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Objective(s): In the treatment of castration-resistant prostate cancer (CRPC) with bone metastases, radium-223 dichloride (Ra-223) is the only bone-targeted drug that shows survival benefits. Completing six courses of Ra-223 treatment is thought to be associated with better patient survival, but this treatment has a relatively high rate of acute adverse events. Methods: This retrospective study included 85 patients from 12 institutions in Japan to investigate the clinical significance of the completion of Ra-223 treatment and acute adverse events in CRPC patients. Results: Six courses of Ra-223 treatment were completed in 65.9% of the patients. Grade 3 or higher acute adverse events were observed in 27.1% of patients. The prostate specific antigen and alkaline phosphatase declined at 26.9% and 87.9%, respectively. The overall survival rates at 12 and 24 months were 80.7% and 63.2%, respectively. Both completion of six courses of Ra-223 treatment and absence of grade 3 or higher acute adverse events were associated with longer overall survival. In univariate analysis, factors related to the history of treatment (five or more hormone therapy agents and cytotoxic chemotherapy) and hematological parameters (Prostate specific antigen (PSA) doubling time, alkaline phosphatase, hemoglobin, albumin, and serum calcium) were associated with completing six courses of Ra-223 treatment without experiencing grade 3 or higher acute adverse events. Multivariate analysis showed that a history of chemotherapy, PSA doubling time, hemoglobin, and serum calcium showed statistical significance. We built a predictive score by these four factors. Patients with lower scores showed higher rates of treatment success (p

Abstract Background Palliative radiotherapy for gastric cancer bleeding has been reported to be a safe and effective treatment, but predictive factors for achievement of hemostasis and overall survival have not been established. Methods In this retrospective study, 120 courses of palliative radiotherapy for gastric cancer bleeding in 117 patients in 4 institutes in Japan were reviewed with approval of the ethical committee in each institute. The rate of achieving hemostasis was evaluated by 50% or more reduction of red blood cell transfusion before and after the start of radiotherapy, elevation of blood hemoglobin concentration in a period of 4 weeks from the start of radiotherapy or improvement of subjective or objective clinical symptoms in a period of 4 weeks from the start of radiotherapy. Predictive factors for overall survival and achieving hemostasis were investigated with the Cox hazards model. Results The median overall survival period was 3.7 months. Multivariate analysis showed that absence of metastatic disease, higher biological effective dose, higher serum albumin level, lower blood urea nitrogen level and lower neutrophil-to-lymphocyte ratio (NLR) were associated with longer overall survival. Elevation of hemoglobin concentration in a period of 4 weeks from the start of radiotherapy (mean concentration: 8.2 g/dL vs. 8.9 g/dL, p = 0.006) and decrease in the amount of red cell transfusion from a 4-week period before to a 4-week period after the start of radiotherapy (mean amount: 716 mL vs. 230 mL, p

Abstract Background In clinical practice, the effect of durvalumab and radiation pneumonitis (RP) on survival after intensity-modulated radiotherapy (IMRT) is not fully understood. The purpose of this retrospective study was to investigate factors related to distant metastasis-free survival (DMFS), progression-free survival (PFS) and overall survival (OS) after IMRT for locally advanced non-small cell lung cancer (LA-NSCLC). Methods All patients who were treated with conventional fractionated IMRT for LA-NSCLC between April 2016 and March 2021 were eligible. Time-to-event data were assessed by using the Kaplan–Meier estimator, and the Cox proportional hazards model was used for prognostic factor analyses. Factors that emerged after the start of IMRT, such as durvalumab administration or the development of RP, were analysed as time-dependent covariates. Results A total of 68 consecutive patients treated with conventional fractionated IMRT for LA-NSCLC were analysed. Sixty-six patients completed radiotherapy, 50 patients received concurrent chemotherapy, and 36 patients received adjuvant durvalumab. During the median follow-up period of 14.3 months, 23 patients died, and tumour progression occurred in 37 patients, including 28 patients with distant metastases. The 1-year DMFS rate, PFS rate and OS rate were 59.9%, 48.7% and 84.2%, respectively. Grade 2 RP occurred in 16 patients, grade 3 in 6 patients and grade 5 in 1 patient. The 1-year cumulative incidences of grade 2 or higher RP and grade 3 or higher RP were 33.8% and 10.3%, respectively. The results of multivariate analyses showed that durvalumab had a significantly lower hazard ratio (HR) for DMFS, PFS and OS (HR 0.31, p

The purpose of this study is to introduce differential dose–volume histogram (dDVH) features into machine learning for radiation pneumonitis (RP) prediction and to demonstrate the predictive performance of the developed model based on integrated cumulative dose–volume histogram (cDVH) and dDVH features. Materials and methods: cDVH and dDVH features were calculated for 153 patients treated for non-small-cell lung cancer with 60–66 Gy and dose bins ranging from 2 to 8 Gy in 2 Gy increments. RP prediction models were developed with the least absolute shrinkage and selection operator (LASSO) through fivefold cross-validation. Results: Among the 152 patients in the patient cohort, 41 presented ≥grade 2 RP. The interdependencies between cDVH features evaluated by Spearman’s correlation were significantly resolved by the inclusion of dDVH features. The average area under curve for the RP prediction model using cDVH and dDVH model was 0.73, which was higher than the average area under curve using cDVH model for 0.62 with statistically significance (p < 0.01). An analysis using the entire set of regression coefficients determined by LASSO demonstrated that dDVH features represented four of the top five frequently selected features in the model fitting, regardless of dose bin. Conclusions: We successfully developed an RP prediction model that integrated cDVH and dDVH features. The best RP prediction model was achieved using dDVH (dose bin = 4 Gy) features in the machine learning process.

PURPOSE:Radiographic severity of radiation-induced lung injury (RILI) has not been well-studied. The goal of this study was to assess the CT appearance pattern and severity of RILI without consideration of the clinical presentation. MATERIAL AND METHODS:A total of 49 patients, 41 with primary lung cancer and 8 with metastatic lung cancer, were treated by 4-fraction stereotactic body radiotherapy (SBRT). RILI after SBRT was separately assessed by two observers. The early and late CT appearance patterns and CT-based severity grading were explored. RESULTS:The median follow-up period was 39.0 months. In the early CT findings of observers 1 and 2, there was diffuse consolidation in 15 and 8, diffuse ground glass opacity (GGO) in 0 and 0, patchy consolidation and GGO in 17 and 20, patchy GGO in 3 and 3, and no changes in 10 and 14, respectively (kappa = 0.61). In late CT findings of observer 1 and 2, there were modified conventional pattern in 28 and 24, mass-like pattern in 8 and 11, scar-like pattern in 12 and 12, and no changes in 1 and 2, respectively (kappa = 0.63). In the results of the CT-based grading by observers 1 and 2, there were grade 0 in 1 and 2, grade 1 in 10 and 14, grade 2 in 31 and 29, grade 3 in 7 and 4, and none of grade 4 or more, respectively (kappa = 0.66). According to multivariate analyses (MVA), the significant predicting factors of grade 2 or more CT-based RILI were age (p = 0.01), oxygen dependence (p = 0.03) and interstitial shadow (p = 0.03). CONCLUSIONS:The agreement of the CT appearance and CT-based grading between two observers was good. These indicators may be able to provide us with more objective information and a better understanding of RILI.

Objective(s): In the treatment of castration-resistant prostate cancer (CRPC) with bone metastases, radium-223 dichloride (Ra-223) is the only bone-targeted drug that shows survival benefits. Completing six courses of Ra-223 treatment is thought to be associated with better patient survival, but this treatment has a relatively high rate of acute adverse events . Methods: This retrospective study included 85 patients from 12 institutions in Japan to investigate the clinical significance of the completion of Ra-223 treatment and acute adverse events in CRPC patients. Results: Six courses of Ra-223 treatment were completed in 65.9% of the patients. Grade 3 or higher acute adverse events were observed in 27.1% of patients. The prostate specific antigen and alkaline phosphatase declined at 26.9% and 87.9%, respectively. The overall survival rates at 12 and 24 months were 80.7% and 63.2%, respectively. Both completion of six courses of Ra-223 treatment and absence of grade 3 or higher acute adverse events were associated with longer overall survival. In univariate analysis, factors related to the history of treatment (five or more hormone therapy agents and cytotoxic chemotherapy) and hematological parameters (Prostate specific antigen (PSA) doubling time, alkaline phosphatase, hemoglobin, albumin, and serum calcium) were associated with completing six courses of Ra-223 treatment without experiencing grade 3 or higher acute adverse events. Multivariate analysis showed that a history of chemotherapy, PSA doubling time, hemoglobin, and serum calcium showed statistical significance. We built a predictive score by these four factors. Patients with lower scores showed higher rates of treatment success (p<0.001) and longer overall survival (p<0.001) with statistical significance. Conclusions: Accomplishing six courses of Ra-223 treatment without grade 3 or higher acute adverse events was a prognostic factor in patients with mCRPC treated with Ra-223. We built a predictive score of treatment success and need future external validation. [ABSTRACT FROM AUTHOR]

Background: Renal atrophy is observed in an irradiated kidney. The aim of this study was to determine dose-volume histogram parameters and other factors that predict renal atrophy after 10-fraction stereotactic body radiotherapy (SBRT) for primary renal cell carcinoma (RCC).Methods: A total of 14 patients (11 males, 3 females) who received SBRT for RCC at Tohoku University Hospital between April 2010 and February 2014 were analyzed. The median serum creatinine level was 1.1 mg/dl and two patients had a single kidney. Nine patients were implanted with fiducial markers. The median tumor diameter was 30 mm. SBRT was delivered at 70 Gy in 10 fractions for 7 tumors, at 60 Gy in 10 fractions for 2 tumors, and at 50 Gy in 10 fractions for 5 tumors with 6 and/or 15 MV X-ray using 5 to 8 multi-static beams. Renal atrophy was assessed using post-SBRT CT images after 12-24 months intervals. Correlations were examined by Spearman rank correlation analysis. Differences between two groups were evaluated by the Mann-Whitney test, and pairwise comparisons were made by the Wilcoxon signed-rank test.Results: The median tumor volume shrunk from 14.8 cc to 10.6 cc (p = 0.12), and the median irradiated kidney volume changed from 160.4 cc to 137.1 cc (p < .01). The median peak creatinine level was 1.6 mg/dl after treatment (p < .01). Percentage volumes of the irradiated kidney receiving at least 10 Gy (V10, p = 0.03), V20 (p < .01), V30(p < .01), V40 (p = 0.01), mean irradiated kidney dose (p < .01), and magnitude of overlap between PTV and kidney volume (p = 0.03) were significantly correlated with post-treatment irradiated kidney volume in percent, and V20-V30 had strong correlation (r < -0.70, p < .01). Patients with implanted fiducial markers showed a significantly lower ratio of renal atrophy (p = 0.02).Conclusions: Significant renal atrophic change was observed. Dose distribution of SBRT at 20-30 Gy had a strong correlation with renal atrophy when irradiation was performed in 10 fractions. [ABSTRACT FROM AUTHOR]

Background: To evaluate the long-term results of chemoradiotherapy (CRT) for stage II-III thoracic esophageal cancer mainly by comparing results of three protocols retrospectively.Methods: Between 2000 and 2012, 298 patients with stage II-III thoracic esophageal cancer underwent CRT. Patients in Group A received two cycles of cisplatin (CDDP) at 70 mg/m(2) (day 1 and 29) and 5-fluorouracil (5-FU) at 700 mg/m(2)/24 h (day 1-4 and 29-32) with radiotherapy (RT) of 60 Gy without a break. Patients in Group B received two cycles of CDDP at 40 mg/m(2) (day 1, 8, 36 and 43) and 5-FU at 400 mg/m(2)/24 h (day 1-5, 8-12, 36-40 and 43-47) with RT of 60 Gy with a 2-week break. Patients in Group C received two cycles of nedaplatin at 70 mg/m(2) (day 1 and 29) and 5-FU at 500 mg/m(2)/24 h (day 1-4 and 29-32) with RT of 60-70 Gy without a break. Differences in prognostic factors between the groups were analyzed by univariate and multivariate analyses.Results: The 5-year overall survival rates for patients in Group A, Group B and Group C were 52.4, 45.2 and 37.2%, respectively. The 5-year overall survival rates for patients in Stage II, Stage III (non-T4) and Stage III (T4) were 64.0, 40.1 and 22.5%, respectively. The 5-year overall survival rates for patients who received 1 cycle and 2 cycles of concomitant chemotherapy were 27.9 and 46.0%, respectively. In univariate analysis, stage, performance status and number of concomitant chemotherapy cycles were significant prognostic factors (p < 0.001, p = 0.008 and p < 0.001, respectively). In multivariate analysis, stage, protocol and number of concomitant chemotherapy cycles were significant factors (p < 0.001, p = 0.043 and p < 0.001, respectively).Conclusions: The protocol used in Group A may be an effective protocol of CRT for esophageal cancer. It may be important to complete the scheduled concomitant chemotherapy with the appropriate intensity of CRT. [ABSTRACT FROM AUTHOR]

Background: Pleural invasion status is known to be a predictor of survival after pulmonary resection for non-small cell lung cancer. Our goal was to determine whether the length of tumor attachment to the pleura on a pretreatment CT image has prognostic value as an alternative to pleural invasion status for stage I non-small cell lung cancer treated with stereotactic body radiotherapy (SBRT). Methods: A total of 90 tumors in 87 patients (males: 68, females: 19) who received SBRT between March 2005 and September 2011 in our institution were reviewed. The median age of the patients was 78 years (range, 48-90 years). The median tumor diameter was 2.2 cm (range, 0.9-4.2 cm). The prescribed dose was typically 48 Gy in 4 fractions, 60 Gy in 8 fractions or 60 Gy in 15 fractions to the isocenter with 6 MV X-ray using 4 non-coplanar and 3 coplanar static beams. The lengths of attachment were measured using pretreatment CT images at the lung window. Cumulative incidence rates were calculated using Kaplan-Meier curves, and univariate and multivariate analyses for in-field tumor control, locoregional control (LRC), freedom from distant metastasis and freedom from progression (FFP) were performed using a Cox proportional hazards model. Results: Of the 90 tumors, 42 tumors were attached to the pleura (median, 14.7 mm; range, 4.3-36.0 mm), 21 tumors had pleural indentation and 27 tumors had no attachment. The median follow-up period for survivors was 46.1 months. The 3-year in-field control, LRC, FFP and overall survival rates were 91.2%, 75.3%, 63.8% and 68.6%, respectively. SBRT dose and tumor diameter were independently significant predictors of in-field control (p = 0.02 and p = 0.04, respectively). Broad attachment to the pleura, the length being more than 14.7 mm, was a negative independent predictor of LRC and FFP (p = 0.02 and p = 0.01, respectively). Conclusions: Pleural attachment status on a pretreatment CT image might be an important predictor of LRC and FFP. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs), which recognize a wide range of microbial pathogens and pathogen-related products, play important roles in innate immunology. Macrophages have a variety of TLRs, and pathogen binding to TLR resulted in the activation of macrophages. R-848, an immune response modifier, is an analog of imidazoquinoline derivative and binds to an endosome-localized TLR to exert an anti-viral response on leukocytes. In the present study, we verified that co-treatment of R-848 with other TLR agonists would enhance immune response. The culture supernatant of Aureobasidium pullulans (A. pullulans, which contains predominantly soluble β-glucan), which binds to cell membrane-localized TLR, and to C-type lectin receptor Dectin-1, was treated together with R-848 to THP-1 macrophages. Compared to R-848 treatment alone, co-treatment of R-848 with A. pullulans culture supernatant significantly augmented TNF-α and IL-12p40 cytokine expression. Next, we investigated whether or not apoptotic cell uptake would be increased by co-treatment of R-848 with A. pullulans culture supernatant. To detect engulfed apoptotic cells, we induced apoptosis in human lymphoma Jurkat cells by 5-fluorouracil and stained them with fluorescent dye 5(6)-carboxytetramethylrhodamine (TAMRA), whereas THP-1 macrophage was labeled with fluorescein isothiocyanate-anti-CD14 and determined the percentage increase in TAMRA-positive THP-1 macrophages by flow cytometric assay. Since R-848 or A. pullulans treatment alone stimulated THP-1 macrophages to induce phagocytosis, co-treatment of R-848 with A. pullulans culture supernatant significantly augmented phagocytosis of apoptotic Jurkat cells. These results suggest that the activation of several different innate immune receptor pathways may enhance the immune response of R-848 significantly.

Background: CD10, also known as neprilysin or enkephalinase exhibiting neutral endopeptidase (NEP) activity, is expressed by B-lineage hematopoietic cells as well as a variety of cells from normal tissues. It cleaves peptides such as cytokines to act for terminating inflammatory responses. Although CD10 molecules of the human pre-B-cell line NALM-6 have 6 consensus N-glycosylation sites, three of them are known to be N-glycosylated by X-ray crystallography., Methods: In order to investigate the role of N-glycans in the full expression of NEP activity, we modified N-glycans by treatment of NALM6 cells with various glycosidases or alter each of the consensus N-glycosylation sites by generating site-directed mutagenesis and compared the NEP activities of the sugar-altered CD10 with those of intact CD10., Results: CD10 of the human B-cell line NALM-6 was dominantly localized in raft microdomains and heterogeneously N-glycosylated. Although neither desialylation nor further degalactosylation caused defective NEP activity, removal of only a small part of N-glycans by treatment with glycopeptidase F under non-denaturing conditions decreased NEP activity completely. All of the three consensus sites of CD10 in HEK293 cells introduced with wild type-CD10 were confirmed to be N-glycosylated. Surface expression of N-glycan at Asn(628)-deleted CD10 by HEK293 cells was greatly decreased as well as it lost entire NEP activities., Conclusions: N-glycosylation at Asn(628) is essential not only for NEP activities, but also for surface expression., General Significance: Quality control system does not allow dysfunctional ecto-type proteases to express on plasma membrane., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Lipid rafts enriched in glycosphingolipids (GSLs), cholesterol and signaling molecules play an essential role not only for signal transduction started by ligand binding, but for intracellular events such as organization of actin, intracellular traffic and cell polarity, but their functions in cleavage division of preimplantation embryos are not well known., Results: Here we show that monosialylGb5Cer (MSGb5Cer)-enriched raft domains are involved in development during the cleavage stage of mouse preimplantation embryos. MSGb5Cer preferentially localizes at the interfaces between blastomeres in mouse preimplantation embryos. Live-imaging analysis revealed that MSGb5Cer localizes in cleavage furrows during cytokinesis, and that by accumulating at the interfaces, it thickens them. Depletion of cholesterol from the cell membrane with methyl-beta-cyclodextrin (MbCD) reduced the expression of MSGb5Cer and stopped cleavage. Extensive accumulation of MSGb5Cer at the interfaces by cross-linking with anti-MSGb5Cer Mab (6E2) caused F-actin to aggregate at the interfaces and suppressed the localization of E-cadherin at the interfaces, which resulted in the cessation of cleavage. In addition, suppression of actin polymerization with cytochalasin D (CCD) decreased the accumulation of MSGb5Cer at the interfaces. In E-cadherin-targeted embryos, the MSGb5Cer-enriched raft membrane domains accumulated heterotopically., Conclusions: These results indicate that MSGb5Cer-enriched raft membrane domains participate in cytokinesis in a close cooperation with the cortical actin network and the distribution of E-cadherin., (© 2011 Sato et al; licensee BioMed Central Ltd.)

The functional maturation of spermatozoa during epididymal transit in mammals accompanies the changes in their plasma membrane due to the binding or removal of proteins or interactions with the proteases, glycosidases and glycosyltransferases present in the epididymis. In order to study the surface changes in spermatozoa during their maturation in the epididymis, we previously established several monoclonal antibodies against the 54kDa sialoglycoprotein of mouse cauda epididymal spermatozoa, which gradually increased the expression of antigenic determinants during epididymal transit. One of these monoclonal antibodies, MC121, reacted with mouse sperm glycoproteins on a polyvinylidene fluoride membrane after desialylation of the glycoproteins, and the treatment of the desialylated sperm glycoproteins with β-N-acetylhexosaminidase greatly decreased the expression of the antigenic determinants. In addition to reacting with mouse cauda epididymal spermatozoa, MC121 reacted with human red blood cells (hRBCs). MC121 induced agglutination of sialidase-treated hRBCs and stained hRBCs fixed with formalin vapor much more heavily than it stained hRBCs fixed with methanol. The thin layer chromatography (TLC) immunostaining of the sialidase-treated lipids of hRBCs with MC121 suggested that the epitope-bearing molecule is a glycosphingolipids (GSL), and that MC121 reacts with a pentaose-GSL. Analysis of sialidase-treated GSLs by TLC-Blot-Matrix Assisted Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI TOF MS) revealed that the GSL bound by MC121 was [HexNAc][HexNAc+Hex][Hex][Hex]-Cer. The lipid band stained with mAb TH2, which is specific for a GSL, GalNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-ceramide. These results indicated that the epitope to which MC121 binds is present in a neolacto-series GSL, IV³GalNAcβ-nLc₄Cer² sequence., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Using dodecyl N-acetylglucosaminide (GlcNAc-C12) as a saccharide primer, we investigated the biosynthetic changes of neolacto-series glycosphingolipids (GSLs) in mouse embryonal carcinoma F9 cells during differentiation induced by retinoic acid plus dibutyryl cyclic AMP (RA/dbcAMP). In the differentiated cells, the glycosylation of GlcNAc-C12 was greatly enhanced. The sugar compositions of glycosylated primers were assigned as Hex-GlcNAc, [Hex](2)-GlcNAc, [Hex](2)[HexNAc]-GlcNAc, and [NeuAc][Hex]-GlcNAc by liquid chromatography-tandem mass spectrometry. The detection of augmented biosynthesis of endogenous sialylparagloboside indicated that [NeuAc][Hex]-GlcNAc was predicted to be the non-reducing end trisaccharide of sialylparagloboside. The transcription of B3gnt5, B4galt1, Ggta1, Fut4 and St3gal6, encoding glycosyltransferases involved in the neolacto-series glycosphingolipids biosynthesis, was increased, whereas that of Fut9 and St6galI was decreased after RA/dbcAMP treatment. Furthermore, the sialyltransferase activity of ST3GalVI sialylating paragloboside was enhanced with the increase in St3gal6 expression. Since most stage-specific embryonic antigen-1 (SSEA-1) active determinants are carried by glycoproteins in F9 cells, the changes in glycolipid metabolism do not seem to be closely related to loss of cell surface SSEA-1 expression upon F9 differentiation. These results indicate that RA/dbcAMP treatment activates the biosynthesis of neolacto-series GSL and enhances sialylation of paragloboside in F9 cells with down-regulation of Fut9 expression.

Here, we report the highly efficient in vitro differentiation of human bone marrow-derived mesenchymal stem/progenitor cells (MPCs) using a novel nanotechnology-based culture plate, nanoculture plate(®) (NCP). The NCP contains uneven microfabrications with diameters of ∼2-3 μm arranged in a honeycomb pattern on its culture surface, which is devoid of animal-derived protein sources. When human MPCs were subjected to three-dimensional (3D) culture using an NCP, they rapidly formed adhesive spheroids. We showed that adipogenic differentiation in NCP-mediated 3D cultures led to more rapid accumulation of triglycerides than that in two-dimensional cultures. During adipogenesis in 3D cultures, the rapid and intense induction of adipocyte-specific gene expressions, such as peroxisome proliferator-activated receptor γ (PPAR-γ), CCAAT-enhancer-binding protein α (C/EBP-α), adipocyte protein 2 (aP2), and adiponectin was observed. Together, these results indicate that this 3D culture system is suitable for the differentiation of human MPCs into adipogenic lineage, and could be applicable to adipose tissue engineering under xeno-free condition.

It is hoped that the gangliosides contained in neuroblastomas (NBs) can be used as outcome predictors. We used liquid chromatography-tandem mass spectrometry (LC-MS) to analyze the gangliosides expressed in 11 NB cell lines. LC-MS analysis detected a number of gangliosides, including acetylated forms, with significantly higher sensitivity than conventional high-performance thin-layer chromatography analysis, and the results revealed that the expression profiles of the gangliosides GD1a, GD2, and acetylated GD2 differed according to the NB cell line. Hierarchical clustering based on the ganglioside expression profiles obtained by LC-MS analysis revealed that the NB cell lines could be classified into three types according to their expression of these three gangliosides: A-type characterized by high expression of GD1a and low or no expression of GD2/acetylated GD2, B-type characterized by low or no expression of GD1a and high expression of GD2/acetylated GD2, and AB-type characterized by expression of both GD1a and GD2/acetylated GD2. Interestingly, all three MYCN non-amplified cell lines were classified into the A-type. The classification was found to be correlated with mRNA expression of ganglioside synthase and neural-differentiation-related genes. The results of this study indicate that LC-MS analysis is useful as a tool for glycosphingolipid research on malignancies.

B cell-activating factor belonging to the tumor necrosis factor superfamily (BAFF) is a crucial factor for B cell development and is involved in the survival of malignant B cells, but its effect on B cell precursors (BCPs) remains unclear. We investigated BCP acute lymphoblastic leukemia (-ALL) cells for BAFF receptor (-R) expression and compared the effect of BAFF on BCP-ALL cells and Burkitt lymphoma (BL) cells. Expression of BAFF-R was detected in some cell lines and some clinical specimens of both BL and BCP-ALL. BAFF acted on both BL and BCP-ALL cells and promoted proliferation by both. BAFF also inhibited apoptosis by BL cells induced by cross-linking of cell surface molecules and anticancer drugs, but failed to inhibit apoptosis by BCP-ALL cells. BAFF induced prompt and obvious activation of the NF-kappaB signaling pathway in BL cells, but only weak and delayed activation of the pathway in BCP-ALL cells. The results of this study indicate that some BCP-ALL cells and some BL cells express BAFF-R, but that the effects of BAFF on BCP-ALL cells are different from its effects on mature B cell malignancies.

With an increase in the importance of umbilical cord blood (CB) as an alternative source of haematopoietic progenitors for allogenic transplantation, donor lymphocyte infusion (DLI) with donor CB-derived activated CD4(+) T cells in the unrelated CB transplantation setting is expected to be of increased usefulness as a direct approach for improving post-transplant immune function. To clarify the characteristics of activated CD4(+) T cells derived from CB, we investigated their mRNA expression profiles and compared them with those of peripheral blood (PB)-derived activated CD4(+) T cells. Based on the results of a DNA microarray analysis and quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR), a relatively high level of forkhead box protein 3 (Foxp3) gene expression and a relatively low level of interleukin (IL)-17 gene expression were revealed to be significant features of the gene expression profile of CB-derived activated CD4(+) T cells. Flow cytometric analysis further revealed protein expression of Foxp3 in a portion of CB-derived activated CD4(+) T cells. The low level of retinoic acid receptor-related orphan receptor gamma isoform t (RORgamma t) gene expression in CB-derived activated CD4(+) T cells was speculated to be responsible for the low level of IL-17 gene expression. Our data indicate a difference in gene expression between CD4(+) T cells from CB and those from PB. The findings of Foxp3 expression, a characteristic of regulatory T cells, and a low level of IL-17 gene expression suggest that CB-derived CD4(+) T cells may be a more appropriate source for DLI.

The expression of granulysin, a cytolytic protein produced by activated T and NK cells, has been revealed to be correlated with the prognosis of some adult cancer patients. By examination on various childhood lymphoma tissues, we found that granulysin level was especially high in systemic anaplastic large cell lymphoma (ALCL) cases, whereas no close correlation with the expression of CD96, a marker for activated T and NK cells, was observed. We further demonstrated that both ALCL cells in biopsy specimens and cell lines established from ALCL express granulysin, indicating some correlation of granulysin with biological features of ALCL.

The CH-296 recombinant fragment of human fibronectin is essential for murine leukemia virus (MLV)-derived retroviral transduction of CD34(+) cells for the purpose of stem cell gene therapy. Although the major effect of CH-296 is colocalization of the MLV-derived retrovirus and target cells at specific adhesion domains of CH-296 mediated by integrins expressed on CD34(+) cells, the precise roles of the integrins are unclear. We examined the kinetics of integrin expression on CD34(+) cells during the course of MLV-derived retrovirus-mediated gene transduction with CH-296. Flow cytometry revealed that the levels of both very late activation protein (VLA)-4 and VLA-5 on CD34(+) cells freshly isolated from cord blood were insufficient for effective MLV-derived retroviral transduction. However, increases were achieved during culture for preinduction and MLV-derived retrovirus-mediated gene transduction in the presence of a cocktail of cytokines. In addition, we confirmed by using specific antibodies that inhibition of the cell adhesion mediated by the integrins significantly reduced transduction efficiency, indicating that integrin expression is indeed important for CH-296-based MLV-derived retroviral transduction. Only a few cytokines are capable of inducing integrin expression, and stem cell factor plus thrombopoietin was found to be the minimal combination that was sufficient for effective transduction of an MLV-derived retrovirus based on CH-296. Our findings should be useful for improving the culture conditions for CH-296-based MLV-derived retroviral transduction in stem cell gene therapy.

Background: The Dickkopf (DKK) family comprises a set of proteins that function as regulators of Wnt/beta-catenin signaling and has a crucial role in development. Recent studies have revealed the involvement of this family in tumorigenesis, however their role in tumorigenesis is still remained unclear., Methodology/principal Findings: We found increased expression of DKK2 but decreased expression of DKK1 in Ewing family tumor (EFT) cells. We showed that EFT-specific EWS/ETS fusion proteins enhance the DKK2 promoter activity, but not DKK1 promoter activity, via ets binding sites (EBSs) in the 5' upstream region. EWS/ETS-mediated transactivation of the promoter was suppressed by the deletion and mutation of EBSs located upstream of the DKK2 gene. Interestingly, the inducible expression of EWS/ETS resulted in the strong induction of DKK2 expression and inhibition of DKK1 expression in human primary mesenchymal progenitor cells that are thought to be a candidate of cell origin of EFT. In addition, using an EFT cell line SK-ES1 cells, we also demonstrated that the expression of DKK1 and DKK2 is mutually exclusive, and the ectopic expression of DKK1, but not DKK2, resulted in the suppression of tumor growth in immuno-deficient mice., Conclusions/significance: Our results suggested that DKK2 could not functionally substitute for DKK1 tumor-suppressive effect in EFT. Given the mutually exclusive expression of DKK1 and DKK2, EWS/ETS regulates the transcription of the DKK family, and the EWS/ETS-mediated DKK2 up-regulation could affect the tumorigenicity of EFT in an indirect manner.

B-cell-activating factor (BAFF) is a survival and maturation factor for B cells belonging to the tumour necrosis factor superfamily. Among three identified functional receptors, the BAFF receptor (BAFF-R) is thought to be responsible for the effect of BAFF on B cells though details of how remain unclear. We determined that a hairy-cell leukaemia line, MLMA, expressed a relatively high level of BAFF-R and was susceptible to apoptosis mediated by either CD20 or B-cell antigen receptor (BCR). Using MLMA cells as an in vitro model of mature B cells, we found that treatment with BAFF could inhibit apoptosis mediated by both CD20 and BCR. We also observed, using immunoblot analysis and microarray analysis, that BAFF treatment induced activation of nuclear factor-kappaB2 following elevation of the expression level of Bcl-2, which may be involved in the molecular mechanism of BAFF-mediated inhibition of apoptosis. Interestingly, BAFF treatment was also found to induce the expression of a series of genes, such as that for CD40, related to cell survival, suggesting the involvement of a multiple mechanism in the BAFF-mediated anti-apoptotic effect. MLMA cells should provide a model for investigating the molecular basis of the effect of BAFF on B cells in vitro and will help to elucidate how B cells survive in the immune system in which BAFF-mediated signalling is involved.

Detergent-insoluble microdomains, or rafts, act as a platform to transduce signals from the extracellular space into the cytoplasm. In the process of developing monoclonal antibodies against raft molecules for the purpose of studying the molecular mechanism of raft-mediated signaling, we observed the uniqueness and certain advantages of immunization with rafts. Simple subcutaneous injection of mice with a phosphate-buffered saline (PBS) suspension of rafts without mixing with Freund's adjuvant made it possible to increase the titer of antiserum reacting with raft components. Interestingly, injection of rafts prepared from certain specific cell lines induced monoglycolipid-specific antibodies. Furthermore, antibodies were produced by raft-immunization of even syngeneic mice. Our findings suggest that this phenomenon does not represent a breakdown of immunological self-tolerance, but typical immune reactions accompanying the class switch from IgM antibodies to IgG antibodies.

Ewing's family tumor (EFT) is a rare pediatric tumor of unclear origin that occurs in bone and soft tissue. Specific chromosomal translocations found in EFT cause EWS to fuse to a subset of ets transcription factor genes (ETS), generating chimeric EWS/ETS proteins. These proteins are believed to play a crucial role in the onset and progression of EFT. However, the mechanisms responsible for the EWS/ETS-mediated onset remain unclear. Here we report the establishment of a tetracycline-controlled EWS/ETS-inducible system in human bone marrow-derived mesenchymal progenitor cells (MPCs). Ectopic expression of both EWS/FLI1 and EWS/ERG proteins resulted in a dramatic change of morphology, i.e., from a mesenchymal spindle shape to a small round-to-polygonal cell, one of the characteristics of EFT. EWS/ETS also induced immunophenotypic changes in MPCs, including the disappearance of the mesenchyme-positive markers CD10 and CD13 and the up-regulation of the EFT-positive markers CD54, CD99, CD117, and CD271. Furthermore, a prominent shift from the gene expression profile of MPCs to that of EFT was observed in the presence of EWS/ETS. Together with the observation that EWS/ETS enhances the ability of cells to invade Matrigel, these results suggest that EWS/ETS proteins contribute to alterations of cellular features and confer an EFT-like phenotype to human MPCs.

Background/aim: Although osteoblasts are thought to be the major component of the hematopoietic stem cell niche in the bone marrow microenvironment, the role of osteoblasts in hematopoiesis is still unclear. The ability of human osteoblasts to support early hematopoiesis was investigated., Methods and Results: Human CD34+ bone marrow cells cultured on human osteoblasts were capable of surviving without addition of cytokines and differentiated into myeloid cells with slight proliferation. The results of immunohistochemical experiments suggested activation of FAK and AKT in hematopoietic cells attached to osteoblasts. When stem cell factor, Flt3-L, and IL-3 were added to the coculture system, each cytokine distinctively enhanced proliferation and differentiation of CD34+ bone marrow cells., Conclusion: The results suggest that human osteoblasts have the ability to support hematopoietic cell development in vitro., (Copyright 2008 S. Karger AG, Basel.)