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Lufenuron is an effective benzoylurea insecticide that inhibits the synthesis of chitin and regulates the growth of insects. However, little is known about the effects of lufenuron treatment on the development of Spodoptera frugiperda (J. E. Smith). In this study, we assessed the toxicity of lufenuron on S. frugiperda and evaluated the effects of lufenuron treatment on the growth and development of S. frugiperda. The results showed that lufenuron exhibits high insecticidal activity against S. frugiperda, with the LC

Mobile colistin resistance (

Salmonella enterica subsp. enterica serovar Abortusequi is a frequently reported pathogen causing abortion in mares. In this study, the preventive and therapeutic effects of phage P(IZ) SAE-01E2 against S. Abortusequi in a mouse model of abortion were investigated. Phage P(IZ) SAE-01E2 was stable at different temperatures (4 to 70°C) and pH values (pH 4 to 10) and could lyse the majority of the Salmonella serogroup O:4 and O:9 strains tested (25/28). There was no lysogeny-related, toxin, or antibiotic resistance-related gene in the genome of P(IZ) SAE-01E2. All of these characteristics indicate that P(IZ) SAE-01E2 has the potential for use in phage therapy. In in vivo experiments, 2 × 10(3) CFU/mouse of S. Abortusequi ATCC 9842 was sufficient to lead to murine abortion (gestational day 14.5) within 48 h. A single intraperitoneal inoculation of P(IZ) SAE-01E2 (10(8) PFU/mouse, multiplicity of infection = 10(5)) 1 h before or after S. Abortusequi challenge provided effective protection to all pregnant mice (10/10). After 24 h of treatment with phage P(IZ) SAE-01E2, the bacterial loads in both the placenta and the uterus of the infected mice were significantly decreased (10(6) CFU/g). In addition, the levels of inflammatory cytokines in the placenta and blood of the mice in the phage administration groups were significantly reduced (P

Bacteriophage endolysin is one of the most promising antibiotic substitutes, but in Gram-negative bacteria, the outer membrane prevents the lysin from hydrolyzing peptidoglycans and blocks the development of lysin applications. The prime strategy for new antibiotic substitutes is allowing lysin to access the peptidoglycan from outside of the bacteria by reformation of the lysin. In this study, the novel Escherichia coli (E. coli) phage lyase lysep3, which lacks outside-in catalytic ability, was fused with the N-terminal region of the Bacillus amyloliquefaciens lysin including its cell wall binding domain D8 through the best manner of protein fusion based on the predicted tertiary structure of lysep3-D8 to obtain an engineered lysin that can lyse bacteria from the outside. Our results showed that lysep3-D8 could lyse both Gramnegative and Gram-positive bacteria, whereas lysep3 and D8 have no impact on bacterial growth. The MIC of lysep3-D8 on E. coli CVCC1418 is 60 μg/ml; lysep3-D8 can inhibit the growth of bacteria up to 12 h at this concentration. The bactericidal spectrum of lysep3-D8 is broad, as it can lyse of all of 14 E. coli strains, 3 P. aeruginosa strains, 1 Acinetobacter baumannii strain, and 1 Streptococcus strain. Lysep3-D8 has sufficient bactericidal effects on the 14 E. coli strains tested at the concentration of 100 μg/ml. The cell wall binding domain of the engineered lysin can destroy the integrity of the outer membrane of bacteria, thus allowing the catalytic domain to reach its target, peptidoglycan, to lyse the bacteria. Lysep3-D8 can be used as a preservative in fodder to benefit the health of animals. The method we used here proved to be a successful exploration of the reformation of phage lysin.

Treatment of infections caused by staphylococci has become more difficult because of the emergence of multidrug-resistant strains as well as biofilm formation. In this study, we observed the ability of the phage lysin LysGH15 to eliminate staphylococcal planktonic cells and biofilms formed by Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis All these strains were sensitive to LysGH15, showing reductions in bacterial counts of approximately 4 log units within 30 min after treatment with 20 μg/ml of LysGH15, and the MICs ranged from 8 μg/ml to 32 μg/ml. LysGH15 efficiently prevented biofilm formation by the four staphylococcal species at a dose of 50 μg/ml. At a higher dose (100 μg/ml), LysGH15 also showed notable disrupting activity against 24-h and 72-h biofilms formed by S. aureus and coagulase-negative species. In the in vivo experiments, a single intraperitoneal injection of LysGH15 (20 μg/mouse) administered 1 h after the injection of S. epidermidis at double the minimum lethal dose was sufficient to protect the mice. The S. epidermidis cell counts were 4 log units lower in the blood and 3 log units lower in the organs of mice 24 h after treatment with LysGH15 than in the untreated control mice. LysGH15 reduced cytokine levels in the blood and improved pathological changes in the organs. The broad antistaphylococcal activity exerted by LysGH15 on planktonic cells and biofilms makes LysGH15 a valuable treatment option for biofilm-related or non-biofilm-related staphylococcal infections.IMPORTANCE Most staphylococcal species are major causes of health care- and community-associated infections. In particular, Staphylococcus aureus is a common and dangerous pathogen, and Staphylococcus epidermidis is a ubiquitous skin commensal and opportunistic pathogen. Treatment of infections caused by staphylococci has become more difficult because of the emergence of multidrug-resistant strains as well as biofilm formation. In this study, we found that all tested S. aureus, S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis strains were sensitive to the phage lysin LysGH15 (MICs ranging from 8 to 32 μg/ml). More importantly, LysGH15 not only prevented biofilm formation by these staphylococci but also disrupted 24-h and 72-h biofilms. Furthermore, the in vivo efficacy of LysGH15 was demonstrated in a mouse model of S. epidermidis bacteremia. Thus, LysGH15 exhibits therapeutic potential for treating biofilm-related or non-biofilm-related infections caused by diverse staphylococci.

Staphylococcus aureus (S. aureus) is a common and dangerous pathogen that causes various infectious diseases. Skin damage, such as burn wounds, are at high risk of Staphylococcus aureus colonization and infection, which increases morbidity and mortality. The phage lysin LysGH15 exhibits highly efficient lytic activity against methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains. Apigenin (api) significantly decreases haemolysis of rabbit erythrocytes caused by S. aureus and shows anti-inflammatory function. LysGH15 and api were added to Aquaphor to form an LysGH15-api-Aquaphor (LAA) ointment. The LAA ointment simultaneously exhibited bactericidal activity against S. aureus and inhibited haemolysis. In an LAA-treated mouse model of an MRSA-infected skin wound, the mean bacterial colony count decreased to approximately 102 CFU/mg at 18 h after treatment (and the bacteria became undetectable at 96 h), whereas the mean count in untreated mice was approximately 105 CFU/mg of tissue. The LAA ointment also reduced the levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IFN-γ) and accelerated wound healing in the mouse model. These data demonstrate the potential efficacy of a combination of LysGH15 and api for use as a topical antimicrobial agent against S. aureus.

Chronic hepatitis delta virus infection (CHD) is a condition arising from super-infection of hepatitis B virus (HBV)-infected patients, resulting in a more rapid advance in liver pathology and hepatocellular carcinoma than is observed for HBV mono-infection. Although hepatitis delta virus (HDV) is structurally simple, its life cycle involves the complex participation of host enzymes, HBV-derived surface antigen (HBsAg), and HDV-auto-ribozyme and hepatitis delta antigen (HDAg) activities. Unsatisfactory clinical trial results with interferon-based therapies are motivating researchers to adjust and redirect the approach to CHD drug development. This new effort will likely require additional structural and functional studies of the viral and cellular/host components involved in the HDV replication cycle. This review highlights recent work aimed at new drug interventions for CHD, with interpretation of key pre-clinical- and clinical trial outcomes and a discussion of promising new technological approaches to antiviral drug design.

As yet, very few vaccine candidates with activity in animals against Mycobacterium tuberculosis infection have been tested as therapeutic postexposure vaccines. We recently described two pools of mycobacterial proteins with this activity, and here we describe further studies in which four of these proteins (Rv1738, Rv2032, Rv3130, and Rv3841) were generated as a fusion polypeptide and then delivered in a novel yeast-based platform (Tarmogen) which itself has immunostimulatory properties, including activation of Toll-like receptors. This platform can deliver antigens into both the class I and class II antigen presentation pathways and stimulate strong Th1 and Th17 responses. In mice this fusion vaccine, designated GI-19007, was immunogenic and elicited strong gamma interferon (IFN-γ) and interleukin-17 (IL-17) responses; despite this, they displayed minimal prophylactic activity in mice that were subsequently infected with a virulent clinical strain. In contrast, in a therapeutic model in the guinea pig, GI-19007 significantly reduced the lung bacterial load and reduced lung pathology, particularly in terms of secondary lesion development, while significantly improving survival in one-third of these animals. In further studies in which guinea pigs were vaccinated with BCG before challenge, therapeutic vaccination with GI-19007 initially improved survival versus that of animals given BCG alone, although this protective effect was gradually lost at around 400 days after challenge. Given its apparent ability to substantially limit bacterial dissemination within and from the lungs, GI-19007 potentially can be used to limit lung damage as well as facilitating chemotherapeutic regimens in infected individuals.

Receptor interacting protein kinase 3 (RIP3) is a critical regulator of programmed necrotic cell death. Here, we observed that RIP3 was significantly down-regulated in esophageal cancer. And its remaining expression was associated with better response to chemotherapy and prolonged survival. Notably, re-expression of kinase-dead RIP3 also restored cisplatin sensitivity, suggesting that some roles of RIP3 beyond necroptosis may be involved in cisplatin-based chemosensitivity. To investigate the mechanisms, a large-scale quantitative proteomics study was performed after cisplatin treatment in RIP3-knockdown cells. In total, approximately 7000 protein groups were confidently identified, with a false discovery rate of 0.21% at the protein level. Of these proteins, 685 displayed RIP3-dependent changes in abundance. Bioinformatics analyses indicated that DNA repair pathway was stimulated after RIP3 depletion. Functional studies showed that deficient RIP3 upregulated FOSL1 and POLD1 through activation of the HSP90/CDC37 complex and ERK phosphorylation in multiple cell lines. Furthermore, via inhibition of the HSP90/CDC37 complex, ERK and FOSL1 reversed the cisplatin resistance phenotype. These results suggest that RIP3 regulates cisplatin sensitivity through both pronecrotic and non-necrotic functions. RIP3 may be a potential marker for predicting chemosensitivity.

In recent years, after the emergence of a large number of multidrug-resistant bacteria, phages and phage-associated products for the prevention and control of bacterial disease have revealed prominent advantages as compared with antibiotics. However, bacteria are susceptible to becoming phage-resistant, thus severely limiting the application of phage therapy. In this study, Escherichia coli cells were incubated with lytic bacteriophages to obtain mutants that were resistant to the lytic phages. Then, bacteriophages against the phage-resistant variants were isolated and subsequently mixed with the original lytic phage to prepare a novel phage cocktail for bactericidal use. The data showed that our phage cocktail not only had notable bactericidal effects, including a widened host range and rapid lysis, but also decreased the generation and mutation frequency of phage-resistant strains in vitro. In addition, we tested our cocktail in a murine bacteremia model. The results suggested that compared with the single phage, fewer phage-resistant bacteria appeared during the treatment of phage cocktail, thus prolonging the usable time of the phage cocktail and improving its therapeutic effect in phage applications. Importantly, our preparation method of phage cocktail was proved to be generalizable. Because the bacteriophage against the phage-resistant strain is an ideal guard that promptly attacks potential phage resistance, this guard-killer dual-function phage cocktail provides a novel strategy for phage therapy that allows the natural ecology to be sustained.

Phage-derived lysins can hydrolyse bacterial cell walls and show great potential for combating Gram-positive pathogens. In this study, the potential of LysEF-P10, a new lysin derived from a isolated Enterococcus faecalis phage EF-P10, as an alternative treatment for multidrug-resistant E. faecalis infections, was studied. LysEF-P10 shares only 61% amino acid identity with its closest homologues. Four proteins were expressed: LysEF-P10, the cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain (LysEF-P10C), the putative binding domain (LysEF-P10B), and a fusion recombination protein (LysEF-P10B-green fluorescent protein). Only LysEF-P10 showed highly efficient, broad-spectrum bactericidal activity against E. faecalis. Several key functional residues, including the Cys-His-Asn triplet and the calcium-binding site, were confirmed using 3D structure prediction, BLAST and mutation analys. We also found that calcium can switch LysEF-P10 between its active and inactive states and that LysEF-P10B is responsible for binding E. faecalis cells. A single administration of LysEF-P10 (5 μg) was sufficient to protect mice against lethal vancomycin-resistant Enterococcus faecalis (VREF) infection, and LysEF-P10-specific antibody did not affect its bactericidal activity or treatment effect. Moreover, LysEF-P10 reduced the number of Enterococcus colonies and alleviated the gut microbiota imbalance caused by VREF. These results indicate that LysEF-P10 might be an alternative treatment for multidrug-resistant E. faecalis infections.

Multidrug-resistant Escherichia coli has seriously threatened antibiotic resources and international public health. Bacteriophage lysin preparations have been widely considered as valid agents for solving multidrug resistances. Many lysins have been derived to treat diseases caused by Gram-positive bacteria, but only a few lysin preparations have been found that successively treat diseases caused by Gram-negative bacteria. The outer membrane of Gram-negative bacteria effectively blocks the interactions between peptidoglycan in the periplasmic space and bacteriophage lysins, which therefore hampers the antimicrobial effects of bacteriophage lysins. In this study, a new fusion protein (Colicin-Lysep3) was constructed by fusing the translocation and receptor binding domains of colicin A with an E. coli phage lysin, which endows Colicin-Lysep3 bactericidal activity against E. coli from outside of Gram-negative bacteria. These results show that Colicin-Lysep3 could lyse the E. coli broadly in vitro and significantly reduce the number of E. coli in an intestinal infection mouse model. Overall, our findings first demonstrated that a colicin A fragment could enable a bacteriophage lysin to lyse E. coli from the outside, promoting the application of phage lysin preparations in control of Gram-negative bacteria.

Purpose: Enzalutamide, a second-generation androgen antagonist, was approved by the U.S. Food and Drug Administration (FDA) for castration-resistant prostate cancer (CRPC) treatment. Immunotherapy has been shown to be a promising strategy for prostate cancer. This study was performed to provide data to support the combination of enzalutamide and immunotherapy for CRPC treatment. Experimental Design: Male C57BL/6 or TRAMP (transgenic adenocarcinoma of the mouse prostate) prostate cancer model mice were exposed to enzalutamide and/or a therapeutic vaccine targeting Twist, an antigen involved in epithelial-to-mesenchymal transition and metastasis. The physiologic and immunologic effects of enzalutamide were characterized. The generation of Twist-specific immunity by Twist-vaccine was assessed. Finally, the combination of enzalutamide and Twist-vaccine to improve TRAMP mice overall survival was evaluated. Results: Enzalutamide mediated immunogenic modulation in TRAMP-C2 cells. In vivo, enzalutamide mediated reduced genitourinary tissue weight, enlargement of the thymus, and increased levels of T-cell excision circles. Because no changes were seen in T-cell function, as determined by CD4+ T-cell proliferation and regulatory T cell (Treg) functional assays, enzalutamide was determined to be immune inert. Enzalutamide did not diminish the ability of Twist-vaccine to generate Twist-specific immunity. Twist was confirmed as a valid tumor antigen in TRAMP mice by immunohistochemistry. The combination of enzalutamide and Twist-vaccine resulted in significantly increased overall survival of TRAMP mice compared with other treatment groups (27.5 vs. 10.3 weeks). Notably, the effectiveness of the combination therapy increased with disease stage, i.e., the greatest survival benefit was seen in mice with advanced-stage prostate tumors. Conclusions: These data support the combination of enzalutamide and immunotherapy as a promising treatment strategy for CRPC. Clin Cancer Res; 19(22); 6205–18. ©2013 AACR.

N-myc down-regulated gene 1 (NDRG1) has been shown to regulate tumor growth and metastasis in various malignant tumors and also to be dysregulated in esophageal squamous cell carcinoma (ESCC). Here, we show that NDRG1 overexpression (91.9%, 79/86) in ESCC tumor tissues is associated with poor overall survival of esophageal cancer patients. When placed in stable transfectants of the KYSE 30 ESCC cell line generated by lentiviral transduction with the ectopic overexpression of NDRG1, the expression of transducin-like enhancer of Split 2 (TLE2) was decreased sharply, however β−catenin was increased. Mechanistically, NDRG1 physically associates with TLE2 and β−catenin to affect the Wnt pathway. RNA interference and TLE2 overexpression studies demonstrate that NDRG1 fails to active Wnt pathway compared with isogenic wild-type controls. Strikingly, NDRG1 overexpression induces the epithelial mesenchymal transition (EMT) through activating the Wnt signaling pathway in ESCC cells, decreased the expression of E-cadherin and enhanced the expression of Snail. Our study elucidates a mechanism of NDRG1-regulated Wnt pathway activation and EMT via affecting TLE2 and β-catenin expression in esophageal cancer cells. This indicates a pro-oncogenic role for NDRG1 in esophageal cancer cells whereby it modulates tumor progression.

GlobeImmune's Tarmogen(®) immunotherapy platform utilizes recombinant Saccharomyces cerevisiae yeast as a vaccine vector to deliver heterologous antigens for activation of disease-specific, targeted cellular immunity. The vaccines elicit immune-mediated killing of target cells expressing viral and cancer antigens in vivo via a CD8(+) CTL-mediated mechanism. Tarmogens are not neutralized by host immune responses and can be administered repeatedly to boost antigen-specific immunity. Production of the vaccines yields stable off-the-shelf products that avoid the need for patient-specific manufacturing found with other immunotherapeutic approaches. Tarmogens for the treatment of chronic hepatitis B and C and various cancers were well tolerated and immunogenic in phase 1 and 2 clinical trials encompassing600 subjects. The platform is being widely utilized in basic vaccine research and the most rapid path to success in these endeavors follows from optimal immunoassay selection and execution. This chapter provides detailed methods for the construction and preclinical immunogenicity testing of yeast-based immunotherapeutic products to support the rapid and efficient use of this versatile technology.

Chronic hepatitis B infection (CHB) is characterized by sub-optimal T cell responses to viral antigens. A therapeutic vaccine capable of restoring these immune responses could potentially improve HBsAg seroconversion rates in the setting of direct acting antiviral therapies. A yeast-based immunotherapy (Tarmogen) platform was used to make a vaccine candidate expressing hepatitis B virus (HBV) X, surface (S), and Core antigens (X-S-Core). Murine and human immunogenicity models were used to evaluate the type and magnitude of HBV-Ag specific T cell responses elicited by the vaccine. C57BL/6J, BALB/c, and HLA-A*0201 transgenic mice immunized with yeast expressing X-S-Core showed T cell responses to X, S and Core when evaluated by lymphocyte proliferation assay, ELISpot, intracellular cytokine staining (ICS), or tumor challenge assays. Both CD4+ and CD8+ T cell responses were observed. Human T cells transduced with HBc18–27 and HBs183–91 specific T cell receptors (TCRs) produced interferon gamma (IFNγ following incubation with X-S-Core-pulsed dendritic cells (DCs). Furthermore, stimulation of peripheral blood mononuclear cells (PBMCs) isolated from CHB patients or from HBV vaccine recipients with autologous DCs pulsed with X-S-Core or a related product (S-Core) resulted in pronounced expansions of HBV Ag-specific T cells possessing a cytolytic phenotype. These data indicate that X-S-Core-expressing yeast elicit functional adaptive immune responses and supports the ongoing evaluation of this therapeutic vaccine in patients with CHB to enhance the induction of HBV-specific T cell responses.

The objective of this study was to assess the clinical and histopathological relationship between pulmonary fibrosis and type 1 diabetes. We examined clinical pulmonary function parameters and transbronchial lung biopsies to assess associated histopathological changes in 12 type 1 diabetic patients presenting with dyspnea. Lung CT images pulmonary function tests from 12 diabetic patients without dyspnea and from 12 matched normal subjects served as controls. A similar histopathological analysis, including cytokine levels and pro-fibrotic markers, was performed on lung tissues in mice after the induction of experimental diabetes in an attempt to strengthen the link between diabetes and pulmonary fibrosis. Pulmonary function parameters (FVC, FEV1, TLC, and DLco/VA) were significantly reduced in diabetic patients with dyspnea and without dyspnea, compared to controls. Both patient groups also had increased lung CT scores and symptoms compared to normal controls, though the greatest increases were in the diabetic patients with dyspnea. Chronic hyperglycemia induced in mice led to histopathological changes in the lungs that were similar to those found in the human diabetic subjects and included alveoli compression by hyperplastic interstitium infiltrated with inflammatory cells and fibrotic in nature. Two inflammatory related genes, TNF-α and PAI-1, and two fibrosis-related genes, CTGF and fibronectin, demonstrated increased mRNA and protein expression in diabetic mouse lungs. In conclusion, there were significant clinical and histopathological correlations between pulmonary fibrosis and the presence of type 1 diabetes. Diabetes was clinically associated with pulmonary fibrosis and dysfunction in humans, and diabetes induction led to a similar pulmonary fibrosis in an experimental model. These clinical and non-clinical data suggest that diabetes is an independent risk factor for pulmonary fibrosis.

Purpose: Saccharomyces cerevisiae, a nonpathogenic yeast, has been used previously as a vehicle to elicit immune responses to foreign antigens, and tumor-associated antigens, and has been shown to reduce tumor burden in mice. Studies were designed to determine if vaccination of human carcinoembryonic antigen (CEA)-transgenic (CEA-Tg) mice (where CEA is a self-antigen) with a recombinant S. cerevisiae construct expressing human CEA (yeast-CEA) elicits CEA-specific T-cell responses and antitumor activity. Experimental Design: CEA-Tg mice were vaccinated with yeast-CEA, and CD4+ and CD8+ T-cell responses were assessed after one and multiple administrations or vaccinations at multiple sites per administration. Antitumor activity was determined by tumor growth and overall survival in both pulmonary metastasis and s.c. pancreatic tumor models. Results: These studies demonstrate that recombinant yeast can break tolerance and that (a) yeast-CEA constructs elicit both CEA-specific CD4+ and CD8+ T-cell responses; (b) repeated yeast-CEA administration causes increased antigen-specific T-cell responses after each vaccination; (c) vaccination with yeast-CEA at multiple sites induces a greater T-cell response than the same dose given at a single site; and (d) tumor-bearing mice vaccinated with yeast-CEA show a reduction in tumor burden and increased overall survival compared to mock-treated or control yeast-vaccinated mice in both pulmonary metastasis and s.c. pancreatic tumor models. Conclusions: Vaccination with a heat-killed recombinant yeast expressing the tumor-associated antigen CEA induces CEA-specific immune responses, reduces tumor burden, and extends overall survival in CEA-Tg mice. These studies thus form the rationale for the incorporation of recombinant yeast-CEA and other recombinant yeast constructs in cancer immunotherapy protocols.

Recombinant Saccharomyces cerevisiae (yeast) represents a unique and attractive vehicle to deliver antigens in vaccine immunotherapy protocols for cancer or infectious disease, in that it has been shown to be extremely safe and can be administered multiple times to hosts. In the studies reported here, we describe the effects of treatment with recombinant yeast on murine immature dendritic cells (DCs). Yeast expressing human carcinoembryonic antigen (CEA) as a model antigen was studied. Injection of mice subcutaneously with yeast-CEA resulted in rapid increases in MHC class II(+) cells and total antigen-presenting cells in draining lymph nodes. Post-treatment with yeast-CEA, DCs rapidly elevated both MHC class I and class II, numerous costimulatory molecules and other DC maturation markers, and secreted a range of Type I inflammatory cytokines. Gene expression arrays also revealed the rapid up-regulation of numerous cytokine and chemokine mRNAs, as well as genes involved in signal transduction and antigen uptake. Functional studies demonstrated enhanced allospecific reactivity of DCs following treatment with yeast-CEA or control yeast. Additionally, treatment of DCs with yeast-CEA resulted in specific activation of CEA-specific CD8(+) T cells in an MHC-restricted manner in vitro. Lastly, vaccination of CEA-transgenic mice with yeast-CEA elicited antigen-specific CD4(+) and CD8(+) immune responses in vivo. Thus, these studies taken together form a scientific rationale for the use of recombinant yeast in vaccination protocols for cancer or infectious diseases.