Injectable, Drug-Eluting Nanocrystals Prevent Fibrosis and Stricture Formation In Vivo.

BACKGROUND & AIMS: Tissue fibrosis results from uncontrolled healing responses leading to excessive mesenchymal cell activation and collagen and other extracellular matrix deposition. In the gastrointestinal tract, fibrosis leads to narrowing of the lumen and stricture formation. A drug treatment to prevent fibrosis and strictures in the gastrointestinal tract would be transformational for patient care. We aimed to develop a stricture treatment with the following characteristics and components: a small molecule with strong antifibrotic effects that is delivered locally at the site of the stricture to ensure correct lesional targeting while protecting the systemic circulation, and that is formulated with sustained-release properties to act throughout the wound healing processes. METHODS: A high-throughput drug screening was performed to identify small molecules with antifibrotic properties. Next, we formulated an antifibrotic small molecule for sustained release and tested its antifibrotic potential in 3 animal models of fibrosis. RESULTS: Sulconazole, a US Food and Drug Administration-approved drug for fungal infections, was found to have strong antifibrotic properties. Sulconazole was formulated as sulconazole nanocrystals for sustained release. We found that sulconazole nanocrystals provided superior or equivalent fibrosis prevention with less frequent dosing in mouse models of skin and intestinal tissue fibrosis. In a patient-like swine model of bowel stricture, a single injection of sulconazole nanocrystals prevented stricture formation. CONCLUSIONS: The current data lay the foundation for further studies to improve the management of a range of diseases and conditions characterized by tissue fibrosis.

Use of Biologics for the Treatment of Inflammatory Conditions of the Pouch: A Systematic Review.

BACKGROUND: Patients with medically-refractory ulcerative colitis or advanced neoplasia are often offered an ileal-pouch-anal anastomosis to restore bowel continuity. However, up to 50% of patients can suffer from inflammatory conditions of the pouch, some of which require biological therapy to treat. The aim of this study was to determine the efficacy of each biological agent for the treatment of inflammatory conditions of the pouch. MATERIALS AND METHODS: A comprehensive literature search was performed in the major databases from inception through February 11, 2020, for studies assessing the efficacy of biologics in chronic antibiotic-refractory pouchitis (CARP) and Crohn's disease (CD) of the pouch. Both prospective and retrospective studies were included. The primary outcomes of interest were complete and partial responses were defined within each study. χ 2 test was used to compare variables. RESULTS: Thirty-four studies were included in the systematic review and meta-analysis. Sixteen studies (N=247) evaluated the use of infliximab (IFX), showing complete response in 50.7% and partial response in 28.1% for CARP, and complete response in 66.7% and partial response in 20% for CD of the pouch. Seven studies (n=107) assessed the efficacy of adalimumab. For CARP, 33.3% of patients had a complete response, and 38.1% had a partial response, whereas for CD of the pouch, 47.7% experienced a complete response, and 24.6% had a partial response. Three studies (n=78) reported outcomes with the use of ustekinumab, showing 50% complete response and 3.8% partial response for CARP. For the CD of the pouch, 5.8% had a complete response and 78.8% had a partial response. Seven studies (n=151) reported the efficacy of vedolizumab, showing 28.4% complete response and 43.2% partial response in patients with CARP, whereas 63% of patients experienced partial response in CD of the pouch. IFX had higher rates of complete response in CARP compared with adalimumab ( P =0.04) and compared with vedolizumab ( P =0.005), but not compared with ustekinumab ( P =0.95). There were no new safety signals reported in any of the studies. CONCLUSIONS: Biologics are safe and efficacious in the treatment of chronic, refractory inflammatory conditions of the pouch. IFX seems to be more efficacious than adalimumab and vedolizumab for CARP. Further prospective, head-to-head evaluations are needed to compare biological therapies in the treatment of CARP and CD of the pouch.

Quaternary nanoparticles enable sustained release of bortezomib for hepatocellular carcinoma.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) represents the third leading cause of cancer-related mortality in the world. Over the past two decades, there has been minimal improvement in therapies as well as clinical outcomes for patients with Barcelona Clinic Liver Cancer (BCLC)-B. These patients are treated with local interventions, including transarterial chemoembolization. Current methodologies only allow sustained intratumoral release measured in hours. Methodologies to allow sustained local release of the drug cargo over days to weeks are acutely needed. We hypothesize that tumor response as well as outcomes of patients with BCLC-B can be improved through utilization of a highly cytotoxic agent delivered with a sustained release platform. APPROACH AND RESULTS: High-throughput drug screening across 40 HCC patient-derived organoids identified bortezomib (BTZ) as a highly cytotoxic small molecule for HCC. We designed and manufactured sustained release BTZ nanoparticles (BTZ-NP) using a flash nanocomplexation/nanoprecipitation process. We quantified the release profile and tested the anti-tumoral effects in vivo. The BTZ-NP formulation demonstrated a sustained release of BTZ of 30 days. This BTZ-NP formulation was highly effective in controlling tumor size and improved survival in vivo in three animal models of HCC, including when delivered via the hepatic artery, as we envision its delivery in patients. In addition, the BTZ-NP formulation was superior to treatment with doxorubicin-drug eluting beads. CONCLUSIONS: The BTZ-NP formulation provides a potent and safe treatment of HCC via a localized delivery approach. These results warrant additional preclinical studies to advance this technology to human clinical trials.

Initial Clinical Outcomes Using Umbilical Cord-Derived Tissue Grafts to Repair Anovaginal Fistula.

BACKGROUND: Perianal fistula is a debilitating disease and challenging condition to treat. Recently, the use of stem cells has been shown to improve healing of fistulas. OBJECTIVE: The aim was to examine the use of an umbilical cord-derived stem cell graft in a pilot study as a novel scaffold/stem inlay implanted into fistula repairs for anovaginal fistula to examine healing rates. DESIGN: This was a pilot study. SETTINGS: This study took place in a colorectal surgery practice. PATIENTS: Patients with anovaginal fistula consented to participate. Cryopreserved umbilical cord tissue graft with viable cells was incorporated as an inlay using a previously reported technique by the authors. Demographic data including history of previous repairs and IBD were included. All patients were followed for a minimum of 6 weeks. MAIN OUTCOME MEASURES: The primary measures were safety and efficacy of novel stem cell graft in the treatment of anovaginal fistula. RESULTS: From September 2017 to September 2019, 15 patients underwent anovaginal fistula repair. Three of these patients underwent a second repair, for a total of 18 repairs. No patient was intentionally diverted, but 3 patients presented for repair with a preexisting stoma. The majority of repairs were previous repair failures (12; 67%), and 7 repairs were performed on 5 patients with IBD. Median follow-up was 30 (6-104) weeks. The safety profile for cryopreserved umbilical cord tissue graft was excellent as no adverse events occurred. Overall complete healing rate was 39%, and 12 (67%) repairs resulted in improvement of symptoms. LIMITATIONS: This was a small pilot study. CONCLUSIONS: This is the largest series using cryopreserved umbilical cord graft for anovaginal fistula repair. The use of umbilical cord was safe and effective at closing defects. Randomized studies are necessary to determine added benefits over current standard of care. See Video Abstract at http://links.lww.com/DCR/B896 . RESULTADOS CLNICOS INICIALES DEL USO DE INJERTOS DE TEJIDO DERIVADO DE PLACENTA PARA REPARACIN DE FSTULAS ANOVAGINALES: ANTECEDENTES:La fístula perianal es una enfermedad debilitante y una afección difícil de tratar. Recientemente, se ha demostrado que el uso de células madre mejora la curación de las fístulas.OBJETIVO:Deseamos examinar el uso de un injerto de células madre derivadas de cordón umbilical en un estudio piloto como una nueva matriz/injerto de células madre implantado en reparaciones de fístula para fístula anovaginal para examinar las tasas de curación.DISEÑO:Este fue un estudio piloto.ESCENARIO:Este estudio se llevó a cabo en una clínica de cirugía colorrectal.PACIENTES:Se obtuvo consentimiento informado de pacientes con fístula anovaginal. El injerto de tejido de cordón umbilical criopreservado con células viables se incorporó como incrustación utilizando una técnica previamente informada por los autores. Se incluyeron datos demográficos que incluían antecedentes de reparaciones previas y enfermedad inflamatoria intestinal. Todos los pacientes fueron seguidos durante un mínimo de 6 semanas.PRINCIPALES MEDIDAS DE RESULTADO:Las principales medidas fueron la seguridad y la eficacia del nuevo injerto de células madre en el tratamiento de la fístula anovaginal.RESULTADOS:Desde 9/2017-9/2019, 15 pacientes fueron sometidas a reparación de fístula anovaginal. Tres de estos pacientes fueron sometidos a una segunda reparación, para un total de 18 reparaciones. Ningún paciente fue derivado intencionalmente mientras que 3 pacientes se presentaron para reparación con un estoma preexistente. La mayoría de las reparaciones fueron fallas de reparaciones previas (12, 67%) y se realizaron siete reparaciones en 5 pacientes con enfermedad inflamatoria intestinal (EII). La mediana de seguimiento fue de 30 semanas (6-104). El perfil de seguridad del injerto de tejido de cordón umbilical criopreservado fue excelente ya que no se produjeron efectos adversos. La tasa general de curación completa fue del 39% y 12 (67%) reparaciones dieron como resultado una mejoría de los síntomas.LIMITACIONES:Este fue un pequeño estudio piloto.CONCLUSIÓNES:Ésta es la serie más grande de utilización de injerto de cordón umbilical criopreservado para la reparación de una fístula anovaginal. La utilización del cordón umbilical resultó segura y eficaz para cerrar defectos. Se necesitan estudios aleatorizados para determinar los beneficios adicionales sobre el estándar de atención actual. Consulte Video Resumen en http://links.lww.com/DCR/B896 . (Traducción-Dr. Jorge Silva Velazco ).

A Novel Patient-like Swine Model of Perianal Crohn's Disease.

BACKGROUND: Perianal Crohn's disease is associated with poor outcomes and high medical costs. It is notoriously difficult to treat despite therapeutic advancements for luminal disease. A large animal model that mimics human perianal disease is needed to test innovative therapies. OBJECTIVE: This study aimed to create a swine model that replicates the inflammatory component and therapeutic challenges found in patients with perianal Crohn's disease. DESIGN: This was an animal preclinical study. SETTINGS: The experiments were performed at the animal laboratory at the Johns Hopkins University. PATIENTS: Four sus scrufus female pigs were included in the study. INTERVENTIONS: Four female pigs underwent creation of 3 surgical perianal fistulas each, 1 rectovaginal and 2 perianal. Size 24 French setons were placed to maintain patency of the fistula tracts for 4 weeks. After removal of the setons, trinitrobenzene sulfonic acid was administered into the fistula tract to create and maintain local inflammation mimicking perianal Crohn's disease. MAIN OUTCOMES MEASURES: An MRI was obtained to assess the fistulas and the pigs were euthanized to review histopathology. RESULTS: Three inflammatory chronic fistula tracts were successfully created in each pig as confirmed by MRI and examination under anesthesia. This is the first report of maintaining patent fistulas in swine 2 weeks after removal of setons. For the first time, we reported that 2 pigs developed branching fistulas and small abscesses reminiscent of human perianal Crohn's disease. The corresponding histopathologic examination found significant chronic active inflammation on standard hematoxylin and eosin staining. LIMITATIONS: The fistulas were surgically induced and did not occur naturally. CONCLUSIONS: A chronic perianal fistula model in pigs that strongly resembles human perianal Crohn's disease was successfully created. This model can be used to test novel therapeutics and techniques to pave the path for human trials. See Video Abstract at http://links.lww.com/DCR/B969 . UN NUEVO MODELO PORCINO SIMILAR A UN PACIENTE DE LA ENFERMEDAD DE CROHN PERIANAL ANTECEDENTES: La enfermedad de Crohn perianal se asocia con malos resultados y altos costos médicos. Es notoriamente difícil de tratar a pesar de los avances terapéuticos para la enfermedad luminal. Se precisa de un modelo animal grande que imite la enfermedad perianal humana para probar terapias innovadoras.OBJETIVO:Nuestro objetivo de este estudio fue crear un modelo porcino que replique el componente inflamatorio y los desafíos terapéuticos que se encuentran en los pacientes con enfermedad de Crohn perianal.DISEÑO:Este fue un estudio preclínico en animales.AJUSTES:Los experimentos se realizaron en el laboratorio de animales de la Universidad Johns Hopkins.PACIENTES:Se incluyeron en el estudio cuatro cerdas sus scrofa.INTERVENCIONES:Cuatro cerdas fueron sometidas a la creación de 3 fístulas perianales quirúrgicas cada una: 1 recto vaginal y 2 perianales. Se colocaron sedales de 24 French para mantener la permeabilidad de los trayectos fistulosos durante 4 semanas. Tras el retiro de los sedales, se administró ácido trinitrobenceno sulfónico en el trayecto de la fístula para crear y mantener la inflamación local simulando la enfermedad de Crohn perianal.PRINCIPALES MEDIDAS DE RESULTADOS:Se obtuvo una resonancia magnética para evaluar las fístulas y los cerdos fueron sacrificados para revisar la histopatología.RESULTADOS:Se crearon de manera exitosa tres trayectos fistulosos inflamatorios crónicos en cada cerdo, confirmados por imágenes de resonancia magnética y examen bajo anestesia. Este es el primer informe de preservación de fístulas permeables en cerdos 2 semanas tras el retiro de los setones. Por primera vez, informamos que dos cerdos desarrollaron fístulas ramificadas y pequeños abscesos que recuerdan a la enfermedad de Crohn perianal humana. El examen histopatológico correspondiente encontró una significativa inflamación crónica activa en la tinción estándar de hematoxilina y eosina.LIMITACIONES:Las fístulas se indujeron quirúrgicamente y no se produjeron de forma natural.CONCLUSIONES:Se logro recrear con éxito un modelo de fístula perianal crónica en cerdos que se asemeja mucho a la enfermedad de Crohn perianal humana. Este modelo se puede utilizar para probar nuevas terapias y técnicas para allanar el camino para los ensayos en humanos. Consulte Video Resumen en http://links.lww.com/DCR/B969 . (Traducción-Dr Osvaldo Gauto).

Extracellular Vesicles in Hepatocellular Carcinoma: Progress and Challenges in the Translation from the Laboratory to Clinic.

Extracellular vesicles (EVs) play critical roles in intercellular communication by transporting bioactive cargo to recipient cells. EVs have been implicated in a range of physiological and pathological processes, including tumor progression, metastasis, immune modulation, and drug resistance. The objective of this review is to present a thorough overview of recent studies focusing on EVs in hepatocellular carcinoma (HCC), with an emphasis on their potential utility as diagnostic biomarkers as well as therapeutic agents. Initially, we explore the utility of EVs as diagnostic biomarkers for HCC, followed by a discussion of their potential as carriers of therapeutic payloads. Additionally, we delve into the emerging field of therapeutic EVs for modulating tumor immune responses. Through this review, our ultimate aim is to provide a comprehensive understanding of the opportunities and challenges in the clinical translation of EV research in the domain of HCC.

Antibody Kinetics after Three Doses of SARS-CoV-2 mRNA Vaccination in Patients with Inflammatory Bowel Disease.

Background: The emergence of new SARS-CoV-2 variants calls for more data on SARS-CoV-2 mRNA vaccine response. Aims: We aimed to assess the response to a third mRNA vaccine dose against SARS-CoV-2 in inflammatory bowel disease (IBD) patients. Methods: This was a single-center, observational prospective study of IBD patients who received a third mRNA vaccine dose against SARS-CoV-2. Antibody titers were taken post-third-dose at one and three months using the Roche Elecsys anti-SARS-CoV-2-S enzyme immunoassay. Titers less than 0.8 units/mL were considered negative according to the manufactures. Titers between 0.8 units/mL and 250 units/mL were considered non-neutralizing. Titers greater than 250 units/mL were considered neutralizing. Results: Eighty-three patients were included, all of whom had detectable antibodies at 3 months post-third dose. A total of 89% showed neutralizing and 11% non-neutralizing titers. Participants with non-neutralizing titers were more likely to be on systemic corticosteroids (p = 0.04). Two participants seroconverted from negative to positive, whereas 86% with non-neutralizing titers boosted to neutralizing levels. Only one participant with neutralizing titers after a third dose had a decrease to a non-neutralizing level within 3 months. Conclusions: Our findings support the ongoing recommendations for additional doses in immunocompromised individuals. However, longitudinal studies with a greater-sized patient population are needed.

Antibody Response 3 Months After 2-Dose SARS-CoV-2 mRNA Vaccination in Patients With Inflammatory Bowel Disease.

INTRODUCTION: The response to SARS-CoV-2 vaccination of patients with inflammatory bowel disease (IBD) on immune-modifying therapies requires further investigation because previous studies indicate that patients on immune therapy might have decreased antibody concentrations. METHODS: We present the antireceptor binding domain antibody response over a period of 3 months in 217 patients with IBD who completed standard 2-dose SARS-CoV-2 mRNA vaccine series. RESULTS: Almost all (98.6%) IBD vaccine recipients had a positive antireceptor binding domain antibody response at least 3 months after vaccination. Decreased antibody titers at 3 months were seen in a subset of patients on antitumor necrosis factor-alpha. Approximately 10% of the participants with high-titer antibodies at 1 month had a decrease to low-positive titers at 3 months, which was mostly observed in those on combination therapy and antitumor necrosis factor-alpha monotherapy. DISCUSSION: Larger longitudinal studies are required to define the response in IBD population and its clinical impact.

Biostimulatory Micro-Fragmented Nanofiber-Hydrogel Composite Improves Mesenchymal Stem Cell Delivery and Soft Tissue Remodeling.

Functional microgels are preferred stem cell carriers due to the ease of delivery through minimally invasive injection and seamless integration with the surrounding host tissue. A biostimulatory nanofiber-hydrogel composite (NHC) has been previously developed through covalently crosslinking a hyaluronic acid hydrogel network with surface-functionalized poly (ε-caprolactone) nanofiber fragments. The NHC mimics the microarchitecture of native soft tissue matrix, showing enhanced cell infiltration, immunomodulation, and proangiogenic properties. Here, injectability of the pre-formed NHC is improved by mechanical fragmentation, making it into micro-fragmented NHC (mfNHC) in a granular gel form as a stem cell carrier to deliver mesenchymal stem cells (MSCs) for soft tissue remodeling. The mfNHC shows a similar storage modulus but a significantly reduced injection force, as compared with the corresponding bulk NHC. When injected subcutaneously in a rat model, mfNHC-MSC constructs initiate an elevated level of host macrophage infiltration, more pro-regenerative polarization, and subsequently, improved angiogenesis and adipogenesis response when compared to mfNHC alone. A similar trend of host cell infiltration and pro-angiogenic response is detected in a swine model with a larger volume injection. These results suggest a strong potential for use of the mfNHC as an injectable carrier for cell delivery and soft tissue remodeling.

Endoscopic-mediated, biliary hydrodynamic injection mediating clinically relevant levels of gene delivery in pig liver.

BACKGROUND AND AIMS: Gene therapy could provide curative therapies to many inherited monogenic liver diseases. Clinical trials have largely focused on adeno-associated viruses (AAVs) for liver gene delivery. These vectors, however, are limited by small packaging size, capsid immune responses, and inability to redose. As an alternative, nonviral, hydrodynamic injection through vascular routes can successfully deliver plasmid DNA (pDNA) into mouse liver but has achieved limited success in large animal models. METHODS: We explored hydrodynamic delivery of pDNA through the biliary system into the liver of pigs using ERCP and a power injector to supply hydrodynamic force. Human factor IX (hFIX), deficient in hemophilia B, was used as a model gene therapy. RESULTS: Biliary hydrodynamic injection was well tolerated without significant changes in vital signs, liver enzymes, hematology, or histology. No off-target pDNA delivery to other organs was detected by polymerase chain reaction. Immunohistochemistry revealed that 50.19% of the liver stained positive for hFIX after hydrodynamic injection at 5.5 mg pDNA, with every hepatic lobule in all liver lobes demonstrating hFIX expression. hFIX-positive hepatocytes were concentrated around the central vein, radiating outward across all 3 metabolic zones. Biliary hydrodynamic injection in pigs resulted in significantly higher transfection efficiency than mouse vascular hydrodynamic injection at matched pDNA per liver weight dose (32.7%-51.9% vs 18.9%, P < .0001). CONCLUSIONS: Biliary hydrodynamic injection using ERCP can achieve higher transfection efficiency into hepatocytes compared with AAVs at magnitudes of less cost in a clinically relevant human-sized large animal. This technology may serve as a platform for gene therapy of human liver diseases.

Intrahepatic Cholangiocarcinoma: Continuing Challenges and Translational Advances.

Intrahepatic cholangiocarcinoma (iCCA) has over the last 10-20 years become the focus of increasing concern, largely due to its rising incidence and high mortality rates worldwide. The significant increase in mortality rates from this primary hepatobiliary cancer, particularly over the past decade, has coincided with a rapidly growing interest among clinicians, investigators, and patient advocates to seek greater mechanistic insights and more effective biomarker-driven targeted approaches for managing and/or preventing this challenging liver cancer. In addition to discussing challenges posed by this aggressive cancer, this review will emphasize recent epidemiological, basic, and translational research findings for iCCA. In particular, we will highlight emerging demographic changes and evolving risk factors, the critical role of the tumor microenvironment, extracellular vesicle biomarkers and therapeutics, intertumoral and intratumoral heterogeneity, and current and emerging targeted therapies regarding iCCA. Specifically, recent evidence linking non-bile duct medical conditions, such as nonalcoholic fatty liver disease and nonspecific cirrhosis, to intrahepatic cholangiocarcinogenesis together with geographic and ethnic variation will be assessed. Recent developments concerning the roles played by transforming growth factor-ß and platelet-derived growth factor D in driving the recruitment and expansion of cancer-associated myofibroblasts within cholangiocarcinoma (CCA) stroma as well as their therapeutic implications will also be discussed. In addition, the potential significance of extracellular vesicles as bile and serum biomarkers and therapeutic delivery systems for iCCA will be described. An integrated systems approach to classifying heterogeneous iCCA subtypes will be further highlighted, and recent clinical trials and emerging targeted therapies will be reviewed, along with recommendations for future translational research opportunities. Established international CCA networks are now facilitating collaborations aimed at advancing iCCA translational and clinical research.

Exosome miR-335 as a novel therapeutic strategy in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common and deadly cancer. Most cases of HCC arise in a cirrhotic/fibrotic liver, indicating that environment may play a paramount role in cancer genesis. Previous studies from our group and others have shown that, in desmoplastic cancers, there is a rich intercellular communication between activated, cancer-associated fibroblasts and cancer cells. Moreover, extracellular vesicles (EVs), or exosomes, have been identified as an important arm of this intercellular communication platform. Finally, these studies have shown that EVs can carry microRNA (miR) species in vivo and deliver them to desmoplastic cancers. The precise role played by activated liver fibroblasts/stellate cells in HCC development is insufficiently known. Based on previous studies, it appears plausible that activated fibroblasts produce signals carried by EVs that promote HCC genesis. In the current study, we first hypothesized and then demonstrated that stellate cell-derived EVs 1) can be loaded with an miR species of choice (miR-335-5p); 2) are taken up by HCC cells in vitro and more importantly in vivo; 3) can supply the miR-335-5p cargo to recipient HCC cells in vitro as well as in vivo; and 4) inhibit HCC cell proliferation and invasion in vitro as well as induce HCC tumor shrinkage in vivo. Finally, we identified messenger RNA targets for miR-335 that are down-regulated after treatment with EV-miR-335-5p. This study informs potential therapeutic strategies in HCC, whereby stellate cell-derived EVs are loaded with therapeutic nucleic acids and delivered in vivo. (Hepatology 2018;67:940-954).

Extracellular vesicles carry microRNA-195 to intrahepatic cholangiocarcinoma and improve survival in a rat model.

The cancer microenvironment plays a central role in cancer development, growth, and homeostasis. This paradigm suggests that cancer fibroblasts support cancers, probably in response to stimuli received from the cancer cells. We aimed at investigating whether extracellular vesicles (EVs) can shuttle microRNA (miR) species between cancer-associated fibroblasts (CAFs) and cancer cells. To this end, we extracted EVs according to published protocols. EVs were studied for their miR content by quantitative reverse-transcription polymerase chain reaction. EVs were transfected with select miR species and utilized in vitro as well as in vivo in a rat model of cholangiocarcinoma (CCA). We found that miR-195 is down-regulated in CCA cells, as well as in adjoining fibroblasts. Furthermore, we report that EVs shuttle miR-195 from fibroblasts to cancer cells. Last, we show that fibroblast-derived EVs, loaded with miR-195, can be administered in a rat model of CCA, concentrate within the tumor, decrease the size of cancers, and improve survival of treated rats. CONCLUSION: EVs play a salient role in trafficking miR species between cancer cells and CAFs in human CCA. Understanding of these mechanisms may allow devising of novel therapeutics. (Hepatology 2017;65:501-514).

Successful liver-directed gene delivery by ERCP-guided hydrodynamic injection (with videos).

BACKGROUND AND AIMS: A simple, safe, targeted, and efficient in vivo DNA delivery system is necessary for clinical-grade liver-targeted gene therapy in humans. Intravascular hydrodynamic gene delivery has been investigated in large animal models, but translation to humans has been hampered by its technical challenges, invasiveness, and potential for significant cardiovascular adverse events. We posited that intrabiliary delivery of DNA plasmids via ERCP-guided hydrodynamic injection could overcome these obstacles. METHODS: Twelve pigs (40-50 kg) were divided into 3 groups (4 per group) and survived 21, 30, or 60 days. ERCP was performed by inflating a balloon catheter in the common hepatic duct and creating a closed space between it and the liver parenchyma. Last, a solution composed of plasmid/sleeping beauty (SB) mix was injected under pressure through the catheter into the closed space. Swine were killed at the 3 different time points and liver tissue harvested. Plasmid DNA expression and functional translated protein expression were assessed. RESULTS: ERCP-guided hydrodynamic delivery of naked plasmid DNA facilitated by pCytomegalovirus-Sleep Beauty (pCMV-SB) transposons was technically feasible and devoid of cardiovascular and local adverse events in all 12 pigs. Furthermore, plasmid DNA (both single and combination) was successfully transferred into swine hepatocytes in all 12 pigs. Additionally, stable integration of the DNA constructs in hepatocyte genomic DNA was reliably noted at all 3 time points. In the 4 swine that were kept alive to 60 days, successful genomic integration and subsequent protein expression was observed in the targeted liver tissue. CONCLUSIONS: ERCP-guided hydrodynamic delivery of gene therapy may usher in the next chapter in gene therapy with the potential to impact a variety of single-gene, complex genetic, and epigenetic liver diseases. It also raises the possibility that other nucleic acid therapeutics (microRNA, lncRNA, siRNA, shRNA) could similarly be delivered.

Whole-Exome Sequencing Analyses of Inflammatory Bowel Disease-Associated Colorectal Cancers.

BACKGROUND & AIMS: A long duration of inflammatory bowel disease (IBD) increases the risk for colorectal cancer. Mutation analysis of limited numbers of genes has indicated that colorectal tumors that develop in patients with IBD differ from those of patients without IBD. We performed whole-exome sequencing analyses to characterize the genetic landscape of these tumors. METHODS: We collected colorectal tumor and non-neoplastic tissues from 31 patients with IBD and colorectal cancer (15 with ulcerative colitis, 14 with Crohn's disease, and 2 with indeterminate colitis) and performed whole-exome sequencing analyses of the microdissected tumor and matched nontumor tissues. We identified somatic alterations by comparing matched specimens. The prevalence of mutations in sporadic colorectal tumors was obtained from previously published exome-sequencing studies. RESULTS: Two specimens had somatic mutations in the DNA proofreading or mismatch repair genes POLE, MLH1, and MSH6 and the tumor cells had a hypermutable phenotype. The remaining tumors had, on average, 71 alterations per sample. TP53 was the most commonly mutated gene, with prevalence similar to that of sporadic colorectal tumors (63% of cases). However, tumors from the patients with IBD had a different mutation spectrum. APC and KRAS were mutated at significantly lower rates in tumors from patients with IBD than in sporadic colorectal tumors (13% and 20% of cases, respectively). Several genes were mutated more frequently or uniquely in tumors from patients with IBD, including SOX9 and EP300 (which encode proteins in the WNT pathway), NRG1 (which encodes an ERBB ligand), and IL16 (which encodes a cytokine). Our study also revealed recurrent mutations in components of the Rho and Rac GTPase network, indicating a role for noncanonical WNT signaling in development of colorectal tumors in patients with IBD. CONCLUSIONS: Colorectal tumors that develop in patients with IBD have distinct genetic features from sporadic colorectal tumors. These findings could be used to develop disease-specific markers for diagnosis and treatment of patients with IBD and colorectal cancer.

MicroRNAs in the biology and diagnosis of cholangiocarcinoma.

Ever since their discovery, microRNAs (miRNAs) have been the subject of intense investigation of their roles in cells and tissues, both normal and disease state. Although some of the precise mechanisms of biogenesis and actions of miRNAs remain debatable, the fact that miRNAs are dysregulated in diseases such as cancer is undisputed. For many miRNA species, computational databases predict often numerous targets; however, experimental verification in vitro and in vivo is still lacking. For some miRNAs, species-specific targets have been validated; nevertheless, the precise mechanisms those targets act in and whether they are the only truly important ones remain to be discovered. The authors take a closer look at the current status of the role of miRNAs in the diagnosis and biology of cholangiocarcinomas where the perhaps biggest impact in the short term comes from the use of biomarkers in the early diagnosis.

Human bile contains microRNA-laden extracellular vesicles that can be used for cholangiocarcinoma diagnosis.

UNLABELLED: Cholangiocarcinoma (CCA) presents significant diagnostic challenges, resulting in late patient diagnosis and poor survival rates. Primary sclerosing cholangitis (PSC) patients pose a particularly difficult clinical dilemma because they harbor chronic biliary strictures that are difficult to distinguish from CCA. MicroRNAs (miRs) have recently emerged as a valuable class of diagnostic markers; however, thus far, neither extracellular vesicles (EVs) nor miRs within EVs have been investigated in human bile. We aimed to comprehensively characterize human biliary EVs, including their miR content. We have established the presence of extracellular vesicles in human bile. In addition, we have demonstrated that human biliary EVs contain abundant miR species, which are stable and therefore amenable to the development of disease marker panels. Furthermore, we have characterized the protein content, size, numbers, and size distribution of human biliary EVs. Utilizing multivariate organization of combinatorial alterations (MOCA), we defined a novel biliary vesicle miR-based panel for CCA diagnosis that demonstrated a sensitivity of 67% and specificity of 96%. Importantly, our control group contained 13 PSC patients, 16 with biliary obstruction of varying etiologies (including benign biliary stricture, papillary stenosis, choledocholithiasis, extrinsic compression from pancreatic cysts, and cholangitis), and 3 with bile leak syndromes. Clinically, these types of patients present with a biliary obstructive clinical picture that could be confused with CCA. CONCLUSION: These findings establish the importance of using extracellular vesicles, rather than whole bile, for developing miR-based disease markers in bile. Finally, we report on the development of a novel bile-based CCA diagnostic panel that is stable, reproducible, and has potential clinical utility.

Stimuli-responsive theragrippers for chemomechanical controlled release.

We report on a therapeutic approach using thermo-responsive multi-fingered drug eluting devices. These therapeutic grippers referred to as theragrippers are shaped using photolithographic patterning and are composed of rigid poly(propylene fumarate) segments and stimuli-responsive poly(N-isopropylacrylamide-co-acrylic acid) hinges. They close above 32 °C allowing them to spontaneously grip onto tissue when introduced from a cold state into the body. Due to porosity in the grippers, theragrippers could also be loaded with fluorescent dyes and commercial drugs such as mesalamine and doxorubicin, which eluted from the grippers for up to seven days with first order release kinetics. In an in vitro model, theragrippers enhanced delivery of doxorubicin as compared to a control patch. We also released theragrippers into a live pig and visualized release of dye in the stomach. The design of such tissue gripping drug delivery devices offers an effective strategy for sustained release of drugs with immediate applicability in the gastrointestinal tract.