Methods for non-proportional hazards in clinical trials: A systematic review.

For the analysis of time-to-event data, frequently used methods such as the log-rank test or the Cox proportional hazards model are based on the proportional hazards assumption, which is often debatable. Although a wide range of parametric and non-parametric methods for non-proportional hazards has been proposed, there is no consensus on the best approaches. To close this gap, we conducted a systematic literature search to identify statistical methods and software appropriate under non-proportional hazard. Our literature search identified 907 abstracts, out of which we included 211 articles, mostly methodological ones. Review articles and applications were less frequently identified. The articles discuss effect measures, effect estimation and regression approaches, hypothesis tests, and sample size calculation approaches, which are often tailored to specific non-proportional hazard situations. Using a unified notation, we provide an overview of methods available. Furthermore, we derive some guidance from the identified articles.

LMO3 reprograms visceral adipocyte metabolism during obesity.

Obesity and body fat distribution are important risk factors for the development of type 2 diabetes and metabolic syndrome. Evidence has accumulated that this risk is related to intrinsic differences in behavior of adipocytes in different fat depots. We recently identified LIM domain only 3 (LMO3) in human mature visceral adipocytes; however, its function in these cells is currently unknown. The aim of this study was to determine the potential involvement of LMO3-dependent pathways in the modulation of key functions of mature adipocytes during obesity. Based on a recently engineered hybrid rAAV serotype Rec2 shown to efficiently transduce both brown adipose tissue (BAT) and white adipose tissue (WAT), we delivered YFP or Lmo3 to epididymal WAT (eWAT) of C57Bl6/J mice on a high-fat diet (HFD). The effects of eWAT transduction on metabolic parameters were evaluated 10 weeks later. To further define the role of LMO3 in insulin-stimulated glucose uptake, insulin signaling, adipocyte bioenergetics, as well as endocrine function, experiments were conducted in 3T3-L1 adipocytes and newly differentiated human primary mature adipocytes, engineered for transient gain or loss of LMO3 expression, respectively. AAV transduction of eWAT results in strong and stable Lmo3 expression specifically in the adipocyte fraction over a course of 10 weeks with HFD feeding. LMO3 expression in eWAT significantly improved insulin sensitivity and healthy visceral adipose tissue expansion in diet-induced obesity, paralleled by increased serum adiponectin. In vitro, LMO3 expression in 3T3-L1 adipocytes increased PPARγ transcriptional activity, insulin-stimulated GLUT4 translocation and glucose uptake, as well as mitochondrial oxidative capacity in addition to fatty acid oxidation. Mechanistically, LMO3 induced the PPARγ coregulator Ncoa1, which was required for LMO3 to enhance glucose uptake and mitochondrial oxidative gene expression. In human mature adipocytes, LMO3 overexpression promoted, while silencing of LMO3 suppressed mitochondrial oxidative capacity. LMO3 expression in visceral adipose tissue regulates multiple genes that preserve adipose tissue functionality during obesity, such as glucose metabolism, insulin sensitivity, mitochondrial function, and adiponectin secretion. Together with increased PPARγ activity and Ncoa1 expression, these gene expression changes promote insulin-induced GLUT4 translocation, glucose uptake in addition to increased mitochondrial oxidative capacity, limiting HFD-induced adipose dysfunction. These data add LMO3 as a novel regulator improving visceral adipose tissue function during obesity. KEY MESSAGES: LMO3 increases beneficial visceral adipose tissue expansion and insulin sensitivity in vivo. LMO3 increases glucose uptake and oxidative mitochondrial activity in adipocytes. LMO3 increases nuclear coactivator 1 (Ncoa1). LMO3-enhanced glucose uptake and mitochondrial gene expression requires Ncoa1.

Transfer and loss of allergen-specific responses via stem cell transplantation: A prospective observational study.

BACKGROUND: Currently, no estimates can be made on the impact of hematopoietic stem cell transplantation on allergy transfer or cure of the disease. By using component-resolved diagnosis, we prospectively investigated 50 donor-recipient pairs undergoing allogeneic stem cell transplantation. This allowed calculating the rate of transfer or maintenance of allergen-specific responses in the context of stem cell transplantation. METHODS: Allergen-specific IgE and IgG to 156 allergens was measured pretransplantation in 50 donors and recipients and at 6, 12 and 24 months in recipients post-transplantation by allergen microarray. Based on a mixed effects model, we determined risks of transfer of allergen-specific IgE or IgG responses 24 months post-transplantation. RESULTS: After undergoing stem cell transplantation, 94% of allergen-specific IgE responses were lost. Two years post-transplantation, recipients' allergen-specific IgE was significantly linked to the pretransplantation donor or recipient status. The estimated risk to transfer and maintain individual IgE responses to allergens by stem cell transplantation was 1.7% and 2.3%, respectively. Allergen-specific IgG, which served as a surrogate marker of maintaining protective IgG responses, was highly associated with the donor's (31.6%) or the recipient's (28%) pretransplantation response. CONCLUSION: Hematopoietic stem cell transplantation profoundly reduces allergen-specific IgE responses but also comes with a considerable risk to transfer allergen-specific immune responses. These findings facilitate clinical decision-making regarding allergic diseases in the context of hematopoietic stem cell transplantation. In addition, it provides prospective data to estimate the risk of transmitting allergen-specific responses via hematopoietic stem cell transplantation.

The Peritoneal Surface Proteome in a Model of Chronic Peritoneal Dialysis Reveals Mechanisms of Membrane Damage and Preservation.

Peritoneal dialysis (PD) fluids are cytotoxic to the peritoneum. Recent studies have shown that alanyl-glutamine (AlaGln) modulates the cellular stress response, improves mesothelial cell survival, reduces submesothelial thickening in experimental models of PD, and in clinical studies improves PD effluent cell stress and immune responses. However, the mechanisms of AlaGln-mediated membrane protection are not yet fully understood. Here, we explore those mechanisms through application of a novel proteomics approach in a clinically relevant in vivo model in rats. Experimental PD was performed for 5 weeks using conventional single-chamber bag (SCB) or neutral dual-chamber bag (DCB), PD fluid (PDF), with or without AlaGln supplementation, via a surgically implanted catheter. Rats subjected to a single dwell without catheter implantation served as controls. The peritoneal surface proteome was directly harvested by detergent extraction and subjected to proteomic analysis by two-dimensional difference gel electrophoresis (2D-DiGE) with protein identification by mass spectrometry. An integrated bioinformatic approach was applied to identify proteins significantly affected by the treatments despite biological variation and interfering high abundance proteins. From 505 of 744 common spots on 59 gels, 222 unique proteins were identified. Using UniProt database information, proteins were assigned either as high abundance plasma proteins, or as cellular proteins. Statistical analysis employed an adapted workflow from RNA-sequencing, the trimmed mean of M-values (TMM) for normalization, and a mixed model for computational identification of significantly differentially abundant proteins. The most prominently enriched pathways after 5 weeks chronic treatment with SCB or DCB, PDFs belonged to clusters reflecting tissue damage and cell differentiation by cytoskeletal reorganization, immune responses, altered metabolism, and oxidative stress and redox homeostasis. Although the AlaGln effect was not as prominent, associated enriched pathways showed mostly regression to control or patterns opposite that of the PDF effect. Our study describes the novel peritoneal surface proteome through combined proteomic and bioinformatic analyses, and assesses changes elicited by chronic experimental PD. The biological processes so identified promise to link molecular mechanisms of membrane damage and protection in the in vivo rat model to pathomechanisms and cytoprotective effects observed in vitro and in clinical PD.

Deviations of the immune cell landscape between healthy liver and hepatocellular carcinoma.

Tumor-infiltrating immune cells are highly relevant for prognosis and identification of immunotherapy targets in hepatocellular carcinoma (HCC). The recently developed CIBERSORT method allows immune cell profiling by deconvolution of gene expression microarray data. By applying CIBERSORT, we assessed the relative proportions of immune cells in 41 healthy human livers, 305 HCC samples and 82 HCC adjacent tissues. The obtained immune cell profiles provided enumeration and activation status of 22 immune cell subtypes. Mast cells were evaluated by immunohistochemistry in ten HCC patients. Activated mast cells, monocytes and plasma cells were decreased in HCC, while resting mast cells, total and naïve B cells, CD4+ memory resting and CD8+ T cells were increased when compared to healthy livers. Previously described S1, S2 and S3 molecular HCC subclasses demonstrated increased M1-polarized macrophages in the S3 subclass with good prognosis. Strong total immune cell infiltration into HCC correlated with total B cells, memory B cells, T follicular helper cells and M1 macrophages, whereas weak infiltration was linked to resting NK cells, neutrophils and resting mast cells. Immunohistochemical analysis of patient samples confirmed the reduced frequency of mast cells in human HCC tumor tissue as compared to tumor adjacent tissue. Our data demonstrate that deconvolution of gene expression data by CIBERSORT provides valuable information about immune cell composition of HCC patients.

Oxidative stress mediates an increased formation of vascular endothelial growth factor in human hepatocarcinoma cells exposed to erlotinib.

The tyrosine kinase inhibitor erlotinib targets the receptor of epidermal growth factor (EGFR) involved in development of hepatocellular carcinoma (HCC). Although inefficient in established HCC, erlotinib has been recently proposed for HCC chemoprevention. Since Cyp3A4 and Cyp1A2 enzymes metabolize erlotinib in the liver, the insights into the mechanisms of erlotinib effects on liver cells with maintained drug metabolizing activity are needed. We applied erlotinib to both commercially available (SNU398, Huh7) and established in Austria HCC cell lines (HCC-1.2, HCC-3). Cyp3A4 and Cyp1A2, microarray gene expression, cell viability, LDH release, DHFC fluorescence were assessed. VEGF expression was analysed by real-time RT-PCR and ELISA. Higher cumulative expression of erlotinib metabolizing enzymes was observed in HCC-1.2 and HCC-3 cells. Gene expression microarray analysis showed upregulation of VEGF signalling by erlotinib. VEGF was increased up to 134 ± 14% (n = 5, p = 0.002) in HCC-1.2, HCC-3 and Huh7 cells. Interventions by Cyp1A2 and Mek2siRNA, MEK inhibitor UO126, diphenylene iodonium, as well as a combination of N-acetylcysteine with selenium all inhibited VEGF upregulation caused by erlotinib. Thus, erlotinib increases VEGF production by mechanisms involving Cyp1A2, oxidative stress and MEK1/2. VEGF may favour angiogenesis and growth of early HCC tumours limiting the therapeutic and chemopreventive effects of erlotinib.

HO-1 inhibits preadipocyte proliferation and differentiation at the onset of obesity via ROS dependent activation of Akt2.

Excessive accumulation of white adipose tissue (WAT) is a hallmark of obesity. The expansion of WAT in obesity involves proliferation and differentiation of adipose precursors, however, the underlying molecular mechanisms remain unclear. Here, we used an unbiased transcriptomics approach to identify the earliest molecular underpinnings occuring in adipose precursors following a brief HFD in mice. Our analysis identifies Heme Oxygenase-1 (HO-1) as strongly and selectively being upregulated in the adipose precursor fraction of WAT, upon high-fat diet (HFD) feeding. Specific deletion of HO-1 in adipose precursors of Hmox1fl/flPdgfraCre mice enhanced HFD-dependent visceral adipose precursor proliferation and differentiation. Mechanistically, HO-1 reduces HFD-induced AKT2 phosphorylation via ROS thresholding in mitochondria to reduce visceral adipose precursor proliferation. HO-1 influences adipogenesis in a cell-autonomous way by regulating events early in adipogenesis, during the process of mitotic clonal expansion, upstream of Cebpα and PPARγ. Similar effects on human preadipocyte proliferation and differentiation in vitro were observed upon modulation of HO-1 expression. This collectively renders HO-1 as an essential factor linking extrinsic factors (HFD) with inhibition of specific downstream molecular mediators (ROS &AKT2), resulting in diminished adipogenesis that may contribute to hyperplastic adipose tissue expansion.

Cardiac extracellular matrix is associated with adverse outcome in patients with chronic heart failure.

AIMS: Accumulation of extracellular matrix (ECM) is known to play a crucial role in the pathophysiology of heart failure (HF). However, its prognostic relevance is poorly investigated. METHODS AND RESULTS: A total of 73 HF patients who underwent LV endomyocardial biopsy were enrolled in our study. ECM area was quantified by TissueFAXS and ImageJ software. Patients were followed-up at 6-month intervals. The study endpoint was defined as hospitalization for a cardiac reason and/or cardiac death. Furthermore, the influence of the ECM on invasively measured haemodynamic parameters was tested. During a median follow-up period of 9.0 months, 34 patients (46.6%) reached the combined endpoint. Median ECM area was 30.5%. Patients with ECM area ≥30.5% experienced significantly more events (67.6% vs. 25.0%, P < 0.001) in comparison with patients with an ECM area <30.5%. ECM area was independently associated with outcome in the total HF cohort [hazard ratio (HR) 1.041, 95% confidence interval (CI) 1.017-1.066, P = 0.001] as well as in HF patients with preserved (HR 1.079, 95% CI 1.001-1.163, P =0 .046) or reduced ejection fraction (HR 1.149, 95% CI 1.036-1.275, P = 0.009). Positive correlations were found between ECM area and LV end-diastolic pressure (P = 0.021, R = 0.303), pulmonary artery wedge pressure (P = 0.042, R = 0.249), mean pulmonary arterial pressure (P = 0.035, R = 0.258), as well as right atrial pressure (P = 0.003, R = 0.353). CONCLUSION: ECM area within the LV myocardium correlates with left and right heart haemodynamics and is associated with clinical course in various non-ischaemic HF types.

Response prediction to neoadjuvant chemotherapy: comparison between pre-therapeutic gene expression profiles and in vitro chemosensitivity assay.

Although the use of (neo-)adjuvant chemotherapy in breast cancer patients has resulted in improved outcome, not all patients benefit equally. We have evaluated the utility of an in vitro chemosensitivity assay in predicting response to neoadjuvant chemotherapy. Pre-therapeutic biopsies were obtained from 30 breast cancer patients assigned to neoadjuvant epirubicin 75 mg/m2 and docetaxel 75 mg/m2 (Epi/Doc) in a prospectively randomized clinical trial. Biopsies were subjected to a standardized ATP-based Epi/Doc chemosensitivity assay, and to gene expression profiling. Patients then received 3 cycles of chemotherapy, and response was evaluated by changes in tumor diameter and Ki67 expression. The efficacy of Epi/Doc in vitro was correlated with differential changes in tumor cell proliferation in response to Epi/Doc in vivo (p = 0.0011; r = 0.73670, Spearmans rho), but did not predict for changes in tumor size. While a pre-therapeutic gene expression signature identified tumors with a clinical response to Epi/Doc, no such signature could be found for tumors that responded to Epi/Doc in vitro, or tumors in which Epi/Doc exerted an antiproliferative effect in vivo. This is the first prospective clinical trial to demonstrate the utility of a standardized in vitro chemosensitivity assay in predicting the individual biological response to chemotherapy in breast cancer.

Identification of novel trophoblast invasion-related genes: heme oxygenase-1 controls motility via peroxisome proliferator-activated receptor gamma.

Invasion of cytotrophoblasts (CTBs) into uterine tissues is essential for placental development. To identify molecules regulating trophoblast invasion, mRNA signatures of purified villous (CTB, poor invasiveness) and extravillous trophoblasts (EVTs) (high invasiveness) isolated from first trimester human placentae and villous explant cultures, respectively, were compared using GeneChip analyses yielding 991 invasion/migration-related transcripts. Several genes involved in physiological and pathological cell invasion, including A disintegrin and metalloprotease-12, -19, -28, as well as Spondin-2, were up-regulated in EVTs. Pathway prediction analyses identified several functional modules associated with either the invasive or noninvasive trophoblast phenotype. One of the genes that was down-regulated in the invasive mRNA pool, heme oxygenase-1 (HO-1), was selected for functional analyses. Real-time PCR analyses, Western blotting, and immunofluorescence of first trimester placentae and differentiating villous explant cultures demonstrated down-regulation of HO-1 in invasive EVTs as compared with CTBs. Modulation of HO-1 expression in loss-of as well as gain-of function cell models (BeWo and HTR8/SVneo, respectively) demonstrated an inverse relationship of HO-1 expression with trophoblast migration in transwell and wound healing assays. Importantly, HO-1 expression led to an increase in protein levels and activity of the nuclear hormone receptor peroxisome proliferator activated receptor (PPAR) gamma. Pharmacological inhibition of PPARgamma abrogated the inhibitory effects of HO-1 on trophoblast migration. Collectively, our results demonstrate that gene expression profiling of EVTs and CTBs can be used to unravel novel regulators of cell invasion. Accordingly, we identify HO-1 as a negative regulator of trophoblast motility acting via up-regulation of PPARgamma.