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Objective: The aim of this study is to characterise the molecular characteristics of NDM-producing Enterobacterales, which have been on the increase in recent years in Japan, where IMP-producing bacteria are dominant among carbapenemase-producing Enterobacterales., Methods: We collected 21 strains of NDM-producing Enterobacterales detected between 2015 and 2022 at five hospitals in Tokyo and performed illumina whole genome sequencing. For the seven selected strains, nanopore long-read sequencing was also performed to characterise the plasmids harbouring bla NDM ., Results: Fourteen strains were Escherichia coli and all carried bla NDM-5 . Among these strains, eight and three were sequence type (ST) 410 and ST167, respectively, and both groups of strains were spread clonally in different hospitals. Two strains of Klebsiella pneumoniae ST147 carrying bla NDM-1 were detected in a hospital, and these strains had also spread clonally. The remainder included Enterobacter hormaechei, Klebsiella quasipneumoniae, Citrobacter amalonaticus, and Klebsiella michiganensis. Plasmid analysis revealed that an identical IncX3 plasmid harbouring bla NDM-5 was shared among four strains of different bacterial species (E. coli, C. amalonaticus, K. michiganensis, and E. hormaechei) detected at the same hospital. In addition, a Klebsiella quasipneumoniae strain detected at a different hospital also carried an IncX3 plasmid with a similar genetic structure., Conclusions: Nosocomial spread of multiple multidrug-resistant global clones and transmission of IncX3 plasmids harbouring bla NDM-5 among multiple species were detected as the major pathways of spread of NDM-producing Enterobacterales in Tokyo. Early detection of carriers and measures to prevent nosocomial spread are important to prevent further spread of NDM-producing organisms., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Although we previously reported that some meningococcal isolates in Japan were resistant to penicillin (PCG) and ciprofloxacin (CIP), the antibiotic susceptibilities of Neisseria meningitidis isolates obtained in Japan remained unclear. In the present study, 290 N . meningitidis isolates in Japan between 2003 and 2020 were examined for the sensitivities to eight antibiotics (azithromycin, ceftriaxone, ciprofloxacin, chloramphenicol, meropenem, minocycline, penicillin, and rifampicin). All isolates were susceptible to chloramphenicol, ceftriaxone, meropenem, minocycline, and rifampicin while two were resistant to azithromycin. Penicillin- and ciprofloxacin-resistant and -intermediate isolates (PCG R , CIP R , PCG I and CIP I , respectively) were also identified. Based on our previous findings from whole genome sequence analysis, approximately 40% of PCG I were associated with ST-11026 and cc2057 meningococci, both of which were unique to Japan. Moreover, the majority of ST-11026 meningococci were CIP R or CIP I . Sensitivities to PCG and CIP were closely associated with genetic features, which indicated that, at least for Japanese meningococcal isolates, PCG R/I or CIP I/R would be less likely to be horizontally conferred from other neisserial genomes by transferring of the genes responsible ( penA and gyrA genes, respectively), but rather that ancestral N. meningitidis strains conferring PCG R/I or CIP I/R phenotypes clonally disseminated in Japan., Competing Interests: The authors declare no conflict of interest.

Neisseria meningitidis causes invasive meningococcal diseases and has also been identified as a causative agent of sexually transmitted infections, including urethritis. Unencapsulated sequence type 11 meningococci containing the gonococcal aniA-norB locus and belonging to the United States N. meningitidis urethritis clade (US_NmUC) are causative agents of urethral infections in the United States, predominantly among men who have sex with men. We identified 2 subtypes of unencapsulated sequence type 11 meningococci in Japan that were phylogenetically close to US_NmUC, designated as the Japan N. meningitidis urethritis clade (J_NmUC). The subtypes were characterized by PCR, serologic testing, and whole-genome sequencing. Our study suggests that an ancestor of US_NmUC and J_NmUS urethritis-associated meningococci is disseminated worldwide. Global monitoring of urethritis-associated N. meningitidis isolates should be performed to further characterize microbiologic and epidemiologic characteristics of urethritis clade meningococci.

Klebsiella variicola is a pathogen that is increasingly recognized as being associated with human infections, but the methods available to clinical microbiology laboratories for accurate identification are limited. In this study, we assessed the accuracy of identification of K. variicola by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry using genetic identification with multiplex PCR as the reference method. Antimicrobial susceptibilities and virulence of K. variicola strains were also investigated. Fifty-five Klebsiella pneumoniae, 26 K. variicola , and 2 Klebsiella quasipneumoniae clinical strains were used for evaluation. Both MALDI Biotyper with library version 9 and Klebsiella MALDI TypeR, a web-based species identification tool using MALDI-TOF data, accurately identified all K. variicola strains. In addition, two strains of K. quasipneumoniae were accurately identified with Klebsiella MALDI TypeR. Whole-genome sequencing confirmed the accurate identification to the subspecies level by Klebsiella MALDI TypeR for four strains (two strains each of K. variicola subsp. variicola and K. quasipneumoniae subsp. similipneumoniae ). While 13 strains, 3 strains, and 1 strain of K. pneumoniae showed nonsusceptibility to ampicillin-sulbactam, ceftriaxone, and meropenem, respectively, all strains of K. variicola were susceptible to all tested antimicrobial agents. Although two K. variicola strains were positive for the string test, no K. variicola strains harbored any of the genes associated with hypervirulence of K. pneumoniae. Accurate identification of the K. pneumoniae complex, including K. variicola , by MALDI-TOF in clinical microbiology laboratories is expected to clarify the clinical characteristics of each species in the future. IMPORTANCE Recent widespread use of bacterial whole-genome sequencing analysis has resulted in the proposal of novel bacterial species and reclassification of taxonomy. Accurate methods for identification of bacterial species in clinical microbiology laboratories are essential to accumulate information on the clinical characteristics of each bacterial species. Klebsiella variicola is a member of the Klebsiella pneumoniae complex, and its association with human infections has been increasingly recognized, but accurate identification methods approved for use in clinical microbiology laboratories have been limited thus far. The findings of the present study suggest that K. variicola can be accurately identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry using updated library or web-based identification tools. Accurate identification will promote exploration of clinical characteristics of K. variicola .

Objectives: This study examined the immediate effects of neuromuscular electrical stimulation (NMES) on the dynamics of oropharyngeal structure and laryngeal vestibular closure (LVC) in healthy subjects., Methods: Ten healthy male volunteers participated in this controlled, before-and-after, videofluoroscopic swallowing pilot study. The study was conducted in four phases (each performed twice): (1) saliva swallow (SS) before evaluation (BEFORE), (2) NMES while at rest with no SS (NMES AT REST), (3) SS during NMES (DURING NMES), and (4) SS to examine the aftereffects of NMES (AFTER). We measured distances that oropharyngeal structures moved in the NMES AT REST phase, and we analyzed the kinematics of saliva swallowing primarily in the BEFORE and AFTER phases., Results: Four changes in the morphology of the oropharyngeal structure caused by NMES AT REST were statistically significant: anterior-upward displacement of the hyoid bone and larynx, stretch of the laryngeal vestibule, and posterior ridge of the tongue root. Regarding the kinematics measured during SS, although there was no significant change in LVC reaction times, LVC duration in the AFTER phase was significantly longer than BEFORE. Regarding maximal displacement of the hyoid bone, there was significantly greater movement AFTER than BEFORE. As additional exploratory outcomes, the velocity of hyoid bone movement was significantly slower, and the hyoid-to-larynx approximation was significantly smaller, DURING NMES than AFTER., Conclusions: Longer duration of LVC might be caused by adaptive learning with NMES-induced structural changes in the oropharynx. Further clinical studies are warranted to determine whether this approach improves dysphagia, which impairs LVC., Competing Interests: CONFLICTS OF INTEREST: The electrical stimulator used for the intervention was on loan from Ampcare, LLC. The electrodes used in the intervention were provided free of charge by Ito Co., Ltd. No funding, gratuity, or labor was provided by Ampcare or Ito, and the study was conducted without influence from the manufacturers., (2022 The Japanese Association of Rehabilitation Medicine.)

Background: Aureobasidium melanigenum is a ubiquitous dematiaceous fungus that rarely causes invasive human infections. Here, we present a case of Aureobasidium melanigenum bloodstream infection in a 20-year-old man with long-term catheter use., Case Presentation: A 20-year-old man receiving home care with severe disabilities due to cerebral palsy and short bowel syndrome, resulting in long-term central venous catheter use, was referred to our hospital with a fever. After the detection of yeast-like cells in blood cultures on day 3, antifungal therapy was initiated. Two identification tests performed at a clinical microbiological laboratory showed different identification results: Aureobasidium pullulans from matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and Cryptococcus albidus from a VITEK2 system. Therefore, we changed the antifungal drug to liposomal amphotericin B. The fungus was identified as A. melanigenum by DNA sequence-based analysis. The patient recovered with antifungal therapy and long-term catheter removal., Conclusion: It is difficult to correctly identify A. melanigenum by routine microbiological testing. Clinicians must pay attention to the process of identification of yeast-like cells and retain A. melanigenum in cases of refractory fungal infection., (© 2022. The Author(s).)

Background: Protothecosis is a rare infection in humans and animals caused by the achlorophyllic algae Prototheca species. More than half of the protothecosis cases are cutaneous infections, and most cases are observed in immunocompromised individuals., Case Presentation: We report a case of Prototheca wickerhamii infection in the mucosa of the pharynx in a 53-year-old immunocompetent woman with an incidentally found mass lesion at the left tongue base. Histopathological findings of the mass lesion suggested cryptococcosis, but P. wickerhamii was identified from the oropharynx scrape culture based on DNA sequencing. After surgical resection, fosfluconazole treatment was initiated, and subsequently, treatment was switched to topical amphotericin B. The residual mass lesion did not deteriorate during the 4-month antifungal treatment and 1-year observational period., Conclusions: Prototheca species can be easily misdiagnosed as yeasts because of their morphological and pathological similarities. Prototheca, in addition to Cryptococcus should be considered if slow-growing, large Gram-positive organisms are encountered. Lactophenol cotton blue staining of the colony helps distinguish these organisms. Further study is needed to determine the appropriate treatment according to the infection focus., (© 2022. The Author(s).)

Although Shewanella spp. are most frequently isolated from marine environments; more rarely, they have been implicated in human infections. Shewanella spp. are also recognized as the origin of genes for carbapenem-hydrolyzing class D β-lactamases. Due to the spread globally among Enterobacterales in recent years, risk assessments of both clinical and environmental Shewanella strains are urgently needed. In this study, we analyzed the whole-genome sequences of 10 clinical isolates and 13 environmental isolates of Shewanella spp. and compared them with those of Shewanella species strains registered in public databases. In addition, the levels of bla OXA-55-like transcription and β-lactamase activity of a carbapenem-resistant Shewanella algae isolate were compared with those of carbapenem-susceptible S. algae clade isolates. All clinical isolates were genetically identified as S. algae clade (S. algae, Shewanella chilikensis, and Shewanella carassii), whereas all but one of the environmental isolates were identified as various Shewanella spp. outside the S. algae clade. Although all isolates of the S. algae clade commonly possessed an approximately 12,500-bp genetic region harboring bla OXA-55-like , genetic structures outside this region were different among species. Among S. algae clade isolates, only one showed carbapenem resistance, and this isolate showed a high level of bla OXA-55-like transcription and β-lactamase activity. Although this study documented the importance of the S. algae clade in human infections and the relationship between enhanced production of OXA-55-like and resistance to carbapenems in S. algae, further studies are needed to elucidate the generalizability of these findings. IMPORTANCE Shewanella spp., which are known to carry chromosomally located bla OXA genes, have mainly been isolated from marine environments; however, they can also cause infections in humans. In this study, we compared the molecular characteristics of clinical isolates of Shewanella spp. with those originating from environmental sources. All 10 clinical isolates were genetically identified as members of the Shewanella algae clade (S. algae, S. chilikensis , and S. carassii ); however, all but one of the 13 environmental isolates were identified as Shewanella species members outside the S. algae clade. Although all the S. algae clade isolates possessed an approximately 12,500-bp genetic region harboring bla OXA-55-like , only one isolate showed carbapenem resistance. The carbapenem-resistant isolate showed a high level of bla OXA-55-like transcription and β-lactamase activity compared with the carbapenem-susceptible isolates. To confirm the clinical significance and antimicrobial resistance mechanisms of the S. algae clade members, analysis involving more clinical isolates should be performed in the future.

Background: Mycobacterium marseillense is a new species of the Mycobacterium avium complex. There has been only a few human infections caused by M. marseillense worldwide., Case Presentation: We report a case of tenosynovitis caused by M. marseillense in an immunocompetent adult in Japan. The isolate was initially identified as M. intracellulare using commercial real time polymerase chain reaction assays and later identified as M. marseillense with sequencing of the the rpoB and hsp65 regions, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)., Conclusions: This is the first case reporting on M. marseillense generating a positive result with commercial real time PCR assays targeting MAC. Human infections associated by M. marseillense might be underreported due to similarities with Mycobacterium intracellulare. To accurately identify M. marseillese, MALDI-TOF MS might provide a rapid and reliable method., (© 2021. The Author(s).)

Background: Staphylococcus schleiferi is a gram-positive pathogenic coccus which causes canine skin and ear infections. Only four cases of human infection caused by Staphylococcus schleiferi subspecies coagulans have been reported. Herein, we present the first case of catheter-related bloodstream infection caused by S. schleiferi subspecies coagulans., Case Presentation: A 62-year-old Japanese man was admitted to our hospital for examination of sigmoid colon tumor. During hospitalization, he had fever, shaking chills, and swelling at the peripheral venous catheter insertion site. Two sets of blood cultures were positive for S. schleiferi subspecies coagulans which was confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), 16S ribosomal RNA sequencing and the coagulase test. The patient was successfully treated without relapse., Conclusion: To our knowledge, this is the first report of catheter-related bloodstream infection caused by S. schleiferi subspecies coagulans. S. schleiferi subsp. coagulans can be pathogenic in humans, and MALDI-TOF MS can contribute to accurate identification of S. schleiferi subspecies coagulans., (© 2021. The Author(s).)

Japan has not been able to eliminate rubella; as a result, the large rubella epidemic has occurred. Considering the complicated history of the vaccine policy in Japan, some susceptible populations became infected with rubella, resulting in an outbreak. We conducted a large serosurveillance against rubella in Chiba city after initiating free rubella-specific antibody testing and an immunization campaign during 2018-2019. The total number of rubella specific antibody tests that was conducted in the nationwide campaign and Chiba city original campaign was 8277 and 6104, respectively. The proportion of participants with an antibody titer of ≤1:16 using the hemagglutination inhibition (HI) test was higher in those in their 20-30s. On the contrary, the proportion of participants with an antibody titer of <1:8 using the HI test was higher in men in their 40-50s. This discrepancy possibly reflects the complicated history of the vaccine policy. The number of participants in the nationwide immunization campaign in this city was 1517, whereas that in the Chiba city campaign was 3607. The Chiba city campaign was effective against women in their 20-30s (child-bearing generation); however, the nationwide campaign was not sufficiently effective against men in their 40-50s because many workers were did not visit medical facilities to receive the measles-rubella vaccine.

Staphylococcus condimenti is a Gram-positive coccus that was first isolated from soy sauce mash. Only four cases of human S. condimenti infections have been reported to date. We herein report the first case of spondylodiscitis caused by S. condimenti. A 72-year-old Japanese man complaining of lower back pain and numbness in his legs was diagnosed with spondylodiscitis. A computed tomography (CT)-guided biopsy was performed. A culture of the intravertebral disc aspirate yielded S. condimenti. The result was confirmed using gene sequencing methods. The patient was successfully treated without relapse. This case shows that S. condimenti can be pathogenic and cause invasive infection.

Liquid marbles (LMs) have recently shown a great promise as microbioreactors to construct self-supported aqueous compartments for chemical and biological reactions. However, the evaporation of the inner aqueous liquid core has limited their application, especially in studying cellular functions. Hydrogels are promising scaffolds that provide a spatial environment suitable for three-dimensional cell culture. Here, we describe the fabrication of redox-responsive hydrogel marbles (HMs) as a three-dimensional cell culture platform. The HMs are prepared by introducing an aqueous mixture of a tetra-thiolated polyethylene glycol (PEG) derivative, thiolated gelatin (Gela-SH), horseradish peroxidase, a small phenolic compound, and human hepatocellular carcinoma cells (HepG2) to the inner aqueous phase of LMs. Eventually, HepG2 cells are encapsulated in the HMs then immersed in culture media, where they proliferate and form cellular spheroids. Experimental results show that the Gela-SH concentration strongly influences the physicochemical and microstructure properties of the HMs. After 6 days in culture, the spheroids were recovered from the HMs by degrading the scaffold, and examination showed that they had reached up to about 180 μm in diameter depending on the Gela-SH concentration, compared with 60 μm in conventional HMs without Gela-SH. After long-term culture (over 12 days), the liver-specific functions (secretion of albumin and urea) and DNA contents of the spheroids cultured in the HMs were elevated compared with those cultured in LMs. These results suggest that the developed HMs can be useful in designing a variety of microbioreactors for tissue engineering applications., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Background: Difficult-to-treat infections caused by rapidly growing mycobacteria (RGM) are increasingly observed in clinical settings. However, studies on antimicrobial susceptibilities and effective treatments against RGM in Japan are limited., Methods: We conducted susceptibility testing of potential antimicrobial agents, including tigecycline and tebipenem, against RGM. Clinical RGM isolates were collected from a university hospital in Japan between December 2010 and August 2013. They were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the sequencing of 16S rRNA, rpoB, and hsp65 genes. The samples were utilized for susceptibility testing using 16 antimicrobials, with frozen broth microdilution panels., Results: Forty-two isolates were obtained: 13, Mycobacterium abscessus complex; 12, Mycobacterium chelonae; 9, Mycobacterium fortuitum; and 8, M. fortuitum group species other than M. fortuitum. Different antimicrobial susceptibility patterns were observed between RGM species. Clarithromycin-susceptible strain rates were determined to be 0, 62, and 100% for M. fortuitum, M. abscessus complex, and M. chelonae, respectively. M. abscessus complex (100%) and >80% M. chelonae isolates were non-susceptible, while 100% M. fortuitum group isolates were susceptible to moxifloxacin. Linezolid showed good activity against 77% M. abscessus complex, 89% M. fortuitum, and 100% M. chelonae isolates. Regardless of species, all tested isolates were inhibited by tigecycline at very low minimal inhibitory concentrations (MICs) of ≤0.5 μg/mL. MICs of tebipenem, an oral carbapenem, were ≤4 μg/mL against all M. fortuitum group isolates., Conclusions: Our study demonstrates the importance of correct identification and antimicrobial susceptibility testing, including the testing of potential new agents, in the management of RGM infections.

Objective: To examine the molecular basis of aromatase expression in stromal cell culture from endometriotic chocolate cysts., Design: Prospective study., Setting: Department of Obstetrics and Gynecology and Department of Biosignaling, Tottori University, Yonago Japan., Patient(s): Thirty women, selected randomly, who underwent laparoscopy (n = 18) or laparotomy (n = 12)., Intervention(s): Endometrial and endometriotic stromal cells were obtained from the uterus and chocolate cyst lining of the ovary., Main Outcome Measure(s): Estradiol concentrations in the culture media were measured by means of enzyme immunoassay. Aromatase expression was examined by quantitative real-time polymerase chain reaction. Promoter usage was examined using unique exon I (PII, I.1, I.3, I.4, I.5, and I.6) and exon II primers. To determine the effect of 5-aza-deoxycytidine on endometrial stromal cells, the cells were treated with the agent for 96 hours., Result(s): Endometriotic cells secreted a marginal level of estradiol into the culture media, but adding testosterone to the culture produced a pronounced level of estradiol. In endometrial cells, estradiol production was far less efficient than in endometriotic cells even after adding testosterone. Real-time polymerase chain reaction analyses demonstrated the up-regulation of aromatase messenger RNA (mRNA) expression in endometriotic cells. Three proximal promoters, PII, 1.3, and 1.6, drove mRNA expression. In endometrial cells where a marginal level of aromatase mRNA expression was observed, the same promoters as those in the endometriotic cells were used. To determine the role of epigenetic modification of aromatase gene expression in endometriotic cells, endometrial cells were treated with 5-aza-deoxycytidine, which markedly enhanced aromatase mRNA expression, depending on the same proximal promoters as those in endometriotic cells., Conclusion(s): An epigenetic disorder may play a role in the pathophysiology of endometriosis.

Objective: To evaluate the influence of peroxisome proliferator-activated receptor-gamma (PPAR gamma) ligand (pioglitazone) on tumor necrosis factor-alpha (TNF-alpha)-induced interleukin-8 (IL-8) expression in endometriotic stromal cells (ESCs) and on proliferation of ESCs., Design: Prospective study., Setting: Department of Obstetrics and Gynecology, Tottori University Hospital, Yonago, Japan., Patient(s): Twenty-seven patients who underwent laparoscopic surgery., Intervention(s): The ESCs were obtained from the chocolate cyst linings of ovaries., Main Outcome Measure(s): The expression of PPAR gamma gene and protein was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry. We determined the effect of pioglitazone on the production of TNF-alpha-induced IL-8 protein in culture supernatant of ESCs using ELISA. The effect of pioglitazone on TNF-alpha-induced proliferation of ESCs was evaluated by 5-bromo-2'-deoxyuridine proliferation assay. The activation of nuclear factor (NF)-kappaB in ESCs was evaluated by Western blot analyses and NF-kappaB transcription factor assays., Result(s): Immunocytochemistry and RT-PCR revealed the expression of PPAR gamma gene and protein in ESCs. The PPAR gamma protein was predominantly located in the cell nucleus. Measurement of IL-8 protein by ELISA showed that adding TNF-alpha (100 pg/mL) significantly increased IL-8 protein. Treating ESCs with 0.1-10 microM of pioglitazone significantly reduced the TNF-alpha-induced IL-8 production. The presence of 0.1-10 microM of pioglitazone significantly suppressed growth of ESCs. The TNF-alpha increased the expression of phosphorylation of inhibitor kappaB (I kappaB). Adding pioglitazone (10 microM) did not influence the expression of phosphorylated inhibitor kappaB (I kappaB). The TNF-alpha markedly increased the intranuclear concentration of p65, and adding pioglitazone (10 microM) significantly reduced the concentration of p65., Conclusion(s): The present study demonstrates for the first time that PPAR gamma is expressed in ESCs, and that pioglitazone reduced IL-8 secretion and the proliferation of ESCs. The PPAR gamma ligand may be an attractive therapeutic agent for endometriosis.

Ovarian surface epithelial cells (OSEs) are considered to be the common source of endometrioma and epithelial ovarian cancer. The present study reveals that keratinocyte growth factor receptor (KGFR) messenger RNA was expressed in OSEs of endometriomas but not in those of normal ovaries, suggesting that autocrine KGF/KGFR and paracrine fibroblast growth factor 10/KGFR signaling loops may be involved with the proliferation in OSEs of endometrioma.

Lipid vesicles are potentially useful as microcapsules for drug and/or gene delivery. We developed cationic lipid vesicles consisting mainly of sorbitan monooleate (Span 80) and cationic peptide lipid (CPL), and evaluated the CPL vesicles as gene transfection vectors. The optimum CPL concentration for gene transfection into HeLa cells was found to be 20 wt % of total lipid, and such CPL vesicles did not exhibit significant cytotoxicity. Co-culture of Poly-L-lysine and plasmids prior to making CPL vesicle-plasmid complexes was effective. Lipofection using LipofectAMINE was suppressed in 10% serum-supplemented medium. The transfection efficiency of 20 wt % CPL vesicles, however, was not affected by serum in the medium when plasmids were treated with poly-L-lysine.

Eucheuma serra agglutinin (ESA) derived from a marine red alga, Eucheuma serra, is a lectin that specifically binds to mannose-rich carbohydrate chains. ESA is a monomeric molecule, with a molecular weight of29,000. ESA induced cell death against several cancer cell lines, such as colon cancer Colo201 cells and cervix cancer HeLa cells. DNA ladder detection and the induction of caspase-3 activity suggested that the cell death induced by ESA against cancer cells was apoptosis. ESA bound to the cell surface of Colo201 cells in the sugar chain dependent manner. This means that the binding of ESA to the cell surface is specific for mannose-rich sugar chains recognized by ESA. The binding of ESA to the cell surface of Colo201 cells was slightly suppressed by the high concentrations of serum because of the competition with serum components possessing the mannose-rich sugar chain motifs. On the other hand, a lipid vesicle is a very useful microcapsule constructed by multilamellar structure,and adopted as drug or gene carrier. ESA was immobilized on the surface of the lipid vesicles to apply the lipid vesicles to cancer specific drug delivery system. ESA-immobilized lipid vesicles were effectively bound to cancer cell lines compared with plane vesicles.

We report an 18-month-old Japanese girl with purely epithelioid leiomyosarcoma presenting as a huge intraabdominal mass. The patient had been well from birth and had shown no signs of immunodeficiency. She was negative for human immunodeficiency virus. Blood examination revealed elevated serum neuron specific enolase (NSE). Histologically, the tumor was comprised of solid growths of round or polygonal cells with vesicular nuclei and often vacuolated cytoplasm rich in glycogen. The tumor cells were positive for vimentin, NSE, and MIC2, and were negative for desmin and neurofilament. The age, clinical presentation, and histologic findings mostly favored Ewing's sarcoma/primitive neuroectodermal tumor. Silver stain, however, demonstrated well-developed reticulin fibers often outlining individual tumor cells. An expanded panel of immunostains showed that the tumor cells were intensely positive for smooth muscle actin, and ultrastructural study revealed abundant fine cytoplasmic filaments with focal subsarcolemmal densities, various amounts of glycogen, and irregularly arranged, thick basal lamina. The diagnosis of epithelioid leiomyosarcoma was made. Following reduction in tumor size by chemotherapy, the serum NSE level was normalized. From the surgical finding, the primary site was presumed to be the urachus or the urinary bladder dome. Although extremely rare, epithelioid leiomyosarcoma should be added in the list of differential diagnoses of pediatric "round cell tumors."