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.

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.

Transcriptional profiling of human mesenchymal stem cells transduced with reporter genes for imaging.

Mesenchymal stem cells (MSCs) can differentiate into osteogenic, adipogenic, chondrogenic, myocardial, or neural lineages when exposed to specific stimuli, making them attractive for tissue repair and regeneration. We have used reporter gene-based imaging technology to track MSC transplantation or implantation in vivo. However, the effects of lentiviral transduction with the fluc-mrfp-ttk triple-fusion vector on the transcriptional profiles of MSCs remain unknown. In this study, gene expression differences between wild-type and transduced hMSCs were evaluated using an oligonucleotide human microarray. Significance Analysis of Microarray identified differential genes with high accuracy; RT-PCR validated the microarray results. Annotation analysis showed that transduced hMSCs upregulated cell differentiation and antiapoptosis genes while downregulating cell cycle, proliferation genes. Despite transcriptional changes associated with bone and cartilage remodeling, their random pattern indicates no systematic change of crucial genes that are associated with osteogenic, adipogenic, or chondrogenic differentiation. This correlates with the experimental results that lentiviral transduction did not cause the transduced MSCs to lose their basic stem cell identity as demonstrated by osteogenic, chondrogenic, and adipogenic differentiation assays with both transduced and wild-type MSCs, although a certain degree of alterations occurred. Histological analysis demonstrated osteogenic differentiation in MSC-loaded ceramic cubes in vivo. In conclusion, transduction of reporter genes into MSCs preserved the basic properties of stem cells while enabling noninvasive imaging in living animals to study the biodistribution and other biological activities of the cells.

Imaging early stage osteogenic differentiation of mesenchymal stem cells.

Stem cells, such as mesenchymal stem cells (MSCs), contribute to bone fracture repair if they are delivered to the injury site. However, it is difficult to assess the retention and differentiation of these cells after implantation. Current options for non-invasively tracking the transplanted stem cells are limited. Cell-based therapies using MSCs would benefit greatly through the use of an imaging methodology that allows cells to be tracked in vivo and in a timely fashion. In this study, we implemented an in vivo imaging methodology to specifically track early events such as differentiation of implanted human MSCs (hMSCs). This system uses the collagen type 1 (Col1α1) promoter to drive expression of firefly luciferase (luc) in addition to a constitutively active promoter to drive the expression of green fluorescent protein (GFP). The resulting dual-promoter reporter gene system provides the opportunity for osteogenic differentiation-specific luc expression for in vivo imaging and constitutive expression of GFP for cell sorting. The function of this dual-promoter reporter gene was validated both in vitro and in vivo. In addition, the ability of this dual-promoter reporter system to image an early event of osteogenic differentiation of hMSCs was demonstrated in a murine segmental bone defect model in which reporter-labeled hMSCs were seeded into an alginate hydrogel scaffold and implanted directly into the defect. Bioluminescence imaging (BLI) was performed to visualize the turn-on of Col1α1 upon osteogenic differentiation and followed by X-ray imaging to assess the healing process for correlation with histological analyses.