Pdx1 and Ngn3 overexpression enhances pancreatic differentiation of mouse ES cell-derived endoderm population

In order to define the molecular mechanisms regulating the specification and differentiation of pancreatic β-islet cells, we investigated the effect of upregulating Pdx1 and Ngn3 during the differentiation of the β-islet-like cells from murine embryonic stem (ES) cell-derived activin induced-endoderm. Induced overexpression of Pdx1 resulted in a significant upregulation of insulin (Ins1 and Ins2), and other pancreas-related genes. To enhance the developmental progression from the pancreatic bud to the formation of the endocrine lineages, we induced the overexpression express of Ngn3 together with Pdx1. This combination dramatically increased the level and timing of maximal Ins1 mRNA expression to approximately 100% of that found in the βTC6 insulinoma cell line. Insulin protein and C-peptide expression was confirmed by immunohistochemistry staining. These inductive effects were restricted to c-kit(+) endoderm enriched EB-derived populations suggesting that Pdx1/Ngn3 functions after the specification of pancreatic endoderm. Although insulin secretion was stimulated by various insulin secretagogues, these cells had only limited glucose response. Microarray analysis was used to evaluate the expression of a broad spectrum of pancreatic endocrine cell-related genes as well as genes associated with glucose responses. Taken together, these findings demonstrate the utility of manipulating Pdx1 and Ngn3 expression in a stage-specific manner as an important new strategy for the efficient generation of functionally immature insulin-producing β-islet cells from ES cells.     

Synthesis of novel heterobranched beta-cyclodextrins having beta-D-N-acetylglucosaminyl-maltotriose on the side chain

From a mixture of N-acetylglucosaminyl-beta-cyclodextrin (GlcNAc-betaCD) and lactose, beta-D-galactosyl-GlcNAc-betaCD (Gal-GlcNAc-betaCD) was synthesized by the transfer action of beta-galactosidase. GlcNAc-maltotriose (Glc3) and Gal-GlcNAc-Glc3 were produced with hydrolysis of GlcNAc-betaCD by cyclodextrin glycosyltransferase, and Gal-GlcNAc-betaCD by bacterial saccharifying alpha-amylase respectively. Finally, GlcNAc-Glc3-betaCD and Gal-GlcNAc-Glc3-betaCD were synthesized in 5.2% and 3.5% yield when Klebsiella pneumoniae pullulanase was incubated with the mixture of GlcNAc-Glc(3) and betaCD, or Gal-GlcNAc-Glc3 and betaCD respectively. The structures of GlcNAc-Glc3-betaCD and Gal-GlcNAc-Glc3-betaCD were analyzed by FAB-MS and NMR spectroscopy and identified as 6-O-alpha-(6(3)-O-beta-D-N-acetylglucosaminyl-maltotriosyl)-betaCD, and 6-O-alpha-(4-O-beta-D-galactopyranosyl-6(3)-O-beta-D-N-acetylglucosaminyl-maltotriosyl)-betaCD respectively.     

Breakdown of mucosal immunity in the gut and resultant systemic sensitization by oral antigens in a murine model for systemic lupus erythematosus

Secreted IgA plays a pivotal role in the mucosal immunity to maintain the front line of body defense. We found that the level of fecal IgA was dramatically decreased in aged (NZB x NZW)F(1) (BWF(1)) mice developing lupus nephritis, whereas levels in similarly aged New Zealand Black (NZB) and New Zealand White (NZW) mice remained unchanged compared with young mice. The number of cells obtained from Peyer's patches was markedly decreased in aged BWF(1) mice. Aged BWF(1) mice showed increased susceptibility to pathogenic bacterial infection. Furthermore, oral administration of OVA failed to inhibit secondary IgG response induced by systemic immunization, suggesting defective oral tolerance in aged BWF(1) mice. A significant amount of orally administered OVA was incorporated directly into the intestinal lamina propria in aged BWF(1) mice whereas it was mainly localized in subepithelial domes and interfollicular region in Peyer's patches in young mice. T cells obtained from renal and pulmonary lymph nodes of aged BWF(1) mice that had been orally administered with OVA showed an Ag-specific T cell proliferation, whereas those from young BWF(1), aged NZB, and aged NZW mice did not. Interestingly, aerosol exposure to OVA of aged BWF(1) mice, which had been orally administered with the same Ag, provoked an eosinophil infiltration in the lung. These results demonstrate that mucosal immunity in the gut is impaired and oral Ags induce systemic sensitization instead of oral tolerance in the development of murine lupus.     

Mutual regulation of protein-tyrosine phosphatase 20 and protein-tyrosine kinase Tec activities by tyrosine phosphorylation and dephosphorylation

PTP20, also known as HSCF/protein-tyrosine phosphatase K1/fetal liver phosphatase 1/brain-derived phosphatase 1, is a cytosolic protein-tyrosine phosphatase with currently unknown biological relevance. We have identified that the nonreceptor protein-tyrosine kinase Tec-phosphorylated PTP20 on tyrosines and co-immunoprecipitated with the phosphatase in a phosphotyrosine-dependent manner. The interaction between the two proteins involved the Tec SH2 domain and the C-terminal tyrosine residues Tyr-281, Tyr-303, Tyr-354, and Tyr-381 of PTP20, which were also necessary for tyrosine phosphorylation/dephosphorylation. Association between endogenous PTP20 and Tec was also tyrosine phosphorylation-dependent in the immature B cell line Ramos. Finally, the Tyr-281 residue of PTP20 was shown to be critical for deactivating Tec in Ramos cells upon B cell receptor ligation as well as dephosphorylation and deactivation of Tec and PTP20 itself in transfected COS7 cells. Taken together, PTP20 appears to play a negative role in Tec-mediated signaling, and Tec-PTP20 interaction might represent a negative feedback mechanism.     

小鼠雌性生殖系统电穿孔与微泡增强超声波转EGFP基因的研究(简报)

非病毒载体转基因法,如注射裸DNA或脂质体转染,不产生细胞毒性,但除了肌肉组织外其他组织的转导效率均不高。电脉冲可使细胞膜产生临时的微孔允许一些分子通过,因此应用此方法可将药物或基因转入动物组织。电穿孔常用于培养的细胞转基因,理论上,低强度、长脉冲,或高强度、短脉冲有利于电转导,选择适合的参数是电转导的关键。本实验比较了不同电压和脉冲时间对小鼠卵巢在体转入绿色荧光蛋白基因的效果,确定了最适的电转导参数,为卵巢疾病的药物、基因治疗和研究卵泡发育中的基因调控提供了实验手段。超声波是临床常用的诊断方法,对人体无害…     

小鼠雌性生殖系统电穿孔与微泡增强超声波转EG即基因的研究（简报）

非病毒载体转基因法。如注射裸DNA或脂质体转染,不产生细胞毒性。但除了肌肉组织外其他组织的转导效率均不高。电脉冲可使细胞膜产生临时的微孔允许一些分子通过。因此应用此方法可将药物或基因转人动物组织。电穿孔常用于培养的细胞转基因,理论上,低强度、长脉冲。或高强度、短脉冲有利于电转导。选择适合的参数是电转导的关键。本实验比较了不同电压和脉冲时间对小鼠卵巢在体转入绿色荧光蛋白基因的效果．确定了最适的电转导参数。为卵巢疾病的药物、基因治疗和研究卵泡发育中的基因调控提供了实验手段。     

Apoptotic cells activate NKT cells through T cell Ig-like mucin-like-1 resulting in airway hyperreactivity

T cell Ig-like mucin-like-1 (TIM-1) is an important asthma susceptibility gene, but the immunological mechanisms by which TIM-1 functions remain uncertain. TIM-1 is also a receptor for phosphatidylserine (PtdSer), an important marker of cells undergoing programmed cell death, or apoptosis. We now demonstrate that NKT cells constitutively express TIM-1 and become activated by apoptotic cells expressing PtdSer. TIM-1 recognition of PtdSer induced NKT cell activation, proliferation, and cytokine production. Moreover, the induction of apoptosis in airway epithelial cells activated pulmonary NKT cells and unexpectedly resulted in airway hyperreactivity, a cardinal feature of asthma, in an NKT cell-dependent and TIM-1-dependent fashion. These results suggest that TIM-1 serves as a pattern recognition receptor on NKT cells that senses PtdSer on apoptotic cells as a damage-associated molecular pattern. Furthermore, these results provide evidence for a novel innate pathway that results in airway hyperreactivity and may help to explain how TIM-1 and NKT cells regulate asthma.     

Naturally arising CD4+CD25+ regulatory T cells suppress the expansion of colitogenic CD4+CD44highCD62L- effector memory T cells

Naturally arising CD4+CD25+ regulatory T (T(R)) cells have been shown to prevent and cure murine T cell-mediated colitis. However, their exact mechanism of controlling colitogenic memory CD4+ T cells in in vivo systems excluding the initial process of naive T cell activation and differentiation has not been examined to date. Using the colitogenic effector memory (T(EM)) CD4+ cell-mediated colitis model induced by adoptive transfer of colitogenic CD4+CD44(high)CD62L(-) lamina propria (LP) T cells obtained from colitic CD4+CD45RB(high) T cell-transferred mice, we have shown in the present study that CD4+CD25+ T(R) cells are able not only to suppress the development of colitis, Th1 cytokine production, and the expansion of colitogenic LP CD4+ T(EM) cells but also to expand these cells by themselves extensively in vivo. An in vitro coculture assay revealed that CD4+CD25+ T(R) cells proliferated in the presence of IL-2-producing colitogenic LP CD4+ T(EM) cells at the early time point (48 h after culture), followed by the acquisition of suppressive activity at the late time point (96 h after culture). Collectively, these data suggest the distinct timing of the IL-2-dependent expansion of CD4+CD25+ T(R) cells and the their suppressive activity on colitogenic LP CD4+ T(EM) cells.     

Pheromone-Responsive Conjugative Vancomycin Resistance Plasmids in Enterococcus faecalis Isolates from Humans and Chicken Feces

The drug resistances and plasmid contents of a total of 85 vancomycin-resistant enterococcus (VRE) strains that had been isolated in Korea were examined. Fifty-four of the strains originated from samples of chicken feces, and 31 were isolated from hospital patients in Korea. Enterococcus faecalis KV1 and KV2, which had been isolated from a patient and a sample of chicken feces, respectively, were found to carry the plasmids pSL1 and pSL2, respectively. The plasmids transferred resistances to vancomycin, gentamicin, kanamycin, streptomycin, and erythromycin to E. faecalis strains at a high frequency of about 10⁻³ per donor cell during 4 hours of broth mating. E. faecalis strains containing each of the pSL plasmids formed clumps after 2 hours of incubation in broth containing E. faecalis FA2-2 culture filtrate (i.e., the E. faecalis sex pheromone), and the plasmid subsequently transferred to the recipient strain in a 10-min short mating in broth, indicating that the plasmids are responsive to E. faecalis pheromones. The pSL plasmids did not respond to any of synthetic pheromones for the previously characterized plasmids. The pheromone specific for pSL plasmids has been designated cSL1. Southern hybridization analysis showed that specific FspI fragments from each of the pSL plasmids hybridized with the aggregation substance gene (asa1) of the pheromone-responsive plasmid pAD1, indicating that the plasmids had a gene homologous to asa1. The restriction maps of the plasmids were identical, and the size of the plasmids was estimated to be 128.1 kb. The plasmids carried five drug resistance determinants for vanA, ermB, aph(3′), aph(6′), and aac(6′)/aph(2′), which encode resistance to vancomycin, erythromycin, kanamycin, streptomycin, and gentamicin/kanamycin, respectively. Nucleotide sequence analyses of the drug resistance determinants and their flanking regions are described in this report. The results described provide evidence for the exchange of genetic information between human and animal (chicken) VRE reservoirs and suggest the potential for horizontal transmission of multiple drug resistance, including vancomycin resistance, between farm animals and humans via a pheromone-responsive conjugative plasmid.     

Proton beam therapy with concurrent chemotherapy is feasible in children with newly diagnosed rhabdomyosarcoma

BackgroundThe optimal treatment for rhabdomyosarcoma (RMS) requires multidisciplinary treatment with chemotherapy, surgery, and radiotherapy. Surgery and radiotherapy are integral to the local control (LC) of RMS. However, postsurgical and radiotherapy-related complications could develop according to the local therapy and tumor location. In this study, we conducted a single-center analysis of the outcomes and toxicity of multidisciplinary treatment using proton beam therapy (PBT) for pediatric RMS.Materials and methodsRMS patients aged younger than 20 years whose RMS was newly diagnosed and who underwent PBT at University of Tsukuba Hospital (UTH) during the period from 2009 to 2019 were enrolled in this study. The patients’ clinical information was collected by retrospective medical record review.ResultsForty-eight patients were included. The 3-year progression-free survival (PFS) and overall survival (OS) rates of all the patients were 68.8% and 94.2%, respectively. The 3-year PFS rates achieved with radical resection, conservative resection, and biopsy only were 65.3%, 83.3%, and 67.6%, respectively (p = 0.721). The 3-year LC rates achieved with radical resection, conservative resection, and biopsy only were 90.9%, 83.3%, and 72.9%, respectively (p = 0.548). Grade 3 or higher mucositis/dermatitis occurred in 14 patients. Although the days of opioid use due to mucositis/dermatitis during the chemotherapy with PBT were longer than those during the chemotherapy without PBT [6.1 and 1.6 (mean), respectively, p = 0.001], the frequencies of fever and elevation of C-reactive protein were equivalent.ConclusionsMultidisciplinary therapy containing PBT was feasible and provided a relatively fair 3-year PFS, even in children with newly diagnosed RMS without severe toxicity.