Iliocostalis Muscle Rotational Flap: A Novel Flap for Esophagopleural Fistula Repair.

Intrathoracic fistulas present major challenges to reconstructive surgeons. Reconstruction with muscle flaps have been shown to improve patient outcomes; however, there are patients for whom one or more of the commonly used muscle flaps is not available for several reasons. We describe the use of an iliocostalis muscle rotational flap for the repair of a caudally located esophagopleural fistula in the setting of definitive chemoradiotherapy for treatment of nonsmall-cell lung cancer and reirradiation with photons for local recurrence 5 years later. Our repair remained intact through the nearly 12-month follow-up period during which the patient tolerated a regular diet. This report demonstrates that the iliocostalis lumborum muscle is a viable option for repair of intrathoracic fistulas that are located in the distal esophagus, even in the setting of previous thoracotomy and radiation, and should be part of the reconstructive surgeon's armamentarium in the management of intrathoracic fistulas.

Oncologic and Functional Outcomes of Surgical and Nonsurgical Treatment of Advanced Squamous Cell Carcinoma of the Supraglottic Larynx.

IMPORTANCE: Nonsurgical treatment of advanced supraglottic laryngeal cancer is widely used as part of a larynx preservation protocol. However, recent studies have suggested that nonsurgical treatment may be associated with inferior survival. Furthermore, it is not clear whether preservation of the larynx provides superior voice or swallowing function in the long term. OBJECTIVE: To test the hypothesis that surgical treatment of advanced-stage squamous cell carcinoma of the supraglottic larynx is associated with superior overall survival (OS), freedom from recurrence (FFR), and noninferior voice and swallowing function. DESIGN, SETTING, AND PARTICIPANTS: Retrospective medical record review of patients treated for stage III or IV squamous cell carcinoma of the supraglottic larynx between January 1990 and June 2013 at a tertiary referral center: 97 patients underwent surgical treatment and 138, nonsurgical treatment. Exclusion criteria included prior definitive treatment for laryngeal cancer or evidence of distant metastatic disease at presentation. The median follow-up for all 235 patients was 63 months. INTERVENTIONS: Surgical or nonsurgical therapy. MAIN OUTCOMES AND MEASURES: Freedom from recurrence (FFR), OS, larynx preservation, voice graded from 1 to 5, and swallowing graded from 1 to 6 using our voice and swallowing function scales. RESULTS: Surgical treatment was associated with superior FFR (5-year FFR: 75% vs 55%; P = .006) but not OS (5-year OS: 52% vs 52%; P = .61). The larynx was preserved in 83% of patients in the nonsurgical group vs 42% of patients in the surgical group (P < .001). Voice function was superior in the nonsurgical group at all time points through 5 years after treatment (mean voice score, 3.8 vs 2.6; P < .001). Swallowing function was comparable between surgical and nonsurgical groups. Multivariable analysis revealed that advanced age (hazard ratio [HR], 1.43 per 10-year increment; 95% CI, 1.19-1.72) and clinical N stage (HR, 1.17 per 1-level increment; 95% CI, 1.05-1.30) were associated with worse OS, while treatment with chemotherapy was associated with superior OS (HR, 0.61; 95% CI, 0.41-0.92). CONCLUSIONS AND RELEVANCE: Compared with surgical treatment, nonsurgical treatment as part of a larynx preservation protocol is associated with a higher likelihood of recurrence but has similar OS and should continue to be viewed as a viable alternative for the treatment of advanced supraglottic laryngeal cancer.

Release of liposomal contents by cell-secreted matrix metalloproteinase-9.

Liposomes have been widely used as a drug delivery vehicle, and currently, more than 10 liposomal formulations are approved by the Food and Drug Administration for clinical use. However, upon targeting, the release of the liposome-encapsulated contents is usually slow. We have recently demonstrated that contents from appropriately formulated liposomes can be rapidly released by the cancer-associated enzyme matrix metalloproteinase-9 (MMP-9). Herein, we report our detailed studies to optimize the liposomal formulations. By properly selecting the lipopeptide, the major lipid component, and their relative amounts, we demonstrate that the contents are rapidly released in the presence of cancer-associated levels of recombinant human MMP-9. We observed that the degree of lipid mismatch between the lipopepides and the major lipid component profoundly affects the release profiles from the liposomes. By utilizing the optimized liposomal formulations, we also demonstrate that cancer cells (HT-29) which secrete low levels of MMP-9 failed to release a significant amount of the liposomal contents. Metastatic cancer cells (MCF7) secreting high levels of the enzyme rapidly release the encapsulated contents from the liposomes.

Mechanistic studies of the triggered release of liposomal contents by matrix metalloproteinase-9.

Matrix metalloproteinases (MMPs) constitute a class of extracellular-matrix-degrading enzymes overexpressed in many cancers and contribute to the metastatic ability of the cancer cells. We have recently demonstrated that liposomal contents can be released when triggered by the enzyme MMP-9. Herein, we report the results of our mechanistic studies of the MMP-9-triggered release of liposomal contents. We synthesized peptides containing the cleavage site for MMP-9 and conjugated them with fatty acids to prepare the corresponding lipopeptides. By employing circular dichroism (CD) spectroscopy, we demonstrated that the lipopeptides, when incorporated into liposomes, are demixed in the lipid bilayers and generate triple-helical structures. MMP-9 cleaves the triple-helical peptides, leading to the release of the liposomal contents. Other MMPs, which cannot hydrolyze triple-helical peptides, fail to release the contents from the liposomes. We also observed that the rate and extent of release of the liposomal contents depend on the mismatch between the acyl chains of the synthesized lipopeptide and phospholipid components of the liposomes. CD spectroscopic studies imply that the observed differences in the release reflect the ability of the liposomal membrane to anneal the defects following the enzymatic cleavage of the liposome-incorporated lipopeptides.

Matrix metalloproteinase-assisted triggered release of liposomal contents.

We offer a novel methodology for formulating liposomes by incorporating sequence-specific collagen-mimetic peptides such that they are specifically "uncorked" by a matrix metalloproteinase, MMP-9. By encapsulating carboxyfluorescein (as a self-quenching fluorescent dye), we demonstrate that the time-dependent release of the dye from liposomes is due to the specific enzymatic cleavage of the surface-exposed collagen-mimetic peptides. The specificity of such cleavage is attested by the fact that the liposomal "uncorking" and their content release occur only by MMP-9 and not by a general proteolytic enzyme, trypsin, despite the fact that the collagen mimetic peptides contain the trypsin cleavage site. The mechanistic details underlying the formulations of liposomes and their enzyme-selective "uncorking" and content release are discussed. Arguments are presented that such liposomes can be fine-tuned to serve as the drug delivery vehicles for the detection and treatment of various human diseases, which occur due to the overexpression of a variety of pathogenic matrix metalloproteinases.

Recognition of isozymes via lanthanide ion incorporated polymerized liposomes.

We report the selective recognition of carbonic anhydrase isozymes based on the excited-state lifetimes of chelated Eu(3+) ions incorporated in polymerized liposomes.

A strategy for designing "multi-prong" enzyme inhibitors by incorporating selective ligands to the liposomal surface.

We offer a novel strategy for designing "multi-prong" inhibitors of enzymes by incorporating selective ligands on the liposomal surface.

Intrinsic selectivity in binding of matrix metalloproteinase-7 to differently charged lipid membranes.

We provide evidence that matrix metalloproteinase-7 (MMP-7) interacts with anionic, cationic and neutral lipid membranes, although it interacts strongest with anionic membranes. While the catalytic activity of the enzyme remains unaffected upon binding to neutral and negatively charged membranes, it is drastically impaired upon binding to the positively charged membranes. The structural data reveal that the origin of these features lies in the "bipolar" distribution of the electrostatic surface potentials on the crystallographic structure of MMP-7.

Purification of recombinant annexins without the use of phospholipids.

Due to their involvement in a variety of physiological and pathological processes, different isoforms of annexins are being utilized as markers of some human diseases and bio-imaging of tissue injury (due to apoptosis), and have been proposed as drug delivery vehicles. These, in addition to extensive biophysical studies on the role of annexins in organizing lipid domains in biological membranes, have necessitated development of an efficient protocol for producing annexins in bulk quantities. In this paper, we report a one-step purification protocol for annexin a5 without using lipid vesicles or involving any column chromatographic step. Depending on the growth and expression condition, a fraction of recombinant annexin a5 (cloned in pET3d vector) was sequestered into inclusion bodies. When these inclusion bodies were dissolved in 6 M urea, subjected to a 10-fold snap dilution in the presence of 5 mM Ca(2+) and stored overnight at 4 degrees C, annexin a5 was precipitated as a homogenous protein as judged by SDS-PAGE. This one-step purification protocol produced about 35 mg of highly purified annexin a5 per liter of bacterial culture. The annexin a5 purified from inclusion bodies exhibited similar properties to that obtained from the soluble fraction using the conventional lipid-partitioning approach. Our purification protocol for annexin a5 elaborated herein is equally effective for purification of annexin A2, and we believe, will serve as general protocol for purifying other annexins in bulk quantities for diagnostic as well as detailed biophysical studies.

Temperature and composition dependence of the interaction of delta-lysin with ternary mixtures of sphingomyelin/cholesterol/POPC.

The kinetics of carboxyfluorescein efflux induced by the amphipathic peptide delta-lysin from vesicles of porcine brain sphingomyelin (BSM), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and cholesterol (Chol) were investigated as a function of temperature and composition. Sphingomyelin (SM)/Chol mixtures form a liquid-ordered (L(o)) phase whereas POPC exists in the liquid-disordered (L(d)) phase at ambient temperature. delta-Lysin binds strongly to L(d) and poorly to L(o) phase. In BSM/Chol/POPC vesicles the rate of carboxyfluorescein efflux induced by delta-lysin increases as the POPC content decreases. This is explained by the increase of delta-lysin concentration in L(d) domains, which enhances membrane perturbation by the peptide. Phase separations in the micrometer scale have been observed by fluorescence microscopy in SM/Chol/POPC mixtures for some SM, though not for BSM. Thus, delta-lysin must detect heterogeneities (domains) in BSM/Chol/POPC on a much smaller scale. Advantage was taken of the inverse variation of the efflux rate with the L(d) content of BSM/Chol/POPC vesicles to estimate the L(d) fraction in those mixtures. These results were combined with differential scanning calorimetry to obtain the BSM/Chol/POPC phase diagram as a function of temperature.