The ototoxic drug cisplatin localises to stress granules altering their dynamics and composition.

Cisplatin is an effective platinum-based chemotherapeutic with several side effects, including ototoxicity. Cochlear cells have low rates of proliferation yet are highly susceptible to cisplatin. We hypothesised that cisplatin ototoxicity might be caused by cisplatin-protein interactions rather than cisplatin-DNA interactions. Two known cisplatin-binding proteins are involved in the stress granule (SG) response. SGs are a pro-survival mechanism involving formation of transient ribonucleoprotein complexes during stress. We examined the effects of cisplatin on SG dynamics and composition in cell lines derived from the cochlea and retinal pigment epithelium. Cisplatin-induced SGs are significantly diminished in size and quantity compared to arsenite-induced SGs and are persistent after 24 h recovery. Additionally, cisplatin pre-treated cells were unable to form a typical SG response to subsequent arsenite stress. Cisplatin-induced SGs had significant reductions in the sequestration of eIF4G and the proteins RACK1 and DDX3X. Live-cell imaging of Texas Red-conjugated cisplatin revealed its localisation to SGs and retention for at least 24 h. We show cisplatin-induced SGs have impaired assembly, altered composition and are persistent, providing evidence of an alternate mechanism for cisplatin-induced ototoxicity via an impaired SG response.

B Lymphocyte-Derived CCL7 Augments Neutrophil and Monocyte Recruitment, Exacerbating Acute Kidney Injury.

Acute kidney injury (AKI) is a serious condition affecting one fifth of hospital inpatients. B lymphocytes have immunological functions beyond Ab production and may produce cytokines and chemokines that modulate inflammation. In this study, we investigated leukocyte responses in a mouse model of AKI and observed an increase in circulating and kidney B cells, particularly a B220low subset, following AKI. We found that B cells produce the chemokine CCL7, with the potential to facilitate neutrophil and monocyte recruitment to the injured kidney. Siglec-G-deficient mice, which have increased numbers of B220low innate B cells and a lower B cell activation threshold, had increased Ccl7 transcripts, increased neutrophil and monocyte numbers in the kidney, and more severe AKI. CCL7 blockade in mice reduced myeloid cell infiltration into the kidney and ameliorated AKI. In two independent cohorts of human patients with AKI, we observed significantly higher CCL7 transcripts compared with controls, and in a third cohort, we observed an increase in urinary CCL7 levels in AKI, supporting the clinical importance of this pathway. Together, our data suggest that B cells contribute to early sterile inflammation in AKI via the production of leukocyte-recruiting chemokines.

Pilot study of a noninvasive real-time optical backscatter probe in liver transplantation.

Transplantation of severely steatotic donor livers is associated with early allograft dysfunction and poorer graft survival. Histology remains the gold standard diagnostic of donor steatosis despite the lack of consensus definition and its subjective nature. In this prospective observational study of liver transplant patients, we demonstrate the feasibility of using a handheld optical backscatter probe to assess the degree of hepatic steatosis and correlate the backscatter readings with clinical outcomes. The probe is placed on the surface of the liver and emits red and near infrared light from the tip of the device and measures the amount of backscatter of light from liver tissue via two photodiodes. Measurement of optical backscatter (Mantel-Cox P < 0.0001) and histopathological scoring of macrovesicular steatosis (Mantel-Cox P = 0.046) were predictive of 5-year graft survival. Recipients with early allograft dysfunction defined according to both Olthoff (P = 0.0067) and MEAF score (P = 0.0097) had significantly higher backscatter levels from the donor organ. Backscatter was predictive of graft loss (AUC 0.75, P = 0.0045). This study demonstrates the feasibility of real-time measurement of optical backscatter in donor livers. Early results indicate readings correlate with steatosis and may give insight to graft outcomes such as early allograft dysfunction and graft loss.

Mitochondrial mechanisms and therapeutics in ischaemia reperfusion injury.

Acute kidney injury (AKI) remains a major problem in critically unwell children and young adults. Ischaemia reperfusion (IR) injury is a major contributor to the development of AKI in a significant proportion of these cases and mitochondria are increasingly recognised as being central to this process through generation of a burst of reactive oxygen species early in reperfusion. Mitochondria have additionally been shown to have key roles in downstream processes including activation of the immune response, immunomodulation, and apoptosis and necrosis. The recognition of the central role of mitochondria in IR injury and an increased understanding of the pathophysiology that undermines these processes has resulted in identification of novel therapeutic targets and potential biomarkers. This review summarises a variety of therapeutic approaches that are currently under exploration and may have potential in ameliorating AKI in children in the future.

Assessment of H2S in vivo using the newly developed mitochondria-targeted mass spectrometry probe MitoA.

Hydrogen sulfide (H2S) is produced endogenously in vivo and has multiple effects on signaling pathways and cell function. Mitochondria can be both an H2S source and sink, and many of the biological effects of H2S relate to its interactions with mitochondria. However, the significance of mitochondrial H2S is uncertain, in part due to the difficulty of assessing changes in its concentration in vivo Although a number of fluorescent H2S probes have been developed these are best suited to cells in culture and cannot be used in vivo To address this unmet need we have developed a mitochondria-targeted H2S probe, MitoA, which can be used to assess relative changes in mitochondrial H2S levels in vivo MitoA comprises a lipophilic triphenylphosphonium (TPP) cation coupled to an aryl azide. The TPP cation leads to the accumulation of MitoA inside mitochondria within tissues in vivo There, the aryl azido group reacts with H2S to form an aryl amine (MitoN). The extent of conversion of MitoA to MitoN thus gives an indication of the levels of mitochondrial H2S in vivo Both compounds can be detected sensitively by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the tissues, and quantified relative to deuterated internal standards. Here we describe the synthesis and characterization of MitoA and show that it can be used to assess changes in mitochondrial H2S levels in vivo As a proof of principle we used MitoA to show that H2S levels increase in vivo during myocardial ischemia.

Localized Liver Injury During Normothermic Ex Situ Liver Perfusion Has No Impact on Short-term Liver Transplant Outcomes.

BACKGROUND: Normothermic ex situ liver perfusion (NESLiP) has the potential to increase organ utilization. Radiological evidence of localized liver injury due to compression at the time of NESLiP, termed cradle compression, is a recognized phenomenon but is poorly characterized. METHODS: A retrospective analysis of a prospectively collected database was performed of transplanted livers that underwent NESLiP and subsequently had a computed tomography performed within the first 14 d posttransplant. The primary study outcome was 1-y graft survival. RESULTS: Seventy livers (63%) were included in the analysis. Radiological evidence of cradle compression was observed in 21 of 70 (30%). There was no difference in rate of cradle compression between donor after circulatory death and donated after brain death donors ( P  = 0.37) or with duration of NESLiP. Univariate analysis demonstrated younger (area under the receiver operating characteristic, 0.68; P = 0.008; 95% confidence interval [CI], 0.55-0.82) and heavier (area under the receiver operating characteristic, 0.80; P  < 0.001; 95% CI, 0.69-0.91) livers to be at risk of cradle compression. Only liver weight was associated with cradle compression on multivariate analysis (odds ratio, 1.003; P  = 0.005; 95% CI, 1.001-1.005). There was no difference in 1-y graft survival (16/17 [94.1%] versus 44/48 [91.6%]; odds ratio, 0.69; P  = 0.75; 95% CI, 0.07-6.62). CONCLUSIONS: This is the first study assessing the impact of cradle compression on outcome. We have identified increased donor liver weight and younger age as risk factors for the development of this phenomenon. Increasing utilization of NESLiP will result in the increased incidence of cradle compression but the apparent absence of long-term sequelae is reassuring. Routine postoperative axial imaging may be warranted.

Long-term results of plaque excision combined with aggressive pharmacotherapy in high-risk patients with advanced peripheral artery disease (SAVE a LEG registry).

BACKGROUND: In United States alone there are more than 12 million people with peripheral artery disease (PAD). Long-term outcomes of plaque excision in high-risk population (patients with diabetes and patients with end stage renal disease on dialysis) are scarce. METHODS: Since November 2003, we treated 225 consecutive patients (138 male, mean age: 66.3 ± 12.4, range: 29-93) with SilverHawk(TM) plaque excision for critical limb ischemia or disabling claudication. A total of 367 procedures were performed treating 832 lesions (157 restenotic, 675 de novo). One hundred fifty-five patients (68.9%) were diabetics, 74 (32.9%) were on dialysis. All patients were treated with statins, clopidogrel, aspirin, and aggressive glycemic control. The primary endpoint for our study was target lesion revascularization (TLR), and the secondary endpoint was an assessment of major adverse events (all cause death, amputation, TLR). RESULTS: The average time of observation was 2.2 ± 1.2 years. Procedural success rate was 99.4% with <30% residual stenosis achieved in 818 (98.9%) lesions. SilverHawk was used alone in 86.7%. No acute limb loss or major perforation occurred. Sixty (26.6%) patients had TLR. Long-term mortality was 16.4%. Seven (3.1%) patients had to undergo major amputations and 7 (3.1%) minor amputations. Seventy (31.1%) patients had a major adverse event. Atorvastatin 80 mg was found to be independent predictor of survival, and major amputation was found to be independent predictor of mortality. CONCLUSIONS: SilverHawk Plaque Excision combined with aggressive pharmacotherapy in this presented high-risk population is associated with promising long-term outcomes that compare favorably with accepted standards of care.

Surgical outcomes of gallbladder cancer: the OMEGA retrospective, multicentre, international cohort study.

Background: Gallbladder cancer (GBC) is rare but aggressive. The extent of surgical intervention for different GBC stages is non-uniform, ranging from cholecystectomy alone to extended resections including major hepatectomy, resection of adjacent organs and routine extrahepatic bile duct resection (EBDR). Robust evidence here is lacking, however, and survival benefit poorly defined. This study assesses factors associated with recurrence-free survival (RFS), overall survival (OS) and morbidity and mortality following GBC surgery in high income countries (HIC) and low and middle income countries (LMIC). Methods: The multicentre, retrospective Operative Management of Gallbladder Cancer (OMEGA) cohort study included all patients who underwent GBC resection across 133 centres between 1st January 2010 and 31st December 2020. Regression analyses assessed factors associated with OS, RFS and morbidity. Findings: On multivariable analysis of all 3676 patients, wedge resection and segment IVb/V resection failed to improve RFS (HR 1.04 [0.84-1.29], p = 0.711 and HR 1.18 [0.95-1.46], p = 0.13 respectively) or OS (HR 0.96 [0.79-1.17], p = 0.67 and HR 1.48 [1.16-1.88], p = 0.49 respectively), while major hepatectomy was associated with worse RFS (HR 1.33 [1.02-1.74], p = 0.037) and OS (HR 1.26 [1.03-1.53], p = 0.022). Furthermore, EBDR (OR 2.86 [2.3-3.52], p < 0.0010), resection of additional organs (OR 2.22 [1.62-3.02], p < 0.0010) and major hepatectomy (OR 3.81 [2.55-5.73], p < 0.0010) were all associated with increased morbidity and mortality. Compared to LMIC, patients in HIC were associated with poorer RFS (HR 1.18 [1.02-1.37], p = 0.031) but not OS (HR 1.05 [0.91-1.22], p = 0.48). Adjuvant and neoadjuvant treatments were infrequently used. Interpretation: In this large, multicentre analysis of GBC surgical outcomes, liver resection was not conclusively associated with improved survival, and extended resections were associated with greater morbidity and mortality without oncological benefit. Aggressive upfront resections do not benefit higher stage GBC, and international collaborations are needed to develop evidence-based neoadjuvant and adjuvant treatment strategies to minimise surgical morbidity and prioritise prognostic benefit. Funding: Cambridge Hepatopancreatobiliary Department Research Fund.

Assessment of left ventricular tissue mitochondrial bioenergetics in patients with stable coronary artery disease.

Recurrent myocardial ischemia can lead to left ventricular (LV) dysfunction in patients with coronary artery disease (CAD). In this observational cohort study, we assessed for chronic metabolomic and transcriptomic adaptations within LV myocardium of patients undergoing coronary artery bypass grafting. During surgery, paired transmural LV biopsies were acquired on the beating heart from regions with and without evidence of inducible ischemia on preoperative stress perfusion cardiovascular magnetic resonance. From 33 patients, 63 biopsies were acquired, compared to analysis of LV samples from 11 donor hearts. The global myocardial adenosine triphosphate (ATP):adenosine diphosphate (ADP) ratio was reduced in patients with CAD as compared to donor LV tissue, with increased expression of oxidative phosphorylation (OXPHOS) genes encoding the electron transport chain complexes across multiple cell types. Paired analyses of biopsies obtained from LV segments with or without inducible ischemia revealed no significant difference in the ATP:ADP ratio, broader metabolic profile or expression of ventricular cardiomyocyte genes implicated in OXPHOS. Differential metabolite analysis suggested dysregulation of several intermediates in patients with reduced LV ejection fraction, including succinate. Overall, our results suggest that viable myocardium in patients with stable CAD has global alterations in bioenergetic and transcriptional profile without large regional differences between areas with or without inducible ischemia.

An emerging role for stress granules in neurodegenerative disease and hearing loss.

Stress granules (SGs) are membrane-less cytosolic assemblies that form in response to stress (e.g., heat, oxidative stress, hypoxia, viral infection and UV). Composed of mRNA, RNA binding proteins and signalling proteins, SGs minimise stress-related damage and promote cell survival. Recent research has shown that the stress granule response is vital to the cochlea's response to stress. However, emerging evidence suggests stress granule dysfunction plays a key role in the pathophysiology of multiple neurodegenerative diseases, several of which present with hearing loss as a symptom. Hearing loss has been identified as the largest potentially modifiable risk factor for dementia. The underlying reason for the link between hearing loss and dementia remains to be established. However, several possible mechanisms have been proposed including a common pathological mechanism. Here we will review the role of SGs in the pathophysiology of neurodegenerative diseases and explore possible links and emerging evidence that they may play an important role in maintenance of hearing and may be a common mechanism underlying age-related hearing loss and dementia.

Cardiva Catalyst II vascular access management device in percutaneous diagnostic and interventional procedures with same-day discharge (Catalyst II trial).

PURPOSE: To present a prospective, single-center trial of the Cardiva Catalyst II, a vascular closure device that provides temporary hemostasis after the procedure and is removed under manual compression, leaving no material behind. METHODS: During a 16-month period between April 2008 and July 2009, 400 procedures (100 interventions, 300 diagnostic procedures) were performed on 351 nonconsecutive patients (185 men; mean age 60.2 ± 12.0 years, range 27-93). All interventions were performed utilizing bivalirudin for anti-thrombin therapy. Initial follow-up was done at a mean 1.3 ± 0.7 days after the index procedure in all patients. Final follow-up, for diagnostic procedures as well as interventions, was performed at a mean 15.4 ± 7.1 days. The primary and secondary endpoints were the rate of major and minor vascular complications, respectively. RESULTS: Successful deployment of the device was reported in 397 (99.3%) procedures, which were primarily in retrograde fashion (97.0%) from the right groin (92.5%). In most cases (309, 77.2%), a 5-F sheath was used. A major vascular complication occurred after 1 intervention; none was noted after diagnostic procedures. Minor vascular complications were recorded after 2 interventions and 5 diagnostic procedures. Overall vascular complication rates were 0.25% for major sequelae and 1.75% for minor events. Mean time to discharge after diagnostic procedures was 145.0 ± 21.2 minutes versus 295.1 ± 44.1 minutes after interventional procedures (p < 0.05). There was no death, stroke, myocardial infarction, or urgent hospital transfer in the study cohort. CONCLUSION: Cardiva Catalyst is safe and effective device in achieving local hemostasis after percutaneous diagnostic procedures and interventions performed under bivalirudin anticoagulation. The use of this device with an appropriate protocol facilitates same-day discharge.

Mitochondria as Therapeutic Targets in Transplantation.

Advances in surgical procedures, technology, and immune suppression have transformed organ transplantation. However, the metabolic changes that occur during organ retrieval, storage, and implantation have been relatively neglected since the developments many decades ago of cold storage organ preservation solutions. In this review we discuss how the metabolic changes that occur within the organ during transplantation, particularly those associated with mitochondria, may contribute to the outcome. We show how a better understanding of these processes can lead to changes in surgical practice and the development of new drug classes to improve the function and longevity of transplanted grafts, while increasing the pool of organs available for transplantation.

Targeting succinate dehydrogenase with malonate ester prodrugs decreases renal ischemia reperfusion injury.

Renal ischemia reperfusion (IR) injury leads to significant patient morbidity and mortality, and its amelioration is an urgent unmet clinical need. Succinate accumulates during ischemia and its oxidation by the mitochondrial enzyme succinate dehydrogenase (SDH) drives the ROS production that underlies IR injury. Consequently, compounds that inhibit SDH may have therapeutic potential against renal IR injury. Among these, the competitive SDH inhibitor malonate, administered as a cell-permeable malonate ester prodrug, has shown promise in models of cardiac IR injury, but the efficacy of malonate ester prodrugs against renal IR injury have not been investigated. Here we show that succinate accumulates during ischemia in mouse, pig and human models of renal IR injury, and that its rapid oxidation by SDH upon reperfusion drives IR injury. We then show that the malonate ester prodrug, dimethyl malonate (DMM), can ameliorate renal IR injury when administered at reperfusion but not prior to ischemia in the mouse. Finally, we show that another malonate ester prodrug, diacetoxymethyl malonate (MAM), is more potent than DMM because of its faster esterase hydrolysis. Our data show that the mitochondrial mechanisms of renal IR injury are conserved in the mouse, pig and human and that inhibition of SDH by 'tuned' malonate ester prodrugs, such as MAM, is a promising therapeutic strategy in the treatment of clinical renal IR injury.

The pharmacodynamics of enoxaparin in percutaneous coronary intervention with precise rapid enoxaparin loading (PEPCI-PRE study).

OBJECTIVE: We evaluated the early pharmacodynamic profile of the combined 30 mg intravenous and 1 mg/kg subcutaneous enoxaparin loading utilized in the TIMI 11B and ExTRACT TIMI 25 trials. BACKGROUND: It has not been reported whether anti-Xa levels appropriate for percutaneous coronary intervention (PCI) can be reliably achieved within 2 h utilizing this regimen. METHODS: Twenty-six patients with acute coronary syndrome (ACS) treated with this regimen had anti-Xa levels measured at 5 min, 2, 4, 6 and 8 h. RESULTS: Seventy-six percent of patients had anti-Xa levels above 0.5 IU/ml at 5 min. Dose-response curves showed all patients to have anti-Xa levels above 0.5 IU/ml within 1 h. Anti-Xa remained in the targeted range for PCI (0.5 to 1.8 IU/ml) at 2, 4, 6 and 8 h in all patients. CONCLUSION: This regimen is well suited for ACS treatment with an invasive strategy, including the rapid transition to early and rescue PCI.

Succinate accumulation drives ischaemia-reperfusion injury during organ transplantation.

During heart transplantation, storage in cold preservation solution is thought to protect the organ by slowing metabolism; by providing osmotic support; and by minimising ischaemia-reperfusion (IR) injury upon transplantation into the recipient1,2. Despite its widespread use our understanding of the metabolic changes prevented by cold storage and how warm ischaemia leads to damage is surprisingly poor. Here, we compare the metabolic changes during warm ischaemia (WI) and cold ischaemia (CI) in hearts from mouse, pig, and human. We identify common metabolic alterations during WI and those affected by CI, thereby elucidating mechanisms underlying the benefits of CI, and how WI causes damage. Succinate accumulation is a major feature within ischaemic hearts across species, and CI slows succinate generation, thereby reducing tissue damage upon reperfusion caused by the production of mitochondrial reactive oxygen species (ROS)3,4. Importantly, the inevitable periods of WI during organ procurement lead to the accumulation of damaging levels of succinate during transplantation, despite cooling organs as rapidly as possible. This damage is ameliorated by metabolic inhibitors that prevent succinate accumulation and oxidation. Our findings suggest how WI and CI contribute to transplant outcome and indicate new therapies for improving the quality of transplanted organs.

A randomized trial comparing compression, Perclose Proglide and Angio-Seal VIP for arterial closure following percutaneous coronary intervention: the CAP trial.

OBJECTIVE: This prospective randomized trial compared the Angio-Seal VIP with Perclose Proglide and to manual compression with respect to time to hemostasis and ambulation, patient satisfaction, and vascular complications following percutaneous coronary intervention (PCI). BACKGROUND: The use of arterial closure devices for the reduction of vascular complications following PCI remains controversial. There have been no head to head trials comparing these most commonly used arterial closure devices following PCI. METHODS: Two hundred patients undergoing PCI were randomized to manual compression, Perclose Proglide or Angio-Seal VIP. Ambulation was allowed 3 hr after Perclose Proglide or Angio-Seal VIP and 6 hr after compression. RESULTS: There were 10 failures to deploy Perclose Proglide and none for Angio-Sealt (P < 0.01). Time to hemostasis was significantly shorter with Angio-Seal VIP compared with Perclose Proglide (5.3 vs. 46.8 min, P < 0.01). Time to ambulation was shorter with Angio-Seal VIP than with Perclose Proglide (261 vs. 334 min, P < 0.05) and the time to ambulation, as expected, was longest with compression (943 min, P < 0.01 vs. Angio-Seal VIP and Perclose Proglide). Delay in ambulation was higher with Perclose Proglide than with Angio-Seal VIP (18 vs. 9, P < 0.01). There was no significant difference in major vascular complications between groups. Compared with compression, patient discomfort was significantly improved with Angio-Seal (1.5 vs. 2.0, P < 0.01), but not with Perclose Proglide. CONCLUSION: The Angio-Seal VIPtrade mark device has a high rate of deployment success, which is significantly better than that of Perclose Proglide. Angio-Seal VIP allows for earlier hemostasis and ambulation compared with both compression and Perclose Proglide and is associated with greater patient satisfaction compared with compression.

Pharmacokinetics/Pharmacodynamics of Dabigatran 75 mg Twice Daily in Patients With Nonvalvular Atrial Fibrillation and Severely Impaired Renal Function.

INTRODUCTION: Dabigatran etexilate is an oral direct thrombin inhibitor. Dabigatran excretion is 80% renal, so exposure increases with severity of renal failure. The US Food and Drug Administration-approved dabigatran etexilate 75 mg twice daily (BID) for patients with nonvalvular atrial fibrillation (NVAF) having severely impaired renal function (creatinine clearance: 15-30 mL/min), based on post hoc pharmacokinetic modeling. We assessed dabigatran exposure at trough and peak levels in patients with NVAF and severely impaired renal function and compared with model predictions. METHODS: Patients received dabigatran etexilate (75 mg BID) for ≥7 days before blood sampling; Cpre,ss (steady-state predose concentration; trough) was taken 10 to 16 hours postdose (prior to next dose), and C2,ss (steady-state concentration; peak) was taken 2 hours (± 30 minutes) postdose. Pharmacodynamic parameters at baseline (Ebase), trough concentrations (Epre,ss), and peak concentrations (E2,ss) were assessed by established coagulation assays. RESULTS: Of the 150 patients screened, 60 were treated, of which 40% were male and 78.3% were white; median age was 84 years. Cpre,ss values (n = 51) were close to pharmacokinetic modeling predictions with a geometric mean (gMean) of 155 ng/mL, geometric coefficient of variation (gCV) of 76.9%, and range of 15.6 to 498 ng/mL. The C2,ss values (n = 59) had a gMean of 202 ng/mL, gCV of 70.6%, and range of 42.0 to 680 ng/mL. Pharmacodynamic effects on coagulation paralleled dabigatran concentrations. Eleven (18.3%) patients had ≥1 adverse event (AE); pharmacokinetic results for these patients versus those without AEs (n = 49) were Cpre, ss: gMean = 206 versus 145 ng/mL, gCV = 64.0% versus 78.3%; C2,ss: gMean = 243 versus 193 ng/mL, gCV = 68.9% versus 70.8%. All bleeding events (8 events in 5 patients) were considered minor by the investigators. CONCLUSION: Dabigatran exposure levels largely confirmed earlier pharmacokinetic predictions, supporting the use of dabigatran etexilate 75 mg BID in patients with NVAF and severely impaired renal function. Pharmacodynamic results were also in agreement with earlier studies. TRIAL REGISTRATION: ClinicalTrials.gov NCT01896297.

Predictors of infarct size after primary coronary angioplasty in acute myocardial infarction from pooled analysis from four contemporary trials.

Determinates of infarct size in patients with acute myocardial infarction (AMI) undergoing percutaneous coronary intervention (PCI) have been incompletely characterized, in part because of the limited sample size of previous studies. Databases therefore were pooled from 4 contemporary trials of primary or rescue PCI (EMERALD, COOL-MI, AMIHOT, and ICE-IT), in which the primary end point was infarct size assessed using technetium-99m sestamibi single-photon emission computed tomographic imaging, measured at the same core laboratory. Of 1,355 patients, infarct size was determined using technetium-99m sestamibi imaging in 1,199 patients (88.5%), at a mean time of 23 +/- 15 days. Median infarct size of the study population was 10% (interquartile range 0% to 23%; mean 14.9 +/- 16.1%). Using multiple linear regression analysis of 18 variables, left anterior descending infarct artery, baseline Thrombolysis In Myocardial Infarction grade 0/1 flow, male gender, and prolonged door-to-balloon time were powerful independent predictors of infarct size (all p <0.0001). Other independent correlates of infarct size were final Thrombolysis In Myocardial Infarction grade <3 flow (p = 0.0001), previous AMI (p = 0.005), symptom-onset-to-door time (p = 0.021), and rescue angioplasty (p = 0.026). In conclusion, anterior infarction, time to reperfusion, epicardial infarct artery patency before and after reperfusion, male gender, previous AMI, and failed thrombolytic therapy were important predictors of infarct size after angioplasty in patients with AMI assessed using technetium-99m sestamibi imaging and should be considered when planning future trials of investigational drugs or devices designed to enhance myocardial recovery.

Validation of Reference Genes for Oral Cancer Detection Panels in a Prospective Blinded Cohort.

BACKGROUND: Reference genes are needed as internal controls to determine relative expression for clinical application of gene expression panels. Candidate constitutively expressed genes must be validated as suitable reference genes in each body fluid and disease entity. Prior studies have predominantly validated oral squamous cell carcinoma associated messenger RNAs (mRNAs) based on quantitative polymerase chain reaction (qPCR) quantification cycle (Cq) values without adjustment for housekeeping genes. METHODS: One hundred sixty eight patients had saliva collected before clinically driven biopsy of oral lesions suspicious for cancer. Seven potential housekeeping mRNAs and six pre-specified oral cancer associated mRNAs were measured with qPCR by personnel blinded to tissue diagnosis. Housekeeping gene stability was determined with the NormFinder program in a training set of 12 randomly selected cancer and 24 control patients. Genes with stability indices <0.02 were then tested in the validation set consisting of the remaining cancer and control patients and were further validated by the geNorm program. Cancer gene delta Cqs were compared in case and control patients after subtracting the geometric mean of the reference gene raw Cqs. RESULTS: B2M and UBC had stability indices >0.02 in the training set and were not further tested. MT-ATP6, RPL30, RPL37A, RPLP0 and RPS17 all had stability indices <0.02 in the training set and in the verification set. The geNorm M values were all ≤1.10. All six pre-specified cancer genes (IL8, IL1, SAT, OAZ1, DUSP1 and S100P) were up-regulated in cancer versus control patients with from nearly twofold to over threefold higher levels (p<0.01 for all based on delta Cq values). CONCLUSIONS: Five reference genes are validated for use in oral cancer salivary gene expression panels. Six pre-specified oral carcinoma associated genes are demonstrated to be highly significantly up-regulated in cancer patients based on delta Cq values. These cancer and reference genes are suitable for inclusion in gene expression panels for research and clinical applications. TRIAL REGISTRATION: ClinicalTrials.gov NCT01587573.