PAD4 Deficiency Improves Bleomycin-induced Neutrophil Extracellular Traps and Fibrosis in Mouse Lung.

Excessive release of neutrophil extracellular traps (NETs) has been implicated in several organ fibrosis, including pulmonary fibrosis. NETs constitute a phenomenon in which decorated nuclear chromatin with cytosolic proteins is released into the extracellular space. PAD4 (peptidylarginine deiminase 4) plays an important role in the formation of NETs. However, the role of NETs in the pathogenesis of pulmonary fibrosis remains undefined. Here, we identified NETs in the alveolar and interstitial lung space of mice undergoing bleomycin (BLM)-induced lung fibrosis, which was suppressed by a pan-PAD inhibitor, Cl-amidine. In vitro, BLM directly induced NETs in blood neutrophils, which was also inhibited by Cl-amidine. Furthermore, Padi4 gene knockout (PAD4-KO) in mice led to the alleviation of BLM-induced NETs and pulmonary fibrosis and to the expression of inflammatory and fibrotic genes. PAD4 deficiency prevented decreases in alveolar epithelial and pulmonary vascular endothelial cell numbers and increases in ACTA2-positive mesenchymal cells and S100A4-positive fibroblasts in the lung. Hematopoietic cell grafts from PAD4-KO mice, not wild-type mice, resolved BLM-induced lung fibrosis and fibrotic gene expression in wild-type and PAD4-KO mice, suggesting that expression of PAD4 in hematopoietic cells may be involved in the development of lung fibrosis. These data suggest that PAD4 deficiency could ameliorate BLM-induced formation of NETs and lung fibrosis, suggesting that this pathway could serve as a therapeutic target for pulmonary fibrosis treatment.

Mucosa-associated lymphoid tissue lymphoma with metachronous involvement of the palpebral conjunctiva and bronchus: A case report.

A 61-year-old woman with a history of palpebral conjunctival mucosa-associated lymphoid tissue (MALT) lymphoma, treated with rituximab, was referred to the authors' hospital after follow-up positron emission tomography/computed tomography revealed 18F-fluoro-2-deoxy-d-glucose uptake in a tumor located in the left main bronchus. The diagnosis of MALT lymphoma was made by pathological and immunohistochemical findings homologous to previous palpebral conjunctival lesion via bronchoscopic biopsy. The disease was controlled with rituximab, cyclophosphamide, oncovin, and prednisolone (i.e., R-COP) chemotherapy. Although MALT lymphoma occurs in several organs, metachronous occurrence in the palpebral conjunctiva and bronchus is especially rare, and careful check-up is required to monitor for occurrence of systemic relapse.

Generation and Characterization of a Novel Small Biologic Alternative to Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Antibodies, DS-9001a, Albumin Binding Domain-Fused Anticalin Protein.

Since it was recently reported that an antibody for proprotein convertase subtilisin/kexin type 9 (PCSK9) reduces the risk of cardiovascular events in a clinical context, PCSK9 inhibition is thought to be an attractive therapy for dyslipidemia. In the present study, we created a novel small biologic alternative to PCSK9 antibodies called DS-9001a, comprising an albumin binding domain fused to an artificial lipocalin mutein (ABD-fused Anticalin protein), which can be produced by a microbial production system. DS-9001a strongly interfered with PCSK9 binding to low-density-lipoprotein receptor (LDL-R) and PCSK9-mediated degradation of LDL-R. In cynomolgus monkeys, single DS-9001a administration significantly reduced the serum LDL-C level up to 21 days (62.4% reduction at the maximum). Moreover, DS-9001a reduced plasma non-high-density-lipoprotein cholesterol and oxidized LDL levels, and their further reductions were observed when atorvastatin and DS-9001a were administered in combination in human cholesteryl ester transfer protein/ApoB double transgenic mice. Additionally, their reductions on the combination of atorvastatin and DS-9001a were more pronounced than those on the combination of atorvastatin and anacetrapib. Besides its favorable pharmacologic profile, DS-9001a has a lower molecular weight (about 22 kDa), yielding a high stoichiometric drug concentration that might result in a smaller administration volume than that in existing antibody therapy. Since bacterial production systems are viewed as more suited to mass production at low cost, DS-9001a may provide a new therapeutic option to treat patients with dyslipidemia. In addition, considering the growing demand for antibody-like drugs, ABD-fused Anticalin proteins could represent a promising new class of small biologic molecules.

Fatal fungemia with Scedosporium prolificans in a patient with acute myeloid leukemia.

Scedosporium prolificans (S. prolificans) is a type of mold, which rarely affects immunocompromised people. We treated a 71-year-old woman with acute myeloid leukemia (AML-M5a) with low-dose cytarabine, acralubicin, and filgrastim as the induction therapy. On day 7 after the initiation of chemotherapy, she became febrile and agranulocytic, and developed anal pain ; therefore, we discontinued the chemotherapy on day 8. Broad-spectrum antibiotics, micafungin, and then liposomal amphotericin B were ineffective. The serum concentration of ß-D-glucan was 525 pg/mL. She died of multiple organ failure on day 17. S. prolificans was detected from the blood culture on day 13. Physicians should consider Scedosporium spp. infection when principal antifungal agents are ineffective and fungal infection is strongly suspected.

Interleukin enhancer-binding factor 3 functions as a liver receptor homologue-1 co-activator in synergy with the nuclear receptor co-activators PRMT1 and PGC-1α.

LRH-1 (liver receptor homologue-1), a transcription factor and member of the nuclear receptor superfamily, regulates the expression of its target genes, which are involved in bile acid and cholesterol homoeostasis. However, the molecular mechanisms of transcriptional control by LRH-1 are not completely understood. Previously, we identified Ku80 and Ku70 as LRH-1-binding proteins and reported that they function as co-repressors. In the present study, we identified an additional LRH-1-binding protein, ILF3 (interleukin enhancer-binding factor 3). ILF3 formed a complex with LRH-1 and the other two nuclear receptor co-activators PRMT1 (protein arginine methyltransferase 1) and PGC-1α (peroxisome proliferator-activated receptor γ co-activator-1α). We demonstrated that ILF3, PRMT1 and PGC-1α were recruited to the promoter region of the LRH-1-regulated SHP (small heterodimer partner) gene, encoding one of the nuclear receptors. ILF3 enhanced SHP gene expression in co-operation with PRMT1 and PGC-1α through the C-terminal region of ILF3. In addition, we found that the small interfering RNA-mediated down-regulation of ILF3 expression led to a reduction in the occupancy of PGC-1α at the SHP promoter and SHP expression. Taken together, our results suggest that ILF3 functions as a novel LRH-1 co-activator by acting synergistically with PRMT1 and PGC-1α, thereby promoting LRH-1-dependent gene expression.

Repression of the promoter activity mediated by liver receptor homolog-1 through interaction with ku proteins.

Nuclear receptor liver receptor homolog-1 (LRH-1; NR5A2) plays a crucial role in the homeostasis of bile acids and cholesterol by controlling the expression of genes central to bile acid synthesis and efflux, reverse cholesterol transport, and high density lipoprotein-remodeling. However, the molecular mechanisms that modulate the transactivation activity of LRH-1 remain unclear. It is proposed that LRH-1's activity is regulated by post-modifications, the binding of small heterodimer partner (SHP), or the binding of coregulators. To search for cofactors that regulate the transactivation activity of LRH-1, we performed a pull-down assay using glutathione S-transferase (GST) fused to the N-terminal portion of LRH-1 and nuclear extracts from HeLa cells, and identified Ku proteins as interacting proteins with LRH-1. We also found that Ku proteins associate with LRH-1 through its DNA-binding domain and hinge region. Luciferase reporter assays revealed that Ku proteins repressed the SHP promoter activity mediated by LRH-1. Furthermore, Ku proteins suppressed the coactivating effect of peroxisome proliferator-activated receptor (PPAR) gamma coactivator-1alpha (PGC-1alpha), an LRH-1 coactivator, on the LRH-1-mediated SHP promoter activity. Previously, we showed that Ku proteins interacted with nuclear receptor farnesoid X receptor (FXR; NR1H4) and decreased the expression of its target gene. In this study, we demonstrated that Ku proteins also interacted with not only LRH-1 but various nuclear receptors, such as the estrogen receptor, PPAR, and Rev-erb. Ku proteins may function as corepressors for various nuclear receptors including LRH-1.

Monitoring kidney safety in drug development: emerging technologies and their implications.

Drug-induced kidney injury is a serious and not uncommon adverse event which needs to be considered during drug development. The current standards used to monitor kidney function, such as blood urea nitrogen and serum creatinine, are late indicators of kidney injury and thus do not allow for timely intervention before loss of function. Improving the diagnosis and monitoring of kidney damage goes hand-in-hand with the identification of new biomarkers and the development of technologies that enable their sensitive and specific measurements. In order to move beyond restriction to internal company decisions, every entity that demonstrates the qualities of a biomarker must gain acceptance by health authorities if it is to be used for regulatory decision making in preclinical studies and clinical trials. This review focuses on the most promising achievements of new technologies applied to monitoring drug-induced nephrotoxicity (eg, gene expression, imaging, in vitro screening, protein assays) and on the use and implications of peripheral biomarkers such as the urinary protein biomarkers glutathione S-transferase-alpha, N-acetyl-beta-d-glucosaminidase, total protein, cystatin C, beta2-microglobulin, KIM-1, lipocalin-2 and serum cystatin C. Finally, the associated regulatory processes for use in clinics are also discussed.

Antiviral sulfated polysaccharide from Navicula directa, a diatom collected from deep-sea water in Toyama Bay.

A sulfated polysaccharide named naviculan was isolated from a diatom, Navicula directa (W. SMITH) RALFS, collected in deep sea water from Toyama Bay. The polysaccharide consisted of fucose, xylose, galactose, mannose, rhamnose and sulfate with an apparent molecular weight of 220000. It showed antiviral activities against herpes simplex viruses type 1 and 2, and influenza A virus with selectivity indices (CC50/IC50) of 270, 510 and 32, respectively. Naviculan also showed an inhibitory effect on cell-cell fusion between CD4-expressing and human immunodeficiency virus (HIV) gp160-expressing cells that was used as a model system of infection with HIV.

Multiplex PCR assay for rapid identification of oculopathogenic adenoviruses by amplification of the fiber and hexon genes.

Eye infections caused by adenovirus (Ad) often result in nosocomial infections and community epidemics with significant rates of morbidity. No antiviral agent effective against Ad is yet available for clinical use. Therefore, early diagnosis is still the mainstay for patient management and the prevention of epidemics. A multiplex PCR assay based on amplification of a combination of the fiber and hexon genes which can identify the six important oculopathogenic serotypes of Ads (Ad serotype 3[Ad3], Ad4, Ad7, Ad8, Ad19, and Ad37) in a single-tube amplification reaction was developed. Ad serotypes could be distinguished by the different amplicon sizes. The assay correctly identified prototype strains as well as isolates in clinical specimens. In comparison with a previously described PCR-restriction fragment polymorphism method, our assay gave unequivocal results for clinical specimens. Our multiplex PCR has the potential to serve as a rapid and cost-effective tool for the typing of important ocular Ads.

Molecular epidemiology of adenovirus infection among pediatric population with diarrhea in Asia.

A total of 3,577 fecal specimens from infants and children with acute gastroenteritis in Japan, Korea and Vietnam during 1998 and 2001 were tested for adenovirus by the ELISA method. Of these, adenovirus was detected in 158 (4.4%). The detection rate of adenovirus was highest in Korea (8.7%, 20/231) followed by 5.0% (100/1,991) in Japan and 2.8% (38/1,355) in Vietnam. All adenoviruses were further serotyped by PCR-RFLP. The diversity of adenovirus serotypes including Ad2, 3, 5, 8, 31, 40 and 41 was demonstrated. Worth of note was a decrease in the rate of isolation of Ad40 (7.6%, 12/158) and a concomitant increase of Ad41 (63.9%, 101/158) to become the predominant serotype. Another interesting feature of the study was the presence of Ad3 (4.0% and 30%) and Ad8 (8.0% and 20%) in Japan and Korea, respectively, which is mainly associated with keratoconjunctivitis worldwide. Our result underscored the importance of adenovirus in association with acute gastroenteritis in Asian countries.

Characterizations of adenovirus type 41 isolates from children with acute gastroenteritis in Japan, Vietnam, and Korea.

Genetic and antigenic characterizations of 70 strains of adenovirus type 41 (Ad41), isolated between 1998 and 2001 from children in Japan, Vietnam, and Korea, were done by DNA restriction enzyme (RE) analysis, sequencing analysis, and monoclonal antibody (MAb)-based enzyme-linked immunosorbent assay (ELISA). Eight genome types were observed in the present study, among which D25, D26, D27, and D28 were novel genome types. These eight genome types were divided into two genome-type clusters (GTCs) based on phylogenetic analysis of the hypervariable regions (HVRs) of the hexon. GTC1 includes D1, D25, D26, D27, and D28, and the GTC2 contains D4, D12, and D22. The amino acid homologies among the members within a GTC were 97 to 100%, whereas between the members of different GTCs the homologies were 92 to 94%. The specificity of the GTC classification was confirmed by ELISA with MAb 1F, which was selected by the Ad41 prototype Tak strain. It was found that only the isolates of GTC1 but not of GTC2 reacted with MAb 1F. These results suggest that Ad41 isolates from the three countries should be classified into two subtypes. The accumulation of amino acid mutations located in HVRs of hexon are indicative for the classification of Ad41 subtype.