Rates of Serious Intracellular Infections in Autoimmune Disease Patients Receiving Initial Glucocorticoid Therapy

Background/Aims

The Japanese National Hospital Organization evidence-based medicine (EBM) Study group for Adverse effects of Corticosteroid therapy (J-NHOSAC) is a Japanese hospital-based cohort study investigating the safety of the initial use of glucocorticoids (GCs) in patients with newly diagnosed autoimmune diseases. Using the J-NHOSAC registry, the purpose of this observational study is to analyse the rates, characteristics and associated risk factors of intracellular infections in patients with newly diagnosed autoimmune diseases who were initially treated with GCs.

Methodology/Principal Findings

A total 604 patients with newly diagnosed autoimmune diseases treated with GCs were enrolled in this registry between April 2007 and March 2009. Cox proportional-hazards regression was used to determine independent risk factors for serious intracellular infections with covariates including sex, age, co-morbidity, laboratory data, use of immunosuppressants and dose of GCs. Survival was analysed according to the Kaplan-Meier method and was assessed by the log-rank test. There were 127 serious infections, including 43 intracellular infections, during 1105.8 patient-years of follow-up. The 43 serious intracellular infections resulted in 8 deaths. After adjustment for covariates, diabetes (Odds ratio [OR]: 2.5, 95% confidence interval [95% CI] 1.1–5.9), lymphocytopenia (≦1000/μl, OR: 2.5, 95% CI 1.2–5.2) and use of high-dose (≧30 mg/day) GCs (OR: 2.4, 95% CI 1.1–5.3) increased the risk of intracellular infections. Survival curves showed lower intracellular infection-free survival rate in patients with diabetes, lymphocytopaenia and high-dose GCs treatments.

Conclusions/Significance

Patients with newly diagnosed autoimmune diseases were at high risk of developing intracellular infection during initial treatment with GCs. Our findings provide background data on the risk of intracellular infections of patients with autoimmune diseases. Clinicians showed remain vigilant for intracellular infections in patients with autoimmune diseases who are treated with GCs.     

Identification of Biological Properties of Intralymphatic Tumor Related to the Development of Lymph Node Metastasis in Lung Adenocarcinoma

Background

Intralymphatic tumors in the extratumoral area are considered to represent the preceding phase of lymph node metastasis. The aim of this study was to clarify the biological properties of intralymphatic tumors susceptible to the development of lymph node metastasis, with special reference to the expression of cancer initiating/stem cell (CIC/CSC) related markers in cancer cells and the number of infiltrating stromal cells.

Material and Methods

Primary lung adenocarcinomas with lymphatic permeation in the extratumoral area were retrospectively examined (n = 107). We examined the expression levels of CIC/CSC related markers including ALDH1, OCT4, NANOG, SOX2 and Caveolin-1 in the intralymphatic cancer cells to evaluate their relationship to lymph node metastasis. Moreover, the number of infiltrating stromal cells expressing CD34, α-smooth muscle actin, and CD204 were also evaluated.

Results

Among the intralymphatic tissues, low ALDH1 expression in cancer cells, high SOX2 expression in cancer cells, and a high number of CD204(+) macrophages were independent predictive factors for lymph node metastasis (P = 0.004, P = 0.008, and P = 0.028, respectively). Among these factors, only low ALDH1 expression in cancer cells was significantly correlated with the farther spreading of lymph node metastasis (mediastinal lymph node, pathological N2) (P = 0.046) and the metastatic lymph node ratio (metastatic/resected) (P = 0.028). On the other hand, in the primary tumors, ALDH1 expression in the cancer cells was not associated with lymph node metastasis. Intralymphatic cancer cells expressing low ALDH1 levels exhibited lower E-cadherin expression levels than cancer cells with high levels of ALDH1 expression (P = 0.015).

Conclusions

Intralymphatic cancer cells expressing low levels of ALDH1 and infiltrating macrophages expressing CD204 have a critical impact on lymph node metastasis. Our study also highlighted the significance of evaluating the biological properties of intralymphatic tumors for tumor metastasis.     

Urinary Fetuin-A Is a Novel Marker for Diabetic Nephropathy in Type 2 Diabetes Identified by Lectin Microarray

We analyzed the urine samples of patients with type 2 diabetes at various stages of diabetic nephropathy by lectin microarray to identify a biomarker to predict the progression of diabetic nephropathy. Japanese patients with type 2 diabetes at various stages of nephropathy were enrolled and we performed lectin microarray analyses (n = 17) and measured urinary excretion of fetuin-A (n = 85). The increased signals of urine samples were observed in Siaα2-6Gal/GalNAc-binding lectins (SNA, SSA, TJA-I) during the progression of diabetic nephropathy. We next isolated sialylated glycoproteins by using SSA-lectin affinity chromatography and identified fetuin-A by liquid chromatography–tandem mass spectrometer. Urinary excretion of fetuin-A significantly increased during the progression of albuminuria (A1, 0.40±0.43; A2, 0.60±0.53; A3 1.57±1.13 ng/gCr; p = 7.29×10⁻⁸) and of GFR stages (G1, 0.39±0.39; G2, 0.49±0.45; G3, 1.25±1.18; G4, 1.34±0.80 ng/gCr; p = 3.89×10⁻⁴). Multivariate logistic regression analysis was employed to assess fetuin-A as a risk for diabetic nephropathy with microalbuminuria or GFR<60 mL/min. Fetuin-A is demonstrated as a risk factor for both microalbuminuria and reduction of GFR in diabetic nephropathy with the odds ratio of 4.721 (1.881–11.844) and 3.739 (1.785–7.841), respectively. Collectively, the glycan profiling analysis is useful method to identify the urine biomarkers and fetuin-A is a candidate to predict the progression of diabetic nephropathy.     

Principal Component Analysis Based Feature Extraction Approach to Identify Circulating microRNA Biomarkers

The discovery and characterization of blood-based disease biomarkers are clinically important because blood collection is easy and involves relatively little stress for the patient. However, blood generally reflects not only targeted diseases, but also the whole body status of patients. Thus, the selection of biomarkers may be difficult. In this study, we considered miRNAs as biomarker candidates for several reasons. First, since miRNAs were discovered relatively recently, they have not yet been tested extensively. Second, since the number of miRNAs is relatively limited, selection is expected to be easy. Third, since they are known to play critical roles in a wide range of biological processes, their expression may be disease specific. We applied a newly proposed method to select combinations of miRNAs that discriminate between healthy controls and each of 14 diseases that include 5 cancers. A new feature selection method is based on principal component analysis. Namely this method does not require knowledge of whether each sample was derived from a disease patient or a healthy control. Using this method, we found that hsa-miR-425, hsa-miR-15b, hsa-miR-185, hsa-miR-92a, hsa-miR-140-3p, hsa-miR-320a, hsa-miR-486-5p, hsa-miR-16, hsa-miR-191, hsa-miR-106b, hsa-miR-19b, and hsa-miR-30d were potential biomarkers; combinations of 10 of these miRNAs allowed us to discriminate each disease included in this study from healthy controls. These 12 miRNAs are significantly up- or downregulated in most cancers and other diseases, albeit in a cancer- or disease-specific combinatory manner. Therefore, these 12 miRNAs were also previously reported to be cancer- and disease-related miRNAs. Many disease-specific KEGG pathways were also significantly enriched by target genes of up−/downregulated miRNAs within several combinations of 10 miRNAs among these 12 miRNAs. We also selected miRNAs that could discriminate one disease from another or from healthy controls. These miRNAs were found to be largely overlapped with miRNAs that discriminate each disease from healthy controls.     

Norovirus Binding to Intestinal Epithelial Cells Is Independent of Histo-Blood Group Antigens

Human noroviruses (NoVs) are a major cause of non-bacterial gastroenteritis. Although histo-blood group antigens (HBGAs) have been implicated in the initial binding of NoV, the mechanism of that binding before internalization is not clear. To determine the involvement of NoVs and HBGAs in cell binding, we examined the localization of NoV virus-like particles (VLPs) and HBGAs in a human intestinal cell line and the human ileum biopsy specimens by immunofluorescence microscopy. The localizations of Ueno 7k VLPs (genogroup II.6) and each HBGA (type H1-, H2- and Le^b-HBGAs) on the human intestinal cell line, Caco-2, were examined by confocal laser-scanning microscopy. To explore any interactions of NoVs and HBGAs in vivo, fresh biopsy specimens from human ileum were directly incubated with NoV VLPs and examined by immunofluorescence microscopy. We found that VLP binding depended on the state of cell differentiation, but not on the presence of HBGAs. In differentiated Caco-2 cells, we detected no type H1 HBGAs, but VLPs bound to the cells anyway. We incubated fresh biopsies of human ileum directly with VLPs, a model that better replicates the in vivo environment. VLPs mainly bound epithelial cells and goblet cells. Although the incubations were performed at 4°C to hinder internalization, VLPs were still detected inside cells. Our results suggest that VLPs utilize molecule(s) other than HBGAs during binding and internalization into cells.     

Cooperative Functions of ZnT1, Metallothionein and ZnT4 in the Cytoplasm Are Required for Full Activation of TNAP in the Early Secretory Pathway

The activation process of secretory or membrane-bound zinc enzymes is thought to be a highly coordinated process involving zinc transport, trafficking, transfer and coordination. We have previously shown that secretory and membrane-bound zinc enzymes are activated in the early secretory pathway (ESP) via zinc-loading by the zinc transporter 5 (ZnT5)-ZnT6 hetero-complex and ZnT7 homo-complex (zinc transport complexes). However, how other proteins conducting zinc metabolism affect the activation of these enzymes remains unknown. Here, we investigated this issue by disruption and re-expression of genes known to be involved in cytoplasmic zinc metabolism, using a zinc enzyme, tissue non-specific alkaline phosphatase (TNAP), as a reporter. We found that TNAP activity was significantly reduced in cells deficient in ZnT1, Metallothionein (MT) and ZnT4 genes (ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells), in spite of increased cytosolic zinc levels. The reduced TNAP activity in ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells was not restored when cytosolic zinc levels were normalized to levels comparable with those of wild-type cells, but was reversely restored by extreme zinc supplementation via zinc-loading by the zinc transport complexes. Moreover, the reduced TNAP activity was adequately restored by re-expression of mammalian counterparts of ZnT1, MT and ZnT4, but not by zinc transport-incompetent mutants of ZnT1 and ZnT4. In ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells, the secretory pathway normally operates. These findings suggest that cooperative zinc handling of ZnT1, MT and ZnT4 in the cytoplasm is required for full activation of TNAP in the ESP, and present clear evidence that the activation process of zinc enzymes is elaborately controlled.     

5-Aminolevulinic Acid: Production by Fermentation,and Agricultural and Biomedical Applications

Abstract

Research involving differentiated embryonic stem (ES) cells may revolutionize the study of liver disease, improve the drug discovery process, and assist in the development of stem-cell-based clinical therapies. Generation of ES cell-derived hepatic tissue has benefited from an understanding of the cytokines, growth factors and biochemical compounds that are essential in liver development, and this knowledge has been used to mimic some aspects of embryonic development in vitro. Although great progress has been made in differentiating human ES cells into liver cells, current protocols have not yet produced cells with the phenotype of a mature hepatocyte. There is a of disease models have been examined concerning whether stem cells can correct liver disease. It is a bit premature to conclude that hepatocytes can be generated from non-hepatic cells in culture that will be clinically useful. Standard criteria will need to be developed to assess the extent to which human stem cell-derived hepatocytes have been produced.