Bringing Molecular Biology to Bear on Adhesion Prevention: Postsurgical Adhesion Reduction Using Intraperitoneal Inoculation of Hyaluronic Acid–Inducing Adenoviral Vector in a Murine Model

Objective: Seprafilm (Genzyme, Cambridge, MA) an absorbable adhesion barrier incorporating hyaluronic acid (HA), a high molecular mass glycosaminoglycan and important component of the extracellular matrix, has been shown to prevent adhesions in both experimental models and human subjects. Yet, the application of HA as a sheet at the time of surgery has several important logistic limitations. Recently, our laboratory has identified and cloned the genes encoding murine hyaluronic acid synthase 2 (mHAS2) and 3 (mHAS3) and engineered adenoviruses incorporating these genes, which, on intraperitoneal injection, significantly increases HA in peritoneal fluid. We hypothesized that intraperitoneal gene therapy with mHAS2 or mHAS3 via an adenoviral vector prior to a standardized cecal abrasion surgery would lead to a reduction in postoperative adhesion severity. Methods: Mice were assigned to one of four groups: (1) intraperitoneal inoculation with adenovirus encoding mHAS2; (2) mHAS3; (3) a control reporter adenovirus (RV) encoding GFP; or (4) intraoperative placement of a commercially available and murine-validated hyaluronic acid adhesion barrier (Seprafilm, SF). An a priori sample size calculation was performed. Mice in groups 1, 2, and 3 underwent injection of 2 x 107 viral particles in 1 ml of fluid on day -1. Sham injection was performed on group 4 SF mice. On day 0, laparotomy was performed in random sequence by surgeon blinded to the experimental group. On day 7, adhesion scores (0-3) were assigned independently by two blinded investigators. Results: Mean adhesion scores (n = 247) were 0.68 (mHAS2), 0.91 (mHAS3), 1.28 (RV), and 0.47 (SF). Pairwise comparisons using Wilcoxon rank-sum test revealed significant reduction in severity of adhesions between mHAS2, mHAS3, and SF compared to RV (p = 0.0004, 0.039, and 0.0001, respectively). Significance persisted despite correction for multiple comparisons (p = 0.0002, Kruskal-Wallis). There was a direct relationship between intraperitoneal HA concentration and adhesion reduction. Only one death (RV) was secondary to adhesive disease; differential risk of death between groups was statistically significant (p = 0.008) (highest in mHAS2 group). Conclusions: In a dose-response relationship, an intraperitoneal gene therapy approach to adhesion prevention in a murine model was successful, with adenoviruses most productive of HA resulting in the most significant reduction in adhesion scores compared to empty virus (RV). Although SF best reduced postoperative adhesions, the adenoviral gene delivery approach may prove to be more effective in clinical use when peritoneal injury is less localized or at laparoscopy where the application of SF is not possible. Further studies to elucidate the reason for the differential death rates (time bias may have played a role) and to validate results are in progress

Elastin-specific MRI of extracellular matrix-remodelling following hepatic radiofrequency-ablation in a VX2 liver tumor model

Hepatic radiofrequency ablation (RFA) induces a drastic alteration of the biomechanical environment in the peritumoral liver tissue. The resulting increase in matrix stiffness has been shown to significantly influence carcinogenesis and cancer progression after focal RF ablation. To investigate the potential of an elastin-specific MR agent (ESMA) for the assessment of extracellular matrix (ECM) remodeling in the periablational rim following RFA in a VX2 rabbit liver tumor-model, twelve New-Zealand-White-rabbits were implanted in the left liver lobe with VX2 tumor chunks from donor animals. RFA of tumors was performed using a perfused RF needle-applicator with a mean tip temperature of 70 degrees C. Animals were randomized into four groups for MR imaging and scanned at four different time points following RFA (week 0 [baseline], week 1, week 2 and week 3 after RFA), followed by sacrifice and histopathological analysis. ESMA-enhanced MR imaging was used to assess ECM remodeling. Gadobutrol was used as a third-space control agent. Molecular MR imaging using an elastin-specific probe demonstrated a progressive increase in contrast-to-noise ratio (CNR) (week 3: ESMA: 28.1 +/- 6.0; gadobutrol: 3.5 +/- 2.0), enabling non-invasive imaging of the peritumoral zone with high spatial-resolution, and accurate assessment of elastin deposition in the periablational rim. In vivo CNR correlated with ex vivo histomorphometry (ElasticaVanGiesson-stain, y=1.2x - 1.8, R-2=0.89, p<0.05) and gadolinium concentrations at inductively coupled mass spectroscopy (ICP-MS, y=0.04x+1.2, R-2=0.95, p<0.05). Laser-ICP-MS confirmed colocalization of elastin-specific probe with elastic fibers. Following thermal ablation, molecular imaging using an elastin-specific MR probe is feasible and provides a quantifiable biomarker for the assessment of the ablation-induced remodeling of the ECM in the periablational rim

A Randomized Trial to Assess Anti-HIV Activity in Female Genital Tract Secretions and Soluble Mucosal Immunity Following Application of 1% Tenofovir Gel

Preclinical and early phase clinical microbicide studies have not consistently predicted the outcome of efficacy trials. To address this gap, candidate biomarkers of microbicide pharmacodynamics and safety were evaluated in a double-blind, placebo-controlled trial of tenofovir gel, the first microbicide to demonstrate significant protection against HIV acquisition.30 women were randomized to apply a single daily dose of tenofovir or placebo gel for 14 consecutive days. Anti-HIV activity was measured in cervicovaginal lavage (CVL) on Days 0, 3, 7, 14 and 21 by luciferase assay as a surrogate marker of pharmacodynamics. Endogenous activity against E. coli and HSV-2 and concentrations of immune mediators were quantified in CVL as candidate biomarkers of safety. Tenofovir levels were measured in CVL and blood.A significant increase in anti-HIV activity was detected in CVL from women who applied tenofovir gel compared to their endogenous anti-HIV activity in genital tract secretions on Day 0 and compared to activity in CVL from women in the placebo group. The activity correlated significantly with CVL concentration of tenofovir (r = 0.6, p<0.001) and fit a sigmoid E(max) pharmacodynamic model. Anti-HIV activity in CVL from women who applied tenofovir persisted when virus was introduced in semen, whereas endogenous anti-HIV activity decreased. Tenofovir did not trigger an inflammatory response or induce sustained loss in endogenous antimicrobial activity or immune mediators.Tenofovir gel had no deleterious impact on soluble mucosal immunity. The increased anti-HIV activity in CVL, which persisted in the presence of semen and correlated with tenofovir concentration, is consistent with the efficacy observed in a recent clinical trial. These results promote quantified CVL anti-HIV activity as a surrogate of tissue pharmacodynamics and as a potential biomarker of adherence to product. This simple, feasible and inexpensive bioassay may promote the development of models more predictive of microbicide efficacy.ClinicalTrials.gov NCT00594373

The Anti-Apoptotic Bcl-xL Protein, a New Piece in the Puzzle of Cytochrome C Interactome

A structural model of the adduct between human cytochrome c and the human anti-apoptotic protein Bcl-xL, which defines the protein-protein interaction surface, was obtained from solution NMR chemical shift perturbation data. The atomic level information reveals key intermolecular contacts identifying new potentially druggable areas on cytochrome c and Bcl-xL. Involvement of residues on cytochrome c other than those in its complexes with electron transfer partners is apparent. Key differences in the contact area also exist between the Bcl-xL adduct with the Bak peptide and that with cytochrome c. The present model provides insights to the mechanism by which cytochrome c translocated to cytosol can be intercepted, so that the apoptosome is not assembled

Integrative Taxonomy for Continental-Scale Terrestrial Insect Observations

Although 21st century ecology uses unprecedented technology at the largest spatio-temporal scales in history, the data remain reliant on sound taxonomic practices that derive from 18th century science. The importance of accurate species identifications has been assessed repeatedly and in instances where inappropriate assignments have been made there have been costly consequences. The National Ecological Observatory Network (NEON) will use a standardized system based upon an integrative taxonomic foundation to conduct observations of the focal terrestrial insect taxa, ground beetles and mosquitoes, at the continental scale for a 30 year monitoring program. The use of molecular data for continental-scale, multi-decadal research conducted by a geographically widely distributed set of researchers has not been evaluated until this point. The current paper addresses the development of a reference library for verifying species identifications at NEON and the key ways in which this resource will enhance a variety of user communities

Ligation of Macrophage Fcγ Receptors Recapitulates the Gene Expression Pattern of Vulnerable Human Carotid Plaques

Stroke is a leading cause of death in the United States. As ∼60% of strokes result from carotid plaque rupture, elucidating the mechanisms that underlie vulnerability is critical for therapeutic intervention. We tested the hypothesis that stable and vulnerable human plaques differentially express genes associated with matrix degradation. Examination established that femoral, and the distal region of carotid, plaques were histologically stable while the proximal carotid plaque regions were vulnerable. Quantitative RT-PCR was used to compare expression of 22 genes among these tissues. Distal carotid and femoral gene expression was not significantly different, permitting the distal carotid segments to be used as a paired control for their corresponding proximal regions. Analysis of the paired plaques revealed differences in 16 genes that impact plaque stability: matrix metalloproteinases (MMP, higher in vulnerable), MMP modulators (inhibitors: lower, activators: higher in vulnerable), activating Fc receptors (FcγR, higher in vulnerable) and FcγR signaling molecules (higher in vulnerable). Surprisingly, the relative expression of smooth muscle cell and macrophage markers in the three plaque types was not significantly different, suggesting that macrophage distribution and/or activation state correlates with (in)stability. Immunohistochemistry revealed that macrophages and smooth muscle cells localize to distinct and non-overlapping regions in all plaques. MMP protein localized to macrophage-rich regions. In vitro, treatment of macrophages with immune complexes, but not oxidized low density lipoprotein, C-reactive protein, or TNF-α, induced a gene expression profile similar to that of the vulnerable plaques. That ligation of FcγR recapitulates the pattern of gene expression in vulnerable plaques suggests that the FcγR → macrophage activation pathway may play a greater role in human plaque vulnerability than previously appreciated

Longitudinal Associations Between Interpersonal Relationship Functioning and Mood Episode Severity in Youth With Bipolar Disorder

This study examined the longitudinal association between mood episode severity and relationships in BP youth. Participants were 413 Course and Outcome of Bipolar Youth study youth, aged 12.6 ± 3.3 years. Monthly ratings of relationships (parents, siblings, and friends) and mood episode severity were assessed by the Adolescent Longitudinal Interval Follow-Up Evaluation (ALIFE) Psychosocial Functioning Schedule (PFS) and Psychiatric Rating Scales (PSR) on average every 8.2 months over 5.1 years. Correlations examined whether participants with increased episode severity also reported poorer relationships, and also examined whether fluctuations in episode severity predicted fluctuations in relationships, and vice versa. Results indicated that participants with greater mood episode severity also had worse relationships. Longitudinally, participants had largely stable relationships. To the extent that there were associations, changes in parental relationships may precede changes in episode severity, although the magnitude of this finding was small. Findings have implications for relationship interventions in BP youth

RACK1 Associates with Muscarinic Receptors and Regulates M2 Receptor Trafficking

Receptor internalization from the cell surface occurs through several mechanisms. Some of these mechanisms, such as clathrin coated pits, are well understood. The M2 muscarinic acetylcholine receptor undergoes internalization via a poorly-defined clathrin-independent mechanism. We used isotope coded affinity tagging and mass spectrometry to identify the scaffolding protein, receptor for activated C kinase (RACK1) as a protein enriched in M2-immunoprecipitates from M2-expressing cells over those of non-M2 expressing cells. Treatment of cells with the agonist carbachol disrupted the interaction of RACK1 with M2. We further found that RACK1 overexpression inhibits the internalization and subsequent down regulation of the M2 receptor in a receptor subtype-specific manner. Decreased RACK1 expression increases the rate of agonist internalization of the M2 receptor, but decreases the extent of subsequent down-regulation. These results suggest that RACK1 may both interfere with agonist-induced sequestration and be required for subsequent targeting of internalized M2 receptors to the degradative pathway