. ×mildei is most likely a hybrid between the paternal O. japonica and the maternal O. vachellii.

Osmunda subgenus Osmunda is variable across its worldwide range. In addition to the three species assigned to the subgenus, there is an enigmatic group of plants in Laos and Myanmar. To reveal the taxonomic and evolutionary status of these plants, we performed taxonomic, cytological, and flow-cytometric analyses, and molecular phylogenetic analyses for 32 samples of subgenus Osmunda for three nuclear DNA markers and the chloroplast rbcL gene. Results show that the enigmatic plants are a tetraploid (n = 44, 2n = 88) of hybrid origin from Osmunda regalis and O. japonica, which is described as Osmunda hybrida sp. nov. This is the first record of a naturally occurring polyploid species in the most primitive leptosporangiate family Osmundaceae, previously thought to be diploid throughout. Molecular data suggest that Indian O. regalis is a putative maternal ancestor and O. japonica is a paternal ancestor. Osmunda hybrida shares the fully dimorphic or partially dimorphic leaves with its parental species...

Introduction: Tyrosine kinase inhibitors (TKIs) have dramatically improved the prognosis of chronic myelogenous leukemia (CML). The treatment with TKIs maintain the depth of response; however, the life-long use of TKI has also been associated with late complications such as cardiovascular events and huge financial burden impairing their quality of life. To overcome these issues, investigators have been attempting to discontinue TKIs after durable molecular remission. However, the optimal timing to stop TKIs remains to be elucidated. We previously demonstrated that CD25 was highly expressed in murine and human CML-leukemia initiating cells (LICs) (Kobayashi CI et al., Blood, 2014). In this study we tried to clarify whether the proportion of CD25-positive cells in hematopoietic stem/progenitor cell fraction of bone marrow cells in CML patients treated with TKIs is associated with their molecular response and could serve as a novel surrogate marker to select patients who are likely to obtain durable treatment-free remission after stopping TKIs. Methods: Bone marrow samples were obtained from the patients with CML in chronic phase who were treated solely with TKIs at Keio University Hospital (Tokyo, Japan). This study was approved by the institutional ethical committee and informed consent was obtained from each patient. Both quantitative and qualitative PCR of BCR-ABL1 was performed using bone marrow mononuclear cells (BMMNCs). The proportion of CD25-positive cells in bone marrow hematopoietic stem/progenitor cell (HSPC; CD34+CD38-) fraction (%CD25+) was evaluated by flow cytometry. The response to TKIs at the time of analysis was determined according to as follows: complete cytogenetic remission (CCyR) defined as Philadelphia chromosome undetectable and quantitative PCR copy numbers >731 among BMMNCs; major molecular remission (MMR) as quantitative PCR copy numbers ≤731, and complete molecular remission (CMR) as undetectable BCR-ABL1 by quantitative and qualitative PCR. Results: Bone marrow samples obtained from 109 patients were evaluated (median age, 52 years; male/female, 76/33). Analysis was performed prior to TKI exposure in 26 patients and under TKI therapy in 64 patients (imatinib, 22; dasatinib, 33; nilotinib, 9). Remaining 19 patients were treatment free because they were enrolled into a clinical trial of TKI discontinuation. At diagnosis (n=26), %CD25+ were significantly correlated with hemoglobin level and platelet count (Table). The %CD25+ was significantly lower in patients with post TKI exposure than those at diagnosis without TKIs (p Conclusion: We confirmed that the expression of CD25 in HSPC fraction of CML patients was significantly correlated with the disease status, and may be useful as a LIC minimal residual disease marker. Disclosures Kasahara: Chugai: Research Funding. Sakurai:Bristol-Myers Squibb K.K.: Speakers Bureau. Kikuchi:Celgene: Speakers Bureau; Takeda: Speakers Bureau; Ono: Speakers Bureau. Shimizu:Bristol-Myers Squibb K.K: Honoraria. Mori:Astella Pharma: Honoraria; Kyowa Hakko Kirin: Honoraria; Novartis Pharma: Research Funding; MSD: Research Funding; MSD: Honoraria; Janssen: Honoraria; SHIONOGI: Honoraria; Taisho Toyama Pharmaceutical Co: Honoraria; Celgene: Honoraria; Ono: Honoraria; Eisai: Honoraria; Novartis Pharma: Honoraria; Shire Japan: Honoraria; CHUGAI: Honoraria; Asahi Kasei: Research Funding; Japan Blood Products Organization: Honoraria; Pfizer: Honoraria. Okamoto:Pfizer Inc.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Eisai Co.,Ltd.: Research Funding; Kyowa Hakko Kirin Co.: Research Funding; Bristol-Myers Squibb K.K.: Honoraria, Research Funding; Teijin Pharma Limited: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Toyama Chemical Co., Ltd.: Research Funding; Alexion Pharmaceuticals, Inc.:: Research Funding; Nippon Shinyaku Co., Ltd: Research Funding; Shionogi & Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Asahi Kasei Pharma Corp.:: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; JCR Pharmaceuticals Co., Ltd.: Research Funding.

While speciation can be found in the presence of gene flow, it is not clear what impact this gene flow has on genome- and range-wide patterns of differentiation. Here we examine gene flow across the entire range of the common sunflower, H. annuus, its historically allopatric sister species H. argophyllus and a more distantly related, sympatric relative H. petiolaris. Analysis of genotypes at 26 microsatellite loci in 1015 individuals from across the range of the three species showed substantial introgression between geographically proximal populations of H. annuus and H. petiolaris, limited introgression between H. annuus and H. argophyllus, and essentially no gene flow between the allopatric pair, H. argophyllus and H. petiolaris. Analysis of sequence divergence levels among the three species in 1420 orthologs identified from EST databases identified a subset of loci showing extremely low divergence between H. annuus and H. petiolaris and extremely high divergence between the sister species H. annuus and H. argophyllus, consistent with introgression between H. annuus and H. petiolaris at these loci. Thus, at many loci, the allopatric sister species are more genetically divergent than the more distantly related sympatric species, which have exchanged genes across much of the genome while remaining morphologically and ecologically distinct.

In order to clarify patterns of hybrid formation in the Asplenium nidus complex, artificial crossing experiments were performed between individuals of genetically differentiated groups based on the sequence of the rbcL gene, including A. australasicum from New Caledonia, A. setoi from Japan and several cryptic species in the A. nidus complex. No hybrid plants were obtained in crosses between nine of the 16 pairs. Even for pairs that generated hybrids, the frequency of hybrid formation was lower than expected given random mating, or only one group was able to act as the maternal parent, when the genetic distance (Kimura's two parameter) between parental individuals was at least 0.006. Sterile hybrids were produced by three pairs that were distantly related but capable of forming hybrids. Considering the results of the crosses together with the genetic distance between the parental individuals, it seems that the frequency of hybrid formation decreases rapidly with increasing divergence. The frequency of hybrid formation has not been previously examined in homosporous ferns, but it seems that a low frequency of hybrid formation can function as an important mechanism of reproductive isolation between closely related pairs of species in the A. nidus complex in addition to hybrid sterility. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 42–63.

Plant species may remain morphologically distinct despite gene exchange with congeners, yet little is known about the genomewide pattern of introgression among species. Here we analyze the effects of persistent gene flow on genomic differentiation between the sympatric sunflower species Helianthus annuus and H. petiolaris. While the species are strongly isolated in testcrosses, genetic distances at 108 microsatellite loci and 14 sequenced genes are highly variable and much lower (on average) than for more closely related but historically allopatric congeners. Our analyses failed to detect a positive association between levels of genetic differentiation and chromosomal rearrangements (as reported in a prior publication) or proximity to QTL for morphological differences or hybrid sterility. However, a significant increase in differentiation was observed for markers within 5 cM of chromosomal breakpoints. Together, these results suggest that islands of differentiation between these two species are small, except in areas of low recombination. Furthermore, only microsatellites associated with ESTs were identified as outlier loci in tests for selection, which might indicate that the ESTs themselves are the targets of selection rather than linked genes (or that coding regions are not randomly distributed). In general, these results indicate that even strong and genetically complex reproductive barriers cannot prevent widespread introgression.

In Japanese Stegnogramma pozoi subsp. mollissima (Fisher ex Kunze) K. Iwats. there is the intrasubspecific variation among rbcL sequences. Northern and southern plants are genetically differentiated for maternally inherited cpDNA. In the present study we examined allozyme polymorphisms to test the hypothesis that northern and southern plants may be separate species. Based on allozyme data, the degree of gene flow among populations was estimated to be large. The artificial crossing experiments between cpDNA haplotypes also suggested that isolation has not developed among these cpDNA haplotypes. However, interpopulation genetic differentiation in cpDNA was observed even in the small area at the foot of Mt. Hakone, and the cpDNA haplotypes appear to have different habitat preferences.

Asplenium nidus is an epiphytic fern with large simple leaves. Because A. nidus lacks the good taxonomic characters available for species recognition, multiple cryptic species may exist within A. nidus. In Mt. Halimun National Park, West Java, Indonesia, three rbcL sequence types of A. nidus were recorded. All plants regardless of rbcL sequence were 2n = 144. Crossing experiments among these rbcL types were conducted, and it was observed that the molecularly distinct types were reproductively isolated because hybrids failed to form between at least two pairs of rbcL types. These results suggest that these rbcL types are cryptic species because they are reproductively isolated but they are difficult to distinguish morphologically. Thus, the characters of DNA sequences information are useful in the discovery of cryptic species in ferns.

Two cytotypes, an apomictic triploid (2n=123) and an apomictic tetraploid (2n=164), were found in Japanese Diplazium doederleinii. The cytotypes are clearly distinguishable from each other in phytogeographical, ecological, and morphological characteristics. The tetraploid is smaller in relation to several morphological characteristics and occurs in more northerly regions than the triploid. The distributions of the two cytotypes do not overlap. The leaves of the triploids are evergreen, while those of the tetraploids are summer‐green. Allozyme analysis indicated that one and four multilocus genotypes are contained in the tetraploid and triploid, respectively. The nucleotide sequence of the rbcL gene did not vary within the same cytotype, except for a single triploid with the same rbcL sequence as the tetraploid. There are only two nucleotide differences between the triploid and tetraploid races. These results indicate that the apomictic triploids are of recurrent origin from an undiscovered or extinct ance...

Introduction: Tyrosine kinase inhibitors (TKIs) have dramatically improved the prognosis of chronic myelogenous leukemia (CML). The treatment with TKIs continues to improve the depth of response and overall survival of CML patients, but the life-long use of TKI is known to be associated with late complications such as cardiovascular events as well as heavy financial burden, and thus impairs the quality of life. To overcome these issues, many studies evaluating the possibility of TKI discontinuation have been ongoing worldwide. In order to achieve durable treatment-free remission, it is crucial to understand the dynamics of CML-leukemia initiating cells (LICs). We previously reported that CD25 was highly expressed in murine and human CML-LICs (Kobayashi CI et al., Blood, 2014). The aim of this study was to assess whether the proportion of CD25 positive cells in hematopoietic stem/progenitor cell fraction of bone marrow cells in CML patients treated with TKIs is associated with their molecular response and could serve as a novel surrogate marker to stop TKI therapy. Methods: Bone marrow samples were obtained from patients with CML in chronic phase who were diagnosed and have been treated solely with TKIs at Keio University Hospital (Tokyo, Japan). This study was approved by the institutional ethical committee and informed consent was obtained from each patient. Both quantitative and qualitative PCR of BCR-ABL was performed using bone marrow mononuclear cells (BMMNCs). The proportion of CD25 positive cells in bone marrow hematopoietic stem/progenitor cell (HSPC; CD34+CD38-) fraction was evaluated by flow cytometry using FITC-labeled anti-CD34, PE- labeled anti-CD38 and APC-labeled anti-CD25 antibodies. The response to TKIs at the time of evaluation was determined according to the previous report (Yoshida C et al., Int J Clin Oncol, 2012): complete cytogenetic remission (CCyR) defined as Philadelphia chromosome undetectable and quantitative PCR copy numbers >731 among BMMNCs; major molecular remission (MMR) as quantitative PCR copy numbers ≤731, and complete molecular remission (CMR) as undetectable BCR-ABL by quantitative and qualitative PCR. Results: Bone marrow samples obtained from 95 patients were evaluated (median age 53 years old; male/female, 67/28). Analysis was performed prior to TKI exposure in nine patients and under TKI therapy including 2nd generation TKI in 64 patients (imatinib, 22; dasatinib, 33; nilotinib, 9). Remaining 22 patients were treatment free because they enrolled in a clinical trial of TKI discontinuation. The proportion of CD25 positive cells in HSPC fraction significantly decreased in patients with prior TKI exposure relative to patients at diagnosis (n=86; Mean 4.2%, SD 7.0% vs n=9; Mean 22.4%, SD 11.3%, P Conclusion: We confirmed that the expression of CD25 in HSPC fraction of CML patients was significantly correlated with the response to TKI therapy, and may serve as an asset to select patients who are likely to achieve durable treatment-free survival. Figure Figure. Disclosures Karigane: Celgene: Honoraria. Sakurai:Celgene: Honoraria. Matsuki:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Nippon Shinyaku: Honoraria. Kikuchi:Celgene: Honoraria; Takeda Pharmaceutical Company: Honoraria; Kyowa Hakko Kirin: Honoraria. Mitsuhashi:LSI Medience: Consultancy. Okamoto:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Asahi Kasei Pharma Corp.: Research Funding; Astellas Pharma Inc.: Research Funding; Shionogi & Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Alexion Pharmaceuticals, Inc.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Teijin Pharma Limited: Research Funding; Bristol-Myers Squibb K.K.: Honoraria, Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Honoraria, Research Funding; JCR Pharmaceuticals Co., Ltd.: Research Funding.

sequences of 11 of 15 extant species of Osmundaceae which represent all three genera, Osmunda, Todea and Leptopteris. Our phylogenetic analysis concluded: 1) Osmunda subg. Osmunda and subg. Plenasium are monophyletic groups, but subg. Osmundastrum is not. The genus Osmunda is not monophyletic because Todea and Leptopteris are positioned within Osmunda. 2) Osmunda cinnamomea is the most basally positioned species in Osmundaceae, and it can be called as “a living fossil” because a fossil species (O. claytoniites) with almost the same morphology as this species was recorded from the Triassic. 3) Osmunda japonica and O. regalis are very closely related with only one nucleotide difference in the rbcL gene. 4) Greater nucleotide variation (5–7 nucleotides) was found between conspecific samples of O. cinnamomea and O. claytoniana collected from Japan and United States. Each of these two species may comprise more than two biologically differentiated species.

sect. Thamnopteris or A. nidus L. complex is defined by the synapomorphic character peculiar to Aspleniaceae, an anastomosing vein near the margin of the simple lamina. Thus, it is easily recognizable and its monophyly is quite clear. In spite of its naturalness as the whole group, species delimitation is very confusing. Three species of sect. Thamnopteris, A. antiquum Makino, A. australasicum (J. Smith) Hooker and A. nidus L. have been recognized in Japan, but the naturalness of each species is still not clear because their morphology is too simple to find good qualitative taxonomical characters. In the present work, we examined the intraspecific variation of allozymes and rbcL sequences in the Japanese plants of sect. Thamnopteris and compared them with those from other paleotropical localities in order to recognize natural units in such morphologically simple plants. We found a large amount of genetic variation in this section and inferred that A. antiquum is a species of ancient origin, though morphologically it is not so different from other species of the sect. Thamnopteris. It was also discovered that the so called “A. australasicum” in Japan has a very different rbcL sequence from A. australasicum sensu Holttum, which is distributed in Australia and South Pacific Islands. Based on these molecular data, we described the Japanese “A. australasicum” as a new species, Asplenium setoi N. Murak. et Seriz.

Stegnogramma pozoi (Lagasca) K. Iwats. subsp.mollissima (Fischer ex Kunze) K. Iwats. is one of the most common ferns in Japan with a very broad geographical distribution and habitat ranges. We examined the intrasubspecific variation of therbcL gene, and found three types in the subspecies (Atami-1, Atami-2, and Tateyama types). The Atami-1 and 2 types shared three apomorphic nucleotides and their geographical distribution is intermingled. Thus, we considered them to have intraspecific variation. In contrast, the Tateyama type had 4–5 different nucleotides than the Atami types, and showed a more northern distribution. Leaf morphology of the Tateyama type is distinct from that of the Atami types, though the leaf shape in Tateyama type varies considerably and in some cases overlaps with other types. Ferns have fewer morphological features that are systematically significant than flowering plants because they lack complex reproductive organ. It is reasonable to expect that species recognition of ferns based only on leaf morphology can be fine tuned by molecular features. Variation in therbcL sequences might be useful as an indication of significant biological units.

In ferns, intra-gametophytic selfing occurs as a mode of reproduction where two gametes from the same gametophyte form a completely homozygous sporophyte. Intra-gametophytic selfing is considered to be prevented by lethal or deleterious recessive genes in several diploid species. In order to investigate the modes and tempo of selection acting different developmental stages, doubled haploids obtained from intra-gametophytic selfing within isolated gametophytes of a putative F1 hybrid between Osmunda japonica and O. lancea were analyzed with EST_derived molecular markers, and the distribution pattern of transmission ratio distortion (TRD) along linkage map was clarified. As the results, the markers with skewness were clustered in two linkage groups. For the two highly distorted regions, gametophytes and F2 population were also examined. The markers skewed towards O. japonica on a linkage group (LG_2) showed skewness also in gametophytes, and the TRD was generated in the process of spore formation or growth of gametophytes. Also, selection appeared to be operating in the gametophytic stage. The markers on other linkage group (LG_11) showed highest skewness towards O. lancea in doubled haploids, and it was suggested that the segregation of LG_11 were influenced by zygotic lethality or genotypic evaluation and that some deleterious recessive genes exist in LG_11 and reduce the viability of homozygotes with O. japonica alleles. It is very likely that a region of LG_11were responsible for the low frequencies of intra-gametophytic selfing in O. japonica.

The feasibility of later-generation hybrid production in ferns has not been previously studied, although it is a significant factor in relation to reproductive isolation. Osmunda × intermedia, a hybrid between O. japonica and O. lancea, is semifertile and has moderate spore germination rates. Under the artificial conditions of this study, F2 and F3 offspring were formed. Some of the F2 offspring showed precocity, and some of the F3 offspring also showed precocity. This fertility suggests that introgressive hybridization might be ongoing in nature. This also indicates a currently unknown genetic control over the timing of fertile frond production in Osmunda.

Rheophilous Osmunda lancea often hybridizes with a dryland ally, Osmunda japonica, to produce O. × intermedia, forming zonation in riverbanks and the adjacent dryland along flooding frequency clines. This study examined the genetic structure of populations consisting of O. × intermedia and the two parental species by analyzing ten nuclear DNA markers [six cleaved amplified polymorphic sequence (CAPS) markers and three simple sequence repeat (SSR) markers developed from an expressed sequence tag (EST) library, and the sequence of the glyceraldehyde-3-phosphate dehydrogenase gene GapCp] and chloroplast DNA sequences. The results suggest that the nuclear genes of O. japonica and O. lancea are genetically differentiated despite shared polymorphism in their chloroplast DNA sequences. This discrepancy may be attributable to natural selection and recent introgression, although it is not evident if introgression occurs between O. japonica and O. lancea in the examined populations. Our findings of putative F2 hybrids in O. × intermedia support its partial reproducibility, and also suggest that formation of later-generation hybrids generates morphological variation in O. × intermedia. O. lancea plants collected from geographically distant localities were genetically very similar, and it is suggested that O. lancea originated monotopically.

We analyzed the factor XII (FXII) gene of a patient with congenital FXII deficiency and identified a novel amino acid substitution (W486C) in the catalytic domain. The proband was an asymptomatic 49-year-old Japanese female with abnormal coagulation test, discovered by chance. The FXII activity and antigen level were both under 10%, suggesting a cross-reacting material-negative FXII deficiency. Sequence analysis of the proband's FXII gene revealed a homozygous nucleotide substitution G --> C in exon 12, resulting in the amino acid substitution W486C in the catalytic domain. We constructed the mutant FXII cDNA in an expression plasmid vector and transfected it into Chinese hamster ovary cells. The recombinant wild-type FXII antigen was detected in the culture medium by immunoprecipitation assay, but the mutant FXII (W486C) was not observed. On the other hand, both the wild-type FXII and W486C cell lysates contained FXII antigen and FXII mRNA, as estimated by western blotting and quantitative reverse transcriptase-polymerase chain reaction. These findings suggest that the W486C substitution of FXII impairs intracellular processing of the protein and/or transport system.

For the taxonomic revision of the problematic species Ceratopteris thalictroides, molecular analyses and crossing tests were conducted for 16 sources in the world. An analysis of allozyme composition of five enzymes revealed the presence of three intraspecific entities, which were called the south type, the north type, and the third type. An analysis of the nucleotide sequences of chloroplast DNA also distinguished the same entities. Crossing tests showed that the south type was completely cross-sterile with the other two types, and that the other two were considerably cross-sterile with each other. These results suggest that the three entities should be regarded as different biological species. Although the south type and the other two meet in several regions, complete cross-sterility between them seems to sustain their genetic distinctiveness in spite of occasional crossing. The results from the present study suggest that widely distributed fern species are apt to comprise several cryptic species.

In various fern species, a large amount of rbcL sequence variation has been reported, and it is possible that these species contain several reproductively isolated cryptic species. In our previous study on Asplenium nidus L., it was suggested that the plants growing in Mt. Halimun National Park, West Java, Indonesia, consist of several cryptic species based on the results of crossing experiments among rbcL sequence types. In this study, we examined allozyme polymorphisms of five rbcL sequence types found in West Java in order to test the hypothesis that the assemblages of A. nidus delimited based on the rbcL sequences are separate Mendelian populations and gene flow is disrupted by reproductive isolation from one another. The calculated fixation indices suggested that the individuals in each rbcL type are randomly crossing at least in the investigated localities. Nevertheless, these rbcL-based assemblages were genetically differentiated in allozymes that are encoded in their nuclear genomes, and it is also suggested that gene flow is disrupted even between sympatrically distributed pairs of rbcL sequence types. Therefore, our findings support the view that the five rbcL sequence types in West Java are potential cryptic species.

Atrial natriuretic peptide (ANP) plays a crucial role in regulating body fluid volume and blood pressure, by promoting natriuresis and vasodilatation and by inhibiting the renin-angiotensin system. Plasma levels of ANP are elevated in heart failure and hypertension, and ANP is thus believed to be involved in the pathogenesis of cardiovascular disorders. Previous case-control studies have shown that a single nucleotide polymorphism in the first exon of ANP gene, 664G/A, is associated with a risk of cerebrovascular disease (CVD) in white populations. Plasma ANP levels, however, were not evaluated in these studies in relation to the 664G/A, although the nucleotide substitution causes an amino-acid change in the propeptide of ANP. In this study, we analyzed the genotype frequencies of the 664G/A in Japanese patients with CVD (n = 199) and age- and gender-matched control subjects(n = 176). Genotypes with the 664A allele in the Japanese control subjects (G/A and A/A 12.5%) were apparently more frequent compared to the published frequency of the white control population (G/A and A/A 6.6%, p = 0.0437). Genotypes with the 664A allele, however, were not significantly different between our CVD patients(15.1%) and controls (12.5% p = 0.4714). In the control group (n = 137), the mean plasma ANP levels were not different between the 664G/G (15.7 +/- 10.7 pg/ml) and 664G/A genotypes (15.6 +/- 6.8 pg/ml, p = 0.9708). These results suggest that there is a racial difference in the allele frequency of 664G/A, and that this polymorphism may not be a major risk factor for CVD in the Japanese, nor is it a major determinant of plasma ANP level.

Asplenium nidus is one of the most common epiphytic fern species in the Old World tropics. It has simple leaves without any particular appendages, and the lack of good species recognition might be due to the lack of good taxonomic characters. Recently, it has become easier to collect DNA nucleotide sequence data from wild plants using PCR and direct sequencing techniques. This kind of molecular information has mostly been used forphylogenetic analyses, but can also be useful for a-taxonomy (recognition of naturally existing species). In this work, we made a detailed analysis of the genetic variation of A. nidus in one locality, Mt. Halimun National Park, West Java, Indonesia. We collected leaf materials from 25 individuals, all identified as A. nidus according to the most recent monograph of the group by Holttum (1974). We found three types of rbcL sequences and there was variation at 30 sites out of 1,227 bp. Moreover, we found good correlation among rbcL types, some morphological characters (leaf shape and size, sorus length) and ecological traits (habitats and altitudes). DNA nucleotide sequence information might be useful as a primary key even for a-taxonomy, especially for morphologically simple organisms like ferns.