The Nuclear Function of IL-33 in Desensitization to DNA Damaging Agent and Change of Glioma Nuclear Structure.

Glioma, the most common subtype of primary brain tumor, is an aggressive and highly invasive neurologically tumor among human cancers. Interleukin-33 (IL-33) is considered as a dual functional cytokine, an alarmin upon tissue damage and a nuclear chromatin-associated protein. Despite that, IL-33 is known to foster the formation of the inflammatory tumor microenvironment and facilitate glioma progression, evidence showing nuclear IL-33 function is still poor. In this study using lentivirus-mediated IL-33 gene knockdown (IL33KD) and IL-33 overexpression (IL33oe) in rat C6 glioma cells and human glioma cell lines (U251MG and U87MG), we found that IL33oe-glioma cells had resistance to the insults of the alkylating agent, temozolomide (TMZ), possibly because of the increased expression of DNA repair genes (i.e., BRCA1, BRCA2, Rad51, FANCB, and FANCD) in IL33oe-glioma cells. Alternatively, examination of glioma nuclear shape from transmission electron microscopy (TEM) imaging analysis and immunofluorescence for histone protein H2A staining showed that IL33KD attenuated the abnormal cancerous nuclear characteristic, such as indentation, long clefts, and multiple nucleoids. Yet, IL33oe promoted the changes in glioma nuclear shapes, such as the formation of multiple lobes. We further found that histone proteins, H2A and H3, were reduced in IL33KD glioma cells. The non-histone DNA-binding nucleoproteins, the high mobility group A1 (HMGA1) and HMGA2, were also downregulated by IL33KD. In contrast, IL33oe increased H2A and H3 proteins and HMGA1 and HMGA2 in glioma cells. Altogether, the upregulation of nuclear IL-33 expression was along with an increase in the expression of DNA repair genes, contributing to the desensitization of glioma cells to DNA damaging agents. Moreover, nuclear IL-33 proteins in cooperation with chromatin-associated proteins regulate glioma nuclear structure, which might be crucial for glioma progression and malignancy.

Chemotherapeutic Regimens and Chemotherapy-Free Intervals Influence the Survival of Patients with Recurrent Advanced Epithelial Ovarian Carcinoma: A Retrospective Population-Based Study.

We aimed to evaluate factors influencing the outcomes of patients with platinum-sensitive recurrent epithelial ovarian carcinoma (EOC). Patients with advanced-stage EOC, who received debulking surgery and adjuvant chemotherapy for recurrence, were obtained from the National Health Insurance Research database of Taiwan between 2000 and 2013. A total of 1038 patients with recurrent advanced-stage EOC were recruited. The platinum + paclitaxel (PT) group had the best five-year overall survival (OS) compared with the other three groups (p < 0.001). The hazard ratios (HRs) of five-year OS for the platinum + liposomal doxorubicin (PD), topotecan (TOP), and pegylated liposomal doxorubicin (PLD) groups were 1.21 (p = 0.07), 1.35 (p = 0.016), and 1.80 (p < 0.001), respectively, compared with the PT group. The PT group also had lower hazard ratios of five-year OS for patients with platinum therapy-free interval (TFIp) between 6 and 12 months compared with the other three groups (p < 0.0001). However, the HRs of five-year OS did not differ between the PT and PD groups in patients with TFIp >12 months. Patients with TFIp >12 months had lower HRs of five-year OS compared with those with TFIp of 6-12 months, regardless of whether they were treated with platinum-based (p = 0.001) or non-platinum-based (p = 0.003) regimens. Chemotherapeutic regimens and TFIp influenced the outcomes of patients with recurrent EOC. For patients with TFIp of 6-12 months, the PT regimen is the first choice based on their best overall survival result. For patients with TFIp >12 months, either platinum-based or non-platinum regimens could be used because of their similar excellent overall survival.

Identification of a new subset of lymph node stromal cells involved in regulating plasma cell homeostasis.

Antibody-secreting plasma cells (PCs) arise rapidly during adaptive immunity to control infections. The early PCs are retained within the reactive lymphoid organ where their localization and homeostasis rely on extrinsic factors, presumably produced by local niche cells. While myeloid cells have been proposed to form those niches, the contribution by colocalizing stromal cells has remained unclear. Here, we characterized a subset of fibroblastic reticular cells (FRCs) that forms a dense meshwork throughout medullary cords of lymph nodes (LNs) where PCs reside. This medullary FRC type is shown to be anatomically, phenotypically, and functionally distinct from T zone FRCs, both in mice and humans. By using static and dynamic imaging approaches, we provide evidence that medullary FRCs are the main cell type in contact with PCs guiding them in their migration. Medullary FRCs also represent a major local source of the PC survival factors IL-6, BAFF, and CXCL12, besides also producing APRIL. In vitro, medullary FRCs alone or in combination with macrophages promote PC survival while other LN cell types do not have this property. Thus, we propose that this FRC subset, together with medullary macrophages, forms PC survival niches within the LN medulla, and thereby helps in promoting the rapid development of humoral immunity, which is critical in limiting early pathogen spread.

Ovarian and endometrial endometrioid adenocarcinomas have distinct profiles of microsatellite instability, PTEN expression, and ARID1A expression.

AIMS: To understand the role of and differences in molecular alterations between endometrial and ovarian endometrioid adenocarcinomas. METHODS AND RESULTS: We investigated the microsatellite status of 26 ovarian endometrioid adenocarcinomas (OVEMs), 42 endometrial endometrioid adenocarcinomas (EMCAs), and 19 concurrent (endometrial and ovarian) endometrioid adenocarcinomas. We evaluated the expression of the mismatch repair proteins, PTEN and ARID1A, and mutations of PTEN, KRAS, CTNNB1, and PIK3CA. High levels of microsatellite instability (MSI-H) were present in one of 26 OVEMs, 12 of 42 EMCAs, and four of 19 concurrent endometrioid adenocarcinomas. Only four of 19 concurrent endometrioid adenocarcinomas showed identical molecular alterations in their endometrial and ovarian components. Loss of ARID1A or loss of PTEN expression, and MSI-H, were more common in EMCAs than OVEMs (P = 0.044, P = 0.004, and P = 0.012, respectively). MSI-H in endometrial endometrioid adenocarcinomas was also related to loss of ARID1A expression (P < 0.001). In the cohort of MSI-H endometrioid adenocarcinomas involving the endometrium (n = 16), MSH6-deficient cases showed higher frequencies of CTNNB1 and PIK3CA mutations (P = 0.008 and P = 0.036, respectively), but lower frequencies of KRAS mutation (P = 0.011), than PMS2-deficient cases. CONCLUSIONS: The different frequencies of molecular genetic alterations between endometrial endometrioid adenocarcinomas and ovarian endometrioid adenocarcinomas imply that distinct processes may be involved in their tumorigenesis or tumour progression.

Chromosome 20q13.2 ZNF217 locus amplification correlates with decreased E-cadherin expression in ovarian clear cell carcinoma with PI3K-Akt pathway alterations.

This study aims to evaluate the relationships between chromosome 20q13.2 zinc finger protein 217 (ZNF217) locus amplification, ZNF217 expression, E-cadherin expression, and PI3K-Akt pathway alterations (activating PIK3CA mutations or loss of phosphatase and tensin homolog [PTEN] expression), and whether these molecular alterations can predict the clinical survival data in ovarian clear cell carcinoma (OCCC) patients. Samples and clinical data of 72 OCCC patients were collected. Chromosome 20q13.2 ZNF217 locus amplification was detected by fluorescence in situ hybridization. ZNF217, E-cadherin and PTEN expression were assessed using immunohistochemical stain. PIK3CA mutation was identified by PCR-amplified gene sequencing. Cox proportional hazard regression model was used to estimate the adjusted hazard ratios of survival. Chromosome 20q13.2 ZNF217 locus amplification was detected in 31% (22/72) of cases, and ZNF217 expression was increased in 40% (27/68) of cases. E-cadherin and PTEN expressions were decreased or lost in 44% (32/72) and 14% (10/72) of cases, respectively. Activating PIK3CA mutations were present in 35% (25/72) of cases. Thirty-three OCCC patients (46%) showed activating PI3K-Akt pathway alterations. Chromosome 20q13.2 ZNF217 locus amplification was significantly associated with decreased E-cadherin expression (P = .001). In contrast, ZNF217 expression was not related to ZNF217 amplification or E-cadherin expression. In OCCC patients with activating PI3k-Akt pathway, decreased E-cadherin expression (P = .033) and advanced Federation of Gynecology and Obstetrics stage (P = .014) predicted shorter overall survival. Two conclusions were raised in our study. First, ZNF217 plays a role in down-regulating E-cadherin expression and is a potential therapeutic target for OCCC patients. Second, E-cadherin expression is a prognostic marker for OCCC patients with activating PI3K-Akt pathway.

Specific fibroblastic niches in secondary lymphoid organs orchestrate distinct Notch-regulated immune responses.

Fibroblast-like cells of secondary lymphoid organs (SLO) are important for tissue architecture. In addition, they regulate lymphocyte compartmentalization through the secretion of chemokines, and participate in the orchestration of appropriate cell-cell interactions required for adaptive immunity. Here, we provide data demonstrating the functional importance of SLO fibroblasts during Notch-mediated lineage specification and immune response. Genetic ablation of the Notch ligand Delta-like (DL)1 identified splenic fibroblasts rather than hematopoietic or endothelial cells as niche cells, allowing Notch 2-driven differentiation of marginal zone B cells and of Esam(+) dendritic cells. Moreover, conditional inactivation of DL4 in lymph node fibroblasts resulted in impaired follicular helper T cell differentiation and, consequently, in reduced numbers of germinal center B cells and absence of high-affinity antibodies. Our data demonstrate previously unknown roles for DL ligand-expressing fibroblasts in SLO niches as drivers of multiple Notch-mediated immune differentiation processes.

Trapping of naive lymphocytes triggers rapid growth and remodeling of the fibroblast network in reactive murine lymph nodes.

Adaptive immunity is initiated in T-cell zones of secondary lymphoid organs. These zones are organized in a rigid 3D network of fibroblastic reticular cells (FRCs) that are a rich cytokine source. In response to lymph-borne antigens, draining lymph nodes (LNs) expand several folds in size, but the fate and role of the FRC network during immune response is not fully understood. Here we show that T-cell responses are accompanied by the rapid activation and growth of FRCs, leading to an expanded but similarly organized network of T-zone FRCs that maintains its vital function for lymphocyte trafficking and survival. In addition, new FRC-rich environments were observed in the expanded medullary cords. FRCs are activated within hours after the onset of inflammation in the periphery. Surprisingly, FRC expansion depends mainly on trapping of naïve lymphocytes that is induced by both migratory and resident dendritic cells. Inflammatory signals are not required as homeostatic T-cell proliferation was sufficient to trigger FRC expansion. Activated lymphocytes are also dispensable for this process, but can enhance the later growth phase. Thus, this study documents the surprising plasticity as well as the complex regulation of FRC networks allowing the rapid LN hyperplasia that is critical for mounting efficient adaptive immunity.

siRNA as a therapy for asthma.

RNAi is the process of sequence-specific, post-transcriptional/transcriptional gene silencing through siRNA. RNAi is a popular method of controlling gene expression and has potential in the development of drugs for several diseases, such as various types of cancer and viral infections. Gene therapy for asthma has already been developed and has demonstrated promising results in animal models. Recent progress in delivering siRNA to the respiratory system has also improved the therapeutic feasibility of RNAi for asthma. In this review, the mechanism, as well as the general strategies and problems associated with the application of RNAi in vivo, are discussed, focusing on the possibility of applying RNAi to alleviate airway inflammation in allergic asthma. Data from studies with siRNA against molecules involved in allergic responses are summarized, and the potential and limitations of RNAi as a novel therapeutic approach are discussed.

Sustained intraspinal delivery of neurotrophic factor encapsulated in biodegradable nanoparticles following contusive spinal cord injury.

Glial cell line derived neurotrophic factor (GDNF) induces neuronal survival and tissue repair after spinal cord injury (SCI). A continuous GDNF supply is believed to gain greater efficacy in the neural restoration of the injured spinal cord. Accordingly, nanovehicle formulation for their efficient delivery and sustained release in injured spinal cord was examined. We first used fluorescence-labeled bovine serum albumin (FBSA) loaded in biodegradable poly(lactic acid-co-glycolic acid) (PLGA) for intraspinal administration after SCI and for in vitro study. Our results showed that the preservation of PLGA-FBSA was observed in the injured spinal cord at 24h, and PLGA-FBSA nanoparticles were well absorbed by neurons and glia, indicating that PLGA as a considerable nanovehicle for the delivery of neuroprotective polypeptide into injured spinal cord. Furthermore, intraspinal injection of GDNF encapsulated in PLGA (PLGA-GDNF) nanoparticles into the injured spinal cord proximal to the lesion center had no effect on gliosis when compared to that observed in SCI rats receiving PLGA injection. However, local administration of PLGA-GDNF effectively preserved neuronal fibers and led to the hindlimb locomotor recovery in rats with SCI, providing a potential strategy for the use of PLGA-GDNF in the treatment of SCI.

Lentiviral-mediated GATA-3 RNAi decreases allergic airway inflammation and hyperresponsiveness.

GATA-3 is the key transcriptional factor for Th2 commitment in T cells and is strongly associated with asthma and allergic disease. We studied the silencing of the GATA-3 gene expression using RNA interference (RNAi) delivered by a lentiviral vector, to evaluate the therapeutic role of GATA-3 short hairpin RNAs (shRNAs) in a murine model of asthma. Mice were sensitized with OVA and instilled intratracheally (IT) with GATA-3 shRNAs lentiviral vector (Lenti-si-GATA-3) once, 48 hours before challenge. After three challenges with the OVA antigen, the mice were assessed for airway hyperresponsiveness (AHR) and inflammation. With infection of Lenti-si-GATA-3 in EL-4 cells, GATA-3 gene expression was abrogated and downstream Th2 cytokines, such as interleukin-4 (IL-4) and IL-5, were also significantly inhibited. IT delivery of Lenti-si-GATA-3 in OVA-immunized mice resulted in a strong inhibition of local GATA-3 gene expression. Treatment with Lenti-si-GATA-3 successfully alleviated OVA-induced airway eosinophilia and Th2 cytokine release. While evaluating AHR by means of enhanced pause (Penh) and pulmonary resistance (R(L)) using body plethysmography, it was found that the administration of Lenti-si-GATA-3 had significantly decreased AHR in OVA-immunized mice. These results suggest that inhibition of GATA-3 gene expression by shRNAs lentiviral vectors strongly attenuates antigen-induced airway inflammation and hyper-responsiveness in mice.

Inhibition of lipopolysaccharide-induced microglial activation by preexposure to neurotrophin-3.

Microglia activated by neural injuries produce proinflammatory mediators, but activated microglia also appear in developing neural tissue to phagocytose cell debris resulting from programmed cell death without inducing tissue damage. Thus, factors associated with the developing CNS may modulate microglial activities. Previously we reported that pretreatment with neurotrophin-3 (NT-3), a factor known to regulate neural development, inhibits the production of proinflammatory mediators, nitric oxide (NO), tumor necrosis factor-alpha, and interleukin-1beta, in BV2 activated by inflammagen lipopolysaccharide (LPS). In this study, the inhibition of proinflammatory mediators by NT-3 pretreatment (preNT-3) in primary microglia with LPS stimulation was corroborated. Moreover, pretreatment of LPS-activated microglia with NT-3 induced a trend of reduction in phagocytotic ability. By using LPS-activated BV2 cells, we further found that reduced expression of inducible NO synthetase by preNT-3 was mediated by MAP kinase and PI3 kinase signaling pathways. Moreover, pretreatment of BV2 cells with NT-3 led to reduced levels of the p65 subunit of nucleus factor-kappaB (NFkappaB) and its DNA binding activity. Accordingly, our results indicate that preexposure of microglia to NT-3 leads to a reduced production of proinflammatory mediators in activated microglia by the induction of MAP kinase and PI3 kinase signaling, which in turn may reduce NFkappaB DNA binding activity. This suggests that an NT-enriched microenvironment may be favorable for preventing the inflammatory reaction of microglia.

Downregulation of inducible nitric oxide synthetase by neurotrophin-3 in microglia.

Microglia activated after many neurological degeneration of the central nervous system (CNS) act as important regulators for neuropathogenesis in the injured CNS via producing proinflammatory mediators, such as nitric oxide (NO), TNF-alpha, and IL-1beta. Neurotrophin-3 (NT-3) is a well-known trophic factor for neural survival, development, and plasticity. Activated microglia are NT-3-producing cells in the injured CNS, and express its receptor-TrkC. However, little is known about the effect of NT-3 on activated microglia. In this study, pre-treatment of a mouse microglial cell line, BV2, with NT-3 for 24 h indicated that NT-3 reduced the inducible form of NO synthase (iNOS), NO, and TNF-alpha in BV2 stimulated with lipopolysaccharide (LPS). NT-3 exerted less effect on the reduction of these proinflammatory mediators when it was added to BV2 cultures either simultaneously with LPS or post LPS treatment. These findings indicate that NT-3 may serve as an anti-inflammatory factor to suppress microglial activation.