American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, Jan 14, 2015
Ischemia-reperfusion (IR)-induced lung injury is one of the major contributing factors of morbidi... more Ischemia-reperfusion (IR)-induced lung injury is one of the major contributing factors of morbidity and mortality after lung transplantation. To determine the IR-induced molecular changes in lung epithelial cells, we developed a cell-culture model that simulates lung preservation and transplantation. Six hours of cold ischemic time (CIT) and reperfusion elicited production of multiple inflammatory cytokines and chemokines and increased expression of endoplasmic reticulum (ER) proteins. Prolonged hypothermic condition (18 h CIT) reduced ER stress protein levels, and induced apoptosis and necrosis (via mechanisms related to mitochondrial permeability transition pore opening). Protein kinase C (PKCδ) was activated during CIT, and its downregulation via small interference (si) (in siRNA) RNA reduced IR-induced cytokine production and apoptotic cell death. δV1-1, a PKCδ peptide inhibitor, reduced translocation of PKCδ and p53 to the mitochondria after 18 h CIT, rescued ER stress protein ...
The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation, Jan 10, 2015
Ex vivo lung perfusion (EVLP) allows normothermic evaluation and treatment of donor lungs not cur... more Ex vivo lung perfusion (EVLP) allows normothermic evaluation and treatment of donor lungs not currently acceptable for transplant and improves organ use. Donor lungs undergo a period of cold preservation before (cold ischemic time [CIT]-1) and after (CIT-2) EVLP. We investigated the effect of an extended CIT-2 on lung function after transplantation. Explanted pig lungs, preserved in low-potassium dextran flush (Perfadex) at 4°C for 10 hours, were subjected to 6 hours of EVLP. They were subsequently allocated to 2 groups: short CIT-2 (CIT-2 = 2 hours; n = 5), and long CIT-2 (CIT-2 = 10 hours; n = 5). In a control group (n = 6), explanted lungs were placed in cold static preservation for 24 hours without EVLP. After the total preservation period, the left lung was transplanted in all groups. After 4 hours of reperfusion, oxygenation function, acute lung injury score, inflammatory markers, and cell death pathway markers were similar between short and long CIT-2 groups. Both EVLP groups...
This paper reports a novel intracellular peptide delivery strategy using gold nanoparticle–peptid... more This paper reports a novel intracellular peptide delivery strategy using gold nanoparticle–peptide hybridization for the treatment of acute lung injury.
Cell-penetrating peptides (CPPs) are commonly used as delivery vehicles for the introduction of a... more Cell-penetrating peptides (CPPs) are commonly used as delivery vehicles for the introduction of a variety of macromolecules into cells. Trans-activator of transcription (TAT) is the most commonly used CPP and, as a delivery vehicle, is assumed to be biologically inert. In this study, we pretreated human lung epithelial cells with TAT prior to stimulation with phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator. Surprisingly, TAT alone inhibited the production of multiple cytokines induced by PKC activation. Furthermore, PKC activation-induced IκBα degradation was partially reduced by TAT. Moreover, TAT treatment alone induced apoptosis in a dose-dependent manner, influenced expression of several B cell lymphoma 2 (Bcl-2) family members and increased caspase 3 cleavage at a high dose. These findings suggest that TAT as a delivery vehicle should be used cautiously, as it may affect the inflammatory response, as well as signals related to apoptosis.
Gene-attenuated replication-competent adenoviruses are emerging as a promising new modality for t... more Gene-attenuated replication-competent adenoviruses are emerging as a promising new modality for the treatment of cancer. For the aim of improving adenoviral vectors for cancer gene therapy, we have constructed genetically attenuated replicating adenoviruses (Ads) with different combinations of E1A & E1B mutant genes and investigated the possibility of enhanced oncolytic and replication effects of these engineered replication-competent Ads. We show here that cytolytic potency of each gene attenuated replicating adenoviruses differed significantly depending on the presence or deletion of E1B55 kDa and E1B19 kDa function. More specifically, among the constructed (Ad-DE1B19, Ad-DE1B55, Ad-DE1B19/55, and Ad-wt), E1B19 kDa-inactivated adenovirus (Ad-DE1B19) was the most potent against all tumor cells tested, inducing the largest-sized plaques and marked CPE. As an additional effort to increase cancer cell-selectivity of replicating adenovirus, we have generated eleven E1A-mutant Ads (Ad-mt#1 ∼#11) with deletion or substitution in retinoblastoma (Rb) binding sites of E1A. Of these viruses, Ad-mt#7 demonstrated significantly improved CPE and viral replication in a cancer cell-specific manner. To further increase the cancer cell-specific cell killing effect of Ad-mt#7 adenovirus, both E1B19 kDa and E1B55 kDa gene was deleted, resulting in an Ad-ΔB7 Ad. As assessed using CPE assay, MTT assay, and Ad fiber immunoblot analysis, Ad-ΔB7 exerted markedly enhanced cancer cell-specific killing effect as well as viral replication in comparison to either Ad-mt#7 or Ad-ΔE1B19/55. Furthermore, the growth of established human cervical carcinoma in nude mice was significantly suppressed by intratumoral injection of Ad-DB7. In summary, we have developed replication competent adenoviruses with significantly improved therapeutic profiles for cancer treatment.
American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, Jan 14, 2015
Ischemia-reperfusion (IR)-induced lung injury is one of the major contributing factors of morbidi... more Ischemia-reperfusion (IR)-induced lung injury is one of the major contributing factors of morbidity and mortality after lung transplantation. To determine the IR-induced molecular changes in lung epithelial cells, we developed a cell-culture model that simulates lung preservation and transplantation. Six hours of cold ischemic time (CIT) and reperfusion elicited production of multiple inflammatory cytokines and chemokines and increased expression of endoplasmic reticulum (ER) proteins. Prolonged hypothermic condition (18 h CIT) reduced ER stress protein levels, and induced apoptosis and necrosis (via mechanisms related to mitochondrial permeability transition pore opening). Protein kinase C (PKCδ) was activated during CIT, and its downregulation via small interference (si) (in siRNA) RNA reduced IR-induced cytokine production and apoptotic cell death. δV1-1, a PKCδ peptide inhibitor, reduced translocation of PKCδ and p53 to the mitochondria after 18 h CIT, rescued ER stress protein ...
The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation, Jan 10, 2015
Ex vivo lung perfusion (EVLP) allows normothermic evaluation and treatment of donor lungs not cur... more Ex vivo lung perfusion (EVLP) allows normothermic evaluation and treatment of donor lungs not currently acceptable for transplant and improves organ use. Donor lungs undergo a period of cold preservation before (cold ischemic time [CIT]-1) and after (CIT-2) EVLP. We investigated the effect of an extended CIT-2 on lung function after transplantation. Explanted pig lungs, preserved in low-potassium dextran flush (Perfadex) at 4°C for 10 hours, were subjected to 6 hours of EVLP. They were subsequently allocated to 2 groups: short CIT-2 (CIT-2 = 2 hours; n = 5), and long CIT-2 (CIT-2 = 10 hours; n = 5). In a control group (n = 6), explanted lungs were placed in cold static preservation for 24 hours without EVLP. After the total preservation period, the left lung was transplanted in all groups. After 4 hours of reperfusion, oxygenation function, acute lung injury score, inflammatory markers, and cell death pathway markers were similar between short and long CIT-2 groups. Both EVLP groups...
This paper reports a novel intracellular peptide delivery strategy using gold nanoparticle–peptid... more This paper reports a novel intracellular peptide delivery strategy using gold nanoparticle–peptide hybridization for the treatment of acute lung injury.
Cell-penetrating peptides (CPPs) are commonly used as delivery vehicles for the introduction of a... more Cell-penetrating peptides (CPPs) are commonly used as delivery vehicles for the introduction of a variety of macromolecules into cells. Trans-activator of transcription (TAT) is the most commonly used CPP and, as a delivery vehicle, is assumed to be biologically inert. In this study, we pretreated human lung epithelial cells with TAT prior to stimulation with phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator. Surprisingly, TAT alone inhibited the production of multiple cytokines induced by PKC activation. Furthermore, PKC activation-induced IκBα degradation was partially reduced by TAT. Moreover, TAT treatment alone induced apoptosis in a dose-dependent manner, influenced expression of several B cell lymphoma 2 (Bcl-2) family members and increased caspase 3 cleavage at a high dose. These findings suggest that TAT as a delivery vehicle should be used cautiously, as it may affect the inflammatory response, as well as signals related to apoptosis.
Gene-attenuated replication-competent adenoviruses are emerging as a promising new modality for t... more Gene-attenuated replication-competent adenoviruses are emerging as a promising new modality for the treatment of cancer. For the aim of improving adenoviral vectors for cancer gene therapy, we have constructed genetically attenuated replicating adenoviruses (Ads) with different combinations of E1A & E1B mutant genes and investigated the possibility of enhanced oncolytic and replication effects of these engineered replication-competent Ads. We show here that cytolytic potency of each gene attenuated replicating adenoviruses differed significantly depending on the presence or deletion of E1B55 kDa and E1B19 kDa function. More specifically, among the constructed (Ad-DE1B19, Ad-DE1B55, Ad-DE1B19/55, and Ad-wt), E1B19 kDa-inactivated adenovirus (Ad-DE1B19) was the most potent against all tumor cells tested, inducing the largest-sized plaques and marked CPE. As an additional effort to increase cancer cell-selectivity of replicating adenovirus, we have generated eleven E1A-mutant Ads (Ad-mt#1 ∼#11) with deletion or substitution in retinoblastoma (Rb) binding sites of E1A. Of these viruses, Ad-mt#7 demonstrated significantly improved CPE and viral replication in a cancer cell-specific manner. To further increase the cancer cell-specific cell killing effect of Ad-mt#7 adenovirus, both E1B19 kDa and E1B55 kDa gene was deleted, resulting in an Ad-ΔB7 Ad. As assessed using CPE assay, MTT assay, and Ad fiber immunoblot analysis, Ad-ΔB7 exerted markedly enhanced cancer cell-specific killing effect as well as viral replication in comparison to either Ad-mt#7 or Ad-ΔE1B19/55. Furthermore, the growth of established human cervical carcinoma in nude mice was significantly suppressed by intratumoral injection of Ad-DB7. In summary, we have developed replication competent adenoviruses with significantly improved therapeutic profiles for cancer treatment.
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