A Novel Ex Vivo Isolation and Expansion Procedure for Chimeric Antigen Receptor Engrafted Human T Cells

Genetically engineered T lymphocytes are a promising option for cancer therapy. Prior to adoptive transfer they have to be expanded in vitro to reach therapeutically sufficient numbers. So far, no universal method exists for selective in vitro expansion of engineered T lymphocytes. In order to overcome this problem and for proof of concept we incorporated a novel unique peptide sequence of ten amino acids as epitope (E-Tag) into the binding domains of two novel chimeric antigen receptors (ECARs) directed against either prostate stem cell antigen (PSCA) for the treatment of prostate cancer (PCa) or CD33 for the treatment of acute myeloide leukemia (AML). The epitope tag then was utilized for expanding ECAR engrafted T cells by triggering the modified T cells via a monoclonal antibody directed against the E-Tag (Emab). Moreover, the E-Tag served as an efficient selection epitope for immunomagnetic isolation of modified T cells to high purity. ECAR engrafted T cells were fully functional and mediated profound anti-tumor effects in the respective models of PCa or AML both in vitro and in vivo. The method can be integrated straightforward into clinical protocols to improve therapeutic efficiency of tumor treatment with CAR modified T lymphocytes.     

Flat Feet,Happy Feet? Comparison of the Dynamic Plantar Pressure Distribution and Static Medial Foot Geometry between Malawian and Dutch Adults

In contrast to western countries, foot complaints are rare in Africa. This is remarkable, as many African adults walk many hours each day, often barefoot or with worn-out shoes. The reason why Africans can withstand such loading without developing foot complaints might be related to the way the foot is loaded. Therefore, static foot geometry and dynamic plantar pressure distribution of 77 adults from Malawi were compared to 77 adults from the Netherlands. None of the subjects had a history of foot complaints. The plantar pressure pattern as well as the Arch Index (AI) and the trajectory of the center of pressure during the stance phase were calculated and compared between both groups. Standardized pictures were taken from the feet to assess the height of the Medial Longitudinal Arch (MLA). We found that Malawian adults: (1) loaded the midfoot for a longer and the forefoot for a shorter period during roll off, (2) had significantly lower plantar pressures under the heel and a part of the forefoot, and (3) had a larger AI and a lower MLA compared to the Dutch. These findings demonstrate that differences in static foot geometry, foot loading, and roll off technique exist between the two groups. The advantage of the foot loading pattern as shown by the Malawian group is that the plantar pressure is distributed more equally over the foot. This might prevent foot complaints.     

Proteomic analysis of Fusarium solani isolated from the Asian longhorned beetle, Anoplophora glabripennis

Wood is a highly intractable food source, yet many insects successfully colonize and thrive in this challenging niche. Overcoming the lignin barrier of wood is a key challenge in nutrient acquisition, but full depolymerization of intact lignin polymers has only been conclusively demonstrated in fungi and is not known to occur by enzymes produced by insects or bacteria. Previous research validated that lignocellulose and hemicellulose degradation occur within the gut of the wood boring insect, Anoplophora glabripennis (Asian longhorned beetle), and that a fungal species, Fusarium solani (ATCC MYA 4552), is consistently associated with the larval stage. While the nature of this relationship is unresolved, we sought to assess this fungal isolate's ability to degrade lignocellulose and cell wall polysaccharides and to extract nutrients from woody tissue. This gut-derived fungal isolate was inoculated onto a wood-based substrate and shotgun proteomics using Multidimensional Protein Identification Technology (MudPIT) was employed to identify 400 expressed proteins. Through this approach, we detected proteins responsible for plant cell wall polysaccharide degradation, including proteins belonging to 28 glycosyl hydrolase families and several cutinases, esterases, lipases, pectate lyases, and polysaccharide deacetylases. Proteinases with broad substrate specificities and ureases were observed, indicating that this isolate has the capability to digest plant cell wall proteins and recycle nitrogenous waste under periods of nutrient limitation. Additionally, several laccases, peroxidases, and enzymes involved in extracellular hydrogen peroxide production previously implicated in lignin depolymerization were detected. In vitro biochemical assays were conducted to corroborate MudPIT results and confirmed that cellulases, glycosyl hydrolases, xylanases, laccases, and Mn- independent peroxidases were active in culture; however, lignin- and Mn- dependent peroxidase activities were not detected While little is known about the role of filamentous fungi and their associations with insects, these findings suggest that this isolate has the endogenous potential to degrade lignocellulose and extract nutrients from woody tissue.     

Proteomic interactions between the parasitoid Diachasmimorpha longicaudata and the oriental fruit fly,Bactrocera dorsalis during host parasitism

The oriental fruit fly, Bactrocera dorsalis, is an important agricultural pest and biological control is one of the most effective control methodologies. We conducted an investigation on the molecular response of the fruit fly to parasitism by the larval parasitoid, Diachasmimorpha longicaudata using two-dimensional gel electrophoresis and mass spectroscopy. We identified 285 differentially expressed protein spots (109 proteins) during parasitism. The molecular processes affected by parasitism varied at different time point during development. Transferrin and muscle specific protein 20 are the only two proteins differentially expressed that play a role in host immunity 24 h after parasitism. Developmental and metabolic proteins from parasitoids (transferrin and enolase) were up-regulated to ensure establishment and early development of parasitoids 48 h post parasitism. 72 h after parasitism, larval cuticle proteins, transferrin and CREG1 were overexpressed to support the survival of parasitoids while host metabolism proteins and parasitoid regulatory proteins were down-regulated. Host development slowed down while parasitoid development went up at 96 h after parasitism. All developmental, regulatory, structural, and metabolic proteins were expressed at their optimum at 120 h post parasitism. Host development was reduced, metabolism and regulatory proteins were strongly involved in the activities. The development deteriorated further at 144 h after parasitism. Enolase and CREG1 were indicators of parasitoid survival. Hexamerin and transferrin from the parasitoid was peaked at 168–216 h after parasitism, strongly indicating that parasitoid would survive. This study represents the first report that reveals the molecular players involved in the interaction between the host and parasitoid.     

DIETARY LUFENURON REDUCES EGG HATCH AND INFLUENCES PROTEIN EXPRESSION IN THE FRUIT FLY Bactrocera latifrons (HENDEL)

Lufenuron (LFN), a chitin synthase inhibitor, impacts the fertility of Ceratitis capitata, Bactrocera dorsalis, B. cucurbitae, and B. latifrons. We posed the hypothesis that LFN curtails egg hatch in the solanaceous fruit fly, B. latifrons. In this study, newly emerged virgin adults were sexed and fed for 12 days with varying concentrations of LFN‐laced agar diets until sexual maturation. Eggs were collected from 12‐d‐old adults and the egg hatch was assessed. Egg hatch decreased in adults reared on LFN‐treated diets. LFN‐treated media did not influence fertility after one gender was reared on experimental and the other on control media before mating. Exposure to LFN‐treated medium after mating led to reduced egg hatch. We infer that LFN is not a permanent sterilant, and reduced egg hatch depends on continuous exposure to dietary LFN after mating. Proteomic analysis identified two differentially expressed proteins, a pheromone binding protein and a chitin binding protein, between adults maintained on LFN‐treated and control diets. Expression of two genes encoding chitin synthase 2, and chitin binding protein, was altered in adults exposed to dietary LFN. LFN treatments also led to increased expression of two odorant binding proteins one in females and one in males. We surmise these data support our hypothesis and provide insight into LFN actions.     

Dosage-dependent impacts of a floral volatile compound on pollinators, larcenists, and the potential for floral evolution in the alpine skypilot Polemonium viscosum

All volatile organic compounds (VOCs) vary quantitatively, yet how such variation affects their ecological roles is unknown. Because floral VOCs are cues for both pollinators and floral antagonists, variation in emission may have major consequences for costs and benefits in plant-pollinator interactions. In Polemonium viscosum, the emission rate for the floral VOC 2-phenylethanol (2PE) spans more than two orders of magnitude. We investigated the ecological and evolutionary impacts of this immense phenotypic variation. The emission rate of 2PE varies independently of nectar rewards and thus is uninformative of profitability. Emission is elevated in flowers that are morphologically vulnerable to ant larcenists, suggesting that chemical deterrence may compensate for weak physical barriers. In nature, plants emitting more 2PE than their neighbors escape ant damage. Flower-damaging ants die when exposed to 2PE in the laboratory, and they avoid high 2PE emitters in the field. High 2PE also reduces bumblebee visitation and pollination, suggesting an ecological cost of defense in pollinator service. However, at more moderate emission rates, 2PE enhances the amount of nectar left in flowers, at no pollination cost. In conclusion, repellency of 2PE is highly sensitive to dosage, giving it a key role in shaping ecological interactions between skypilot plants and their floral visitors.