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The composition of human skin microbiome profoundly impacts host skin health and disease. However, the relationship between skin homeostasis or the development of skin diseases and daily changes in skin microbial composition is poorly understood. Longitudinal samplings at more frequent intervals would address this issue, while conventional sampling methods have technical difficulties, leading to limitations in sampling opportunities. Here, we developed a simple and stable tape-stripping method regardless of the operator's skill. Our method enables skin microbial sampling within 30 seconds and taking multiple skin microbial samples from the same body site. The amount of microbial DNA among multiple sampling sites could be measured within 13.5%. The sequencing results of multiple sampling showed high consistency, Pearson's correlation coefficient between multiple samples of 0.98. Furthermore, these results were comparable to those collected by the conventional swabbing method. These results demonstrate that our tape-stripping method enables simple microbiome collection and highly reliable quantitative skin microbiome analysis. These features of our method would lead to a further understanding of skin disease development or diagnosis of skin conditions in clinical research by increasing the opportunities for microbial sampling., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Myoblast fusion is essential for skeletal muscle development, growth, and regeneration. However, the molecular mechanisms underlying myoblast fusion and differentiation are not fully understood. Previously, we reported that interleukin-4 (IL-4) promotes myoblast fusion; therefore, we hypothesized that IL-4 signaling might regulate the expression of the molecules involved in myoblast fusion. In this study, we showed that in addition to fusion, IL-4 promoted the differentiation of C2C12 myoblast cells by inducing myoblast determination protein 1 (MyoD) and myogenin, both of which regulate the expression of myomerger and myomaker, the membrane proteins essential for myoblast fusion. Unexpectedly, IL-4 treatment increased the expression of myomerger, but not myomaker, in C2C12 cells. Knockdown of IL-4 receptor alpha (IL-4Rα) in C2C12 cells by small interfering RNA impaired myoblast fusion and differentiation. We also demonstrated a reduction in the expression of MyoD, myogenin, and myomerger by knockdown of IL-4Rα in C2C12 cells, while the expression level of myomaker remained unchanged. Finally, cell mixing assays and the restoration of myomerger expression partially rescued the impaired fusion in the IL-4Rα-knockdown C2C12 cells. Collectively, these results suggest that the IL-4/IL-4Rα axis promotes myoblast fusion and differentiation via the induction of myogenic regulatory factors, MyoD and myogenin, and myomerger.

The skin microbiota has been recognized to play an integral role in the physiology and pathology of the skin. The crosstalk between skin and the resident microbes has been extensively investigated using two-dimensional (2D) and three-dimensional (3D) cell cultures in vitro ; however, skin colonization by multiple species and the effects of interspecific interactions on the structure and function of skin remains to be elucidated. This study reports the establishment of a mixed infection model, incorporating both commensal ( Staphylococcus epidermidis ) and pathogenic ( Staphylococcus aureus ) bacteria, based on a 3D human epidermal model. We observed that co-infecting the 3D epidermal model with S. aureus and S. epidermidis restricted the growth of S. aureus . In addition, S. aureus induced epidermal cytotoxicity, and the release of proinflammatory cytokines was attenuated by the S. aureus - S. epidermidis mixed infection model. S. epidermidis also inhibited the invasion of the deeper epidermis by S. aureus , eliciting protective effects on the integrity of the epidermal barrier. This 3D culture-based mixed infection model would be an effective replacement for existing animal models and 2D cell culture approaches for the evaluation of diverse biotic and abiotic factors involved in maintaining skin health., Competing Interests: Author KK, XL, NS, RN, MI, and AI were employed by company Toyota Motor Corporation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kohda, Li, Soga, Nagura, Duerna, Nakajima, Nakagawa, Ito and Ikeuchi.)

Background: The signal transducer and activator of transcription 6 (STAT6) transcription factor plays a vitally important role in immune cells, where it is activated mainly by interleukin-4 (IL-4). Because IL-4 is an essential cytokine for myotube formation, STAT6 might also be involved in myogenesis as part of IL-4 signaling. This study was conducted to elucidate the role of STAT6 in adult myogenesis in vitro and in vivo., Methods: Myoblasts were isolated from male mice and were differentiated on a culture dish to evaluate the change in STAT6 during myotube formation. Then, the effects of STAT6 overexpression and inhibition on proliferation, differentiation, and fusion in those cells were studied. Additionally, to elucidate the myogenic role of STAT6 in vivo, muscle regeneration after injury was evaluated in STAT6 knockout mice., Results: IL-4 can increase STAT6 phosphorylation, but STAT6 phosphorylation decreased during myotube formation in culture. STAT6 overexpression decreased, but STAT6 knockdown increased the differentiation index and the fusion index. Results indicate that STAT6 inhibited myogenin protein expression. Results of in vivo experiments show that STAT6 knockout mice exhibited better regeneration than wild-type mice 5 days after cardiotoxin-induced injury. It is particularly interesting that results obtained using cells from STAT6 knockout mice suggest that this STAT6 inhibitory action for myogenesis was not mediated by IL-4 but might instead be associated with p38 mitogen-activated protein kinase phosphorylation. However, STAT6 was not involved in the proliferation of myogenic cells in vitro and in vivo., Conclusion: Results suggest that STAT6 functions as an inhibitor of adult myogenesis. Moreover, results suggest that the IL-4-STAT6 signaling axis is unlikely to be responsible for myotube formation.

Objective: This study examined the chronic effect of increased physical activity on postprandial triglycerides in older women., Methods: Twenty-six women, aged 72 ± 5 years (mean ± SD), participated in this study. Participants in the physical activity group (n = 11) were asked to increase their activities above their usual lifestyle levels for 12 weeks. Participants in the control group (n = 15) maintained their usual lifestyle for 12 weeks. All participants rested and consumed a standardized breakfast after a 24-h period of physical activity avoidance at baseline, 4 weeks, and 12 weeks. Blood samples were collected in the fasted state (0 h) and at 2, 4, and 6 h after breakfast., Results: The average increased time spent in self-selected activities per day was 1.1 ± 19.3 min over the 12 weeks compared with the baseline in the physical activity group. There was no difference in the postprandial time-averaged triglyceride area under the curve at baseline (1.59 ± 0.81 vs. 1.39 ± 0.67 mmol/L, p  = 0.515) or over the 12-week intervention (1.78 ± 1.00 vs. 1.31 ± 0.67 mmol/L, p  = 0.212) between the physical activity and control groups., Conclusion: Postprandial triglyceride concentrations were not reduced after performing self-selected activities under free-living conditions in older women when these responses were determined 24 h after the last physical activity bout. (Trial registration ID: UMIN000037420)., Competing Interests: None., (© 2020 The Society of Chinese Scholars on Exercise Physiology and Fitness. Published by Elsevier (Singapore) Pte Ltd.)

Transient receptor potential vanilloid 4 (TRPV4) channel is a polymodal receptor activated by moderate heat and hypoosmolarity. TRPV4 expressed in the skin area contributes to several skin functions as a barrier to maintain internal body physiology and a transporter of external stimuli. The skin condition such as skin temperature and osmolarity varies with internal and external changes, and may influence the activity of TRPV4 contributing to skin physiology, thermal sensation, and thermoregulation. However, the combination effect of skin conditions such as temperature and osmolarity on the activity of TRPV4 has not been examined. In the current study, we investigated the effect of temporal adaptation (5-10 min) to different temperature (25-35 °C) and osmolarity (250-350 mOsm) conditions on the heat response (until 40 °C) of human TRPV4 in cultured cells using Ca 2+ imaging. The temperature to activate TRPV4 increased with elevation of the adaptation temperature, and decreased with the adaptation to hypoosmolarity in the range of 25-35 °C. In addition, the heat response was inhibited with the adaptation to hyperosmolarity in the range of 25-35 °C. Thus, we demonstrated that the activation temperature of TRPV4 varied with the temporal sensory adaptation to different temperature and osmolarity conditions. These findings may contribute to gaining better understanding of the variation in several TRPV4-mediated skin functions., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Synapse formation is achieved by various synaptic organizers. Although this process is highly regulated by neuronal activity, the underlying molecular mechanisms remain largely unclear. Here we show that Cbln1, a synaptic organizer of the C1q family, is released from lysosomes in axons but not dendrites of cerebellar granule cells in an activity- and Ca 2+ -dependent manner. Exocytosed Cbln1 was retained on axonal surfaces by binding to its presynaptic receptor neurexin. Cbln1 further diffused laterally along the axonal surface and accumulated at boutons by binding postsynaptic δ2 glutamate receptors. Cbln1 exocytosis was insensitive to tetanus neurotoxin, accompanied by cathepsin B release, and decreased by disrupting lysosomes. Furthermore, overexpression of lysosomal sialidase Neu1 not only inhibited Cbln1 and cathepsin B exocytosis in vitro but also reduced axonal bouton formation in vivo. Our findings imply that co-release of Cbln1 and cathepsin B from lysosomes serves as a new mechanism of activity-dependent coordinated synapse modification., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Long-term depression (LTD) of AMPA-type glutamate receptor (AMPA receptor)-mediated synaptic transmission has been proposed as a cellular substrate for learning and memory. Although activity-induced AMPA receptor endocytosis is believed to underlie LTD, it remains largely unclear whether LTD and AMPA receptor endocytosis at specific synapses are causally linked to learning and memory in vivo. Here we developed a new optogenetic tool, termed PhotonSABER, which enabled the temporal, spatial, and cell-type-specific control of AMPA receptor endocytosis at active synapses, while the basal synaptic properties and other forms of synaptic plasticity were unaffected. We found that fiberoptic illumination to Purkinje cells expressing PhotonSABER in vivo inhibited cerebellar motor learning during adaptation of the horizontal optokinetic response and vestibulo-ocular reflex, as well as synaptic AMPA receptor decrease in the flocculus. Our results demonstrate that LTD and AMPA receptor endocytosis at specific neuronal circuits were directly responsible for motor learning in vivo. VIDEO ABSTRACT., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: This multicenter cooperative study aimed to analyze the adverse events (AEs) associated with tyrosine kinase inhibitors (TKIs) used as initial treatment for chronic-phase chronic myeloid leukemia (CML-CP) and their impact on outcome., Methods: We retrospectively evaluated 450 patients with CML-CP who received TKIs between 2004 and 2014., Results: The 5-year overall survival (OS) and event-free survival (EFS) rates were 95.1% and 89.0%, respectively. Patients with comorbidities (46.4%) and aged ≥60 years (50.4%) at diagnosis had significantly inferior OS to those without comorbidities and aged <60. Patients achieved higher rates of major molecular response (MMR) at 6 and 12 months after initial treatment with dasatinib or nilotinib compared to imatinib, but final MMR rates were almost the same. Sixty-six percent of patients required treatment modifications from first-line TKI therapy; the main reasons were AEs (48.4%) and failure (18%). Grade III-IV AEs in first-line TKI therapy were significantly correlated to inferior OS/EFS compared to grade 0-II AEs., Conclusion: Although long-term outcomes were similar in CML-CP patients treated with each TKI regardless of first-line TKI selection, severe AEs in first-line TKI therapy decreased their survival rates. Early change in TKIs is recommended, when faced with severe AEs of specific TKIs., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Pluripotent human embryonic stem cells (hESCs) can differentiate into multiple cell lineages, thus, providing one of the best platforms to study molecular mechanisms during cell differentiation. Recently, we have reported rapid and efficient differentiation of hESCs into functional neurons by introducing a cocktail of synthetic mRNAs encoding five transcription factors (TFs): NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2. Here we further tested a possibility that even single transcription factors, when expressed ectopically, can differentiate hESCs into neurons. To this end, we established hESC lines in which each of these TFs can be overexpressed by the doxycycline-inducible piggyBac vector. The overexpression of any of these five TFs indeed caused a rapid and rather uniform differentiation of hESCs, which were identified as neurons based on their morphologies, qRT-PCR, and immunohistochemistry. Furthermore, calcium-imaging analyses and patch clamp recordings demonstrated that these differentiated cells are electrophysiologically functional. Interestingly, neural differentiations occurred despite the cell culture conditions that rather promote the maintenance of the undifferentiated state. These results indicate that over-expression of each of these five TFs can override the pluripotency-specific gene network and force hESCs to differentiate into neurons., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Astrocytes regulate synaptic transmission through controlling neurotransmitter concentrations around synapses. Little is known, however, about their roles in neural circuit development. Here we report that Bergmann glia (BG), specialized cerebellar astrocytes that thoroughly enwrap Purkinje cells (PCs), are essential for synaptic organization in PCs through the action of the l-glutamate/l-aspartate transporter (GLAST). In GLAST-knockout mice, dendritic innervation by the main ascending climbing fiber (CF) branch was significantly weakened, whereas the transverse branch, which is thin and nonsynaptogenic in control mice, was transformed into thick and synaptogenic branches. Both types of CF branches frequently produced aberrant wiring to proximal and distal dendrites, causing multiple CF-PC innervation. Our electrophysiological analysis revealed that slow and small CF-evoked excitatory postsynaptic currents (EPSCs) were recorded from almost all PCs in GLAST-knockout mice. These atypical CF-EPSCs were far more numerous and had significantly faster 10-90% rise time than those elicited by glutamate spillover under pharmacological blockade of glial glutamate transporters. Innervation by parallel fibers (PFs) was also affected. PF synapses were robustly increased in the entire dendritic trees, leading to impaired segregation of CF and PF territories. Furthermore, lamellate BG processes were retracted from PC dendrites and synapses, leading to the exposure of these neuronal elements to the extracellular milieus. These synaptic and glial phenotypes were reproduced in wild-type mice after functional blockade of glial glutamate transporters. These findings highlight that glutamate transporter function by GLAST on BG plays important roles in development and maintenance of proper synaptic wiring and wrapping in PCs., Competing Interests: The authors declare no conflict of interest.

The progress of microenvironment-mediated tumor progression in an artificial extracellular matrix explores the design criteria to understand the cancer progression mechanism and metastatic potential. This study was aimed at examining the combination of both surface topographies (fiber alignments) and different stiffness of polymeric substrates (PLLA and PCL) to evaluate the effects on the cellular morphologies, proliferation, motility, and gene expression regarding epithelial to mesenchymal transition (EMT) of two different types of breast cancer cells (MDA-MB-231 and MCF-7). The cellular morphologies (roundness and nuclear elongation factor), E-cadherin and vimentin expression, and cellular motility in terms of cellular migration speed, persistent time, and diffusivity have been comprehensively discussed. We demonstrated that the microenvironment of cell culture substrates influences cancer progression and metastatic potential.

Efficient differentiation of human pluripotent stem cells (hPSCs) into neurons is paramount for disease modeling, drug screening, and cell transplantation therapy in regenerative medicine. In this manuscript, we report the capability of five transcription factors (TFs) toward this aim: NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2. In contrast to previous methods that have shortcomings in their speed and efficiency, a cocktail of these TFs as synthetic mRNAs can differentiate hPSCs into neurons in 7 days, judged by calcium imaging and electrophysiology. They exhibit motor neuron phenotypes based on immunostaining. These results indicate the establishment of a novel method for rapid, efficient, and footprint-free differentiation of functional neurons from hPSCs.

Background: Mogamulizumab, a defucosylated humanized monoclonal antibody targeting C-C chemokine receptor 4, recently became available for the treatment of adult T-cell leukemia/lymphoma (ATL). We conducted a multicenter retrospective study of the efficacy of mogamulizumab in ATL treatment in patients on Hokkaido Island, Japan., Materials and Methods: A total of 125 patients with ATL treated from January 2010 to December 2014 in 20 hospitals affiliated with the Hokkaido Hematology Study Group were enrolled in the present retrospective study., Results: Of the 125 ATL patients, 62 (46.6%) presented with the acute type, 51 (38.3%) with the lymphoma type, and 12 (9.0%) with the chronic type; the latter group included 7 unfavorable chronic cases. The median age at diagnosis was 68 years (range, 35-86 years). The median survival for those with acute, lymphoma, and unfavorable chronic types was 302, 279, and 921 days, respectively. Advanced age, high lactate dehydrogenase level, poor performance status (3-4), and the existence of B symptoms were unfavorable prognostic factors for overall survival (OS). Survival rate calculated from the day of diagnosis was significantly higher in patients treated with mogamulizumab. The OS of individuals receiving hematopoietic stem cell transplantation (HSCT) was superior to that of the non-HSCT group. The median interval between the last mogamulizumab dose and allogeneic HSCT was 38 days (range, 21-53 days). Of the 22 HSCT recipients who were not treated with mogamulizumab, overall acute graft-versus-host disease (aGVHD) and grade III-IV aGVHD occurred in 12 (54.5%) and 3 (13.6%) patients, respectively. However, overall aGVHD and grade III-IV aGVHD developed in 8 (88.9%) and 3 (33.3%) of the 9 HSCT recipients treated with mogamulizumab, respectively., Conclusion: Mogamulizumab improves OS in patients with ATL, although its use in HSCT patients might trigger severe GVHD. Determining the optimal pre-HSCT mogamulizumab treatment regimen is thus a priority., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The cerebellum is thought to be involved in cognitive functions in addition to its well established role in motor coordination and motor learning in humans. Cerebellin 1 (Cbln1) is predominantly expressed in cerebellar granule cells and plays a crucial role in the formation and function of parallel fiber-Purkinje cell synapses. Although genes encoding Cbln1 and its postsynaptic receptor, the delta2 glutamate receptor (GluD2), are suggested to be associated with autistic-like traits and many psychiatric disorders, whether such cognitive impairments are caused by cerebellar dysfunction remains unclear. In the present study, we investigated whether and how Cbln1 signaling is involved in non-motor functions in adult mice. We show that acquisition and retention/retrieval of cued and contextual fear memory were impaired in Cbln1-null mice. In situ hybridization and immunohistochemical analyses revealed that Cbln1 is expressed in various extracerebellar regions, including the retrosplenial granular cortex and the hippocampus. In the hippocampus, Cbln1 immunoreactivity was present at the molecular layer of the dentate gyrus and the stratum lacunosum-moleculare without overt mRNA expression, suggesting that Cbln1 is provided by perforant path fibers. Retention/retrieval, but not acquisition, of cued and contextual fear memory was impaired in forebrain-predominant Cbln1-null mice. Spatial learning in the radial arm water maze was also abrogated. In contrast, acquisition of fear memory was affected in cerebellum-predominant Cbln1-null mice. These results indicate that Cbln1 in the forebrain and cerebellum mediates specific aspects of fear conditioning and spatial memory differentially and that Cbln1 signaling likely regulates motor and non-motor functions in multiple brain regions., Significance Statement: Despites its well known role in motor coordination and motor learning, whether and how the cerebellum is involved in cognitive functions remains less clear. Cerebellin 1 (Cbln1) is highly expressed in the cerebellum and serves as an essential synaptic organizer. Although genes encoding Cbln1 and its receptor are associated with many psychiatric disorders, it remains unknown whether such cognitive impairments are caused by cerebellar dysfunction. Here, we show that Cbln1 is also expressed in the forebrain, including the hippocampus and retrosplenial granular cortex. Using forebrain- and cerebellum-predominant conditional Cbln1-null mice, we show that Cbln1 in the forebrain and cerebellum mediates specific aspects of fear conditioning and spatial memory differentially, indicating that Cbln1 signaling regulates both motor and non-motor functions in multiple brain regions., (Copyright © 2016 the authors 0270-6474/16/3611801-16$15.00/0.)

Ionotropic glutamate receptor (iGluR) family members are integrated into supramolecular complexes that modulate their location and function at excitatory synapses. However, a lack of structural information beyond isolated receptors or fragments thereof currently limits the mechanistic understanding of physiological iGluR signaling. Here, we report structural and functional analyses of the prototypical molecular bridge linking postsynaptic iGluR δ2 (GluD2) and presynaptic β-neurexin 1 (β-NRX1) via Cbln1, a C1q-like synaptic organizer. We show how Cbln1 hexamers "anchor" GluD2 amino-terminal domain dimers to monomeric β-NRX1. This arrangement promotes synaptogenesis and is essential for D: -serine-dependent GluD2 signaling in vivo, which underlies long-term depression of cerebellar parallel fiber-Purkinje cell (PF-PC) synapses and motor coordination in developing mice. These results lead to a model where protein and small-molecule ligands synergistically control synaptic iGluR function., (Copyright © 2016, American Association for the Advancement of Science.)

A 58-year-old man was diagnosed with accelerated phase chronic myelogenous leukemia (CML). He was treated with dasatinib and followed-up; 6 months later, he achieved a complete molecular response. Seventeen months after this therapy, he developed pancytopenia, and was examined. His diagnosis was Ph-negative acute myeloid leukemia (AML) with no karyotype abnormalities. He was administered two courses of induction chemotherapy, and during the first remission, he received allogeneic hematopoietic stem cell transplantation. Treatment with a tyrosine kinase inhibitor (TKI) achieved a successful outcome. However, approximately 10% of CML cases develop clonal cytogenetic changes in Ph-negative cells during TKI treatment, and rarely, cases of Ph-negative myelodysplastic syndrome or AML are reported. Furthermore, similar to our case, CML patients developing AML with Ph-negative and normal chromosome abnormalities have been reported. We suggest vigilant monitoring during TKI therapy and stress the importance of further analysis based on similar accumulated cases.

Neuronal networks are dynamically modified by selective synapse pruning during development and adulthood. However, how certain connections win the competition with others and are subsequently maintained is not fully understood. Here, we show that C1ql1, a member of the C1q family of proteins, is provided by climbing fibers (CFs) and serves as a crucial anterograde signal to determine and maintain the single-winner CF in the mouse cerebellum throughout development and adulthood. C1ql1 specifically binds to the brain-specific angiogenesis inhibitor 3 (Bai3), which is a member of the cell-adhesion G-protein-coupled receptor family and expressed on postsynaptic Purkinje cells. C1ql1-Bai3 signaling is required for motor learning but not for gross motor performance or coordination. Because related family members of C1ql1 and Bai3 are expressed in various brain regions, the mechanism described here likely applies to synapse formation, maintenance, and function in multiple neuronal circuits essential for important brain functions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Central venous catheter-related blood stream infections (CR-BSIs) are a serious complication in patients with hematological malignancies. However, it remains unclear whether there is a difference in the rate of CR-BSI associated with the conventional type of central venous catheters (cCVCs) and peripherally inserted CVCs (PICCs) in such patients. To address this question, we retrospectively investigated the incidence of CR-BSIs associated with PICCs versus cCVCs in patients with hematological malignancies. We used PICCs in all consecutive patients requiring CVC placement between February 2009 and February 2013. We compared the CR-BSI rate in patients with PICCs with that in patients with cCVCs treated between September 2006 and January 2009 (control group). Eighty-four patients received PICCs and 85 received cCVCs. The most common reason for removal due to catheter-related complications was CR-BSI. The CR-BSI rate in the PICC group was significantly lower than that in the cCVC group (PICCs: 1.23/1000 catheter days; cCVCs: 5.30/1000 catheter days; P < 0.01). Catheter-related complications other than CR-BSIs occurred at an extremely low rate in the PICC group. The median catheter-related complication-free survival duration was significantly longer in the PICC group than in the cCVC group. Our study shows that PICCs are useful in patients with hematological malignancies.

Following the introduction of rituximab, the long-term overall survival (OS) rate of advanced-stage follicular lymphoma (FL) cases was expected to improve after the introduction of rituximab: however, there is a lack of large-scale survey data in Asia due to the relatively low incidence of FL. We conducted a retrospective survey to assess the treatment outcomes in patients with newly diagnosed advanced-stage FL in 29 institutions in Hokkaido from January 2001 to December 2010. The total number of patients was 443 (men 47.6%, women 52.4%), with a median age of 55 years (range 20-80 years). Of the cases examined, 42.2% had stage III and 57.8% had stage IV disease. Furthermore, 62.5, 19.7, 9.2, 5.2, and 3.4% had performance statuses of 0, 1, 2, 3, and 4, respectively. The 5-year OS was 91.2%, and no survival plateau was observed. Seventeen patients experienced secondary malignancies (six hematological diseases and 11 solid cancers; 5-year probability, 4.2%). Eighteen patients experienced transformation (5-year probability, 4.5%). The overall survival at 5 years after therapy for transformation was 50%. Before the introduction of rituximab, the 5- to 10-year OS of advanced-stage FL patients in Japan was reported to be about 30-60%. Although these data are limited, improvement in OS has been observed in Japan during the rituximab era.