ABSTRACT
Biolistic transmission of mRNA provides transient gene therapy to in vivo organs. This study documents particle mediated mRNA transmission to a solid organ and wound healing model using the mRNA of Green Fluorescent Protein to determine optimal delivery parameters. Renal function, bullet penetration, cellular injury, and Green Fluorescent Protein synthesis were quantified. Chimeric human epidermal growth factor-FLAG epitope cDNA or mRNA was transmitted to wounds in normal or steroid treated animals. Wound bursting strength, human epidermal growth factor-FLAG, and collagen synthesis were determined. Injury and bullet penetration correlated with the delivery velocity and bullet size. Optimal delivery parameters were established which provided widespread Green Fluorescent Protein synthesis. Human epidermal growth factor-FLAG treatment significantly increased collagen content and wound breaking strength in normal and steroid treated animals. FLAG protein synthesis was evident in mRNA treated fascia following treatment. We found the gene gun provides a novel method for efficient, in vivo delivery of mRNA-based therapeutic strategies to mammalian organs with minimal histologic damage allowing transient expression of protein in in vivo target tissues. Co-delivery of Green Fluorescent Protein mRNA may provide a useful positive control to determine effective transmission. Biolistic transmission of human epidermal growth factor-FLAG mRNA provides increased tissue epidermal growth factor levels and accelerates wound healing in normal and steroid exposed animals.
Subject(s)
Genetic Therapy/methods , Kidney/physiology , Luminescent Proteins , RNA, Messenger/pharmacology , Wounds and Injuries/therapy , Animals , Biological Availability , Disease Models, Animal , Drug Delivery Systems , Epidermal Growth Factor/pharmacology , Gene Transfer Techniques , Green Fluorescent Proteins , Humans , Immunohistochemistry , Male , Rats , Rats, Sprague-Dawley , Regeneration , Sensitivity and Specificity , Wound Healing/physiology , Wounds and Injuries/genetics , Wounds and Injuries/pathologyABSTRACT
BACKGROUND: Acute lung injury is common after shock and sepsis, but the pathophysiology is unclear. Lipid hydroperoxide products including 4-hydroxynonenal (HNE) increase significantly during these insults and may induce apoptosis. This study investigates the role of pathophysiologic concentrations of HNE on isolated lung biophysical function and apoptosis. METHODS: Male Sprague-Dawley rat lungs were isolated and perfused with Krebs-Henseleit buffered solution for 120 minutes. Hydroxynonenal (50 micromol/L) or vehicle was added to the perfusate at 60 minutes. Lung elastance and perfusion pressure were determined. Perfusate glutathione and lactate dehydrogenase were determined at 30-minute intervals. Genomic DNA was extracted for electrophoretic determination of apoptotic laddering. RESULTS: There were no differences in any parameter measured before HNE infusion. Lung edema increased significantly with HNE infusion; a trend increase in lung elastance and perfusion pressure was noted. DNA laddering characteristic of apoptosis was noted in HNE-treated lungs that was absent in control animals. CONCLUSION: Lipid hydroperoxide products formed during shock or sepsis may be causally related to lung injury. Low concentrations of a candidate metabolite, HNE, appear to induce significant lung injury and apoptosis, which may partially mediate lung injury during shock and sepsis.
Subject(s)
Aldehydes/pharmacology , Apoptosis/drug effects , Cysteine Proteinase Inhibitors/pharmacology , Respiratory Distress Syndrome/etiology , Animals , Lung/cytology , Lung/pathology , Male , Organ Culture Techniques , Rats , Rats, Sprague-Dawley , Respiratory Distress Syndrome/pathology , Respiratory Distress Syndrome/physiopathologyABSTRACT
Apoptosis is well described in invertebrates and recently documented in mammals. The prevalence and pathophysiology of mammalian apoptosis is unknown and may have clinical ramifications. The aim of this study is to investigate the apoptotic response during kidney ischemia-reperfusion (I/R) injury. Kidney I/R was initiated in anesthetized rats by occlusion of the renal pedicle for 45 min with or without pretreatment with .2 mg/kg verapamil: control animals received sham exposure. Flow was re-established after ischemia and the animals were allowed to recover for 24 h. Bilateral kidneys were harvested for DNA electrophoresis, Western analysis for p53, Northern analysis for c-myc expression, and light and electron microscopic analysis. Kidney I/R caused characteristic DNA laddering in the clamped kidney, and less extensive laddering was seen in the contralateral kidney. Light and electron microscopic analysis confirmed apoptotic morphology in the reperfused tissues. Verapamil pretreatment completely abolished DNA laddering and attenuated the microscopic evidence of apoptosis. p53 levels were increased by I/R in the ischemic kidney and moderately increased in the contralateral organ. c-myc mRNA levels were increased by the I/R insult. Kidney I/R injury may induce global apoptosis, which seems to be associated with an alteration in calcium homeostasis. The increase in p53 and c-myc mRNA levels seen with I/R may facilitate apoptosis. Calcium modulation seems to reduce apoptosis during I/R and may have therapeutic implications.
Subject(s)
Apoptosis/drug effects , Calcium Channel Blockers/pharmacology , Kidney/pathology , Reperfusion Injury/physiopathology , Animals , Apoptosis/genetics , Blotting, Northern , Calcium/metabolism , DNA/chemistry , Electrophoresis, Agar Gel , Genes, myc , Homeostasis , Kidney/drug effects , Male , RNA, Messenger/drug effects , Rats , Rats, Sprague-Dawley , Reperfusion Injury/drug therapy , Tumor Suppressor Protein p53/drug effects , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Surfactant functional effectiveness is dependent on phospholipid compositional integrity; sepsis decreases this through an undefined mechanism. Sepsis-induced hypothyroidism is commensurate and may be related. This study examines the effect of 3,3',5-triiodo-L-thyronine (T3) supplementation on surfactant composition and function during sepsis. Male Sprague-Dawley rats underwent sham laparotomy (Sham) or cecal ligation and puncture (CLP) with or without T3 supplementation [CLP/T3 (3 ng/h)]. After 6, 12, or 24 h, surfactant was obtained by lavage. Function was assessed by a pulsating bubble surfactometer and in vivo compliance studies. Sepsis produced a decrease in surfactant phosphatidylglycerol and phosphatidic acid, with an increase in lesser surface-active lipids phosphatidylserine and phosphatidylinositol. Phosphatidylcholine content was not significantly changed. Sepsis caused an alteration in the fatty acid composition and an increase in saturation in most phospholipids. Hormonal replacement attenuated these changes. Lung compliance and surfactant adsorption were reduced by sepsis and maintained by T3 treatment. Thyroid hormone may have an active role in lung functional preservation through maintenance of surfactant homeostasis during sepsis.
Subject(s)
Infections/metabolism , Lung Diseases/metabolism , Phospholipids/metabolism , Pulmonary Surfactants/metabolism , Triiodothyronine/pharmacology , Animals , Fatty Acids/metabolism , Infections/physiopathology , Lung/physiopathology , Lung Compliance , Lung Diseases/physiopathology , Male , Rats , Rats, Sprague-Dawley , Surface TensionABSTRACT
BACKGROUND: Long surfactant phospholipids are altered during sepsis; the role of surfactant apoproteins is unknown. This study investigates the effect of cecal ligation and puncture (CLP) on surfactant functional effectiveness and apoprotein transcriptional activity with or without T3 replacement. METHODS: Male Sprague Dawley rats underwent sham laparotomy or CLP with or without T3 replacement. Lung compliance, surfactant adsorption, and surface tension were measured with a surfactometer. Surfactant apoproteins A, B, and C (SP-A, SP-B, SP-C) mRNA was quantified by Northern blot analysis. RESULTS: Lung compliance was significantly decreased by sepsis; initial surface tension and adsorption values in CLP animals reflected apoprotein dysfunction. Sepsis decreased SP-A mRNA levels and increased SP-B mRNA; SP-C mRNA were unchanged. T3 treatment improved compliance, adsorption, and ST isotherms in septic animals. CONCLUSION: T3 attenuated sepsis-induced surfactant dysfunction and SP-A and SP-B transcriptional changes during sepsis. This suggests an interaction between the thyroid, surfactant apoproteins, and lung surfactant functional effectiveness and requires further study.
Subject(s)
Proteolipids/analysis , Pulmonary Surfactants/analysis , RNA, Messenger/analysis , Sepsis/drug therapy , Triiodothyronine/therapeutic use , Animals , Blotting, Northern , Disease Models, Animal , Drug Evaluation, Preclinical , Lung Compliance , Male , Proteolipids/drug effects , Pulmonary Surfactant-Associated Protein A , Pulmonary Surfactant-Associated Proteins , Pulmonary Surfactants/drug effects , Rats , Rats, Sprague-Dawley , Transcription, GeneticABSTRACT
In order to describe the telephone management of gastroenteritis by family practice residents, audiotapes and transcripts of telephone calls by 31 family practice residents were analysed with respect to clinical content, temporal patterns, and physicians' utterance form. The study hypothesis were that year 3 residents would have shorter calls, take more complete histories, and score better on other performance measures than year 1 residents, but no significant differences were demonstrated in the study. The mean duration of telephone calls was 4.6 minutes. Residents talked three times longer than the caller. History taking completeness was highly correlated with telephone call duration and particularly with time spent talking by the caller. Significant questions, such as asking about hydration status, were frequently omitted. Most questions were closed (84 percent). Time spent talking by the caller was longer, and more listener feedback was given, with year 1 than with year 3 residents.
