Inhibition or deficiency of cathepsin B leads defects in HIV-1 Gag pseudoparticle release in macrophages and HEK293T cells

Human immunodeficiency virus type 1 (HIV-1) egresses from infected cells through utilizing the host membrane budding mechanisms. Assembly of HIV-1 Gag particles occurs on membranes where the Gag multimers subsequently bud off and form enveloped viral particles. In certain cell types such as macrophages, HIV-1 Gag particles have shown to be released into intracellular virus containing compartments (VCC) such as late endosomes, multivesicular bodies (MVBs) or invaginated plasma membrane pockets. Here, we showed that macrophages or HEK293T cells treated with the cathepsin B (CTSB)-specific inhibitor CA-074Me or cells deficient in CTSB failed to release HIV-1 Gag pseudoparticles into the extracellular environment. Based on immunofluorescence and electron microscopy, these cells retained the pseudoparticles in heterogeneous intracellular VCC. CA-074Me was also able to inhibit propagation of two enveloped viruses, herpes simplex virus and influenza A virus, but not non-enveloped enterovirus. These results suggest that CTSB is required for the efficient release of HIV-1 Gag pseudoparticles and targeting CTSB can be a new therapeutic strategy for inhibiting egress of HIV-1 and other enveloped viruses.     

A bacterial flagellin, Vibrio vulnificus FlaB, has a strong mucosal adjuvant activity to induce protective immunity

Flagellin, the structural component of flagellar filament in various locomotive bacteria, is the ligand for Toll-like receptor 5 (TLR5) of host cells. TLR stimulation by various pathogen-associated molecular patterns leads to activation of innate and subsequent adaptive immune responses. Therefore, TLR ligands are considered attractive adjuvant candidates in vaccine development. In this study, we show the highly potent mucosal adjuvant activity of a Vibrio vulnificus major flagellin (FlaB). Using an intranasal immunization mouse model, we observed that coadministration of the flagellin with tetanus toxoid (TT) induced significantly enhanced TT-specific immunoglobulin A (IgA) responses in both mucosal and systemic compartments and IgG responses in the systemic compartment. The mice immunized with TT plus FlaB were completely protected from systemic challenge with a 200x minimum lethal dose of tetanus toxin. Radiolabeled FlaB administered into the nasal cavity readily reached the cervical lymph nodes and systemic circulation. FlaB bound directly to human TLR5 expressed on cultured epithelial cells and consequently induced NF-kappaB and interleukin-8 activation. Intranasally administered FlaB colocalized with CD11c as patches in putative dendritic cells and caused an increase in the number of TLR5-expressing cells in cervical lymph nodes. These results indicate that flagellin would serve as an efficacious mucosal adjuvant inducing protective immune responses through TLR5 activation.     

MicroRNA‐135a‐3p as a promising biomarker and nucleic acid therapeutic agent for ovarian cancer

Ovarian cancer is the most lethal gynecologic malignancy. Recently, several molecularly targeted anticancer agents have been developed for ovarian cancer; however, its prognosis remains extremely poor. The development of molecularly targeted therapy, as well as companion diagnostics, is required to improve outcomes for patients with ovarian cancer. In this study, to identify microRNAs (miRNAs) involved in the progression of ovarian cancer we analyzed serum miRNAs in patients with ovarian cancer using miRNA array and quantitative RT‐PCR and examined the anticancer properties of miRNA expression in ovarian cancer cells. In patients with ovarian cancer, high amount of miR‐135a‐3p in serum samples was significantly associated with favorable clinical prognosis. The amount of miR‐135a‐3p was significantly decreased in patients with ovarian cancer compared with patients with ovarian cysts or normal ovaries. In SKOV‐3 and ES‐2 human ovarian cancer cells, enhanced expression of miR‐135a‐3p induced drug sensitivity to cisplatin and paclitaxel and suppressed cell proliferation and xenograft tumor growth. These findings suggest that miR‐135a‐3p may be considered as a biomarker and a therapeutic agent in ovarian cancer.     

Dihydroavenanthramide D prevents UV‐irradiated generation of reactive oxygen species and expression of matrix metalloproteinase‐1 and ‐3 in human dermal fibroblasts

Ultraviolet B (UVB) radiation induces photoageing by upregulating the expression of matrix metalloproteinases (MMPs) in human skin cells. Dihydroavenanthramide D (DHAvD) is a synthetic analog to naturally occurring avenanthramide, which is the active component in oats. Although anti‐inflammatory, anti‐atherosclerotic and antioxidant effects have been reported, the antiphotoageing effects of DHAvD are yet to be understood. In this study, we investigated the inhibitory effects of DHAvD on UVB‐induced production of reactive oxygen species (ROS) and expression of MMPs, and its molecular mechanism in UVB‐irradiated human dermal fibroblasts. Western blot and real‐time PCR analyses revealed that DHAvD inhibited UVB‐induced MMP‐1 and MMP‐3 expression. It also significantly blocked UVB‐induced ROS generation in fibroblasts. Additionally, DHAvD attenuated UVB‐induced phosphorylation of MAPKs, activation of NF‐κB and AP‐1. DHAvD regulates UVB‐irradiated MMP expression by inhibiting ROS‐mediated MAPK/NF‐κB and AP‐1 activation. DHAvD may be a useful candidate for preventing UV light‐induced skin photoageing.     

Use of the Ion AmpliSeq Cancer Hotspot Panel in clinical molecular pathology laboratories for analysis of solid tumours: With emphasis on validation with relevant single molecular pathology tests and the Oncomine Focus Assay

Targeted application of next-generation sequencing (NGS) technology allows detection of specific mutations that can provide treatment opportunities for cancer patients. We evaluated the applicability of the Ion AmpliSeq Cancer Hotspot Panel V2 (CHV2) using formalin-fixed, paraffin-embedded (FFPE) tissue of clinical specimens.Thirty-five FFPE tumour samples with known mutational status were collected from four different hospitals and sequenced with CHV2 using an Ion Chef System and Ion S5 XL system. Out of 35 cases, seven were sequenced with Oncomine focus Assay Panel for comparison. For the limit of detection test, we used an FFPE reference standard, a cell line that included an engineered 50% EGFR T790?M in an RKO cell line background. Coverage analysis results including number of mapped reads, on target percent, mean depth, and uniformity were not different according to hospitals. Sensitivity for mutation detection down to 3% was demonstrated. NGS results showed 100% concordance with the results from single molecular pathology tests Assay in 30 cases with 24 known positive mutations and 14 known negative mutations, and another NGS panel of the Oncomine focus in seven cases.The CHV2 NGS test for solid tumours using Ion chef system and S5 XL system in clinical molecular pathology laboratories for analysis of solid tumours could be routinely used and could replace some single molecular pathology tests after a stringent and thorough validation process.     

Neither nefopam nor acetaminophen can be used as postoperative analgesics in a rat model of ischemic stroke

Analgesics such as opioid agonists are usually not given during the postoperative phase of experimental stroke because they are susceptible to interfere with the evaluation of neuroprotective therapies. Here, we investigate the potential of acetaminophen and nefopam, two nonopioid analgesic drugs, to exert an analgesic effect without inducing neuroprotection in a murine model of ischemic stroke. We demonstrate that acetaminophen (200 mg/kg, PO) induces a significant decrease in the infarct volume, particularly in the cortex (VEHICLE: 200.1 mm³ vs. ACETAMINOPHEN: 140.9 mm³, P < 0.05), while nefopam (2, 20 or 40 mg/kg, IM), administered at the end of middle cerebral artery occlusion (MCAO), do not influence the infarct size (VEHICLE: 268.6 mm³ vs. NEFOPAM 2: 248.8 mm³, NEFOPAM 20: 250.6 mm³ and NEFOPAM 40: 215.9 mm³, P > 0.05). Moreover, we find that nefopam administration (20 mg/kg, IM) in the acute postoperative phase do not change the level of neuroprotection induced by MK801 (3 mg/kg, IV), a well‐known neuroprotectant (VEHICLE: 268.6 mm³ vs. MK801: 194.4 mm³ and vs. MK801 + NEFOPAM 20: 195.2 mm³). On the other hand, although nefopam induces analgesia in healthy animals, it is not the case when administered during MCAO (behavior scores at 5 min: HEALTHY: 2.1 vs. HEALTHY + NEFOPAM 20: 0.6, P < 0.5; IR: 0.40 vs. IR + NEFOPAM 20: 0.67, P > 0.05). Our data suggest that neither acetaminophen nor nefopam can be used as analgesic agents to meet the needs of limiting rodent pain and distress during experimental stroke surgery.     

Early treatment with atorvastatin exerts parenchymal and vascular protective effects in experimental cerebral ischaemia

Background and Purpose

From the clinical and experimental data available, statins appear to be interesting drug candidates for preventive neuroprotection in ischaemic stroke. However, their acute protective effect is, as yet, unconfirmed.

Experimental Approach

Male C57Bl6/JRj mice were subjected to middle cerebral artery occlusion and treated acutely with atorvastatin (10–20 mg·kg⁻¹ day⁻¹; 24 or 72 h). Functional recovery (neuroscore, forelimb gripping strength and adhesive removal test) was assessed during follow‐up and lesion volume measured at the end. Vasoreactivity of the middle cerebral artery (MCA), type IV collagen and FITC‐dextran distribution were evaluated to assess macrovascular and microvascular protection. Activated microglia, leucocyte adhesion and infiltration were chosen as markers of inflammation.

Key Results

Acute treatment with atorvastatin provided parenchymal and cerebral protection only at the higher dose of 20 mg·kg⁻¹·day⁻¹. In this treatment group, functional recovery was ameliorated, and lesion volumes were reduced as early as 24 h after experimental stroke. This was associated with vascular protection as endothelial function of the MCA and the density and patency of the microvascular network were preserved. Acute atorvastatin administration also induced an anti‐inflammatory effect in association with parenchymal and vascular mechanisms; it reduced microglial activation, and decreased leucocyte adhesion and infiltration.

Conclusions and Implications

Acute atorvastatin provides global cerebral protection, but only at the higher dose of 20 mg·kg⁻¹·day⁻¹; this was associated with a reduction in inflammation in both vascular and parenchymal compartments. Our results suggest that atorvastatin could also be beneficial when administered early after stroke.

Abbreviations

AT

atorvastatin

IR

ischaemia‐reperfusion

MCA

middle cerebral artery

MCAO

middle cerebral artery occlusion

Phe

phenylephrine

     

Aortic thrombosis resolved with enoxaparin in a patient treated with cisplatin-based regimen for small cell lung cancer

We present a rare case of a thrombus at the aortic arch found 1 month after cisplatin-based chemotherapy in a 50-year-old patient with a diagnosis of small cell lung cancer; there were no symptoms related to the thrombus. This patient did not have any predisposing factors for the development of an aortic thrombus before the chemotherapy was initiated. After immediate treatment with low molecular weight heparin (enoxaparin), the thrombus improved without any additional complications. Thus, we suggest that a hypercoagulable status possibly created by both the malignancy and cisplatin-based chemotherapy may have played a significant role in the development of the aortic thrombus.     

Mutational analysis of MED12 exon 2 in uterine leiomyoma and other common tumors

Recurrent somatic mutations in MED12 exon 2 have recently been reported in uterine leiomyomas. The recurrent nature of the mutations strongly suggests that the mutations may play important roles in the pathogenesis of uterine leiomyomas. The aim of our study was to see whether MED12 exon 2 mutations occur in other human tumors besides uterine leiomyomas. We also attempted to confirm occurrence of the MED12 mutations in uterine leiomyomas of Korean patients. For this, we analyzed 1,862 tumor tissues, including a variety of carcinomas, leukemias and stromal tumors by single-strand conformation polymorphism analysis. We found MED12 mutations in 35 uterine leiomyomas (35/67; 52.2%) and one colon carcinoma (0.3%), but none in other tumors. The MED12 mutations consisted of missense (77%) and inframe insertion-deletion (23%) mutations, the pattern of which was similar to the earlier report. Our data indicate that MED12 exon 2 mutations may be tissue-specific to uterine leiomyoma and rare in other tumors. Our study suggests that the MED12 mutations play unique roles in the pathogenesis of uterine leiomyomas and mutated MED12 could be therapeutically targeted in uterine leiomyomas.     

p53 mutations and microsatellite instabilities in the subtype of intestinal metaplasia of the stomach

To investigate the potential implication of the subtype of intestinal metaplasia in the progression to the gastric carcinoma, we analyzed the mutations of the p53 gene and microsatellite instability (MSI) both in the complete type (type I) and in the sulphomucin-secreting incomplete type (type III) intestinal metaplasia located adjacent to the gastric carcinoma. p53 mutations were observed in 13.3% of type I, in 6.6% of type III intestinal metaplasia, and in 40% of gastric carcinoma. The difference between p53 mutations observed in type I and type III intestinal metaplasia was not statistically significant. No identical mutation of the p53 gene was found in the intestinal metaplasia and carcinoma specimens from the patients. There was no case of intestinal metaplasia showing MSI. In gastric carcinomas, MSI was observed in six cases (40%). The cases harboring BAT-26 instability did not have the mutation of the p53 gene. These data suggest that intestinal metaplasia adjacent to gastric carcinoma, irrespective of its subtype, do not have the genetic alterations as showing in their carcinoma tissues.