A 9-year retrospective evaluation of 102 pressure ulcer reconstructions.

OBJECTIVE: Several pressure ulcer (PU) risk factors including paralysis and age greater than 70 have been identified, while others such as nutrition are debated. The object of this study is to identify perioperative risk factors that may predict improved outcomes and reduced complications in primary and recurrent PU reconstructions. METHOD: A retrospective chart review of patients treated surgically for PUs from 2004 to 2013 at the University of Toledo Medical Center, Toledo, Ohio, US, was completed. Data collected included ulcer and medical history, as well as risk factors, complications and postoperative outcome. Data were statistically analysed for perioperative variances between primary and recurrent ulcers and closure status. RESULTS: A total of 49 patients with 102 reconstructions were reviewed. Spinal cord injured patients accounted for 90% receiving flap coverage of ulcers. Numerous differences between primary and recurrent ulcers were identified, including ulcer location, patient nutritional status, wound infection, postoperative course and recurrence. Multivariate analysis revealed a flap reconstruction prediction model using creatinine, haematocrit, haemoglobin, and prealbumin that is able to successfully predict closure outcome in 83.6% of cases. CONCLUSION: Many factors play a role in the development, course and treatment of PUs. It is vital to understand the role of patient risk factors in the development of PUs, to direct subsequent management and reconstruction, and to prevent future recurrences. DECLARATION OF INTEREST: The authors have no conflicts of interest to disclose.

Involvement of proteasome alpha-subunit PSMA7 in hepatitis C virus internal ribosome entry site-mediated translation.

Ribozymes are small catalytic RNA molecules that can be engineered to enzymatically cleave RNA transcripts in a sequence-specific fashion and thereby inhibit expression and function of the corresponding gene product. With their simple structures and site-specific cleavage activity, they have been exploited as potential therapeutic agents in a variety of human disorders, including hepatitis C virus (HCV) infection. We have designed a hairpin ribozyme (Rz3'X) targeting the HCV minus-strand replication intermediate at position 40 within the 3'X tail. Surprisingly, Rz3'X was found to induce ganciclovir (GCV)-resistant colonies in a bicistronic cellular reporter system with HCV internal ribosome entry site (IRES)-dependent translation of herpes simplex virus thymidine kinase (TK). Rz3'X-transduced GCV-resistant HeLa reporter cells showed substantially reduced IRES-mediated HCV core protein translation compared with control vector-transduced cells. Since these reporter systems do not contain the HCV 3'X tail sequences, the results indicate that Rz3'X probably exerted an inhibitory effect on HCV IRES activity fortuitously through another gene target. A novel technique of ribozyme cleavage-based target gene identification (cleavage-specific amplification of cDNA ends) (M. Krüger, C. Beger, P. J. Welch, J. R. Barber, and F. Wong-Staal, Nucleic Acids Res. 29:e94, 2001) revealed that human 20S proteasome alpha-subunit PSMA7 mRNA was a target RNA recognized and cleaved by Rz3'X. We then showed that additional ribozymes directed against PSMA7 RNA inhibited HCV IRES activity in two assay systems: GCV resistance in the HeLa IRES TK reporter cell system and a transient transfection assay performed with a bicistronic Renilla-HCV IRES-firefly luciferase reporter in Huh7 cells. In contrast, ribozymes were inactive against IRES of encephalomyocarditis virus and human rhinovirus. Additionally, proteasome inhibitor MG132 exerted a dose-dependent inhibitory effect on HCV IRES-mediated translation but not on cap-dependent translation. These data suggest a principal role for PSMA7 in regulating HCV IRES activity, a function essential for HCV replication.

C-SPACE (cleavage-specific amplification of cDNA ends): a novel method of ribozyme-mediated gene identification.

A hairpin ribozyme, RzCR2A, directed against position 323 of the hepatitis C virus 5'-untranslated region (HCV 5'-UTR) was used to establish and validate a novel method for the detection of cellular target molecules for hairpin ribozymes, termed C-SPACE (cleavage-specific amplification of cDNA ends). For C-SPACE, HeLa mRNA containing the transcript of interest was subjected to in vitro cleavage by RzCR2A in parallel with a control ribozyme, followed by reverse transcription using a modified SMART cDNA amplification method and cleavage-specific PCR analysis. C-SPACE allowed identification of the RzCR2A target transcript from a mixture containing the entire cellular mRNA while only requiring knowledge of the ribozyme binding sequence for amplification. In a similar approach, C-SPACE was used successfully to identify human 20S proteasome alpha-subunit PSMA7 mRNA as the cellular target RNA of Rz3'X, a ribozyme originally designed to cleave the negative strand HCV 3'-UTR. Rz3'X was found to substantially inhibit HCV internal ribosome entry site (IRES) activity and PSMA7 was subsequently confirmed to be involved in HCV IRES-mediated translation. Thereby, C-SPACE was validated as a powerful tool to rapidly identify unknown target RNAs recognized and cleaved by hairpin ribozymes.

Identification of Id4 as a regulator of BRCA1 expression by using a ribozyme-library-based inverse genomics approach.

Expression of the breast and ovarian cancer susceptibility gene BRCA1 is down-regulated in sporadic breast and ovarian cancer cases. Therefore, the identification of genes involved in the regulation of BRCA1 expression might lead to new insights into the pathogenesis and treatment of these tumors. In the present study, an "inverse genomics" approach based on a randomized ribozyme gene library was applied to identify cellular genes regulating BRCA1 expression. A ribozyme gene library with randomized target recognition sequences was introduced into human ovarian cancer-derived cells stably expressing a selectable marker [enhanced green fluorescence protein (EGFP)] under the control of the BRCA1 promoter. Cells in which BRCA1 expression was upregulated by particular ribozymes were selected through their concomitant increase in EGFP expression. The cellular target gene of one ribozyme was identified to be the dominant negative transcriptional regulator Id4. Modulation of Id4 expression resulted in inversely regulated expression of BRCA1. In addition, increase in Id4 expression was associated with the ability of cells to exhibit anchorage-independent growth, demonstrating the biological relevance of this gene. Our data suggest that Id4 is a crucial gene regulating BRCA1 expression and might therefore be important for the BRCA1 regulatory pathway involved in the pathogenesis of sporadic breast and ovarian cancer.

Myelodysplastic syndrome and a nonrandom chromosomal abnormality t(1;19): an indolent pathologic entity.

The chromosomal abnormality t(1;19) is an infrequent finding in adult hematopoietic malignancies. This is only the second report of t(1;19) in association with myelodysplastic syndrome in which there was an apparent excellent response to oral cyclosporin A and a very indolent clinical course.

Identification of eIF2Bgamma and eIF2gamma as cofactors of hepatitis C virus internal ribosome entry site-mediated translation using a functional genomics approach.

The 5'-untranslated region of hepatitis C virus (HCV) is highly conserved, folds into a complex secondary structure, and functions as an internal ribosome entry site (IRES) to initiate translation of HCV proteins. We have developed a selection system based on a randomized hairpin ribozyme gene library to identify cellular factors involved in HCV IRES function. A retroviral vector ribozyme library with randomized target recognition sequences was introduced into HeLa cells, stably expressing a bicistronic construct encoding the hygromycin B phosphotransferase gene and the herpes simplex virus thymidine kinase gene (HSV-tk). Translation of the HSV-tk gene was mediated by the HCV IRES. Cells expressing ribozymes that inhibit HCV IRES-mediated translation of HSV-tk were selected via their resistance to both ganciclovir and hygromycin B. Two ribozymes reproducibly conferred the ganciclovir-resistant phenotype and were shown to inhibit IRES-mediated translation of HCV core protein but did not inhibit cap-dependent protein translation or cell growth. The functional targets of these ribozymes were identified as the gamma subunits of human eukaryotic initiation factors 2B (eIF2Bgamma) and 2 (eIF2gamma), respectively. The involvement of eIF2Bgamma and eIF2gamma in HCV IRES-mediated translation was further validated by ribozymes directed against additional sites within the mRNAs of these genes. In addition to leading to the identification of cellular IRES cofactors, ribozymes obtained from this cellular selection system could be directly used to specifically inhibit HCV viral translation, thereby facilitating the development of new antiviral strategies for HCV infection.

A novel functional genomics approach identifies mTERT as a suppressor of fibroblast transformation.

As a tool for functional genomics, a hairpin ribozyme gene library with randomized target recognition sequences was constructed in a retroviral vector. This library has the potential to target and cleave any possible RNA substrate. Mouse fibroblasts transduced with this ribozyme gene vector library were selected in a focus formation assay to isolate in vivo functional ribozymes that promote cell transformation in tissue culture. After two successive rounds of selection by focus formation assay, a transforming ribozyme (Rz007) was identified. The sequence of this ribozyme was used to identify the putative target genes responsible for the transformation. A candidate gene target for Rz007 encodes telomerase reverse transcriptase (mTERT). Both mRNA level and enzymatic activity of mTERT were down-regulated in Rz007-transformed cells. Furthermore, newly designed ribozymes, recognizing other potential ribozyme cleavage sites unique to the mTERT mRNA, also cause cell transformation, thus validating the role of mTERT in suppressing the transformation phenotype. These surprising results suggest that the commonly accepted role of telomerase in maintaining cellular immortalization is more complicated than previously thought. These studies also demonstrate the utility of this novel 'reverse' functional genomics approach, enabling the targeted discovery of genes, whether previously known or not, that are involved in any selectable phenotype.

Identification and validation of a gene involved in anchorage-independent cell growth control using a library of randomized hairpin ribozymes.

We have developed a library of hairpin ribozyme genes that can be delivered and expressed in mammalian cells with the purpose of identifying genes involved in a specific phenotype. By applying the appropriate phenotypic selection criteria in tissue culture, we can enrich for ribozymes that knock down expression of an unknown gene or genes in a particular pathway. Once specific ribozymes are selected, their target binding sequence is used to identify and clone the target gene. We have applied this technology to identify a putative tumor suppressor gene that has been activated in HF cells, a nontransformed revertant of HeLa cells. Using soft agar growth as the selection criteria for gain of transformation, we have isolated ribozymes capable of triggering anchorage-independent growth. Isolation of one of these ribozymes, Rz 568, led to the identification and cloning of the human homologue of the Drosophila gene ppan, a gene involved in DNA replication, cell proliferation, and larval development. This novel human gene, PPAN, was verified as the biologically relevant target of Rz 568 by creating five additional "target validation" ribozymes directed against additional sites in the PPAN mRNA. Rz 568 and all of the target validation ribozymes reduced the level of PPAN mRNA in cells and promoted anchorage-independent growth. Exogenous expression of PPAN in HeLa and A549 tumor cells reduced their ability to grow in soft agar, underscoring its role in regulating anchorage-dependent growth. This study describes a novel method for gene discovery where the intracellular application of hairpin ribozyme libraries was used to identify a novel gene based solely on a phenotype.

Management of ventricular arrhythmias: a trial-based approach.

Sudden cardiac death accounts for approximately 300,000 deaths annually in the U.S., and most of these are secondary to ventricular tachycardia (VT) and fibrillation in patients with coronary artery disease. Most patients with cardiac death die before reaching the hospital, which brought about a tremendous amount of research focused at identifying patients at high risk. Several trials were initiated to test the effectiveness of various therapeutic measures in these high-risk patients. A history of myocardial infarction, depressed left ventricular function and nonsustained VT have all been identified as independent risk factors for future arrhythmic death. Similarly, patients with a history of sustained VT or a history of sudden cardiac death are a high-risk group and should be aggressively evaluated and treated. The purpose of this article is to discuss risk stratification and primary prevention of sustained ventricular arrhythmias. We also review the recent secondary prevention trials and discuss the options available in the management of patients with sustained ventricular arrhythmias.

Effect of radiofrequency ablation on atrial mechanical function in patients with atrial flutter.

Atrial stunning, as assessed by left atrial appendage emptying and increased spontaneous echo contrast, is known to occur following direct-current cardioversion of atrial fibrillation (AF) and atrial flutter (AFI). Little is known on atrial mechanical function and the time course of atrial recovery following radiofrequency ablation of AFI. Fourteen patients undergoing radiofrequency ablation of persistent typical counterclockwise AFI were enrolled. Two-dimensional and pulse Doppler transesophageal echocardiography (TEE) were performed before ablation and immediately following restoration of sinus rhythm. Left atrial spontaneous echo contrast grades, left atrial appendage emptying fractions, and peak left atrial appendage emptying velocities were measured. Transthoracic echocardiography (TTE) was performed immediately after ablation, then repeated after 1 day, 1 week, and 6 weeks to measure peak transmitral velocities and percent atrial contribution to ventricular filling. Left atrial appendage emptying velocities decreased significantly following AFI termination (44 +/- 23 cm/s before ablation vs 25 +/- 14 cm/s after ablation, p = 0.01). Left atrial appendage emptying fractions also decreased significantly (0.48 +/- 0.1 preablation vs 0.34 +/- 0.17 postablation, p = 0.02). New spontaneous echo contrast developed in 4 patients (29%) after ablation. Four patients had complete atrial standstill after ablation, and 1 patient developed a new left atrial appendage thrombus. The percent atrial contribution to ventricular filling recovered progressively over 6 weeks with significant improvement in peak transmitral velocities at day 7. Thus, atrial stunning occurs after catheter ablation of AFI and may lead to rapid formation of thrombus in the left atrial appendage. Significant improvement in left atrial function occurs in 7 days.

The effect of biphasic defibrillation on the immediate pacing threshold of a dedicated bipolar, steroid-eluting lead.

It is apparent that pacing threshold increases following an ICD shock, although the degree of change observed is dependent on the method used to assess pacing and the lead design used. We previously demonstrated a rise in postshock pacing threshold using a lead with integrated bipolar pacing in which the distal shocking coil also serves as the pacing anode. In this study, we sought to investigate whether the postshock pacing threshold increased significantly in an endocardial, steroid-eluting lead with dedicated bipolar pacing electrodes. Twenty patients (16 men, 4 women; median age 73, ejection fraction [EF] 0.17-0.58) were studied during pectoral ICD implantation (Medtronic active can model 7221Cx or 7223Cx with model 6932-65 lead). The diastolic pulse width pacing threshold at 1 or 2 V was determined. Pacing rate was set > or = 100/min at twice diastolic threshold output to assess pacing immediately following the first DFT test shock. For subsequent shocks, the output was adjusted to establish postshock thresholds as 1, 2, 3, or 4 times the diastolic threshold. The postshock threshold was defined as the output yielding 100% capture > or = 2.5 seconds following a shock. In 8 of 20 patients (ratio 0.40 +/- 0.11), a rise in the post-shock threshold was shown by failure of consistent capture when pacing at 2 times diastolic threshold > or = 2.5 seconds after a DFT test shock. Two of these patients failed at 3 times threshold, but none failed at 4 x threshold. Five of 12 patients with successful capture of 2 times threshold failed to capture at threshold. The postshock threshold increased by a mean factor of 2.83 +/- 0.83 in the group of patients with a threshold rise. Following ICD shock in an active can, steroid-eluting lead system with dedicated bipolar pacing, the post-shock threshold increases significantly. Our studies suggest a need for postshock pacing to be set at least 4 x threshold regardless of the lead design.

Reflex control of sympathetic activity during simulated ventricular tachycardia in humans.

BACKGROUND: Ventricular tachyarrhythmias present a unique set of stimuli to arterial and cardiopulmonary baroreceptors by increasing cardiac filling pressures and decreasing arterial pressure. The net effect on the control of sympathetic nerve activity (SNA) in humans is unknown. The purpose of this study was to determine the relative roles of cardiopulmonary and arterial baroreceptors in controlling SNA and arterial pressure during ventricular pacing in humans. METHODS AND RESULTS: Two experiments were performed in which SNA and hemodynamic responses to ventricular pacing were compared with nitroprusside infusion (NTP) in 12 patients and studied with and without head-up tilt or phenylephrine to normalize the stimuli to either the arterial or cardiopulmonary baroreceptors in 9 patients. In experiment 1, the slope of the relation between SNA and mean arterial pressure was greater during NTP (-4.7+/-1.4 U/mm Hg) than during ventricular pacing (-3.4+/-1.1 U/mm Hg). Comparison of NTP doses and ventricular pacing rates that produced comparable hypotension showed that SNA increased more during NTP (P=0.03). In experiment 2, normalization of arterial pressure during pacing resulted in SNA decreasing below baseline (P<0.05), whereas normalization of cardiac filling pressure resulted in a greater increase in SNA than pacing alone (212+/-35% versus 189+/-37%, P=0. 04). Conclusions--These data demonstrate that in humans arterial baroreflex control predominates in mediating sympathoexcitation during ventricular tachyarrhythmias and that cardiopulmonary baroreceptors contribute significant inhibitory modulation.

Interaction of a commercial heart rate monitor with implanted pacemakers.

Dry-electrode heart rate monitors allow display of heart rate by transmitting a signal to the receiving device, which typically is on the wrist or exercise machine, but due to the potential for electromagnetic interference, their use has been contraindicated in patients with pacemakers. In 12 patients, we found no adverse effect on pacemaker function; in addition, the monitors generally were accurate in measuring heart rate during pacing.

Effects of repeated electrical defibrillations on cardiac troponin I levels.

Multiple endocardial countershocks applied during intraoperative endocardial implantable cardioverter-defibrillator testing for the purpose of defibrillation threshold determination resulted in detectable myocardial injury in 5 of 12 patients, as indicated by elevations in cardiac troponin I levels. This injury was not associated with acute changes on the surface electrocardiogram.

Chimeric DNA-RNA hammerhead ribozyme to proliferating cell nuclear antigen reduces stent-induced stenosis in a porcine coronary model.

BACKGROUND: Stent-induced coronary restenosis is a major clinical and public health problem. Proliferating cell nuclear antigen (PCNA) is an important regulator of cell division, and blocking of its expression after angioplasty may limit intimal proliferation. METHODS AND RESULTS: We cloned the porcine PCNA gene and constructed a chimeric hammerhead ribozyme to a segment of the gene with human homology. In vitro studies with both cultured porcine and human vascular smooth muscle cells demonstrated uptake of ribozyme within the nucleus and significant inhibition of cellular proliferation. The ribozyme was then delivered locally into pig coronaries in a stent model. At 30 days, histomorphometric analysis showed neointimal thickness of 0.51+/-0.20 mm in the ribozyme group versus 0.71+/-0.27 and 0.66+/-0.25 mm in stent controls and scrambled ribozyme control, respectively (P=0.002, P=0.03). Quantitative angiographic analysis showed late loss of 1.4+/-0.5 mm for ribozyme versus 1.9+/-0.4 and 2.0+/-0.4 mm for the controls (P=0.05 and P=0. 02). CONCLUSIONS: Chimeric hammerhead ribozyme to PCNA inhibits smooth muscle cell proliferation in vitro and reduces both histomorphometric and angiographic restenosis in the porcine coronary stent model when delivered locally.

Expression of ribozymes in gene transfer systems to modulate target RNA levels.

The possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications. In the therapeutics area, they have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials. More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation.

Ribozyme gene therapy for hepatitis C virus infection.

BACKGROUND: The development of antiviral drugs for hepatitis C virus (HCV) infection represents a substantial challenge. Similar to human immunodeficiency virus (HIV), HCV is highly prone to mutation. It is, therefore, expected that potential HCV therapeutics currently under development, such as protease inhibitors, will suffer from the same shortcomings of HIV therapeutic drugs; the emergence of drug resistant viral mutants. Ribozymes (Rz) are enzymatic RNA molecules that can be engineered to specifically target any given RNA molecule. A therapeutic Rz can be manufactured and administered as a drug, or a Rz gene can be delivered and expressed intracellularly by gene therapy. For HCV therapeutics, we favour the gene therapy approach as delivery and in vivo expression of Rz genes will result in a constant and continuous supply of multiple intracellular Rz, offering less opportunity for the development of drug-resistant viral variants. OBJECTIVES: To utilise direct intravenous injection of hepatotropic viral vectors to transfer Rz genes directly into the hepatocytes of HCV-infected patients, resulting in degradation of the HCV positive strand RNA genome, the viral mRNAs, and even the negative strand RNA replication intermediate. We plan to circumvent the emergence of drug-resistant viral mutants by targeting multiple, highly conserved HCV RNA sequences simultaneously with multiple Rz genes expressed from a single vector. STUDY DESIGN: Rzs targeting conserved regions of the HCV positive and negative RNAs were transcribed in vitro and used to cleave HCV target RNAs. The most effective Rzs identified were then incorporated into adeno associated viral (AAV) vectors and adenoviral (AV) vectors and tested for their ability to inhibit HCV core expression in a tissue culture model. RESULTS: Several Rzs targeting highly conserved HCV sequences effectively degraded positive and negative strands of HCV RNA in vitro. Furthermore, substantial inhibition of HCV gene expression was observed in tissue culture using viral vectors to deliver and express Rz genes. CONCLUSIONS: Rz gene therapy has potential for the production of anti-viral drugs directed against HCV. Initial studies employing Rz gene therapy to produced anti-viral drugs against HCV have proved successful. Rz gene therapy may be a useful approach to overcome problems associated with anti-HCV drug design, such as the emergence of drug-resistant mutants.

Intracardiac shunts resulting from transseptal catheterization for ablation of accessory pathways in otherwise normal hearts.

In a series of 14 patients undergoing transseptal catheterization for ablation of left-sided accessory pathways, hydrogen appearance time was used to detect left-to-right shunting after removal of the catheter. Six of the 12 patients who had no evidence of shunt before catheterization had evidence of shunting after the procedure.

Initial experience with a high-impedance tined endocardial pacemaker lead: evidence for increased lead failure.

We report our initial clinical experience with a new tined ventricular endocardial pacemaker lead, the Medtronic model 5034. This lead has a reduced electrode tip size, which provides a higher impedance. Based on early evidence of elevation of pacing lead threshold, we compared our clinical experience with the performance of this lead with that of other similar models with larger surface area (Medtronic models 4024 and 5024). Of 17 implant procedures performed at our institution with the model 5034 lead, two (11.2%) developed high thresholds, versus 0% in 121 implant procedures with models 4024 or 5024 leads (p = 0.014). We conclude that there is evidence of increased failure caused by elevation of pacing threshold in this lead. This increased failure rate needs to be confirmed in a multicenter observational study or randomized trial.

Intracellular application of hairpin ribozyme genes against hepatitis B virus.

HBV, a partially double-stranded DNA virus, replicates through a pregenomic RNA (pgRNA) intermediate, which provides a therapeutic opportunity for a novel antiviral gene therapy based on ribozyme RNA cleavage. Three hairpin ribozymes (Rzs) were designed which have the potential to disrupt HBV replication by targeting the pgRNA as well as specific mRNAs encoding the HBV surface antigen (HBsAg), the polymerase and the X protein. The ability of each ribozyme to cleave approximately 0.3 kb HBV subgenomic RNA fragments was tested in vitro. Two of the three Rzs tested (BR1 and BR3) were capable of cleaving their respective RNA substrates, while their catalytically disabled mutated counterpart Rzs were not. Structural modifications were performed on these two Rzs, with the goal of increasing catalytic efficiency both in vitro and in cells. To determine the Rz activities in liver cells, the cDNAs for each of the anti-HBV Rzs (and their catalytically disabled negative controls) were cloned into retroviral vectors. Unmodified ribozymes co-expressed with HBV in human liver Huh7 cells reduced the level of viral particle production by up to 66% based on the endogenous polymerase assay, while the structurally modified ribozymes inhibited HBV production up to 83%. These encouraging results indicate the feasibility of ribozyme-mediated gene therapy for the treatment of HBV infections.