Expressions of Mast Cell Tryptase and Brain Natriuretic Peptide in Myocardium of Sudden Death due to Hypersensitivity and Coronary Atherosclerotic Heart Disease.

OBJECTIVES: To explore the value of mast cell tryptase and brain natriuretic peptide（BNP） in the differential diagnostic of sudden death due to hypersensitivity and coronary atherosclerotic heart disease. METHODS: Totally 30 myocardial samples were collected from the autopsy cases in the Department of Forensic Pathology, Shanxi Medical University during 2010-2015. All samples were divided into three groups： death of craniocerebral injury group, sudden death of hypersensitivity group and sudden death of coronary atherosclerotic heart disease group, 10 cases in each group. Mast cell tryptase and BNP in myocardium were detected by immunofluorescence staining and Western Blotting. RESULTS: Immunofluorescence staining showed that the positive staining mast cell tryptase appeared in myocardium of sudden death of hypersensitivity group and coronary atherosclerotic heart disease group. Among the three groups, the expression of mast cell tryptase showed significantly differences through pairwise comparison （P<0.05）; The expression level of BNP in sudden death of coronary atherosclerotic heart disease group were significantly higher than the sudden death of hypersensitivity group and death of craniocerebral injury group （P<0.05）. The difference of the expression level of BNP between the sudden death of hypersensitivity group and the death of craniocerebral injury group had no statistical significance （P>0.05）. CONCLUSIONS: The combined detection of the mast cell tryptase and BNP in myocardium is expected to provide help for the forensic differential diagnosis of sudden death due to hypersensitivity and coronary atherosclerotic heart disease.

More than 10 years survival with sequential therapy in a patient with advanced renal cell carcinoma: a case report

Although radical nephrectomy alone is widely accepted as the standard of care in localized treatment for renal cell carcinoma (RCC), it is not sufficient for the treatment of metastatic RCC (mRCC), which invariably leads to an unfavorable outcome despite the use of multiple therapies. Currently, sequential targeted agents are recommended for the management of mRCC, but the optimal drug sequence is still debated. This case was a 57-year-old man with clear-cell mRCC who received multiple therapies following his first operation in 2003 and has survived for over 10 years with a satisfactory quality of life. The treatments given included several surgeries, immunotherapy, and sequentially administered sorafenib, sunitinib, and everolimus regimens. In the course of mRCC treatment, well-planned surgeries, effective sequential targeted therapies and close follow-up are all of great importance for optimal management and a satisfactory outcome.

More than 10 years survival with sequential therapy in a patient with advanced renal cell carcinoma: a case report.

Although radical nephrectomy alone is widely accepted as the standard of care in localized treatment for renal cell carcinoma (RCC), it is not sufficient for the treatment of metastatic RCC (mRCC), which invariably leads to an unfavorable outcome despite the use of multiple therapies. Currently, sequential targeted agents are recommended for the management of mRCC, but the optimal drug sequence is still debated. This case was a 57-year-old man with clear-cell mRCC who received multiple therapies following his first operation in 2003 and has survived for over 10 years with a satisfactory quality of life. The treatments given included several surgeries, immunotherapy, and sequentially administered sorafenib, sunitinib, and everolimus regimens. In the course of mRCC treatment, well-planned surgeries, effective sequential targeted therapies and close follow-up are all of great importance for optimal management and a satisfactory outcome.

A combination of cilostazol and Ginkgo biloba extract protects against cisplatin-induced Cochleo-vestibular dysfunction by inhibiting the mitochondrial apoptotic and ERK pathways.

Cisplatin (cis-diammine-dichloroplatinum; CDDP) is an anticancer drug that induces significant hearing loss and balance dysfunction as side effects. Cilostazol (CS, 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl) butoxy]-3, 4-dihydro-2-(1H)-quinolinone) has neuroprotective and antioxidant effects, whereas Ginkgo biloba extract (GbE) has preventive effects on CDDP-induced hearing loss in rats, and GbE enhances the antiatherogenic effect of CS by inhibiting the generation of reactive oxygen species (ROS). The purpose of this study was to investigate the effects of renexin (RXN), which contains GbE and CS, against CDDP-induced cochleo-vestibular dysfunction in rats and to elucidate the mechanism underlying the protective effects of RXN on auditory cells. Rats intraperitoneally injected with CDDP exhibited an increase in hearing threshold and vestibular dysfunction, which agreed with hair cell damage in the Organ of Corti and otoliths. However, these impairments were significantly prevented in a dose-dependent manner by pre- and co-treatment with RXN, and these preventive effects in RXN-treated rats were more prominent than those in GbE-treated rats. In a CDDP pharmacokinetic study, platinum concentration was very similar between CDDP-only treated and RXN+CDDP cotreated rats. RXN markedly attenuated CDDP-induced intracellular ROS and significantly reduced CDDP-activated expression of p-extracellular regulated kinase (ERK), BAX, cytochrome c, cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase, but increased BCL-XL expression. These results show that RXN may have a synergistic effect by strongly protecting hearing and vestibular dysfunction induced by CDDP by inhibiting ROS production, mitochondrial pathways and the ERK pathway, without interfering with CDDP pharmacokinetics. Therefore, RXN could potentially be used to reduce CDDP-related hearing loss and dizziness.

Development of a fluorescent-intercalating-dye-based reverse transcription loop-mediated isothermal amplification assay for rapid detection of seasonal Japanese B encephalitis outbreaks in pigs.

The standardization and validation of a one-step, single-tube, accelerated fluorescent-intercalating-dye-based reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the NS3 gene of Japanese B encephalitis virus (JEV) is described for rapid, simple, and high-throughput detection of JEV. The amplification can be completed in 35 min under isothermal conditions at 63°C by employing a set of six primers targeting the NS3 gene of JEV. The RT-LAMP assay described demonstrated high sensitivity for detecting JEV, with a detection limit in swine samples of 8.13 PFU/ml. The specificity of the selected primer sets was established by cross-reactivity studies with pathogens that exhibit similar clinical signs and testing of samples from healthy animals. The clinical applicability of the RT-LAMP assay was validated using either spiked samples or samples from seasonal outbreaks. The comparative evaluation of the RT-LAMP assay revealed 79.59 % concordance with conventional RT-PCR targeting the E gene of JEV. The RT-LAMP assay reported here is a valuable tool for rapid real-time and high-throughput seasonal infection surveillance and quarantine after outbreak through blood sampling by using ordinary real-time PCR thermocyclers without purchasing an expensive Loopamp real-time turbidimeter.

Orally delivered foot-and-mouth disease virus capsid protomer vaccine displayed on T4 bacteriophage surface: 100% protection from potency challenge in mice.

An orally delivered foot-and-mouth disease (FMD) vaccine has not previously been reported. By using a T4 bacteriophage nanoparticle surface gene-protein display system (T4-S-GPDS), we created a foot-and-mouth disease virus (FMDV) entire capsid protein vaccine candidate. On the T4 phage surface SOC site, a full length FMDV capsid precursor polyprotein (P1, 755 aa) and proteinase 3C (213 aa) derived from an infected pig of serotype O strain GD-10 (1999), were separately displayed on different T4 phage particle surfaces through inserting their coding region DNAs into the T4 phage genome, yielding phage strains T4-P1 and T4-3C. We also constructed a series of FMDV sub-full length capsid structural protein (subunit) containing T4 phage recombinant vaccines. Both sucking and young BALB/c mice were used as two kinds of FMDV vaccine potency evaluation models. Many groups of both model mice were vaccinated orally or by subcutaneous injection with varying FMDV-T4 phage recombinant vaccines, with and without addition of adjuvant, then challenged with a lethal dose of cattle source virulent FMDV. In the case of immunization with a mixture of phage T4-P1 and phage T4-3C particles without any adjuvant added, all mice were 100% protected following either oral or injection immunization, whereas 100% of the control, non-immunized mice and mice immunized with only T4 phage vector Z1/Zh(-) or wild-type T4(+)D phage died; in contrast, with FMDV subunit vaccine, less than 75% protection followed the same potency challenge in both mice model groups. In addition, two pigs immunized with a phage T4-P1 and phage T4-3C mix were protected upon housing together with infected pigs. This study represents a clear example of how FMD and other pathogenic disease vaccines can be prepared by a simple and efficient bacteriophage route.

Comparison of human immunodeficiency virus type 1 Pr55(Gag) and Pr160(Gag-pol) processing intermediates that accumulate in primary and transformed cells treated with peptidic and nonpeptidic protease inhibitors.

Human immunodeficiency virus type 1 (HIV-1) produces two polyproteins, Pr55(Gag) and Pr160(Gag-Pol), that are cleaved into mature functional subunits by the virally encoded protease. Drugs that inhibit this protease are an important part of anti-HIV therapy. We studied the ordered accumulation of Gag and Gag-Pol processing intermediates by variably blocking the protease with HIV-1 protease inhibitors (PIs). Variable protease inhibition caused accumulation of a complex pattern of processing intermediates, which was the same after incubating HIV-1-infected cells with increasing concentrations of either one of the peptidomimetic inhibitors indinavir, saquinavir (SQV), ritonavir (RTV), nelfinavir, and SC-52151 or one of the nonpeptidomimetic inhibitors DMP450, DMP323, PNU-140135, and PNU-109112 for 3 days. The patterns of Gag and Gag-Pol processing intermediate accumulation were nearly identical when the following were compared: cell- versus virion-associated proteins, HIV-1-infected transformed cell lines versus primary human peripheral blood mononuclear cells (PBMCs) and HIV-1(MN) versus HIV-1(IIIB) virus strains. RTV was a more potent inhibitor of p24 production in PBMCs than SQV by approximately 7-fold, whereas SQV was a more potent inhibitor in transformed cells than RTV by approximately 30-fold. Although the antiretroviral potency of HIV-1 PIs may change as a function of cell type, the polyprotein intermediates that accumulate with increasing drug concentrations are the same. These results support sequential processing of Gag and Gag-Pol polyproteins by the HIV-1 protease and may have important implications for understanding common cross-resistance pathways.

Interaction of the human immunodeficiency virus type 1 nucleocapsid with actin.

The nucleocapsid (NC) domain of the retrovirus Gag protein plays several important roles in the viral life cycle, including virus assembly, viral genomic RNA encapsidation, primer tRNA placement, and enhancement of viral reverse transcription. In this study, deletion of NC domain of human immunodeficiency virus type 1 (HIV-1) Gag was found to drastically reduce virus particle production in CD4(+) T cells. Cellular fractionation experiments showed that although most of the uncleaved wild-type HIV-1 Gag, unmyristylated Gag, and p6(Gag) domain-truncated Gag molecules copurified with the host cell cytoskeleton, most of the mutant Gag molecules lacking both the NC and p6(Gag) domains failed to cofractionate with cytoskeleton. In wild-type virus-infected cells, in which the viral protease was active, the cleaved NCp7 copurified with the cytoskeleton, whereas most of the MAp17 and CAp24 did not. Monoclonal antibody against actin coimmunoprecipitated full-length Gag and p6(Gag) domain-truncated Gag molecules from cell lysates but failed to precipitate the truncated mutant Gag molecules lacking NC plus p6(Gag). Purified recombinant NCp7, but not CAp24, was able to bind F-actin in cosedimentation experiments. Furthermore, wild-type NCp7 and a zinc finger mutant NCp7(F16A), like a cellular actin-binding protein (the villin headpiece), bound F-actin in a dose-dependent fashion in vitro. Taken together, these results suggest that HIV-1 NCp7 can bind F-actin directly and that interaction between HIV-1 Gag and the actin cytoskeleton through the NC domain may play an important role in HIV-1 assembly and/or other steps of the viral life cycle.

A bipartite membrane-binding signal in the human immunodeficiency virus type 1 matrix protein is required for the proteolytic processing of Gag precursors in a cell type-dependent manner.

It is unclear whether proteolytic processing of the human immunodeficiency virus type 1 (HIV-1) Gag protein is dependent on virus assembly at the plasma membrane. Mutations that prevent myristylation of HIV-1 Gag proteins have been shown to block virus assembly and release from the plasma membrane of COS cells but do not prevent processing of Gag proteins. In contrast, in HeLa cells similar mutations abolished processing of Gag proteins as well as virus production. We have now addressed this issue with CD4(+) T cells, which are natural target cells of HIV-1. In these cells, myristylation of Gag proteins was required for proteolytic processing of Gag proteins and production of extracellular viral particles. This result was not due to a lack of expression of the viral protease in the form of a Gag-Pol precursor or a lack of interaction between unmyristylated Gag and Gag-Pol precursors. The processing defect of unmyristylated Gag was partially rescued ex vivo by coexpression with wild-type myristylated Gag proteins in HeLa cells. The cell type-dependent processing of HIV-1 Gag precursors was also observed when another part of the plasma membrane binding signal, a polybasic region in the matrix protein, was mutated. The processing of unmyristylated Gag precursors was inhibited in COS cells by HIV-1 protease inhibitors. Altogether, our findings demonstrate that the processing of HIV-1 Gag precursors in CD4(+) T cells occurs normally at the plasma membrane during viral morphogenesis. The intracellular environment of COS cells presumably allows activation of the viral protease and proteolytic processing of HIV-1 Gag proteins in the absence of plasma membrane binding.

Mutations of the human immunodeficiency virus type 1 p6Gag domain result in reduced retention of Pol proteins during virus assembly.

One of the crucial steps in the assembly of the human immunodeficiency virus type 1 (HIV-1) and other retroviruses is the incorporation and retention of all the key viral enzymes in released virions. The viral enzymes protease, reverse transcriptase, and integrase of HIV-1 are initially synthesized as Gag-Pol fusion polyproteins. It has been shown that the incorporation of Gag-Pol polyproteins during virus assembly requires the Gag domains that are shared by the Gag and Gag-Pol precursors. We now report that truncation of the C-terminal p6 domain of HIV-1 Gag, which is present in the Gag precursor but not in the Gag-Pol precursor, drastically reduced the amount of Pol proteins in the mutant virions. Mutations in the lentivirus conserved motif P(T/S)APP in p6 also drastically reduced the amount of Pol proteins in mutant virions. The steady-state levels of Gag-Pol precursors and cleaved Pol proteins in the transfected cells were not affected by mutations in p6. The incorporation of unprocessed Gag-Pol precursors into p6 mutant virions was detected when the viral protease was mutated, suggesting that the interactions among mutant Gag molecules and Gag-Pol precursors were not significantly affected. These results suggest that the p6 domain of HIV-1 Gag may play an important role in recruiting or retaining cleaved Pol proteins during virus assembly.