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Biomedical subjects

Richard C Hunt

Publications and source records attributed to Richard C Hunt.

14 recordsLinked to original sources
PubMed2004

Alternative splicing of the APC gene in the neural retina and retinal pigment epithelium.

PURPOSE: Hypertrophy and hyperplasia of the retinal pigment epithelium (RPE) is associated with an inherited predisposition to human familial adenomatous polyposis coli, suggesting that expression of the adenomatous polyposis coli (APC) tumor suppressor may regulate RPE proliferation/differentiation. Distinctive APC isoforms exist in different cell types due to alternative splicing of the APC transcripts. We hypothesize that differences in expression patterns of APC protein isoforms are critical to RPE proliferation/differentiation. METHODS: To investigate these relationships, APC gene expression was characterized in the retinas and RPE from fetal and adult human and mouse, and in the epiretinal membranes (ERM) from 5 patients with proliferative vitreoretinopathy (PVR). Expression patterns of alternative splice-forms of APC transcripts were evaluated by comparative quantitative RT-PCR. Exon 1 of APC encodes a heptad repeat that confers the ability of APC to homodimerize. APC protein isoforms containing or lacking this heptad were characterized by western blot analysis and immunohistochemistry. RESULTS: Comparative quantitative RT-PCR demonstrated a predominant exon 1 containing, conventional APC splice-form in the early developing fetal RPE and retina, and in all the tested ERM samples from patients with PVR. This method also demonstrated an increased level of exon 1 lacking APC splice-form in the mature RPE and retina. Western blot analysis and immunofluorescence microscopy demonstrated the conventional APC only in the RPE, and the APC isoform without the first heptad repeat in both the retina and RPE. Immunofluorescence microscopy also demonstrated only the conventional APC in the ERM samples tested. CONCLUSIONS: These results suggest that alternative splicing of APC leads to differential APC expression with potentially unique functions. APC isoform without the first heptad repeat may play a role in cell cycle cessation in the adult retina and RPE, and the down regulation of this APC isoform may contribute to the potential of RPE to migrate and proliferate.

Adenomatous Polyposis Coli Protein↗
PubMed2004

Small interfering RNA (siRNA) inhibits the expression of the Her2/neu gene, upregulates HLA class I and induces apoptosis of Her2/neu positive tumor cell lines.

Silencing of a specific mRNA using double stranded RNA oligonucleotides represents one of the newest technologies for suppressing a specific gene product. Small interfering RNA (siRNA) are 21 nucleotides long, double stranded RNA fragments that are identical in sequence to the target mRNA. We designed 3 such siRNA against the Her2/neu (HER2) gene. The HER2 gene is known to play an important role in the oncogenesis of several types of cancers, such as breast, ovarian, colon and gastric cancers. Introduction of the siRNA into HER2 positive tumor lines in vitro greatly reduced the cell surface expression of the HER2 protein. Concurrently, a range of effects on cell physiology, such as growth inhibition or apoptosis, was observed. The expression of HLA class I was observed to be upregulated when HER2 was silenced with siRNA. Treatment of SKBr3 and MCF7/HER2 tumor cell lines with the HER2 siRNA resulted in growth arrest of cells in the late G(1)/S-phase. Our results suggest that siRNA may be an effective method of abrogating the effect of HER2 in tumorigenesis.

Antibodies, Monoclonal↗
PubMed2003

Vitreous induces components of the prostaglandin E2 pathway in human retinal pigment epithelial cells.

PURPOSE: To investigate the alterations in gene expression when human retinal pigment epithelial (RPE) cells in culture are treated with vitreous as a model for the changes that occur in proliferative vitreoretinopathy. METHODS: Human RPE cells were cultured with or without human vitreous or collagen. RNA was extracted and reverse transcribed. The RNAs expressed were compared by using DNA macroarrays. Messenger RNA levels were also measured using real-time reverse transcription polymerase chain reaction. Protein expression was examined by immunoblot analysis. Immunoassays were used to determine levels of prostaglandin E(2). RESULTS: Vitreous treatment of RPE cells resulted in increased expression of two critical enzymes in the synthesis of prostaglandin E(2): membrane-associated prostaglandin E-synthase (mPGES) and cyclooxygenase (COX)-2. Increased levels of mPGES RNA and protein were still present at 48 hours of treatment, but the increase in COX-2 mRNA and protein was transient. The increase in the expression of mPGES was associated with an increase in the production of prostaglandin E(2) that was observed at 12 and 24 hours of treatment but not at 48 hours. Treatment with 100 microg collagen I per ml medium did not cause increased expression of mPGES and COX-2, even though both collagen- and vitreous-treatment caused a morphologic change in the RPE cells to a more fibroblast-like phenotype. CONCLUSIONS: Treatment of human RPE cells with vitreous induces changes in gene expression that are indicative of an inflammatory response.

Cells, Cultured↗
PubMed2002

HGF regulation of RPE proliferation in an IL-1beta/retinal hole-induced rabbit model of PVR.

PURPOSE: To understand molecular events that lead to retinal pigment epithelial (RPE) cell proliferation and migration during the early phases of proliferative vitreoretinopathy (PVR) in a rabbit model. METHODS: Retinal holes were created and interleukin-1beta(IL-1beta) was injected intravitreally. Eyes were examined by indirect ophthalmoscopy and eyecup pieces containing retinal holes were analyzed at different times after the surgery up to 4 weeks. RPE proliferation and migration were examined by immunohistochemistry. Tyrosine phosphorylation of extracellular signal regulated kinase (ERK) and hepatocyte growth factor receptor (HGFR or c-met) was determined by immunoprecipitation and western blot analysis. Tyrosine phosphorylation of c-met and morphological studies was performed on vitreous treated ARPE-19 cells. Expression of c-jun was determined by Northern blot analysis. Matrix metalloproteinase (MMP) content in vitreous was assessed by zymography. RESULTS: Indirect ophthalmoscopy identified formation of epiretinal membrane and immunohistochemistry identified proliferative and migratory RPE and other cells in the posterior segment containing retinal holes at 4 weeks post-surgery. Tyrosine phosphorylation of ERK and c-met occurred in this segment within 30 min of surgery. ARPE-19 cells treated with vitreous from the 24 h post-surgical eyes, but not with control vitreous or IL-1beta, showed morphological changes and tyrosine phosphorylation of c-met. Northern blot analysis in this segment identified upregulation of c-jun within 30 min of surgery and the expression peaked at 72 h. Zymographic analysis of vitreous identified MMP-9 in 12-72 h post-surgery. CONCLUSIONS: These data suggest that the presence of retinal holes and IL-1beta may lead to activation of HGF, mitogen activated protein kinases (MAPK), c-jun and extracellular matrix remodeling, resulting in proliferative and migratory cells in the wounded retina.

Animals↗
PubMed2002

MAP kinase and beta-catenin signaling in HGF induced RPE migration.

PURPOSE: Hepatocyte growth factor (HGF) has been implicated in retinal pigment epithelial (RPE) cell proliferation and migration that occurs in proliferative retinal diseases such as proliferative vitreoretinopathy (PVR). The aim of this study is to investigate HGF induced signaling pathways that lead to RPE cell migration. METHODS: Localization of beta-catenin was determined by immunofluorescence. HGF induced migration of ARPE-19 cells was studied using a quantitative migration assay after wounding in the presence of a DNA polymerase inhibitor, and in the presence or absence of a mitogen activated protein kinase (MAP kinase) kinase inhibitor. C-jun expression was determined by semi-quantitative RT-PCR and by Northern blot analysis. P42/p44 MAP kinase activity was determined by western blot and by an immunoprecipitation kinase assay. Tyrosine phosphorylation of the HGF receptor (HGFR or c-met) and beta-catenin was determined by immunoprecipitation and western blot analysis. Transactivation activity of beta-catenin was determined by luciferase reporter gene analysis. RESULTS: Beta-catenin and E-cadherin were co-localized on the basal surface of the RPE in vivo. Diffusion of the cell surface-localized beta-catenin occurs in migratory cells in vitro in the presence of HGF. HGF induced a MAP kinase dependent ARPE-19 cell migration, which is accompanied with a transient increase of c-jun expression and concomitant increases of MAP kinase activity, tyrosine phosphorylation of HGFR and beta-catenin, increased cytosolic levels of beta-catenin, and transactivation activity of beta-catenin. Tyrosine phosphorylation of HGFR and beta-catenin occurs in the primary or passaged RPE cultures or proliferative ARPE-19 cells, but not freshly isolated RPE or differentiated ARPE-19 cells. CONCLUSIONS: This study defines the signal transduction pathways activated by HGF in RPE cells, leading to an increase in the MAP kinase activity and free pool of beta-catenin, and changes in gene expression. These findings are consistent with the hypothesis that both beta-catenin and MAP kinases are components of the HGF induced RPE migration that occurs in proliferative retinal diseases.

Aged↗
PubMed2002

Metallothionein protects retinal pigment epithelial cells against apoptosis and oxidative stress.

The retina expresses metallothionein (MT) which has been reported to protect cells against oxidative stress and apoptosis. The types of MT expressed by human retinal cells were identified by laser capture microdissection and RT--PCR and it was found that MT-2a is expressed by retinal pigment epithelial (RPE) cells, photoreceptor cells, inner nuclear layer cells and ganglion cells while MT-1a is expressed by RPE cells and MT-3 by cells of the neural retina. MT is induced in cultured human RPE cells under stress conditions such as the presence of glucocorticoids, interleukin-1/TNF alpha, oxygen and TGF beta 1. Cultured human D407 RPE cells were transfected with plasmids that allowed the expression of MT to be controlled via the tet operator protein by the level of tetracycline in the medium. These experiments showed that elevation of MT levels by transfection of RPE cells protects them against toxic levels of cadmium, heme- and iron-induced oxidation and UV light-induced apoptosis.

Apoptosis↗
PubMedDate not supplied

Utilization and impact of ambulance diversion at the community level.

OBJECTIVE: To evaluate the utilization and impact of ambulance diversion in the metropolitan area of Syracuse, New York. METHODS: This was a retrospective review of the ambulance diversion system operated by the hospitals of Syracuse, New York. This system allows each emergency department to divert incoming ambulances during periods of extreme overcrowding. Data collected included numbers of hours on ambulance diversion by hospital, numbers of hours when all four hospitals were on diversion simultaneously, and numbers of ambulances received while the hospitals were on and off diversion. RESULTS: For three of the five years evaluated, ambulance diversion hours were most numerous during the period between January and March. For the most recent year studied (2000), ambulance diversion hours did not decline after the first quarter. During periods of diversion, hospital emergency departments received 30%-50% fewer ambulances than they did while open. CONCLUSION: This study demonstrated that, in Syracuse, New York, ambulance diversion was once a seasonal phenomenon, but is increasingly occurring throughout the year because of staff and resource limitations. It also demonstrated that ambulance diversion can be employed to reduce numbers of incoming transports.

Ambulances↗
PubMedDate not supplied

Eliminating errors in emergency medical services: realities and recommendations.

Errors in health care can have serious consequences, not only for patients but for society as a whole, given the considerable national expenditures required to address these errors. Because of the number of patients treated and the acuity of emergency situations, eliminating errors should be a priority in emergency medical services (EMS) systems. In a recent report, the Institute of Medicine called for improvements in patient safety, which it defined as freedom from accidental injury. Recent efforts have focused on integrating EMS systems into error analyses of the total health care system. However, EMS systems must take the initiative in addressing their own major error-prone areas using the best and most current data available. Unfortunately, addressing the problem of medical errors in EMS systems still suffers from a paucity of data, owing to a lack of organized, funded programs backed by legislation and dedicated government coordination. We recommend that EMS medical directors consider specific error audits to decrease sources of errors and to be better able to identify EMS providers who would benefit from retraining. Error audits might first be focused on the following potentially serious errors: equipment malfunction, failure to check oxygen saturation, failure to immobilize the patient, use of incorrect protocol or algorithm, failure to check glucose levels, failure to recognize patient deterioration, failure to detect misplaced endotracheal tubes, and use of wrong drug or drug dose.

Emergency Medical Services↗
PubMedDate not supplied

Emerging communication technologies in emergency medical services.

Advances in communication technologies are changing the face of emergency medical services (EMS). Two communication technologies in particular--cellular-enhanced 9-1-1 service and automatic crash notification (ACN)--will have a considerable impact on EMS. Although enhanced 9-1-1 service from land-line phones is now available in nearly every EMS system across the country, enhanced 9-1-1 service from cell phones currently does not exist. With more and more emergency calls made from cell phones, the implementation of this service is a clear priority. Automatic crash notification, a quickly emerging technology, can potentially provide EMS systems with invaluable data within seconds of when a collision occurs. However, many issues with regard to ACN systems need to be addressed, including liability concerns, access to ACN data, ability of drivers to understand the ACN system, and ACN system architecture. The potential interfaces between ACN systems and EMS systems create significant opportunities to enhance EMS resource management and care of patients involved in motor vehicle crashes. With both cellular-enhanced 9-1-1 and ACN in their infancy, EMS physicians and administrators have a valuable opportunity to provide leadership in the development of these services.

Accidents, Traffic↗
PubMedDate not supplied

Alternative cardiopulmonary resuscitation devices.

Cardiopulmonary resuscitation (CPR) involving manual external chest compression combined with artificial respiration was first described in 1960 by Kouwenhoven et al. (Kouwenhoven W, Jude JR, Knickerbocker GG. Closed-chest cardiac massage. JAMA. 1960; 173:1064-7). In the four decades since then, there have been no widely accepted alternatives for this technique. Even with the subsequent worldwide adoption of CPR and other advanced cardiac life support measures, long-term survival after prehospital cardiac arrest is still typically only 5%, to 10%. The performance of CPR must therefore be improved to increase the rate of long-term survival. Currently under development are new, alternative techniques such as interposed abdominal compression (IAC), active compression-decompression (ACD), pneumatic and nonpneumatic circumferential chest compression, and minimally invasive cardiac massage. Many of these newer techniques, compared with standard manual CPR, appear to provide superior vital organ blood flow and increased blood pressure. To date, only IAC (in-hospital only) and ACD have been shown to improve long-term survival in clinical studies. Circumferential chest compression and minimally invasive cardiac massage, on the other hand, have not yet been adequately tested in large clinical trials. Despite the difficulty and expense in studying these CPR techniques, additional research is necessary to evaluate their effectiveness in improving survival after sudden cardiac arrest.

Advanced Cardiac Life Support↗
PubMedDate not supplied

Reducing ambulance diversion: a multihospital approach.

OBJECTIVE: To evaluate the impact of procedures for reducing ambulance diversion in the metropolitan area of Syracuse, New York. METHODS: This was a retrospective review of procedures for reducing ambulance diversion at the system-wide and hospital-specific levels between January 1, 2001, and June 30, 2002. System-wide procedures involved exchange of information concerning diversion. Hospital-specific procedures involved implementation of additional planning and criteria for implementing ambulance diversion and development of additional patient care resources within the respective hospitals. RESULTS: Between 2000 and 2001, hours on ambulance diversion for the combined Syracuse hospitals declined by 24.8%. Between January and June 2001 and 2002, hours on diversion declined by 33.6%. These reductions in diversion hours occurred as numbers of ambulance transports increased by 7%, and numbers of emergency department visits increased by 5%. CONCLUSION: The study demonstrated that a combination of approaches can produce meaningful reductions of ambulance diversion at the community-wide and hospital-specific levels. It also demonstrated that substantial amounts of diversion time remained after these efforts, which must be addressed by payers and consumers.

Ambulances↗
PubMedDate not supplied

Blast lung injury.

Current trends in global terrorism mandate that emergency medical services, emergency medicine and other acute care clinicians have a basic understanding of the physics of explosions, the types of injuries that can result from an explosion, and current management for patients injured by explosions. High-order explosive detonations result in near instantaneous transformation of the explosive material into a highly pressurized gas, releasing energy at supersonic speeds. This results in the formation of a blast wave that travels out from the epicenter of the blast. Primary blast injuries are characterized by anatomical and physiological changes from the force generated by the blast wave impacting the body's surface, and affect primarily gas-containing structures (lungs, gastrointestinal tract, ears). "Blast lung" is a clinical diagnosis and is characterized as respiratory difficulty and hypoxia without obvious external injury to the chest. It may be complicated by pneumothoraces and air emboli and may be associated with multiple other injuries. Patients may present with a variety of symptoms, including dyspnea, chest pain, cough, and hemoptysis. Physical examination may reveal tachypnea, hypoxia, cyanosis, and decreased breath sounds. Chest radiography, computerized tomography, and arterial blood gases may assist with diagnosis and management; however, they should not delay diagnosis and emergency interventions in the patient exposed to a blast. High flow oxygen, airway management, tube thoracostomy in the setting of pneumothoraces, mechanical ventilation (when required) with permissive hypercapnia, and judicious fluid administration are essential components in the management of blast lung injury.

Blast Injuries↗