Hyun Joon Paek

Induction of tumor-specific protective immunity by in situ Langerhans cell vaccine

Although anti-tumor immunity is inducible by dendritic cell (DC)–based vaccines, time- and cost-consuming “customizing” processes required for ex vivo DC manipulation have hindered broader clinical applications of this concept. Epidermal Langerhans cells (LCs) migrate to draining lymph nodes and undergo maturational changes on exposure to reactive haptens. We entrapped these migratory LCs by subcutaneous implantation of ethylene–vinyl–acetate (EVA) polymer rods releasing macrophage inflammatory protein (MIP)-3β (to create an artificial gradient of an LC-attracting chemokine) and topical application of hapten (to trigger LC emigration from epidermis). The entrapped LCs were antigen-loaded in situ by co-implantation of the second EVA rods releasing tumor-associated antigens (TAAs). Potent cytotoxic T-lymphocyte (CTL) activities and protective immunity against tumors were induced efficiently with each of three tested TAA preparations. Thus, tumor-specific immunity is inducible by the combination of LC entrapment and in situ LC loading technologies. Our new vaccine strategy requires no ex vivo DC manipulation and thus may provide time and cost savings.

Sequestration and Synthesis: The Source of Insulin in Cell Clusters Differentiated from Murine Embryonic Stem Cells

The source of insulin released from insulin‐releasing cell clusters (IRCCs) differentiated from embryonic stem cells remains unclear. Rajagopal et al. have suggested that IRCCs do not synthesize but secrete insulin that had been absorbed from media during the multistep protocol. We report here further data relevant to this controversy. No radioisotopic labeling of insulin was observed when IRCCs were incubated in a medium containing 35S‐cysteine. Less than 1% of the extra‐cellular stoichiometric C‐peptide equivalent to insulin was secreted during glucose stimulation. However, intracellular immunostaining and immunogold labeling were both positive for C‐peptide. Finally, a mass balance calculation showed that simple equilibration of IRCCs by Fickian diffusion from media accounted for at most 4% of secreted insulin. These findings and further analysis of the results of others suggest that the mechanism of insulin secretion by IRCCs is a combination of sequestration and de novo synthesis.

In vitro characterization of TGF-beta1 release from genetically modified fibroblasts in Ca(2+)-alginate microcapsules.

This study was undertaken to develop an in situ source of transforming growth factor-β1 (TGF-β1), one of several molecules potentially useful for a tissue-engineered bioartificial cartilage. Primary human fibroblasts and murine NIH 3T3 cells were genetically modified via viral transfection to express human TGF-β1. Two viral constructs were used, one expressing a gene encoding for the latent and the other for the constitutively active form of the growth factor. Unmodified cells served as controls. Four genetically modified cohorts and two controls were separately encapsulated in a 1.8% alginate solution using a vibrating nozzle and 0.15M calcium chloride crosslinking bath. Diameter of the spherical capsules was 410 ± 87 &mgr;m. In vitro release rate measured over 168 hours varied with cell types and ranged from 2–17 pg/(milligram of capsules·24 h) or 2-17 ng/(106 cells·24 h). None of the formulations exhibited a large initial bolus release. Even when serum-supplemented medium was not replenished, cell viabilities remained over 55% after 1 week for all cell types. Microencapsulated genetically modified cells were capable of a constitutive synthesis and delivery of biologically significant quantity of TGF-β1 for at least 168 hours and thus are of potential utility for artificial cartilage and other orthopedic tissue engineering applications.