Ingestion of heat-killed pathogens confers transgenerational immunity to the pathogens via the vitellogenin-hypopharyngeal gland axis in honeybees.

Honeybee diseases are a serious threat to beekeeping and pollination. Transgenerational immune priming (TGIP) has been attracting increasing attention as a promising strategy to protect honeybee colonies from infections. This study investigated whether feeding honeybees (Apis mellifera) with a heat-killed pathogen cocktail can provide them with transgenerational immunity to these pathogens. We found that vitellogenin (Vg) and defensin-1 were highly upregulated in nurse bees upon feeding them with a cocktail of heat-killed Ascosphaera apis and Paenibacillus larvae (A + P cocktail). Pathogen-pattern-recognition receptor genes in the Toll signaling pathway were upregulated in nurse bees upon ingestion of the A + P cocktail. In the nurse bees of the hives supplied with the A + P cocktail, Vg was upregulated in the fat body, and the defensin-1 expression and Vg uptake in the hypopharyngeal glands were induced. Consequently, the major proteins in royal jelly were upregulated. In addition, defensin-1 was upregulated in the queen larvae and young worker larvae in these hives. In correlation, the young worker larvae showed high pathogen resistance to P. larvae infection. Thus, our findings imply that introduction of a heat-killed pathogen cocktail into hives is an efficient strategy for conferring honeybees with social immunity through TGIP.

Identification of waprin and its microbicidal activity: A novel protein component of honeybee (Apis mellifera) venom.

Bee venom is a rich source of biologically and pharmacologically active proteins. Waprin is a protein component of venoms; however, waprin has yet to be identified in bee venom. Moreover, the biological functions of waprin in venoms remain poorly characterized. Thus, in this study, we have identified and characterized waprin: a novel protein component from the venom of honeybees (Apis mellifera). The waprin in A. mellifera venom (Amwaprin) was found to consist of an 80-amino acid mature peptide, in which the whey acidic protein domain contains four conserved disulfide bonds. We discovered the presence of the Amwaprin protein in secreted venom by using an antibody against recombinant Amwaprin produced in baculovirus-infected insect cells. Recombinant Amwaprin exhibited inhibitory activity against microbial serine proteases and elastases but not thrombin or plasmin. It recognized carbohydrates in the microbial cell wall molecules and bound to the live microbial surfaces. The binding action of Amwaprin produced its microbicidal activity by inducing structural damage to bacterial and fungal cell walls. In addition, recombinant Amwaprin is heat-stable and contains no hemolytic activity. These findings demonstrate that Amwaprin acts as a microbicidal and anti-elastolytic agent.

Antimicrobial Activity of Apidermin 2 from the Honeybee Apis mellifera.

Apidermins (APDs) are known as structural cuticular proteins in insects, but their additional roles are poorly understood. In this study, we characterized the honeybee, Apis mellifera, APD 2 (AmAPD 2), which displays activity suggesting antimicrobial properties. In A. mellifera worker bees, the AmAPD 2 gene is transcribed in the epidermis, hypopharyngeal glands, and fat body, and induced upon microbial ingestion. Particularly in the epidermis of A. mellifera worker bees, the AmAPD 2 gene showed high expression and responded strongly to microbial challenge. Using a recombinant AmAPD 2 peptide, which was produced in baculovirus-infected insect cells, we showed that AmAPD 2 is heat-stable and binds to live bacteria and fungi as well as carbohydrates of microbial cell wall molecules. This binding action ultimately induced structural damage to microbial cell walls, which resulted in microbicidal activity. These findings demonstrate the antimicrobial role of AmAPD 2 in honeybees.

Bee Venom Induces Acute Inflammation through a H2O2-Mediated System That Utilizes Superoxide Dismutase.

Venoms from venomous arthropods, including bees, typically induce an immediate local inflammatory response; however, how venoms acutely elicit inflammatory response and which components induce an inflammatory response remain unknown. Moreover, the presence of superoxide dismutase (SOD3) in venom and its functional link to the acute inflammatory response has not been determined to date. Here, we confirmed that SOD3 in bee venom (bvSOD3) acts as an inducer of H2O2 production to promote acute inflammatory responses. In mouse models, exogenous bvSOD3 rapidly induced H2O2 overproduction through superoxides that are endogenously produced by melittin and phospholipase A2, which then upregulated caspase-1 activation and proinflammatory molecule secretion and promoted an acute inflammatory response. We also showed that the relatively severe noxious effect of bvSOD3 elevated a type 2 immune response and bvSOD3 immunization protected against venom-induced inflammation. Our findings provide a novel view of the mechanism underlying bee venom-induced acute inflammation and offer a new approach to therapeutic treatments for bee envenoming and bee venom preparations for venom therapy/immunotherapy.

Dual function of a bumblebee (Bombus ignitus) serine protease inhibitor that acts as a microbicidal peptide and anti-fibrinolytic venom toxin.

In bee venoms, low-molecular-weight peptides, including serine protease inhibitors (SPIs), exhibit multifunctional activities. Although SPIs in bee venoms are relatively well known, those that function in both the body and secreted venom of bees are not well-characterized. In this study, we identified a bumblebee (Bombus ignitus) SPI (BiSPI) that displays microbicidal and anti-fibrinolytic activities. BiSPI was found to consist of a trypsin inhibitor-like domain containing a P1 site and ten cysteine residues. We observed that the BiSPI gene was ubiquitously transcribed in the body, including the venom glands. In correlation, the BiSPI protein was detected both in the body and secreted venom by using an antibody against a recombinant BiSPI peptide produced in baculovirus-infected insect cells. Recombinant BiSPI exhibited inhibitory activity against trypsin but not chymotrypsin and inhibited microbial serine proteases and plasmin but not elastase or thrombin. Moreover, recombinant BiSPI recognized carbohydrates and bound to fungi and gram-negative and gram-positive bacteria. Consistent with these properties, recombinant BiSPI exhibited microbicidal activities against bacteria and fungi through induction of structural damage by binding to the microbial surfaces. Additionally, recombinant BiSPI inhibited the plasmin-mediated degradation of human fibrin and was thus concluded to exhibit anti-fibrinolytic activity. Moreover, the peptide showed no effect on hemolysis. These findings demonstrate the dual function of BiSPI, which acts as a microbicidal peptide and anti-fibrinolytic venom toxin.

Differential Expression of Major Royal Jelly Proteins in the Hypopharyngeal Glands of the Honeybee Apis mellifera upon Bacterial Ingestion.

Honeybee vitellogenin (Vg) transports pathogen fragments from the gut to the hypopharyngeal glands and is also used by nurse bees to synthesize royal jelly (RJ), which serves as a vehicle for transferring pathogen fragments to the queen and young larvae. The proteomic profile of RJ from bacterial-challenged and control colonies was compared using mass spectrometry; however, the expression changes of major royal jelly proteins (MRJPs) in hypopharyngeal glands of the honeybee Apis mellifera in response to bacterial ingestion is not well-characterized. In this study, we investigated the expression patterns of Vg in the fat body and MRJPs 1-7 in the hypopharyngeal glands of nurse bees after feeding them live or heat-killed Paenibacillus larvae. The expression levels of MRJPs and defensin-1 in the hypopharyngeal glands were upregulated along with Vg in the fat body of nurse bees fed with live or heat-killed P. larvae over 12 h or 24 h. We observed that the expression patterns of MRJPs and defensin-1 in the hypopharyngeal glands and Vg in the fat body of nurse bees upon bacterial ingestion were differentially expressed depending on the bacterial status and the time since bacterial ingestion. In addition, the AMP genes had increased expression in young larvae fed heat-killed P. larvae. Thus, our findings indicate that bacterial ingestion upregulates the transcriptional expression of MRJPs in the hypopharyngeal glands as well as Vg in the fat body of A. mellifera nurse bees.

Real-time in vivo monitoring of viable stem cells implanted on biocompatible scaffolds.

PURPOSE: Three-dimensional fibrous scaffolds provide an environment that enhances transplanted stem cell survival in vivo and facilitates imaging their localization, viability, and growth in vivo. To assess transplanted stem cell viability on biocompatible polymer scaffolds in vivo, we developed in vivo imaging systems for evaluation of implanted viable neural stem cells (NSC) and mesenchymal stem cells (MSC) on scaffolds using luciferase or sodium/iodide symporter (NIS) genes. METHODS: Firefly luciferase stably expressing-C6 cell was established (C6-Fluc). The human neural stem cell, F3, was infected with adenoviral vector carrying luciferase gene (F3-Fluc) and MSC expressing NIS controlled by ubiquitin C promoter using lentiviral vector was established by treating blasticidine for 2 weeks (MSC-NIS). Chitosan and poly L-lactic acid (PLLA) scaffolds were used for in vivo image. In vivo expression of luciferase and human NIS was examined by bioluminescence image or (99m)Tc-pertechnetate gamma camera image, respectively. The cell/scaffold complex was implanted into subcutaneous or abdominal area of BALB/C nude mouse. For quantitative evaluation of cell viability, regions of interest were drawn on (99m)Tc-pertechnetate scintigraphy by manual. RESULTS: The gradual increase of luciferase activity was observed in C6-Fluc seeded with chitosan according to the increase in the number of cells. C6-Fluc/chitosan complex subcutaneously implanted into nude mice showed longitudinal bioluminescence image until 34 days. Luciferase image of abdominal-injected C6-Fluc/PLLA complex was saturated in only 14 days, showing great cell growth due to abundant nutrients. F3 cells showed well-incorporated pattern with fibrous chitosan scaffold using scanning electron microscopy. F3 infected with Ad-Fluc showed >100-fold higher luciferase activity than luciferase activity in F3. Cell-number-dependent increase of luciferase activity was shown in F3-Fluc seeded on chitosan. F3-Fluc incorporation into chitosan after abdominal injection was clearly visible on bioluminescence image up to 11 days. Radionuclide imaging showed higher uptake by MSC-NIS on PLLA scaffolds than by MSC-NIS not seeded on a scaffold. Quantitative data showed significantly better survival of MSC-NIS on PLLA scaffolds than without scaffold at 72 h post-implantation, which concurred with histologic findings. CONCLUSION: These results suggest that NSC-Fluc and MSC-NIS cells incorporated within polymer scaffolds can be monitored on a long-term basis by serial in vivo imaging. We believe that a biocompatible scaffold-based imaging system could be used to assess stem cell viabilities in a non-invasive way to aid the development of regenerative therapeutics.

Development of a sodium/iodide symporter (NIS)-transgenic mouse for imaging of cardiomyocyte-specific reporter gene expression.

UNLABELLED: Development of a small animal imaging system for differentiated cell-specific reporter gene expression will enable us to image cellular differentiation in vivo. In this study, we developed a sodium/iodide symporter (NIS)-transgenic mouse in which NIS is constitutively expressed as an imaging reporter gene only in cardiomyocytes. METHODS: To express NIS gene in cardiomyocytes, alpha-myosin heavy chain (alpha-MHC)-NIS was constructed and used for the production of NIS-transgenic mice. Twelve lines of positive founder were obtained. The adequacy of the transgenic mouse model was tested by in vivo scintigraphy, microPET, and a biodistribution study. RESULTS: The myocardium of transgenic mice showed rapid and intense uptake of 131I, which was much higher than that of the thyroid, and also showed long retention by gamma-camera pinhole imaging. The relative uptake ratio of the heart of transgenic mice was 4.6 +/- 1.5, which was 3.8 +/- 1.2 times higher than that of control wild-type mice. The uptake of the heart was completely blocked by oral administration of KClO4, an NIS inhibitor. The heart of transgenic mouse was also clearly and intensely visualized on microPET using 124I. Biodistribution data of these mice showed the uptake of 40-160 %ID/g (percentage injected dose per gram of tissue) of (99m)Tc-pertechnetate in the heart compared with 40-60 %ID/g in the stomach, respectively. NIS expression in the myocardium was confirmed by immunohistochemistry using a NIS-specific antibody. CONCLUSION: We developed a transgenic mouse model to image cardiomyocytes with a gamma-camera and microPET using an alpha-MHC promoter and NIS. The transgenic mouse can be used as an imaging model for cardiomyocyte-specific reporter gene expression and cellular differentiation into cardiomyocytes after cardiac stem or progenitor cell transplantation.

Reversing the silencing of reporter sodium/iodide symporter transgene for stem cell tracking.

UNLABELLED: To track neural stem cells transfected with reporter gene, perpetual stem cell transgene expression is required. Referring to the knowledge about epigenetic modulation, we succeeded in reversing the silencing of sodium/sodide symporter (hNIS) transgenes transfected into human neural stem (HB1.F3) cells. METHODS: hNIS and hygromycin resistance gene were linked with IRES (Internal Ribosome Entry Site) under control of cytomegalovirus promoter, and this construct was transfected into HB1.F3 cells to yield the F3-NIS cell lines. hNIS transgene expression was examined by (125)I uptake and reverse transcriptase polymerase chain reaction (RT-PCR). The iodide uptake of F3-NIS III cells was initially higher by up to 12.9-fold than that of nontransfected HB1.F3 cells. However, repeated passage gradually silenced hNIS expression. The recovery of hNIS transgene expression by demethylating agent (5-azacytidine) or histone deacetylase inhibitor (trichostatin A; TSA) treatment was investigated. RESULTS: As hNIS transgene was gradually silenced in F3-NIS III cells, after the eighth passage its iodide uptake was 1.9-fold higher than that of nontransfected HB1.F3 cells. 5-azacytidine treatment (up to 40 micromol/L) for 24 h in F3-NIS III cells increased iodide uptake and hNIS messenger RNA (mRNA) 1.8- and 1.9-fold versus nontreated F3-NIS cells, respectively. Moreover, after TSA treatment (up to 62.5 nmol/L) for 24 h, iodide uptake and hNIS mRNA in F3-NIS III cells increased 36- and 1.9-fold versus nontreated F3-NIS III cells, respectively. The synergistic effect of demethylation and histone deacetylation inhibition was significant at high-dose 5-azacytidine and low-dose of TSA treatment. After treating F3-NIS III cells in vitro for 24 h with 62.5 nmol/L TSA, the cells were implanted into BALB/c nude mice. The TSA-treated F3-NIS III cells were clearly visible on gamma-camera imaging using (99m)Tc-pertechnetate as compared with F3-NIS III cells not treated with TSA. CONCLUSION: These results suggest that 2 well-known mechanisms of epigenetic modulation synergistically are involved in silencing reporter hNIS transgene in a neural stem cell line. Transgene silencing was reversed using demethylation and histone deacetylation inhibition. We conclude that silenced reporter transgenes once successfully expressed in stem cells might be awakened by pharmacologic treatment before infusion to track stem cells in vivo.