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Introduction: Real-world data (RWD) can contextualize findings from single-arm trials when randomized comparative trials are unethical or unfeasible. Findings from single-arm trials alone are difficult to interpret and a comparison, when feasible and meaningful, to patient-level information from RWD facilitates the evaluation. As such, there have been several recent regulatory applications including RWD or other external data to support the product's efficacy and safety. This paper summarizes some lessons learned from such contextualization from 20 notable new drug or biologic licensing applications in oncology and rare diseases., Methods: This review focuses on 20 notable new drug or biologic licensing applications that included patient-level RWD or other external data for contextualization of trial results. Publicly available regulatory documents including clinical and statistical reviews, advisory committee briefing materials and minutes, and approved product labeling were retrieved for each application. The authors conducted independent assessments of these documents focusing on the regulatory evaluation, in each case. Three examples are presented in detail to illustrate the salient issues and themes identified across applications., Results: Regulatory decisions were strongly influenced by the quality and usability of the RWD. Comparability of cohort attributes such as endpoints, populations, follow-up, index and censoring criteria, as well as data completeness and accuracy of key variables appeared to be essential to ensure the quality and relevance of the RWD. Given adequate sample size of the clinical trials or external control, the use of appropriate analytic methods to properly account for confounding, such as regression or matching, and pre-specification of these methods while blinded to patient outcomes seemed good strategies to address baseline differences., Discussion: Contextualizing single-arm trials with patient-level RWD appears to be an advance in regulatory science; however, challenges remain. Statisticians and epidemiologists have long focused on analytical methods for comparative effectiveness but hurdles in use of RWD have often occurred upstream of the analyses. More specifically, we noted hurdles in evaluating data quality, justifying cohort selection or initiation of follow-up, and demonstrating comparability of cohorts and endpoints., (© 2022. The Drug Information Association, Inc.)

α-Mangostin (MAN) is a bioactive compound isolated from the inedible pericarp of a tropical fruit mangosteen (Garcinia mangostana Linn). It exhibits notable therapeutic potentials on lung cancers, but the underlying mechanisms are still largely unknown. This study was designed to further explore the mechanisms involved in cytotoxicity of MAN on A549 cells. Apoptosis and cell cycle distribution were analyzed by flow cytometry methods. The fluorescent probes DCFH-DA and JC-1 were used to assess the intracellular reactive oxidative species (ROS) and mitochondrial membrane potential statuses, respectively. The regulation of MAN on relevant pathways was investigated by immunoblotting assays. The results obtained indicated that MAN caused significant apoptosis and cell cycle arrest in A549 cells, which eventually resulted in inhibition on cell proliferation in vitro. All these phenomena were synchronized with escalated oxidative stress and downregulation of nicotinamide phosphoribosyltransferase/nicotinamide adenine dinucleotide (NAMPT/NAD). Supplementation with nicotinamide mononucleotide (NMN) and N-acetylcysteine (NAC) efficiently eased MAN-induced ROS accumulation, and potently antagonized MAN-elicited apoptosis and cell cycle arrest. The pro-apoptotic effect of MAN was further confirmed by increased expressions of cleaved caspase 3, 6, 7, and 9, and its effect on cell cycle progression was validated by the altered expressions of p-p38, p-p53, CDK4, and cyclin D1. The immunoblotting assays also demonstrated that NAC/NMN effectively restored these molecular changes elicited by MAN treatment. Collectively, this study revealed a unique anti-tumor mechanism of MAN by provoking ROS production through downregulation of NAMPT/NAD signaling and further validated MAN as a potential therapeutic reagent for lung cancer treatment.

Implementation of the first Generic Drug User Fee Amendments of 2012 (GDUFA I) provided funding to the US Food and Drug Administration (FDA) for modernizing review of the FDA/CDER Generic Drug Program. Under GDUFA I, FDA agreed to reduce the backlog of pending generic Abbreviated New Drug Applications (ANDAs), improve the efficiency of generic drug review, and reduce the number of review cycles with the goal of reducing overall time to approval. This study presents a preliminary analysis of initial filing and regulatory first actions on ANDAs during GDUFA I cohort year 3 (CY3) and cohort year 4 (CY4). It highlights initial successes and areas of improvement in the ANDA review process for both FDA and ANDA applicants to improve the efficiency of providing the public with high-quality, affordable generic drugs.

This work was to investigate potential roles of HMGB1-mediated ERK pathway in the healing process of bone fracture. Rat tibial fracture models were established and divided into control (rats with normal saline), HMGB1 (rats with HMGB1), and HMGB1+ PD98059 groups (rats with HMGB1 and 1 mg/kg of ERK1/2 inhibitor PD98059) with 30 rats per each. The healing of rats' fracture was observed by X-ray films, the morphological changes of bone fractures by HE staining, the callus formation by micro-CT and biomechanical test, and the expression of osteogenesis-related genes, HMGB1 and ERK-related proteins by qRT-PCR and Western blot. Rats in the HMGB1 group was increased in X-ray scores, peak torque, torsional stiffness, and the bone volume fraction (bone volume/total volume, BV/TV); meanwhile, those rats presented elevations in osteogenesis-related genes and HMGB1 expressions, as well as p-ERK/ERK ratio. However, rats in the HMGB1+ PD98059 group was significantly reduced in X-ray score, peak torque, torsional stiffness, and BV/TV, as well as the expression of osteogenesis-related genes and the ratio of p-ERK/ERK, as compared to those from HMGB1 group. HMGB1 could promote the expressions of osteogenesis-related genes and accelerate the healing process of fracture via activation of the ERK signaling pathway., (© 2019 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia on behalf of Kaohsiung Medical University.)

α-Mangostin (MG) is a bioactive compound isolated from mangosteen. This study was aimed to investigate effects of MG on adjuvant-induced arthritis (AA) in rats and decipher the underlying mechanisms. Clinical severity of AA was evaluated by paw oedema, arthritis score, and hematological parameters. Digital radiography (DR) and histological examinations were employed to assess joints destructions. Immune functions were evaluated by T cell subsets distribution. Effects on NF-κB pathway were investigated by immunohistochemical, western-blot and immunofluorescence methods both in vivo and vitro. It was found MG possessed superior anti-inflammatory effects in vivo, suggested by attenuated paw swelling, reduced inflammatory cells infiltration and decreased the secretion of TNF-α and IL-1β in serum. Meanwhile MG inhibited fibrous hyperplasia, synovial angiogenesis, cartilage and bone degradation in AA rats. Although MG exerted little effects on CD4 + population, it greatly decreased IFN-γ positive cells and promoted expression of FOXP3 in immune organs, indicating restoration of Th1/Treg cells ratio and recovery of immune homeostasis in vivo. Inhibition of NF-κB induced by MG was indicated by reduced the expression of p-p65 and VEGF in synovium. In vitro experiments found MG at 10 μg/ml significantly suppressed the expression and phosphorylation of key proteins implicated in NF-κB pathway and inhibited nucleus translocation of p65. These changes led to increased apoptosis and proliferation inhibition of HFLS-RA cells. The results demonstrated regulation of immune functions was deeply involved in the therapeutic actions of MG on AA, and it's inhibition on NF-κB in fibroblast-like synoviocytes was associated to the protective effects on joints., (Copyright © 2018 Elsevier B.V. All rights reserved.)

1,7-Dihydroxy-3,4-dimethoxyxanthone (XAN) is a bioactive compound isolated from Securidaca inappendiculata Hassk. and exerts the inhibitory effects on fibroblast-like synoviocytes by targeting NF-κB and p38. This study was designed to elucidate mechanisms underlying the divergent regulation on the two pathways in HFLS-RA cells by XAN. Expressions of hallmark proteins and transcription of GADD45α mRNA were determined by Western-blot and RT-qPCR methods, respectively. Fluorescence staining was employed to evaluate intracellular oxidative stress. Effects of XAN and N-acetyl-l-cysteine (NAC) on the proliferation of cells were investigated by MTT assay, and pro-apoptotic effects of XAN were assessed by Annexin V-FITC/PI method. It was found XAN blocked NF-κB signaling in HFLS-RA cells shortly after treatment. Moreover, it up-regulated both transcription and expression of GADD45α, and subsequently activated p38 pathway. As time went on, XAN significantly promoted the generation of reactive oxygen species (ROS), which accompanied with sustained up-regulation of p-p38 and increased apoptosis. 48H later, dual-effects of XAN on NF-κB and p38 were reversed. As activation of p38 and increased apoptosis induced by XAN were antagonized by NAC, they were deemed as ROS mediated effects. Furthermore, the accumulated ROS should also account for the activation of NF-κB in the late stage of treatments via interfering in p38/MSK1/NF-κB feedback. Altogether, these findings suggested XAN-induced ROS contributed great importance to the proliferation inhibition of HFLS-RA cells by mediating NF-κB/p38 feedback loop and apoptosis, which provided us a panoramic view of potential target in the therapy of RA by XAN., (Copyright © 2017. Published by Elsevier Masson SAS.)

Previous studies demonstrated that astragaloside IV (ASIV) is a potential P‑glycoprotein (P‑gp)‑mediated multidrug resistance (MDR) reversal agent through mechanisms involving downregulation of the gene expression of mdr1. In order to investigate whether the c‑Jun N‑terminal kinase (JNK) signaling pathway is involved in the mechanism underlying ASIV‑induced downregulated the expression of mdr1, the present study used 5‑fluorouracil‑resistant Bel‑7402/FU human hepatic cancer cells as target cells. ASIV (0.1 mM) decreased the protein expression of phosphorylated (p)‑JNK and p‑c‑Jun in the Bel‑7402/FU cells, as determined using western blot analysis. Treatment with the JNK pathway inhibitor, SP600125, at a concentration of 11 µM, decreased the mRNA expression levels of mdr1 and P‑gp, as determined using reverse transcription‑quantitative polymerase chain reaction and western blot analyses, and similar effects were observed following exposure to ASIV. Furthermore, electrophoretic mobility shift assays demonstrated that the DNA‑binding activity of activator protein‑1 (AP‑1) was decreased by 0.1 mM ASIV or 11 µM SP600125. Flow cytometric analysis revealed that 0.1 mM ASIV or 11 µM SP600125 increased the intracellular accumulation of fluorescent P‑gp substrates, including rhodamine 123. Taken together, these results indicated that ASIV reversed the drug resistance of Bel‑7402/FU cells by downregulating the expression of mdr1 via inhibition of the JNK/c‑Jun/AP‑1 signaling pathway.

Objectives: Anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitides (AAV) are a group of systemic autoimmune disorders characterized by necrotizing inflammation of small- to medium-sized blood vessels. The pathogenesis of patients with AAV are still in investigation. In this study, we explored the involvement of LL-37 and nucleic acids in AAV., Methods: 15 patients with AAV diagnosed according to the Chapel Hill definition between October 2014 and July 2015 in the department of Nephrology of Huangdao, affiliated Hospital of Qingdao University were enrolled. 16 patients with chronic bronchitis (CB) were selected  as disease control group. 15 cases of healthy people from Medical Healthy Center were as healthy control group. Peripheral blood mononuclear cells (PBMCs) were collected from these groups and stimulated by LL-37and (or) two types of CpG-ODN for 7 days. The IFN-α and ANCA in vitro were measured by ELISA. The serum IFN-α, LL-37 and ANCA were measured also., Results: The serum level of IFN-α in AAV group was much higher than that in CB group (692.13 ± 407.28 vs 397.07 ± 211.62 pg/ml, p = 0.019), and that in healthy control group (692.13 ± 407.28 vs 251.54 ± 190.46 pg/ml, p < 0.001). The serum level of LL-37 in AAV group was much higher than that in CB group (101.18 ± 66.59 vs 40.23 ± 13.51 ng/ml, p < 0.001, and that in healthy control group (101.18 ± 66.59 vs 27.80 ± 16.86 ng/ml, p < 0.001). Also the level of IFN-α showed a significant positive relationship with ANCA in AAV group both in serum and in supernatant of cultured PBMCs stimulated by LL-37 and (or) CpG-ODN (r = 0.783, p = 0.001; r = 0.575, p = 0.064; r = 0.649, p = 0.031; r = 0.806, p = 0.003). In patients with AAV, the supernatant levels of IFN-α in cultured PBMCs stimulated by LL-37 and (or) CpG-ODN were higher than that without stimulating factor (p < 0.05). The supernatant level of IFN-α in cultured PBMC stimulated by LL-37 alone was lower than that stimulated by CpGA alone (699.57 ± 476.26 vs 2342.63 ± 2025.11 pg/ml, p = 0.001). But the supernatant level of IFN-α in cultured PBMCs stimulated by LL-37 alone was higher than in that stimulated by CpGB alone (699.57 ± 476.26 vs 153.35 ± 78.08 pg/ml, p < 0.001). The supernatant level of IFN-α in cultured PBMCs stimulated by both LL-37 and CpG-ODN was higher than that stimulated by LL-37 or CpG-ODN alone (2550.57 ± 2217.41 vs 699.57 ± 476.26 pg/ml, p = 0.003; 2550.57 ± 2217.41 vs 153.35 ± 78.08 pg/ml, p = 0.001; 2660.95 ± 391.31 vs 699.57 ± 476.26 pg/ml, p < 0.001; 2660.95 ± 391.31 vs 153.35 ± 78.08 pg/ml, p < 0.001). Either it is stimulated by LL-37 or CpG-ODN or both, the supernatant level of IFN-α in cultured PBMCs in AAV patients was the highest, that in healthy controls was the lowest. Either stimulated by LL-37 or CPG-ODN, or both, the levels of ANCA production in vitro in AAV groups were statistically significantly higher than those in CB group and healthy control group., Conclusions: There were higher serum levels of LL-37 and IFN-α in patients with AAV. IFN-α could reach a higher level stimulated by LL-37 and nucleic acids both of which are related to infection. Patients with AAV have ANCA-producing B lymphocytes in the circulation even in remission stage. Infections could induce the relapse of AAV.

Objective: To investigate the effect of Nogo-66 receptor (NgR) silencing with specific small interfering RNA (siRNA) on brain injury repair in preterm rats with brain injury caused by intrauterine infection and related mechanism of action., Methods: The pregnant Sprague-Dawley rats (with a gestational age of 15 days) were selected, and premature delivery was induced by RU486 or lipopolysaccharide (LPS). The preterm rats delivered by those treated with RU486 were selected as the control group. The preterm rats with brain injury caused by intrauterine infection induced by LPS were divided into model, empty vector, and NgR-siRNA groups, with 36 rats in each group. The rats in the control and model groups were given routine feeding only, and those in the empty vector and NgR-siRNA groups were given an injection of lentiviral empty vector or NgR-siRNA lentivirus via the lateral ventricle on postnatal day 1 (P1) and then fed routinely. On P3, P7, and P14, 8 rats in each group were randomly selected and sacrificed to harvest the brain tissue. RT-PCR was used to measure the mRNA expression of NgR. Western blot was used to to measure the protein expression of active RhoA. The immunofluorescence histochemistry was used to determine the degree of activation of microglial cells and the morphology of oligodendrocyte precursor cells (OPCs). Hematoxylin and eosin staining was used to observe the pathological changes in brain tissue. The behavioral score was evaluated on P30., Results: On P3, the NgR-siRNA group had significantly lower mRNA expression of NgR and protein expression of active RhoA in brain tissue than the model and empty vector groups (P<0.05). In each group, the mRNA expression of NgR was positively correlated with the protein expression of active RhoA (P<0.05). The results of immunofluorescence histochemistry showed that on P3, the NgR-siRNA group had a significantly reduced fluorescence intensity of the microglial cells labeled with CD11b compared with the model and empty vector groups (P<0.05). The OPCs labeled with O4 antibody in the four groups were mainly presented with tripolar cell morphology. The results of pathological examination showed a normal structure of white matter with clear staining in the periventriclar area in the control group, a loose structure of white matter with disorganized fibers and softening lesions in the model and empty vector groups, and a loose structure of white matter with slightly disorganized fibers, slight gliocyte proliferation, and no significant necrotic lesions in the NgR-siRNA group. As for the behavioral score, compared with the model and empty vector groups, the NgR-siRNA group had a higher score in the suspension test, a longer total activity distance, and greater mean velocity and number of squares crossed, as well as a shorter time of slope test and a shorter time and distance of activity in the central area (P<0.05), while there were no significant differences in these parameters between the NgR-siRNA and control groups (P>0.05)., Conclusions: NgR silencing with specific siRNA can effectively silence the expression of NgR in pertem rats with brain injury caused by interauterine infection and has a significant neuroprotective effect in brain injury repair.

To evaluate the time- and dose-dependent toxicity of clofarabine in mice and to further define the chronotherapy strategy of it in leukemia, we compared the mortality rates, LD50s, biochemical parameters, histological changes and organ indexes of mice treated with clofarabine at various doses and time points. Plasma clofarabine levels and pharmacokinetic parameters were monitored continuously for up to 8 hours after the single intravenous administration of 20 mg/kg at 12:00 noon and 12:00 midnight by high performance liquid chromatography (HPLC)-UV method. Clofarabine toxicity in all groups fluctuated in accordance with circadian rhythms in vivo. The toxicity of clofarabine in mice in the rest phase was more severe than the active one, indicated by more severe liver damage, immunodepression, higher mortality rate, and lower LD50. No significant pharmacokinetic parameter changes were observed between the night and daytime treatment groups. These findings suggest the dosing-time dependent toxicity of clofarabine synchronizes with the circadian rhythm of mice, which might provide new therapeutic strategies in further clinical application., (Copyright © 2016. Published by Elsevier Taiwan.)

1,7-Dihydroxy-3,4-dimethoxyxanthone (XAN) is a bioactive compound isolated from Securidaca inappendiculata Hassk. and validated with antiproliferative activities on a panel of cancer cell lines. This study was designed to investigate its growth inhibitory effects on multidrug resistance (MDR) non-small cell lung carcinoma (NSCLC) cell line A549/Taxol and explore the possible linkage between modulation of MAPKs and the bioactivities. Its growth inhibitory potency on the cells was estimated by MTT assay, and flow cytometric analysis was employed to investigate its potential cell cycle arrest and proapoptosis effects. Expressions of hallmark proteins were assessed by Western-Blot method. The results showed A549/Taxol cells were sensitive to XAN. XAN inhibited the proliferation of A549/Taxol cells in the time and concentration dependent manners. It acted as a potent inducer of apoptosis and cell cycle arrest in the cells. Western-Blot investigation validated the proapoptosis and cell cycle arrest activities of XAN and the potential of MDR reversion. Upregulation of p38 by XAN, which accounted for the cell cycle arrest at G2 phase, and the downregulation of ERK associated with the proapoptosis activity were also revealed. Further analysis found p53 may be the central role mediated the bioactivities of MAPKs in A549/Taxol cells. Based on these evidences, a conclusion has been deduced that XAN could be a potential agent for MDR NSCLC therapy targeting specifically MAPKs.

Background: Conducting clinical trials across multiple regions of the world has become common practice. A multiregional clinical trial (MRCT) presents opportunities as well as challenges. However, regional differences of treatment effects appear in many MRCTs, which make the interpretation of clinical trial results difficult and presents challenges for clinical trial design. Alzheimer disease (AD) is a progressive neurodegenerative disorder that affects approximately 5 million people in the United States and is the sixth leading cause of death in the country. In 2014, AD cost the United States $214 billion, and the cost is expected to rise to $1.2 trillion by 2050., Methods: In this article, we utilize data from New Drug Applications (NDAs) that have been approved for the treatment of AD to study whether there are differences in treatment effect between US and non-US study sites. Using an analysis of covariance (ANCOVA) model and forest plot, we analyze the treatment difference by region (US and non-US) from 3 separate perspectives: by region for each trial, by region for each endpoint, and by region and trial for each endpoint., Results: Overall, the analyses indicate that treatment effects in clinical trials for AD are generally in the expected direction in both US and non-US sites. There was no clear evidence of heterogeneity in treatment effects between US and non-US sites., Conclusions: It appears that there is no clear evidence to suggest that MRCTs should not be used to study AD.

Background: The gut microbiota plays an important role in human health. It is essential to understand how the composition of the gut microbiota in neonates is established., Objectives: To investigate the nature of the microbial community in the first feces of newborn infants compared with the mothers' placentae and vaginas., Methods: One infant who was delivered via Cesarean section was compared with an infant who was delivered vaginally. Bar-coded pyro-sequencing of 16S ribosomal RNA genes was used to investigate the bacterial community composition and structure of each site., Results: Neonatal feces of both infants had similar bacterial communities, and they were similar to the mother's placenta regardless of the method of delivery. The vaginal bacterial community differed between the two mothers, but not different sites within the vagina. The bacteria in the neonatal feces and the mothers' placentae demonstrated considerably higher diversity compared with the vaginas. The family Lactobacillaceae dominated in the vaginal samples, while the most abundant family in the fecal and placental samples was Micrococcineae., Conclusions: These results may provide new directions for the study of infant gut microbial formation.

Extra-nodal NK/T lymphoma (ENKTL) is rare and more frequently encountered in East Asia. The role of high-dose therapy and autologous stem cell transplantation (HDT-ASCT) for ENKTL is unclear. Twenty-eight evaluable patients who had undergone HDT-ASCT in Europe from 2000-2009 were studied. The median age was 47 years and patients had received a median of two lines of prior therapy. Some 57% of patients were not in complete remission or beyond first complete remission at HDT-ASCT. The 1-year non-relapse mortality (NRM) was 11%; 2-year progression-free survival (PFS) and overall survival (OS) rates were 41% and 52%, respectively. Notably, the 2-year PFS and OS for those with stage III/IV disease were 33% and 40%, respectively, with no relapses beyond 1-year post-HDT-ASCT. This is the largest analysis of HDT-ASCT for patients with ENKTL reported from the Western hemisphere. Survival is comparable to East Asian cohorts and outcomes are encouraging for patients with advanced disease.

Background: The aim of this study was to investigate the impact of the high-dose regimen on the outcome of patients with follicular lymphoma (FL) having had autologous stem-cell transplantation (ASCT) in a recent time period., Patients: Between 1995 and 2007, 2233 patients with FL had their first ASCT with either a total body irradiation (TBI)-containing regimen or carmustin, etoposide, cytarabine and melphalan (BEAM), of which 47% were autografted in first remission., Results: After a median observation time of 73 months (interquartile range 30-107), 5- and 10-year non-relapse mortality (NRM) was similar (6% and 10% in both groups). No significant NRM differences became evident after multivariate adjustment for confounders. Secondary malignancies were observed in 9.7% and 7.9% of the patients after TBI and BEAM (P = 0.19), which were treatment-related myelodysplastic syndromes/acute myelogenous leukaemia (t-MDS/AML) in 3.4% and 2.8% (P = 0.57). The median time to t-MDS/AML was around 50 months in both groups. Because of a lower relapse incidence, TBI was associated with better event-free survival reaching statistical significance in the patients transplanted in first remission but not in those transplanted beyond first remission., Conclusions: In patients with FL who received TBI-based ASCT after 1995 increased NRM and t-MDS/AML risks did not emerge compared with BEAM while disease control was at least equivalent., (© The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is the standard of care for patients with relapsed Hodgkin's lymphoma (HL). However, there is currently little information on the predictors of outcome for patients whose disease recurs after ASCT., Methods: Five hundred and eleven adult patients with relapsed HL after ASCT from EBMT-GITMO databases were reviewed., Results: Treatments administered following ASCT failure included conventional chemotherapy and/or radiotherapy in 294 (64%) patients, second ASCT in 35 (8%), and alloSCT in 133 (29%). After a median follow-up of 49 months, overall survival (OS) was 32% at 5 years. Independent risk factors for OS were early relapse (<6 months) after ASCT, stage IV, bulky disease, poor performance status (PS), and age ≥50 years at relapse. For patients with no risk factors OS at 5 years was 62% compared with 37% and 12% for those having 1 and ≥2 factors, respectively. This score was also predictive for outcome in each group of rescue treatment after ASCT failure., Conclusion(s): Early relapse, stage IV, bulky disease, poor PS, and age ≥50 years at ASCT failure are relevant factors for outcome that may help to understand the results of different therapeutic approaches.

Enteropathy-associated T-cell lymphoma (EATL) is a rare subtype of peripheral T-cell lymphomas with a poor prognosis. Autologous stem cell transplantation (ASCT) was retrospectively evaluated as a consolidation or salvage strategy for EATL. The analysis included 44 patients who received ASCT for EATL between 2000 and 2010. Thirty-one patients (70%) were in first complete or partial remission at the time of the ASCT. With a median follow-up of 46 months, relapse incidence, progression-free survival, and overall survival were 39%, 54%, and 59% at 4 years, respectively, with only one relapse occurring beyond 18 months posttransplant. There was a trend for better survival in patients transplanted in first complete or partial remission at 4 years (66% vs 36%; P = .062). ASCT is feasible in selected patients with EATL and can yield durable disease control in a significant proportion of the patients.

Background: Limited experience is available on the feasibility and efficacy of autologous stem-cell transplantation (ASCT) in patients with mantle cell lymphoma (MCL) beyond 65 years., Design and Methods: We analysed 712 patients with MCL treated with ASCT from 2000 to 2007 and reported to the European Group for Blood and Marrow Transplantation registry. Patients>65 years were compared with patients<65 years for the end points non-relapse mortality (NRM), relapse incidence, progression-free survival (PFS), and overall survival (OS)., Results: Seventy-nine patients were ≥65 years old. Median time from diagnosis to ASCT was longer in the elderly patients (11 versus 9 months, P=0.005); they had more commonly received at least two treatment lines (62.0% versus 47.9%, P=0.02) and were less commonly in first complete remission at ASCT (35.4% versus 51.2%, P=0.002). Median follow-up after ASCT was 19 and 25 months, respectively. NRM was comparable at 3 months (3.8% versus 2.5%) and at 5 years (5.6% versus 5.0%). There were no differences in relapse rate (66% versus 55% at 5 years), PFS (29% versus 40%) and OS (61% versus 67%) between both populations of patients., Conclusion: ASCT beyond 65 years of age is feasible in selected patients with MCL and results in similar disease control and survival as in younger patients.

Purpose: To analyze the outcome, including nonrelapse mortality (NRM), relapse rate (RR), progression-free survival (PFS), and overall survival (OS), of patients with diffuse large B-cell non-Hodgkin's lymphoma (DLBCL) relapsed after an autologous stem-cell transplantation (ASCT) and treated with an allogeneic stem-cell transplantation (allo-SCT)., Patients and Methods: The European Group for Blood and Marrow Transplantation database was scanned for a first allo-SCT in relapsed DLBCL after a previous ASCT between 1997 and 2006. Other inclusion criteria were age at allo-SCT ≥ 18 years and availability of an HLA-identical sibling or a matched unrelated donor. A total of 101 patients (57 males; median age, 46 years) were included. Median follow-up for survivors was 36 months., Results: Myeloablative conditioning regimen was used in 37 patients and reduced intensity conditioning (RIC) was used in 64 patients. Three-year NRM was 28.2% (95% CI, 20% to 39%), RR was 30.1% (95% CI, 22% to 41%), PFS was 41.7% (95% CI, 32% to 52%), and OS was 53.8% (95% CI, 44% to 64%). NRM was significantly increased in patients ≥ 45 years (P = .01) and in those with an early relapse (< 12 months) after ASCT (P = .01). RR was significantly higher in refractory patients (P = .03). A time interval to relapse after ASCT of < 12 months was associated with lower PFS (P = .03). The use of RIC regimens was followed by a trend to a lower NRM (P = .1) and a trend to a higher RR (P = .1), with no differences in PFS and OS. No differences were seen between HLA-identical siblings and matched unrelated donors., Conclusion: Allo-SCT in relapsed DLBCL after ASCT is a promising therapeutic modality. Patients with a long remission after ASCT and with sensitive disease at allo-SCT are the best candidates for this approach.

Objective: To compare the antioxidant activity amongst the extract of Ginkgo biloba (EGb) and its main components, flavonoids and terpenoids., Methods: The induction of EGb, flavonoids and terpenoids on a typical antioxidant enzyme, glutamate cysteine ligase catalytic subunit (GCLC), in cell lines was detected by Western-blot. The effects of EGb, flavonoids and terpenoids on superoxide anion radical (O2*(-)), hydroxyl radical (OH*), rat erythrocyte hemolysis and lipid peroxidation of rat liver homogenate were determined by respective activity methods., Results: EGb and flavonoids but not terpenoids were demonstrated significantly to induce the antioxidant enzyme (GCLC), directly scavenge O2*(-), OH* and inhibit rat erythrocyte hemolysis and lipid peroxidation of rat liver homogenate. Compared these antioxidant activities between EGb and flavonoids, the activities of flavonoids were weaker than those of EGb, which contains similar dose of flavonoids., Conclusion: EGb has stronger antioxidant activities than flavonoids, but terpenoids did not show antioxidant activity in this research.

Many bipolar affective disorder (BD) susceptibility loci have been identified but the molecular mechanisms responsible for the disease remain to be elucidated. In the locus 4p16, several candidate genes were identified but none of them was definitively shown to be associated with BD. In this region, the PPP2R2C gene encodes the Bgamma-regulatory subunit of the protein phosphatase 2A (PP2A-Bgamma). First, we identified, in two different populations, single nucleotide polymorphisms and risk haplotypes for this gene that are associated to BD. Then, we used the Bgamma subunit as bait to screen a human brain cDNA library with the yeast two-hybrid technique. This led us to two new splice variants of KCNQ2 channels and to the KCNQ2 channel itself. This unusual K+ channel has particularly interesting functional properties and belongs to a channel family that is already known to be implicated in several other monogenic diseases. In one of the BD populations, we also found a genetic association between the KCNQ2 gene and BD. We show that KCNQ2 splice variants differ from native channels by their shortened C-terminal sequences and are unique as they are active and exert a dominant-negative effect on KCNQ2 wild-type (wt) channel activity. We also show that the PP2A-Bgamma subunit significantly increases the current generated by KCNQ2wt, a channel normally inhibited by phosphorylation. The kinase glycogen synthase kinase 3 beta (GSK3beta) is considered as an interesting target of lithium, the classical drug used in BD. GSK3beta phosphorylates the KCNQ2 channel and this phosphorylation is decreased by Li+.

Background: People in the United States are using complementary and alternative medicine (CAM) increasingly while they are also receiving conventional care. National population-based surveys and studies in primary care settings have documented inadequate communication about CAM between patients and their conventional healthcare providers. Most studies about CAM communication have surveyed urban practices and focused on physicians. Information about how physicians and non-physician in rural areas clinicians communicate with their patients about CAM is needed to develop strategies for improving the quality of care for patients in rural areas., Objective: To investigate how primary care clinicians in the Kentucky Ambulatory Network (KAN) communicate with patients about CAM and to determine interest in additional education about CAM., Methods: A self-administered survey was mailed to 112 community clinicians in a research network of largely rural practices. KAN members include primary care physicians, nurse practitioners, certified nurse midwives, and physician assistants practicing in 32 counties in central and eastern Kentucky., Results: Of 102 deliverable surveys, 65 (64%) were returned. Sixty-one (94%) clinicians reported patient CAM use. Few clinicians consistently asked patients about CAM. A positive attitude toward patient CAM use was associated with clinician comfort in advising patients. Most clinicians recommended CAM to patients. Seventy percent of KAN clinicians expressed interest in continuing education about CAM., Conclusions: Kentucky primary care clinicians are aware of their patients' CAM use and are motivated to learn more about CAM so that they can appropriately advise their patients. They need evidence-based, clinically relevant education about CAM to provide better patient care.

A map of 191 single-nucleotide polymorphism (SNPs) was built across a 5-Mb segment from chromosome 13q34 that has been genetically linked to schizophrenia. DNA from 213 schizophrenic patients and 241 normal individuals from Canada were genotyped with this marker set. Two 1,400- and 65-kb regions contained markers associated with the disease. Two markers from the 65-kb region were also found to be associated to schizophrenia in a Russian sample. Two overlapping genes G72 and G30 transcribed in brain were experimentally annotated in this 65-kb region. Transfection experiments point to the existence of a 153-aa protein coded by the G72 gene. This protein is rapidly evolving in primates, is localized to endoplasmic reticulum/Golgi in transfected cells, is able to form multimers and specifically binds to carbohydrates. Yeast two-hybrid experiments with the G72 protein identified the enzyme d-amino acid oxidase (DAAO) as an interacting partner. DAAO is expressed in human brain where it oxidizes d-serine, a potent activator of N-methyl-D-aspartate type glutamate receptor. The interaction between G72 and DAAO was confirmed in vitro and resulted in activation of DAAO. Four SNP markers from DAAO were found to be associated with schizophrenia in the Canadian samples. Logistic regression revealed genetic interaction between associated SNPs in vicinity of two genes. The association of both DAAO and a new gene G72 from 13q34 with schizophrenia together with activation of DAAO activity by a G72 protein product points to the involvement of this N-methyl-d-aspartate receptor regulation pathway in schizophrenia.

Nonobese diabetic (NOD) mice develop spontaneous autoimmune diabetes that results from the destruction of insulin secreting beta cells by diabetogenic T cells. The time and location of the encounter of autoantigen(s) by naive autoreactive T cells in normal NOD mice are still elusive. To address these issues, we analyzed diabetes development in mice whose spleen or pancreatic lymph nodes (panLNs) had been removed. Excision of panLNs (panLNx) at 3 wk protected mice against insulin autoantibodies (IAAs), insulitis, and diabetes development almost completely, but had no effect when performed at 10 wk. The protection afforded by panLNx at weaning was not due to modifications of the immune system, the absence of autoreactive T cells, or the increase in the potency of regulatory T cells. That panLNs are dispensable during adult life was confirmed by the capacity of 10-wk-old panLNx irradiated recipients to develop diabetes upon transfer of diabetogenic T cells. In contrast, splenectomy had no effect at any age. Partial excision of mesenteric LN at 3 wk did not prevent accelerated diabetes by cyclophosphamide as panLNx did. Thus, in normal NOD mice, autoreactive T cell initial priming occurs in LNs draining the target organ of the disease from 3 wk of age.

Activated lymphocytes of autoimmune non-obese diabetic (NOD) mice exhibit an increased resistance to programmed cell death (PCD) following withdrawal of interleukin-2 (IL-2). In the present study, we found that resistance of NOD T lymphocytes to PCD was increased as early as 1 week of age, hence several weeks before the invasion of the pancreas by inflammatory cells, which is compatible with a role of the NOD apoptotic phenotype in the autoimmune susceptibility of this strain. In the thymus, mature single positive but not double positive or double negative thymocytes were more resistant to PCD in NOD compared to B6 mice. Moreover, in both NOD and B6 mice, CD4+ T cells were more resistant to PCD induced by IL-2 deprivation than CD8+ cells. As a result, NOD CD4+ T cells were remarkably resistant to cell death induced in this manner. In relation with this increased resistance to apoptosis, expression of the anti-apoptotic Bcl-x protein was upregulated in activated T cells of NOD mice, most notably after 24 h of IL-2 deprivation. These results should help us to understand the relationship of the NOD apoptotic phenotype to the emergence of the NOD mouse autoimmune disease.

A quantitative trait locus for increased IgG serum levels in the NOD mouse strain was mapped to distal chromosome 1, close to the fcgr2 locus encoding the low-affinity type II receptor for the Fc portion of IgG (Fc gamma RII). Expression of membrane-inserted (b2) and soluble (b3) isoforms of Fc gamma RII was strongly decreased in macrophages of NOD compared with C57BL/6 (B6) mice. In contrast, B cell-specific (Fc gamma RIIb1) isoform was only slightly decreased and Fc gamma RIII was not altered. This Fc gamma RII regulatory defect was cis-encoded by fcgr2 or by a closely linked locus, occurred at the mRNA level, and was associated with multiple mutations in the fcgr2 gene promoter. In relation with this defect, binding of IgG1- and IgG2b- but not IgG2a-opsonized RBC by macrophages of NOD and congenic B6.NOD-fcgr2 mice was severely impaired, but was normal in macrophages of NOD.B6-fcgr2 congenic mice, indicating that Fc gamma RII plays a nondispensable role in binding of IgG1 and IgG2b isotypes. Likewise, serum levels of IgG1 and IgG2b but not IgG2a were up-regulated in NOD compared with NOD.B6-fcgr2 congenic mice. These findings indicate that macrophage Fc gamma RII may regulate serum IgG1 and IgG2b through their catabolism, and validate the NOD strain as a model to investigate the functions of Fc gamma RII isoforms.

The non-obese diabetic (NOD) mouse strain provides a remarkable model for investigating the mechanisms of autoimmunity. Independent genetic analyses of this model have previously shown that chromosome 1-linked loci were involved in the control of periinsulitis and sialitis on the one hand and of insulitis and diabetes on the other hand. In the present work, analysis of a [NOD x (NOD x C57BL/6)F1] backcross progeny allowed us to clearly dissociate two genetic regions: one was associated with periinsulitis and mapped to the middle region of chromosome 1, in the vicinity of the Bcl-2 gene; the other was associated with insulitis and mapped to the proximal part of the chromosome. Three intermediate markers D1Mit18, D1Mit5 and D1Mit19 covering at least 25 centiMorgans between these two regions, were associated with neither periinsulitis nor insulitis. The role of the Bcl-2-linked region in the immune anomalies of NOD mice was further investigated in a (NOD x C57BL/6)F2 cross where the Bcl-2nod haplotype was linked to elevated serum levels of IgG (p < 0.0005). The middle region of chromosome 1 is, therefore, involved in the control of three phenotypes, including periinsulitis, sialitis and hyperIgG, pointing to Bcl-2 as a good candidate for a cause of the NOD mouse disease. Consistent with the anti-apoptotic function of the Bcl-2 gene product, activated T lymphocytes from NOD mice showed a markedly increased resistance to induction of apoptosis following deprivation of interleukin-2 when compared to those from non-autoimmune strains. After the recent observation of the Fas gene alterations in the lpr and lprcg mutations, these findings indicate that deregulation of lymphoid cell apoptosis may be a general pathogenetic mechanism in autoimmune diseases.

The nonobese diabetic mouse in a model of spontaneous development of autoimmune type I diabetes. The disease can be induced in young, irradiated recipients by injecting splenic T-cells from diabetic donors. The adoptive transfer of diabetes requires the presence of both CD4+ and CD8+ splenic T-cell subsets. To test whether diabetogenic cells distribute in other lymphoid organs of diabetic mice, we first analyzed lymph node cells. Lymph node cells were much less efficient in transferring diabetes than splenocytes. This inefficacious transfer was not attributable to the absence of hematopoietic precursors or a lack of macrophages. Lymph node cells did not protect from the transfer of diabetes by splenocytes, indicating the absence of suppressor cells. Although CD8+ lymph node T-cells seemed functionally comparable to CD8+ splenocytes, CD4+ lymph node T-cells failed to cooperate with CD8+ splenocytes to transfer diabetes. Our study suggests that diabetogenic cells are not evenly distributed in the different lymphoid organs. This may reflect a differential migration pattern of pathogenic T-cells in this animal model.

It is well established that the NOD mouse develops T-cell-dependent autoimmune type I diabetes that is abolished by neonatal Tx and enhanced by Tx at weaning. In a previous study, we demonstrated that the NOD thymus displays various abnormalities in the microenvironmental compartment, including abnormal distribution of epithelial cell subsets, precocious decline in thymic hormone production and formation of giant PVS. These latter structures present an internal ECM-containing network filled with T-cells and to a lesser extent B-cells. Herein we have investigated further the giant PVS and particularly the origin of the T-cells that colonize these structures. The thymic origin of intra-PVS T-cells was ascertained by distinct protocols. First, sublethal X-ray irradiation or HC treatment leading to cortical thymocyte depletion showed that intra-PVS lymphocytes were resistant, similar to medullary thymocytes. Second, adoptive transfer experiments that used newborn or adult irradiated Thy-1 congenic recipients demonstrated that intra-PVS accumulation of T-cells did not result from the reentry of peripheral mature T-cells into the thymus. Third, kinetic studies that used BrdUrd pulse chase revealed that labeled intra-PVS cells appear late, simultaneously with medullary thymocytes, and remain only transiently within the PVS. Thus, the kinetics of T-cell reconstitution of PVS was compatible with the progressive differentiation of T-cell precursors originating from the thymic cortex. In this respect, the giant PVS of the NOD mouse thymus may represent a useful model to study the relationships between trafficking thymocytes and ECM proteins.