Controlled release of bioactive IL-2 from visible light photocured biodegradable elastomers for cancer immunotherapy applications.

Biodegradable elastomeric controlled-release poly (decane-co-tricarballylate) (PDET) based matrices capable of maintaining the stability and bioactivity of Interleukin-2 (IL-2) through the utilization of visible-light curing and solvent-free loading of the cytokine are reported. The elastomeric devices were fabricated by intimately mixing lyophilized IL-2 powder with the acrylated prepolymer before photocrosslinking. The bioactivity of the released protein was assessed by its ability to stimulate the proliferation of the C57BL/6 mouse cytotoxic T lymphocyte, and its concentration was analysed using ELISA. The influence of changes in the polymer's physicochemical and mechanical properties on IL-2 release kinetics and bioactivity were also studied. The increase in the device's surface area and the incorporation of trehalose in the loaded lyophilized mix increased the IL-2 release rate with drug release proceeding via typical zero-order release kinetics. Moreover, the decrease in the degree of acrylation of the prepared devices increased the IL-2 release rate. The bioactivity assay showed that IL-2 retained over 94% of its initial bioactivity throughout 28 days of the release period. A new protein delivery vehicle composed of biodegradable PDET elastomers was demonstrated to be promising and effective for linear, constant, and sustained osmotic-driven release of bioactive IL-2 and other sensitive proteins and hormones.

Histone deacetylase inhibition induces odor preference memory extension and maintains enhanced AMPA receptor expression in the rat pup model.

Histone deacetylase (HDAC) plays a role in synaptic plasticity and long-term memory formation. We hypothesized that trichostatin-A (TSA), an HDAC inhibitor, would promote long-term odor preference memory and maintain enhanced GluA1 receptor levels that have been hypothesized to support memory. We used an early odor preference learning model in neonate rat pups that normally produces only 24-h memory to test behavior and examine receptor protein expression. Our behavioral studies showed that intrabulbar infusion of TSA, prior to pairing of the conditioned stimulus (peppermint odor) with the unconditioned stimulus (tactile stimulation), prolonged 24-h odor preference memory for at least 9 d. The prolonged odor preference memory was selective for the paired odor and was also observed using a specific HDAC6 inhibitor, tubacin, supporting a role for histone acetylation in associative memory. Immunoblot analysis showed that GluA1 receptor membrane expression in the olfactory bulbs of the TSA-treated group was significantly increased at 48 h unlike control rats without TSA. Immunohistochemistry revealed significant increase of GluA1 expression in olfactory bulb glomeruli 5 d after training. These results extend previous evidence for a close relationship between enhanced GluA1 receptor membrane expression and memory expression. Together, these findings provide a new single-trial appetitive model for understanding the support and maintenance of memories of varying duration.

Internalization-dependent and -independent requirements for transforming growth factor beta receptor signaling via the Smad pathway.

Members of the transforming growth factor beta (TGF-beta) family of proteins signal through cell surface transmembrane serine/threonine protein kinases known as type I and type II receptors. The TGF-beta signal is extended through phosphorylation of receptor-associated Smad proteins by the type I receptor. Although numerous investigations have established the sequence of events in TGF-beta receptor (TGF-beta R) activation, none have examined the role of the endocytic pathway in initiation and/or maintenance of the signaling response. In this study we investigated whether TGF-beta R internalization modulates type I receptor activation, the formation of a functional receptor/Smad/SARA complex, Smad2/3 phosphorylation or nuclear translocation, and TGF-beta-dependent reporter gene activity. Our data provide evidence that, whereas type I receptor phosphorylation and association of SARA and Smad2 with the TGF-beta R complex take place independently of clathrin lattice formation, Smad2 or Smad3 activation and downstream signaling only occur after endocytic vesicle formation. Thus, TGF-beta R endocytosis is not simply a way to dampen the signaling response but instead is required to propagate signaling via the Smad pathway.

Tgf-beta induced Erk phosphorylation of smad linker region regulates smad signaling.

The Transforming Growth Factor-Beta (TGF-ß) family is involved in regulating a variety of cellular processes such as apoptosis, differentiation, and proliferation. TGF-ß binding to a Serine/Threonine kinase receptor complex causes the recruitment and subsequent activation of transcription factors known as smad2 and smad3. These proteins subsequently translocate into the nucleus to negatively or positively regulate gene expression. In this study, we define a second signaling pathway leading to TGF-ß receptor activation of Extracellular Signal Regulated Kinase (Erk) in a cell-type dependent manner. TGF-ß induced Erk activation was found in phenotypically normal mesenchymal cells, but not normal epithelial cells. By activating phosphotidylinositol 3-kinase (PI3K), TGF-ß stimulates p21-activated kinase2 (Pak2) to phosphorylate c-Raf, ultimately resulting in Erk activation. Activation of Erk was necessary for TGF-ß induced fibroblast replication. In addition, Erk phosphorylated the linker region of nuclear localized smads, resulting in increased half-life of C-terminal phospho-smad 2 and 3 and increased duration of smad target gene transcription. Together, these data show that in mesenchymal cell types the TGF-ß/PI3K/Pak2/Raf/MEK/Erk pathway regulates smad signaling, is critical for TGF-ß-induced growth and is part of an integrated signaling web containing multiple interacting pathways rather than discrete smad/non-smad pathways.

Troubleshooting tissue specificity and antibody selection: Procedures in immunohistochemical studies.

Optimal antigen detection and identification is dependent on the tissue of interest, the method of fixation, processing, and antibody specificity. We evaluated specific antigens in frozen middle cerebral artery (MCA) sections from rat brains under various conditions of fixation and differing primary and secondary antibody concentrations. Fresh MCAs were frozen, cryosectioned (8 microm), and adhered to chrom-alum coated slides. The effects of different fixation and antigen retrieval/pretreatments were tested for detection of enzymes and receptors involved in MCA tone regulation. Antigen localization was determined with specific primary antibodies and detected using fluorochrome-conjugated secondary antibodies. Spatial distribution of localized antigens was imaged using confocal microscopy. Frozen sections preserved the morphology of the endothelium and/or vessel wall within the tissue in a manner comparable to formalin-fixed sections. Fixation and tissue processing methods were modified based on the primary antibody used. Optimal antigen detection was obtained using fixatives such as 4% paraformaldehyde, 100% acetone or 100% methanol. Pretreatments, such as 1% SDS, enzymatic digestion using 0.1% trypsin, or application of heat were used to optimize antigen-antibody interaction. Stringent background and control checks were performed to ensure specificity of staining in both single and multiple labeling techniques. In a research setting where epitope detection is not used for diagnostic purposes, there is more latitude in tissue fixation. Frozen samples offer a more versatile method of linking the appropriate fixation and tissue processing to the primary antibody's unique needs. At the same time, it stabilizes the tissue in a format that allows for later analysis of multiple antigens with specific detection requirements in same tissue.

Synthesis, characterization and cytocompatibility of a poly(diol-tricarballylate) visible light photo-cross-linked biodegradable elastomer.

The synthesis, characterization and in vitro cytocompatibility of a new family of photo-cross-linked amorphous poly(diol-tricarballylate) (PDT) biodegradable elastomeric polyesters are reported. The synthesis was based on the polycondensation reaction between tricarballylic acid and alkylene diols, followed by acrylation. The prepared and acrylated poly(diol-tricarballylate) (APDT) was characterized by means of FT-IR, (1)H-NMR, GPC and DSC. Liquid-to-solid photo-curing was carried out by exposing the APDT to visible light in the presence of camphorquinone as a photoinitiator. The thermal properties, mechanical characteristics, sol content, long-term in vitro degradation and cytocompatibility of the prepared PDT elastomers were also reported. The mechanical and degradation properties of this new photocurable elastomer can be precisely controlled by varying the density of acrylate moieties in the matrix of the polymer, and through changes in the pre-polymer chain length. The use of visible light cross-linking, possibility of solventless drug loading, controllable mechanical properties and cytocompatibility of these new elastomers make them excellent candidates for use in controlled implantable drug-delivery systems of protein drugs and other biomedical applications.

Human kallikrein related peptidases 6 and 13 in combination withCA125 is a more sensitive test for ovarian cancer than CA125 alone.

The current biomarker for ovarian cancer, CA125, lacks the sensitivity and specificity required to detect early stage ovarian cancers. Since several Kallikreins (KLKs) are up regulated in ovarian cancer, they represent a potential pool of biomarkers for ovarian cancer. The purpose of this study is to determine if elevated expression levels of Muc16 (CA125 gene), KLK6 and KLK13 represent a more sensitive test for detection of early stage ovarian cancer than Muc16 alone. Using quantitative real-time PCR, 106 sporadic ovarian tumors and 8 normal ovaries were evaluated for mRNA expression. Analysis for increased expression levels, above controls, of either KLK6, KLK13 or Muc16 improved overall sensitivity to 93%, from 82% for Muc16 alone. Likewise, the negative predictive value increased from 27% to 50% (Muc16 alone compared to combined). With early stage cancers (n=32), both sensitivity increased 50-56% (individually) to 72% (combined), and negative predictive value increased (30% Muc16 to 58% combined). These results show a combined panel of KLK6, KLK13, and Muc16, is a more sensitive test to detect early stage ovarian cancer than Muc16 alone, indicating assaying for several kallikrein-related peptidases, in addition to CA125, could provide a significant advantage to detect ovarian cancer in the early stages.

Endurance exercise facilitates relearning of forelimb motor skill after focal ischemia.

Endurance exercise (i.e. running), by up-regulating brain-derived neurotrophic factor (BDNF) and other modulators of synaptic plasticity, improves attention and learning, both critical components of stroke rehabilitation. We hypothesized that, following middle cerebral artery occlusion in male Sprague-Dawley rats, endurance exercise would act synergistically with a challenging skilled forelimb task to facilitate motor recovery. Animals were randomly assigned to one of four rehabilitation conditions: no rehabilitation, running only, reach training only, and reach training preceded by running (run/reach training) for 5 weeks beginning 5 days after stroke. The behavioral outcome, morphological change and mRNA expression of proteins implicated in neuroplasticity (BDNF, synapsin I and microtubule-associated protein 2) were compared. Endurance exercise on a motorized running wheel, prior to reach training, enhanced recovery of skilled reaching ability but did not transfer to gross motor skills such as postural support (forelimb asymmetry test) and gait (ladder rung walking test). Microtubule-associated protein 2 staining density in the run/reach group was slightly enhanced in the contralateral motor cortex compared with the contralateral sensory and ipsilateral cingulate cortices, suggesting that running preceding reach training may have resulted in more dendritic branching within the motor cortex in this group. No significant differences in mRNA levels were detected among the training paradigms; however, there was a trend toward greater BDNF and synapsin I mRNA in the reaching groups. These findings suggest that exercise facilitates learning of subsequent challenging reaching tasks after stroke, which has the potential to optimize outcomes in patients with stroke.