Targeted gene delivery, transfection efficiency, and toxicity concerns remain a challenge for effective gene therapy. In this study, we dimerized the HIV-1 TAT peptide and formulated a nanoparticle vector (dTAT NP) to leverage the efficiency of this cell-penetrating strategy for tumor-targeted gene delivery in the setting of intratracheal administration. Expression efficiency for dTAT NP–encapsulated luciferase or angiotensin II type 2 receptor (AT2R) plasmid DNA (pDNA) was evaluated in Lewis lung carcinoma (LLC) cells cultured in vitro or in vivo in orthotopic tumor grafts in syngeneic mice. In cell culture, dTAT NP was an effective pDNA transfection vector with negligible cytotoxicity. Transfection efficiency was further increased by addition of calcium and glucose to dTAT/pDNA NP. In orthotopic tumor grafts, immunohistochemical analysis confirmed that dTAT NP successfully delivered pDNA to the tumor, where it was expressed primarily in tumor cells along with the bronchial epithelium. Notably, gene expression in tumor tissues persisted at least 14 days after intratracheal administration. Moreover, bolus administration of dTAT NP–encapsulated AT2R or TNF-related apoptosis-inducing ligand (TRAIL) pDNA markedly attenuated tumor growth. Taken together, our findings offer a preclinical proof-of-concept for a novel gene delivery system that offers an effective intratracheal strategy for administering lung cancer gene therapy. Cancer Res; 72(8); 2057–67. ©2012 AACR.

Background: Pancreatic cancer is one of the most aggressive human malignancies, with a very poor prognosis. To evaluate the effect of angiotensin II (Ang II) type 2 receptor (AT₂) expression in the host's body on the growth of pancreatic carcinoma, we have investigated the growth of mouse pancreatic ductal carcinoma grafts in syngeneic wild type and AT₂receptor-deficient (AT₂-KO) mice.Methods: The role of AT₂receptor-signaling in stromal cells on the growth of murine pancreatic carcinoma cells (PAN02) was studied using variousin vitroandin vivoassays.In vivocell proliferation, apoptosis, and vasculature in tumors were monitored by Ki-67 immunostaining, TUNEL assay, and von Willebrand factor immunostaining, respectively. In the co-culture study, cell proliferation was measured by MTT cell viability assay. All the data were analyzed using t-test and data were treated as significant whenp< 0.05.Results: Our results show that the growth of subcutaneously transplanted syngeneic xenografts of PAN02 cells, mouse pancreatic ductal carcinoma cells derived from the C57/BL6 strain, was significantly faster in AT₂-KO mice compared to control wild type mice. Immunohistochemical analysis of tumor tissue revealed significantly more Ki-67 positive cells in xenografts grown in AT₂-KO mice than in wild type mice. The index of apoptosis is slightly higher in wild type mice than in AT₂-KO mice as evaluated by TUNEL assay. Tumor vasculature number was significantly higher in AT₂-KO mice than in wild type mice.In vitroco-culture studies revealed that the growth of PAN02 cells was significantly decreased when grown with AT₂receptor gene transfected wild type and AT₂-KO mouse-derived fibroblasts. Faster tumor growth in AT₂-KO mice may be associated with higher VEGF production in stromal cells.Conclusions: These results suggest that Ang II regulates the growth of pancreatic carcinoma cells through modulating functions of host stromal cells; Moreover, Ang II AT₂receptor signaling is a negative regulator in the growth of pancreatic carcinoma cells. These findings indicate that the AT₂receptor in stromal fibroblasts is a potentially important target for chemotherapy for pancreatic cancer.

Although Salmonella enterica can produce life-threatening colitis in horses, certain serotypes are more commonly associated with clinical disease. Our aim was to evaluate the proportional morbidity attributed to different serotypes, as well as the phenotypic and genotypic antimicrobial resistance (AMR) of Salmonella isolates from patients at an equine referral hospital in the southern United States. A total of 255 Salmonella isolates was obtained from clinical samples of patients admitted to the hospital between 2007 and 2015. Phenotypic resistance to 14 antibiotics surveilled by the U.S. National Antimicrobial Resistance Monitoring System was determined using a commercially available panel. Whole-genome sequencing was used to identify serotypes and genotypic AMR. The most common serotypes were Salmonella enterica serotype Newport (18%), Salmonella enterica serotype Anatum (15.2%), and Salmonella enterica serotype Braenderup (11.8%). Most ( n = 219) of the isolates were pansusceptible, while 25 were multidrug resistant (≥3 antimicrobial classes). Genes encoding beta-lactam resistance, such as bla _CMY-2 , bla _SHV-12 , bla _CTX-M-27 , and bla _TEM-1B , were detected. The qnr B2 and aac(6′)-Ib-cr genes were present in isolates with reduced susceptibility to ciprofloxacin. Genes encoding resistance to gentamicin ( aph(3′)-Ia , aac(6′)-IIc ), streptomycin ( str A and str B), sulfonamides ( sul1 ), trimethoprim ( dfrA ), phenicols ( catA ), tetracyclines [ tet (A) and tet (E)], and macrolides [ ere (A)] were also identified. The main predicted incompatibility plasmid type was I1 (10%). Core genome-based analyses revealed phylogenetic associations between isolates of common serotypes. The presence of AMR Salmonella in equine patients increases the risk of unsuccessful treatment and causes concern for potential zoonotic transmission to attending veterinary personnel, animal caretakers, and horse owners. Understanding the epidemiology of Salmonella in horses admitted to referral hospitals is important for the prevention, control, and treatment of salmonellosis. IMPORTANCE In horses, salmonellosis is a leading cause of life-threatening colitis. At veterinary teaching hospitals, nosocomial outbreaks can increase the risk of zoonotic transmission, lead to restrictions on admissions, impact hospital reputation, and interrupt educational activities. The antimicrobials most often used in horses are included in the 5th revision of the World Health Organization's list of critically important antimicrobials for human medicine. Recent studies have demonstrated a trend of increasing bacterial resistance to drugs commonly used to treat Salmonella infections. In this study, we identify temporal trends in the distribution of Salmonella serotypes and their mechanisms of antimicrobial resistance; furthermore, we are able to determine the likely origin of several temporal clusters of infection by using whole-genome sequencing. These data can be used to focus strategies to better contain the dissemination and enhance the mitigation of Salmonella infections and to provide evidence-based policies and guidelines to steward antimicrobial use in veterinary medicine.

GPCR subtypes possess distinct functional and pharmacological profiles, and thus development of subtype-selective ligands has immense therapeutic potential. This is especially the case for the angiotensin receptor subtypes AT1R and AT2R, where a functional negative control has been described and AT2R activation highlighted as an important cancer drug target. We describe a strategy to fine-tune ligand selectivity for the AT2R/AT1R subtypes through electronic control of ligand aromatic-prolyl interactions. Through this strategy an AT2R high affinity (K_i = 3 nM) agonist analogue that exerted 18,000-fold higher selectivity for AT2R versus AT1R was obtained. We show that this compound is a negative regulator of AT1R signaling since it is able to inhibit MCF-7 breast carcinoma cellular proliferation in the low nanomolar range.