Delivery of plasmid DNA through intratumoral infusion and electroporation

We investigated DNA transport in the interstitial space and across cell membrane facilitated by intratumoral infusion and in vivo electroporation, respectively. In the study, a rat fibrosarcoma was perfused ex vivo, and apparent hydraulic conductivity (Kapp) was quantified under different perfusion conditions. In addition, three plasmid DNA vectors were infused into solid tumors. Immediately after infusion, tumors were treated with or without electric pulses. Gene expression and tumor growth delay were determined at different time points after electroporation. We found that Kapp was very sensitive to the perfusion pressure, presumably due to perfusion-induced tissue deformation. Treatment of tumors with electric pulse facilitated gene expression in vivo. The growth of tumors treated with plasmid DNA encoding interleukin 12 (IL-12) and electric pulses was slower than those treated with IL-12 or electric pulses alone. These data suggest that gene delivery in solid tumors could be improved significantly through combination of intratumoral infusion and in vivo electroporation

Delivery of plasmid DNA through intratumoral infusion and electroporation

We investigated DNA transport in the interstitial spaceand across cell membrane facilitated by intratumoral infusionand in vivo electroporation, respectively. In the study, a ratfibrosarcoma was perfused ex vivo, and apparent hydraulicconductivity (Kaap) was quantified under different perfusionconditions. In addition, three plasmid DNA vectors wereinfused into solid tumors. Immediately after infusion, tumorswere treated with or without electric pulses. Gene expressionand tumor growth delay were determined at different timepoints after electroporation. We found that K m was verysensitive to the perfusion pressure, presumably due toperfusion-induced tissue deformation. Treatment of tumorswith electric pulse facilitated gene expression in vivo. Thegrowth of tumors treated with plasmid DNA encodinginterleukin 12 (IL-12) and electric pulses was slower thanthose treated with IL-12 or electric pulses alone. These datasuggest that gene delivery in solid tumors could be improvedsignificantly through combination of intratumoral infusion andin vivo electroporation

Targeted delivery of murine IFN-γ using a recombinant fowlpox virus: NK cell recruitment to regional lymph nodes and priming of tumor-specific host immunity

Interferon-γ (IFN-γ) is a proinflammatory cytokine that also acts as a potent immunomodulatory agent. In this study, a replication-deficient recombinant avian (fowlpox) virus was engineered to express the murine IFN-γ gene (rF-MuIFN-γ) with the rationale of delivering concentrated levels of the cytokine to a local tissue microenvironment. Subcutaneous (s.c.) rF-MuIFN-γ administration resulted in IFN-γ production that (1) was restricted to the tissue microenvironment of the injection site and (2) was biologically active, as evidenced by a significant increase of class I MHC expression levels in s.c. growing tumors following rF-MuIFN-γ administration. Infection of a highly tumorigenic murine cell line, MC38, with rF-MuIFN-γ functioned as an effective tumor cell-based vaccine by protecting mice from the formation of primary tumors and from subsequent tumor challenge. The cell-based vaccine was completely ineffective if mice were vaccinated with MC38 cells either pretreated with rIFN-γ or infected with the wild-type fowlpox virus (FP-WT). Analysis of the regional lymph nodes draining the site of injection of the rF-MuIFN-γ- based tumor cell vaccine revealed the presence of tumor-specific cell lysis (CTL) as well as a significant amount of lysis directed at natural killer (NK)-sensitive YAC-1 cells. Flow cytometric analyses coupled with functional assays confirmed the sustained presence of NK1.1+ cells within those draining lymph nodes for up to 5 days after rF-MuIFN-γ injection. Mice treated with NK cell-depleting antibodies prior to the injection of the rF-MuIFN-γ- infected MC38 tumor cells were not protected from primary tumor growth; analysis of the lymph nodes draining the injection site in NK-depleted mice revealed an accompanying loss of the tumor-specific CTL activity. The findings provide evidence that NK cells, known for their contributions to host innate immunity, also provide immunoregulatory signals required for the development of an adaptive immune response, which, in turn, protected vaccinated mice against tumor growth. © 2008 Mary Ann Liebert, Inc

Data from: The evolution of complexity in the visual systems of stomatopods: insights from transcriptomics

Stomatopod crustaceans have complex visual systems containing up to 16 different spectral classes of photoreceptors, more than described for any other animal. A previous molecular study of this visual system focusing on the expression of opsin genes found many more transcripts than predicted on the basis of physiology, but was unable to fully document the expressed opsin genes responsible for this diversity. Furthermore, questions remain about how other components of phototransduction cascades are involved. This study continues prior investigations by examining the molecular function of stomatopods’ visual systems using new whole eye 454 transcriptome datasets from two species, Hemisquilla californiensis and Pseudosquilla ciliata. These two species represent taxonomic diversity within the order Stomatopoda, as well as variations in the anatomy and physiology of the visual system. Using an evolutionary placement algorithm to annotate the transcriptome, we identified the presence of nine components of the stomatopods’ G-protein-coupled receptor (GPCR) phototransduction cascade, including two visual arrestins, subunits of the heterotrimeric G-protein, phospholipase C, transient receptor potential channels, and opsin transcripts. The set of expressed transduction genes suggests that stomatopods utilize a Gq-mediated GPCR-signaling cascade. The most notable difference in expression between the phototransduction cascades of the two species was the number of opsin contigs recovered, with 18 contigs found in retinas of H. californiensis, and 49 contigs in those of P. ciliata. Based on phylogenetic placement and fragment overlap, these contigs were estimated to represent 14 and 33 expressed transcripts, respectively. These data expand the known opsin diversity in stomatopods to clades of arthropod opsins that are sensitive to short wavelengths and ultraviolet wavelengths and confirm the results of previous studies recovering more opsin transcripts than spectrally distinct types of photoreceptors. Many of the recovered transcripts were phylogenetically placed in an evolutionary clade of crustacean opsin sequences that is rapidly expanding as the visual systems from more species are investigated. We discuss these results in relation to the emerging pattern, particularly in crustacean visual systems, of the expression of multiple opsin transcripts in photoreceptors of the same spectral class, and even in single photoreceptor cells

Intravesical Immunotherapy of Superficial Bladder Cancer with Chitosan/Interleukin-12

Intravesical bacillus Calmette-Guerin (BCG) has been used successfully to treat superficial bladder cancer for 3 decades. However, 20–30% of patients will fail initial BCG therapy and 30–50% of patients will develop recurrent tumors within 5 years. Alternative or complementary strategies for the management of superficial bladder cancer are needed. IL-12 is a potent T(H)1 cytokine with robust antitumor activity and the ability to potentiate immunological memory. Unfortunately, intravesical IL-12 did not demonstrate anti-tumor efficacy in a recent clinical study of patients with recurrent superficial bladder cancer. We hypothesized that coformulation of IL-12 with chitosan – a biocompatible, mucoadhesive polysaccharide – could improve intravesical IL-12 delivery and provide an effective and durable alternative for the treatment of superficial bladder cancer. In antitumor studies, 88 to 100% of mice bearing orthotopic bladder tumors were cured after 4 intravesical treatments with chitosan/IL-12. In contrast, only 38 to 60% of mice treated with IL-12 alone, and 0% treated with BCG, were cured. Antitumor responses following chitosan/IL-12 treatments were durable and provided complete protection from intravesical tumor rechallenge. Urinary cytokine analysis showed that chitosan/IL-12 induced multiple T(H)1 cytokines at levels significantly higher than either IL-12 alone or BCG. Immunohistochemistry revealed moderate to intense tumor infiltration by T cells and macrophages following chitosan/IL-12 treatments. Bladder submucosae from cured mice contained residual populations of immune cells that returned to baseline levels after several months. Intravesical chitosan/IL-12 is a well tolerated, effective immunotherapy that deserves further consideration for testing in humans for the management of superficial bladder cancer

Fasta file of Hemisquilla californiensis phototransduction contigs from retinal transcriptome

This data file contains 22 contigs identified as transcripts potentially involved in phototransduction. The contig sequences are from 454 sequencing, assembled using Newbler v2.3, and identified using targeted BLAST and phylogenetic methods. Retinal tissue was taken from a single male individual of Hemisquilla californiensis, collected by dreading off the coast of Orange County, California, USA