Identification, Pathogenicity, and Culture Conditions of a New Isolate of Cordyceps javanica (Hypocreales: Cordycipitaceae) From Soil.

This study decribes a highly effective insecticidal isolate of Cordyceps javanica (Frieder. & Bally) (Hypocreales: Cordycipitaceae) named IJ-tg19, which was isolated from soil. Spray bioassays were performed with IJ-tg19 on Myzus persicae (Sulzer) (Hemiptera: Aphididae) adults, third-instar nymphs of Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae), and third-instar larvae of Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae) to determine the pathogenicity of the isolate. The corrected mortality rates for all three pests were 100% when the conidia concentration was 1 × 106 conidia/ml, the lowest concentration in this study, and the median survival times (MST) were 4, 4, and 3 d. The MST shortens with increasing conidia concentration. The effects of laboratory culture conditions on the sporulation and growth of the isolate were also studied. This isolate had the greatest conidia production and fastest growth rate on malt extract agar medium at 25°C. The amount of conidia produced had positive correlation to light duration, with the highest production at 24 hr light. The growth of mycelium can adapt to a moderately alkaline environment, but the optimum conidial production occurred at the pH of 7. Our finding and research will be useful in biocontrol programs that are considering using the new isolate of C. javanica against greenhouse pests.

Toxicity of crude toxin protein produced by Cordyceps fumosorosea IF-1106 against Myzus persicae (Sulze).

The entomopathogenic fungus Cordyceps fumosorosea IF-1106 is a potential biocontrol agent with high pathogenicity to the aphid Myzus persicae. We extracted the crude toxin protein from a liquid culture broth of an isolated C. fumosorosea strain using the ammonium sulfate precipitation method, and its toxicity to Myzus persicae was measured by injection, oral exposure, and topical exposure. The crude toxin protein of C. fumosorosea had insecticidal activity against M. persicae. Body cavity injection and oral exposure had significantly higher insecticidal activity against adults than contact sprays. The highest cumulative corrected mortality of adults after injection was 81.85 ± 13.45 %, and the highest cumulative corrected mortality of adults after ingestion was 85.45 ± 11.88 %. The proportion of plasmatocytes in adult blood lymphocytes reached the highest at 3 days after injection and feeding, and the proportion of granulocytes was the highest at 2 days after injection and feeding. These data confirmed the toxicity of the crude toxin protein of C. fumosorosea toxin to M. persicae and helped clarify the pathogenic mechanism of the strain. Population management of M. persicae may be possible by using a natural toxic compound produced by C. fumosorosea that is selective to this pest species.

Knockdown of LINC01694 inhibits growth of gallbladder cancer cells via miR-340-5p/Sox4.

PURPOSE: The indispensable role of long non-coding RNAs (lncRNAs) in tumorigenesis has been increasingly reported. In the present study, LINC01694 was found to regulate the proliferation, invasion, as well as apoptosis in gallbladder cancer (GBC) cells through sponging miR-340-5p. METHODS: LINC01694 level in GBC cells was quantified by quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation, invasion, and apoptosis were determined by Cell Counting Kit-8 (CCK-8), Transwell, and flow cytometry, respectively. The expression of Sry-related high-mobility group box 4 (Sox4) was detected by Western blot (WB). The interaction between LINC01694 and miR-340-5p was measured by dual-luciferase reporter (DLR) assay, RNA immunoprecipitation (RIP) test, and RNA pull-down. Tumor formation was examined by in vivo experiment. RESULTS: qRT-PCR illustrated that cancerous tissues had higher LINC01694 than normal tissues. Survival analysis demonstrated that the prognosis of patients with high LINC01694 was significantly poorer than those with low LINC01694. Down-regulation of LINC01694 slowed down the proliferation and invasion in GBC cells and accelerated the apoptosis. DLR assay indicated that LINC01694 elevated Sox4 expression by regulating miR-340-5p. LINC01694 functioned as miR-340-5p sponge to inhibit Sox4 expression. CONCLUSION: LINC01694 level is elevated in GBC by regulating miR-340-5p/Sox4 axis, which indicates the poor prognosis of the patients.

Mumps caused hemophagocytic syndrome: a rare case report.

Mumps-associated hemophagocytic syndrome (HPS) is exceptionally rare. Here, we report a fatal case of concurrent mumps and HPS. A previously healthy 21-year-old male patient was admitted to the Department of Infectious Diseases on October 18, 2011,with complaints of parotid gland pain for 30 days and persistent fever (38.3°C-40°C) for 15 days. Admission examinations showed severe pancytopenia, liver dysfunction, hyperferritinemia, fibrinopenia, elevated lactalase dehydrase, bilateral pulmonary inflammation and pleural effusion, abdominal lymphadenopathy, and splenomegaly. The patient was accordingly suspected to have mumps-associated HPS and received nutrition support and hormonal therapies as well as platelet transfusions. On hospitalization day 3, the fever stayed high, and painful swelling in the right testicle was reported. A bone marrow biopsy evaluation and serological tests were then performed. Histiocytic hyperplasia and hemophagocytic macrophage infiltration were demonstrated in the bone marrow and antimumps virus immunoglobulin M was detected positive, but bacteria, Epstein-Barr virus, cytomegalovirus, and herpes simplex virus were detected negative in the peripheral blood. The initial diagnosis of mumps-associated HPS was eventually confirmed. Treatments with high doses of methylprednisolone, intravenous immunoglobulin, and etoposide were continued. By hospitalization day 20, patient's condition was improved, his body temperature and blood counts were almost normal, and the pain and swelling in his parotid glands and right testicle subsided considerably. On hospitalization day 28, however, patient's condition deteriorated rapidly, and pancytopenia became evident again. On hospitalization day 33, the patient died of multiple organ dysfunction.

Carbohydrate residues downstream of the terminal Galalpha(1,3)Gal epitope modulate the specificity of xenoreactive antibodies.

Carbohydrates are involved in many immunological responses including the rejection of incompatible blood, tissues and organs. Carbohydrate antigens with Galalpha(1,3)Gal epitopes are recognized by natural antibodies in humans and pose a major barrier for pig-to-human xenotransplantation. Genetically modified pigs have been established that have no functional alpha1,3-galactosyltransferase (alpha1,3GT), which transfers alphaGal to N-acetyllactosamine (LacNAc) type oligosaccharides. However, a low level of Galalpha(1,3)Gal is still expressed in alpha1,3GT knockout animals in the form of a lipid, isoglobotrihexosylceramide (iGb3), which is produced by iGb3 synthase on lactose (Lac) type core structures. Here, we define the reactivity of a series of monoclonal antibodies (mAb) generated in alpha1,3GT-/- mice immunized with rabbit red blood cells (RbRBC), as a rich source of lipid-linked antigens. Interestingly, one mAb (15.101) binds weakly to synthetic and cell surface-expressed Galalpha(1,3)Gal on LacNAc, but strongly to versions of the antigen on Lac cores, including iGb3. Three-dimensional models suggest that the terminal alpha-linked Gal binds tightly into the antibody-binding cavity. Furthermore, antibody interactions were predicted with the second and third monosaccharide units. Collectively, our findings suggest that although the terminal carbohydrate residues confer most of the binding affinity, the fine specificity is determined by subsequent residues in the oligosaccharide.

[Relationship between cytokine gene polymorphism and development of gastric adenocarcinoma].

OBJECTIVE: To investigate the relationship between the single nucleotide polymorphisms (SNP) of tumor necrosis factor-alpha (TNF-alpha) promoter genes and susceptibility to gastric adenocarcinoma or gastric adenocarcinoma with helicobacter pylori (Hp) infection. METHODS: The SNPs of the TNF-alpha (-238G/A and -308G/A) were determined by gene chip in 130 patients with gastric adenocarcinoma and 142 healthy controls. The sera concentrations of IgG, IgM and IgA of Hp antibodies were measured by ELISA in all cases and controls. RESULTS: H. pylori infection was detected in 69.2% of the 130 patients and 46.5% of the 142 controls (P < 0.01). The frequencies of TNF-alpha-238GA genotype and A allele in the patients with gastric adenocarcinoma were significantly higher than that in the healthy controls (P < 0.01, P < 0.01). The frequencies of TNF-alpha-238GA genotype and A allele in the patients with gastric adenocarcinoma with Hp infection were significantly higher than that in the Hp-negative patients with gastric adenocarcinoma (P < 0.05, P < 0.01). No association was found between any of the other polymorphisms and the patients with gastric adenocarcinoma or patients with gastric adenocarcinoma with Hp infection. CONCLUSION: TNF-alpha-238GA genotype and A allele are significantly related to susceptibility to gastric adenocarcinoma or susceptibility to gastric adenocarcinoma with Hp infection.

Enhanced immunogenicity of heat shock protein 70 peptide complexes from dendritic cell-tumor fusion cells.

We have developed a molecular chaperone-based tumor vaccine that reverses the immune tolerance of cancer cells. Heat shock protein (HSP) 70 extracted from fusions of dendritic (DC) and tumor cells (HSP70.PC-F) possess superior properties such as stimulation of DC maturation and T cell proliferation over its counterpart from tumor cells. More importantly, immunization of mice with HSP70.PC-F resulted in a T cell-mediated immune response including significant increase of CD8 T cells and induction of the effector and memory T cells that was able to break T cell unresponsiveness to a nonmutated tumor Ag and provide protection of mice against challenge with tumor cells. By contrast, the immune response to vaccination with HSP70-PC derived from tumor cells is muted against such nonmutated tumor Ag. HSP70.PC-F complexes differed from those derived from tumor cells in a number of key manners, most notably, enhanced association with immunologic peptides. In addition, the molecular chaperone HSP90 was found to be associated with HSP70.PC-F as indicated by coimmunoprecipitation, suggesting ability to carry an increased repertoire of antigenic peptides by the two chaperones. Significantly, activation of DC by HSP70.PC-F was dependent on the presence of an intact MyD88 gene, suggesting a role for TLR signaling in DC activation and T cell stimulation. These experiments indicate that HSP70-peptide complexes (PC) derived from DC-tumor fusion cells have increased their immunogenicity and therefore constitute an improved formulation of chaperone protein-based tumor vaccine.

Delivery of tumor associated antigens to antigen presenting cells using penetratin induces potent immune responses.

Cytoplasmic delivery of proteins or CTL epitopes is crucial for the presentation of antigen for the generation of CTL. We previously described the use of the 16-amino acid peptide penetratin from the Drosophila Antennapedia domain (Int) to transport CTL epitopes into cells. Here we show that, Int, incorporating MUC1 CTL epitopes in tandem is able to facilitate their rapid uptake by macrophages and dendritic cells (DC) in an energy-dependent endocytic pathway. We also demonstrate for the first time that Int conjugated proteins are also able to be efficiently taken up by DC. Furthermore, C57BL/6 and HLA-A2 transgenic mice immunized with the Int-peptides or Int-proteins induce strong IFN-gamma secreting T cells and weak IgG1 antibodies. Immunized C57BL/6 mice were protected against the growth of a MUC1(+) tumor cell line.

Mannan-MUC1-pulsed dendritic cell immunotherapy: a phase I trial in patients with adenocarcinoma.

PURPOSE: Tumor antigen-loaded dendritic cells show promise for cancer immunotherapy. This phase I study evaluated immunization with autologous dendritic cells pulsed with mannan-MUC1 fusion protein (MFP) to treat patients with advanced malignancy. EXPERIMENTAL DESIGN: Eligible patients had adenocarcinoma expressing MUC1, were of performance status 0 to 1, with no autoimmune disease. Patients underwent leukapheresis to generate dendritic cells by culture ex vivo with granulocyte macrophage colony-stimulating factor and interleukin 4 for 5 days. Dendritic cells were then pulsed overnight with MFP and harvested for reinjection. Patients underwent three cycles of leukapheresis and reinjection at monthly intervals. Patients with clinical benefit were able to continue with dendritic cell-MFP immunotherapy. RESULTS: Ten patients with a range of tumor types were enrolled, with median age of 60 years (range, 33-70 years); eight patients were of performance status 0 and two of performance status 1. Dendritic cell-MFP therapy led to strong T-cell IFNgamma Elispot responses to the vaccine and delayed-type hypersensitivity responses at injection sites in nine patients who completed treatments. Immune responses were sustained at 1 year in monitored patients. Antibody responses were seen in three patients only and were of low titer. Side effects were grade 1 only. Two patients with clearly progressive disease (ovarian and renal carcinoma) at entry were stable after initial therapy and went on to further leukapheresis and dendritic cell-MFP immunotherapy. These two patients have now each completed over 3 years of treatment. CONCLUSIONS: Immunization produced T-cell responses in all patients with evidence of tumor stabilization in 2 of the 10 advanced cancer patients treated. These data support further clinical evaluation of this dendritic cell-MFP immunotherapy.

Pim-1 kinase stability is regulated by heat shock proteins and the ubiquitin-proteasome pathway.

Elevated expression of the serine/threonine kinase Pim-1 increases the incidence of lymphomas in Pim-1 transgenic mice and has also been found to occur in some human cancers. Pim-1 acts as a cell survival factor and may prevent apoptosis in malignant cells. It was therefore of interest to understand to what extent maintenance and degradation of Pim-1 protein is affected by heat shock proteins (Hsp) and the ubiquitin-proteasome pathway in K562 and BV173 human leukemic cells. The half-life of Pim-1 protein in these cells was found to increase from 1.7 to 3.1 hours when induced by heat shock or by treating the cells with the proteasome inhibitor PS-341 (bortezomib). The Hsp90 inhibitor geldanamycin prevented the stabilization of Pim-1 by heat shock. Using immunoprecipitation, it was determined that Pim-1 is targeted for degradation by ubiquitin and that Hsp70 is associated with Pim-1 under these circumstances. Conversely, Hsp90 was found to protect Pim-1 from proteasomal degradation. A luminescence-based kinase assay showed that Pim-1 kinase bound to Hsp70 or Hsp90 remains active, emphasizing the importance of its overall cellular levels. This study shows how Pim-1 levels can be modulated in cells through degradation and stabilization.

Characterization of a CD46 transgenic pig and protection of transgenic kidneys against hyperacute rejection in non-immunosuppressed baboons.

Human membrane cofactor protein (CD46) controls complement activation and when expressed sufficiently as a transgene protects xenografts against complement-mediated rejection, as shown here using non-immunosuppressed baboons and heterotopic CD46 transgenic pig kidney xenografts. This report is of a carefully engineered transgene that enables high-level CD46 expression. A novel CD46 minigene was validated by transfection and production of a transgenic pig line. Pig lymphocytes were tested for resistance to antibody and complement-mediated lysis, transgenic tissues were characterized for CD46 expression, and kidneys were transplanted to baboons without immunosuppression. Absorption of anti-Galalpha(1,3)Gal epitope (anti-GAL) serum antibodies was measured. Transgenic pigs expressed high levels of CD46 in all tissues, especially vascular endothelium, with stable expression through three generations that was readily monitored by flow cytometry of transgenic peripheral blood mononuclear cells (PBMC). Transgenic PBMC pre-sensitized with antibody were highly resistant to human complement-mediated lysis which readily lysed normal pig PBMC. Normal pig kidneys transplanted without cold ischemia into non-immunosuppressed adult baboons survived a median of 3.5 h (n = 7) whereas transgenic grafts (n = 9), harvested at approximately 24-h intervals, were either macroscopically normal (at 29, 48 and 68 h) or showed limited macroscopic damage (median > 50 h). Microscopic assessment of transplanted transgenic kidneys showed only focal tubular infarcts with viable renal tissue elsewhere, no endothelial swelling or polymorph adherence and infiltration by lymphocytes beginning at 3 days. Coagulopathy was not a feature of the histology in four kidneys not rejected and assessed at 48 h or later after transplantation. Baboon anti-GAL serum antibody titers were high before transplantation and, in one extensively analyzed recipient, reduced approximately 8-fold within 5.5 h. The data demonstrate that a single CD46 transgene controls hyperacute kidney graft rejection in untreated baboons despite the presence of antibody and complement deposition. The expression levels, tissue distribution and in vitro functional tests indicate highly efficient CD46 function, controlling both classical and alternative pathway complement activation, which suggests it might be the complement regulator of choice to protect xenografts.

Pim-1 associates with protein complexes necessary for mitosis.

The proto-oncogene pim-1 is a serine/threonine kinase the over-expression of which promotes lymphoma formation. Neither the normal function of Pim-1 nor the biochemical mechanism for cancer development mediated by the gene has been delineated, although recent studies have provided compelling evidence that Pim-1 is involved in differentiation and cell survival. We now provide the first evidence that Pim-1 may be involved in the proliferative process. By confocal microscopy, we observed a dynamic redistribution of Pim-1 during the cell cycle, the protein moving from the nucleus and cytoplasm in interphase to the spindle poles during mitosis. From a computer search for putative substrates of Pim-1 that are located in the spindle poles, we discovered that the nuclear mitotic apparatus (NuMA) protein has two peptide sequences that contain preferred phosphorylation sites for Pim-1 kinase. Recombinant glutathione-S-transferase-Pim-1 also readily phosphorylates immunoprecipitated NuMA. By confocal microscopy and co-immunoprecipitation we showed the interaction of the Pim-1 and NuMA proteins in HeLa cells that had been arrested during mitosis with nocodazole. Pim-1 also appeared to interact with heterochromatin-associated protein 1beta (HP1beta) and the cytoplasmic proteins dynein and dynactin via complex formation with NuMA. In our studies, overexpressed wild-type-Pim-1-GFP (green fluorescent protein) fusion protein was found to co-localize in the spindle pole with NuMA during mitosis. In contrast, the 'kinase-dead' mut-Pim-1-GFP fusion protein did not co-localize with NuMA, and appeared to promote apoptosis. Further evidence for apoptotic cell death was the observed blebbing and fragmentation of the chromosomes and a decrease in the level of NuMA protein detected by confocal microscopy. These results strongly suggest that Pim-1 kinase plays a role, most likely by phosphorylation, in promoting complex formation between NuMA, HP1beta, dynein and dynactin, a complex that is necessary for mitosis.