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Multiple Myeloma (MM) patients suffer disease relapse due to the development of therapeutic resistance. Increasing evidence suggests that immunotherapeutic strategies can provide durable responses. Here we evaluate the possibility of adoptive cell transfer (ACT) by generating ex vivo T cells from peripheral blood mononuclear cells (PBMCs) isolated from MM patients by employing our previously devised protocols. We designed peptides from antigens (Ags) including cancer testis antigens (CTAs) that are over expressed in MM. We exposed PBMCs from different healthy donors (HDs) to single peptides. We observed reproducible Ag-specific cluster of differentiation 4+ (CD4+) and CD8+ T cell responses on exposure of PBMCs to different single peptide sequences. These peptide sequences were used to compile four different peptide cocktails. Naive T cells from PBMCs from MM patients or HDs recognized the cognate Ag in all four peptide cocktails, leading to generation of multiclonal Ag-specific CD4+ and CD8+ effector and central memory T (TEM and TCM, respectively) cells which produced interferon-gamma (IFN-γ), granzyme B and perforin on secondary restimulation. Furthermore, this study demonstrated that immune cells from MM patients are capable of switching metabolic programs to induce effector and memory responses. Multiple peptides and cocktails were identified that induce IFN-γ+, T1-type, metabolically active T cells, thereby paving the way for feasibility testing of ACT in phase I clinical trials.

A key immunomodulatory cytokine, IL-10 (interleukin-10), has been shown to be dysregulated in preeclampsia, a pregnancy-specific hypertensive disorder, further characterized by multi-system involvement. However, studies have reported inconsistent findings about circulating IL-10 levels in preeclamptic versus normotensive pregnancies. The aim of the present systematic review and meta-analysis was to assess circulating IL-10 levels in preeclamptic and normotensive pregnancies at 2 time points: before, and at the time of preeclampsia diagnosis. PubMED, EMBASE, and Web of Science databases were searched to include all published studies examining circulating IL-10 levels in preeclamptic and normotensive pregnancies. Differences in IL-10 levels were evaluated by standardized mean differences. Of 876 abstracts screened, 56 studies were included in the meta-analysis. Circulating IL-10 levels were not different before the time of active disease (standardized mean differences, −0.01 [95% CI, −0.11 to 0.08]; P =0.76). At the time of active disease, women with preeclampsia (n=1599) had significantly lower IL-10 levels compared with normotensive controls (n=1998; standardized mean differences, −0.79 [95% CI, −1.22 to −0.35]; P =0.0004). IL-10 levels were lower in both early/severe and late/mild forms of preeclampsia. Subgroup analysis revealed that IL-10 measurement methodology (ELISA or multiplex bead array) and the sample type (plasma or serum) significantly influenced the observed differences, with the use of sera paired with ELISA technology providing the best distinction in IL-10 levels between preeclamptic and normotensive pregnancies. These findings support the role of decreased IL-10 levels in the pathophysiology of preeclampsia. Future studies should address the therapeutic potential of IL-10 in preeclampsia.

Background Glucagon serves as an important regulatory hormone for regulating blood glucose concentration with tight feedback control exerted by insulin and glucose. There are critical gaps in our understanding of glucagon kinetics, pancreatic α cell function and intra-islet feedback network that are disrupted in type 1 diabetes. This is important for translational research applications of evolving dual-hormone (insulin + glucagon) closed-loop artificial pancreas algorithms and their usage in type 1 diabetes. Thus, it is important to accurately measure glucagon kinetics in vivo and to develop robust models of glucose-insulin-glucagon interplay that could inform next generation of artificial pancreas algorithms. Methods Here, we describe the administration of novel 13C15N heavy isotope-containing glucagon tracers—FF glucagon [(Phe 6 13C9,15N; Phe 22 13C9,15N)] and FFLA glucagon [(Phe 6 13C9,15N; Phe 22 13C9,15N; Leu 14 13C6,15N; Ala 19 13C3)] followed by anti-glucagon antibody-based enrichment and LC–MS/MS based-targeted assays using high-resolution mass spectrometry to determine levels of infused glucagon in plasma samples. The optimized assay results were applied for measurement of glucagon turnover in subjects with and without type 1 diabetes infused with isotopically labeled glucagon tracers. Results The limit of quantitation was found to be 1.56 pg/ml using stable isotope-labeled glucagon as an internal standard. Intra and inter-assay variability was Conclusions The methods described in this study for simultaneous detection and quantitation of glucagon tracers have clinical utility for investigating glucagon kinetics in vivo in humans.

Introduction: Current standard electrocardiogram analysis algorithms cannot predict the presence and extent of coronary artery disease (CAD), especially in stable patients. Hypothesis: A novel artificial intelligence algorithm (ECGio) is able to predict the presence, location, and severity of coronary artery lesions in a minimally selected stable patient population as verified by coronary angiography. Methods: A cohort of 1659 stable outpatients were randomly divided into training (86%) and validation (14%) subsets, maintaining population characteristics. ECGio was trained and then validated using electrocardiograms paired with angiograms collected from electronic medical records. Coronary artery lesions were evaluated by a continuous measure of stenosis ranging from 0% - 100%. The prediction was then compared to the angiogram result (worst diameter stenosis in each vessel) with the error calculated per patient and per vessel. Results: In the primary analysis of the validation cohort, 22 had no angiographic CAD and were grouped with 56 patients with mild CAD (DS ≤30%), 31 had moderate CAD (DS 30-70%), and 113 had severe CAD (DS ≥70%). On a vessel level ECGio was able to predict stenosis severity with an overall average error of 16% as well as vessel specific errors of 18% in the LAD, 19% in the LCX, 18% in the RCA, and 8% in the Left Main. Absolute Error and Left Main error can be seen in the Figure. Conclusion: This validation study strongly suggests that it is possible to utilize an artificial intelligence algorithm to determine the presence and severity of CAD in stable patients using data from a standard 12- lead electrocardiogram.

The polycystin–1 (PC1), polycystin–2 (PC2) and fibrocystin proteins, the respective products of the PKD1, PKD2 and PKHD1 genes, are abundant in urinary exosome–like vesicles (ELVs) where they form the polycystin complex (PCC). ELVs are 100 nm diameter membrane vesicles shed into the urine by the cells lining the nephron. Using MS/MS analysis of ELVs from individuals with PKD1 mutations and controls, we show that in addition to the well-described GPS/GAIN cleavage event in PC1 at 3048 aa and the proprotein convertase cleavage (PPC) event in fibrocystin at 3616 aa, there are multiple other cleavage events in these proteins. The C–terminal 11 transmembrane portion of PC1 undergoes three cleavage events in vivo. The absence of peptides from the C–terminal cytoplasmic tail of fibrocystin implies a cleavage event close to its single TM domain prior to loading onto the ELVs. There is also evidence that the C–terminal tail of PC2 is also cleaved in ELVs. Native gel analysis of the PCC shows that the entire complex is > 2 MDa in size and that N–terminal GPS/GAIN cleaved PC1 and PPC cleaved fibrocystin ectodomains can be released under non-reducing conditions and resolve at 300 kDa. This paper shows that the three major human cystogene proteins are detectable in human urinary ELVs and that all three undergo post-translational proteolytic processing. Human urinary ELVs may be a useful source of material in the search for proteins that interact with the PCC.

Multiple Myeloma (MM) patients suffer disease relapse due to the development of therapeutic resistance. Increasing evidence suggests that immunotherapeutic strategies can provide durable responses. Here we evaluate the possibility of adoptive cell transfer (ACT) by generating ex vivo T cells from peripheral blood mononuclear cells (PBMCs) isolated from MM patients by employing our previously devised protocols. We designed peptides from antigens (Ags) including cancer testis antigens (CTAs) that are over expressed in MM. We exposed PBMCs from different healthy donors (HDs) to single peptides. We observed reproducible Ag-specific cluster of differentiation 4+ (CD4+) and CD8+ T cell responses on exposure of PBMCs to different single peptide sequences. These peptide sequences were used to compile four different peptide cocktails. Naïve T cells from PBMCs from MM patients or HDs recognized the cognate Ag in all four peptide cocktails, leading to generation of multiclonal Ag-specific CD4+ and CD8+ effector and central memory T (TEM and TCM, respectively) cells which produced interferon-gamma (IFN-γ), granzyme B and perforin on secondary restimulation. Furthermore, this study demonstrated that immune cells from MM patients are capable of switching metabolic programs to induce effector and memory responses. Multiple peptides and cocktails were identified that induce IFN-γ+, T1-type, metabolically active T cells, thereby paving the way for feasibility testing of ACT in phase I clinical trials.

We have recently developed a novel isotope dilution method using nonradioactive, stable human Glucagon (Phe 6 13C9, 15N; Phe 22 13C9, 15N) tracer to measure glucagon turnover. In this study we combined this method with organ catheterization technique to estimate the effects of increasing circulating glucagon concentrations, spanning the physiological range, on splanchnic and leg glucagon extraction. We present data from the first 6 healthy subjects (age 25±3.6 yrs; BMI 26.7±4.5 kg/m2; fasting glucose 4.6±0.5 mM; HbA1c 5.1±0.3%) completed thus far. After IRB approval and informed consent, subjects were admitted in the morning after an overnight fast to Interventional Radiology, where catheters were placed in the right femoral artery and vein under local anesthesia with aseptic precautions. Under fluoroscopic guidance, a hepatic venous catheter was positioned through the femoral vein. Glucagon tracer was infused intravenously and Indocyanine Green infused via the femoral artery sheath to measure splanchnic and femoral plasma flows. After a 90 min period for basal sampling, exogenous glucagon was infused at 0.65 ng/kg/min for 60 minutes (low) followed by 1.5 ng/kg/min (high) for another 60 minutes. Plasma samples were collected periodically from femoral artery, femoral vein and hepatic vein for concentrations of Glucagon tracer (Tandem Mass Spectrometry), glucagon, glucose and Indocyanine Green (Spectrophotometry) concentrations. Arterial glucagon concentrations were 45.2±9.6 pg/ml (basal), 56.3±13.3 pg/ml (low) and 81.4±15.5 pg/ml (high). While fractional splanchnic glucagon extraction remained relatively unchanged (24.0±12.5% vs. 27.7±15.6% vs. 23.5±10.1%: basal vs. low vs. high), fractional leg glucagon extraction tended to increase (13.5±3.8% vs. 16.0±7.2% vs. 18.5±9.4%: basal vs. low vs. high) with increasing glucagon concentrations. These initial data suggest differing mechanisms of glucagon clearance across the splanchnic and leg tissues in humans. Disclosure F. Ruchi: None. Y.R. Yadav: None. D. Romeres: None. S. Sawleh: None. L.M. Benson: None. K.L. Johnson: None. C. Dalla Man: Research Support; Self; Sanofi-Aventis Deutschland GmbH. C. Cobelli: None. D.J. McCormick: None. L.R. Wilkins: None. R. Basu: Consultant; Self; GENFIT. Research Support; Self; AstraZeneca. A. Basu: Consultant; Spouse/Partner; GENFIT. Research Support; Spouse/Partner; AstraZeneca. Funding National Institutes of Health (R01DK029953 to R.B.), (R01DK085516 to A.B.), (DK059637, DK020593)

Glucagon metabolism in humans is poorly understood and is currently under active investigation. We have recently developed a new isotope dilution method using nonradioactive, stable human glucagon (Phe 6 13C9, 15N; Phe 22 13C9, 15N) tracer to measure glucagon fluxes. To estimate postprandial glucagon turnover using this novel technique, we present data from the first 5 healthy subjects (3 males, age 26.2±7.9 years, BMI 25.4±2.6 kg/m2, fasting plasma glucose 4.7±0.2 mM, HbA1c 5.1±0.2 %) done thus far. After IRB approval and informed consent, subjects presented to the clinical research unit in the morning after an overnight fast. A catheter was inserted in a forearm vein for glucagon tracer infusion and another catheter placed in the contralateral hand and the hand kept in a heated box for periodic draws of arterialized venous samples for measurements of glucagon tracer (tandem mass spectrometry), glucagon and glucose concentrations during the study. A mixed meal (75 grams carb, 15% protein, 35% fat; 8 kcal/kg) was ingested at time 0 and the glucagon tracer infusion rate adjusted to mimic the anticipated changes in postprandial circulating glucagon concentrations for 6 hours. Systemic glucagon appearance rate (Ra glucagon) was calculated by the isotope dilution method. Fig 1 shows the Ra glucagon in all subjects. These initial data show that postprandial glucagon turnover can be calculated in humans applying this novel technique. Disclosure F. Ruchi: None. Y.R. Yadav: None. D. Romeres: None. S. Sawleh: None. L.M. Benson: None. K.L. Johnson: None. M. Schiavon: None. C. Dalla Man: Research Support; Self; Sanofi-Aventis Deutschland GmbH. C. Cobelli: None. D.J. McCormick: None. R. Basu: Consultant; Self; GENFIT. Research Support; Self; AstraZeneca. A. Basu: Consultant; Spouse/Partner; GENFIT. Research Support; Spouse/Partner; AstraZeneca. Funding National Institutes of Health (R01DK029953 to R.B.), (R01DK085516 to A.B.), (DK059637, DK020593)

Glucagon metabolism in humans is poorly understood and is currently under active investigation. We have recently developed a novel isotope dilution method using nonradioactive, stable human glucagon (Phe 6 13C9, 15N; Phe 22 13C9, 15N) tracer to measure glucagon turnover. In this study we combined this method with organ catheterization technique to estimate splanchnic and leg glucagon extraction and uptake. We present data from the first 6 healthy subjects (age 25±3.6 yrs; BMI 26.7±4.5 kg/m2; fasting glucose 4.6±0.5 mM; HbA1c 5.1±0.3%) completed thus far. After IRB approval and informed consent, subjects were admitted in the morning after an overnight fast to Interventional Radiology, where catheters were placed in the right femoral artery and vein under local anesthesia with aseptic precautions. Under fluoroscopic guidance, a hepatic venous catheter was positioned through the femoral vein. Glucagon tracer was infused intravenously and Indocyanine Green infused via the femoral artery sheath to measure splanchnic and femoral plasma flows. Plasma samples were collected periodically from femoral artery, femoral vein and hepatic vein for concentrations of glucagon tracer (Tandem Mass Spectrometry), glucagon, glucose and Indocyanine Green (Spectrophotometry) concentrations. Fractional splanchnic glucagon extraction was 24.0±12.5 % while total splanchnic glucagon uptake was 214.3±56.1 ng/kg total body weight/min. Net splanchnic glucagon balance was -34.4±10.0 ng/kg total body weight/min implying net splanchnic glucagon production. Fractional leg glucagon extraction was 13.5±3.8% while total leg glucagon uptake was 53.0±21.2 pg/kg total body weight/min. To the best of our knowledge, these initial data provide the first direct assessment of regional glucagon metabolism in healthy humans in the post absorptive fasting state. Disclosure F. Ruchi: None. Y.R. Yadav: None. D. Romeres: None. S. Sawleh: None. L.M. Benson: None. K.L. Johnson: None. C. Dalla Man: Research Support; Self; Sanofi-Aventis Deutschland GmbH. C. Cobelli: None. D.J. McCormick: None. L.R. Wilkins: None. R. Basu: Consultant; Self; GENFIT. Research Support; Self; AstraZeneca. A. Basu: Consultant; Spouse/Partner; GENFIT. Research Support; Spouse/Partner; AstraZeneca. Funding National Institutes of Health (R01DK029953 to R.B.), (R01DK085516 to A.B.), (DK059637, DK020593)

Introduction The rapid increase of patients of heart failure (HF) is a major health burden worldwide. Most importantly is the need to develop innovative new drugs for treatment of HF, such as sacubitril/valsartan which in part functions by enhancing the natriuretic peptides (NPs). We engineered NPA7 as a novel 30 amino acid bispecific designer peptide which activates the particulate guanylyl cyclase A receptor (pGC-A)/cGMP and for which the NPs both ANP and BNP are ligands and the Mas-receptor (MasR)/cAMP pathways for Angiotensin 1–7 (Ang1–7) is the endogenous ligand. We previously reported that acute intravenous (IV) administration of NPA7 shows cardiorenal protective and renin-aldosterone suppressing actions that go beyond the native peptides, BNP or Ang 1–7, which may have therapeutic potential for HF. Purpose To support the clinical development of NPA7 as a potential therapy in HF which promotes NP and MasR pathways, we investigated the actions and stability of subcutaneous (SQ) administration of NPA7 in normal canines. We also defined NPA7's peptide stability and metabolites in canine plasma. Methods Plasma and urinary cGMP, cardiorenal and renin-aldosterone responses to SQ injection (10μg/kg) were determined over 4 hours in normal canines (n=5) in vivo. Ex vivo, we established stability of NPA7 and key metabolites in canine serum using liquid chromatography-mass spectrometry (LC-MS). Data are expressed as mean ± SEM. * P Results In vivo, SQ NPA7 resulted in a sustained increase at 2 hours in plasma (BL: 10±3; 120 min: 30±6* pmol/ml) and urinary (BL: 1033±198; 120 min: 5792±857* pmol/min) cGMP, GFR (BL: 29±6; 120 min: 70±12* ml/min) and sodium excretion (BL: 18±10; 120 min: 144±33* ueq/min). We observed a gradual reduction in BP at 60 min (BL: 109±4; 60 min: 99±7* mmHg) with a sustained decrease in PCWP at 4 hours (BL: 5±0.9; 240 min: 3.1±0.6* mmHg). SQ NPA7 also suppressed plasma renin and aldosterone up to 3 hours after SQ injection. LC-MS revealed that NPA7 was highly stable with both the pGC-A and MasR activating moieties intact ex vivo in canine serum with a disappearance time of 2 hours. We also identified 2 major NPA7 metabolites NPA71–27 and NPA71–28. Conclusions SQ NPA7 possesses cGMP activating, cardiac unloading, diuretic, natriuretic, and renin-aldosterone suppressing actions in normal canines. NPA7 is also highly stable in serum. These studies support SQ administration as an effective delivery strategy for NPA7, a first-in-class innovative bispecific dual pGC-A/MasR activator now in preclinical development for HF.

Objectives The aim of this study was to compare trends and outcomes of 3 approaches to carotid revascularization in the coronary artery bypass graft (CABG) population when performed during the same hospitalization. Background The optimal approach to managing coexisting severe carotid and coronary disease remains controversial. Carotid endarterectomy (CEA) or carotid artery stenting (CAS) are used to decrease the risk of stroke in patients with carotid disease undergoing CABG surgery. Methods The authors conducted a serial, cross-sectional study with time trends of 3 revascularization groups during the same hospital admission: 1) combined CEA+CABG; 2) staged CEA+CABG; and 3) staged CAS+CABG from the Nationwide Inpatient Sample database 2004 to 2012. The primary composite endpoints were in-hospital all-cause death, stroke, and death/stroke. Results During the 9-year period, 22,501 concurrent carotid revascularizations and CABG surgeries during the same hospitalization were performed. Of these, 15,402 (68.4%) underwent combined CEA+CABG, 6,297 (28.0%) underwent staged CEA+CABG, and 802 (3.6%) underwent staged CAS+CABG. The overall rate of CEA+CABG decreased by 16.1% (ptrend = 0.03) from 2004 to 2012, whereas the rate of CAS+CABG did not significantly change during these years (ptrend = 0.10). The adjusted risk of death was greater, whereas risk of stroke was lower with both combined CEA+CABG (death odds ratio [OR]: 2.08, 95% confidence interval [CI]: 1.08 to 3.97; p = 0.03; stroke OR: 0.65, 95% CI: 0.42 to 1.01; p = 0.06) and staged CEA+CABG (death OR: 2.40, 95% CI: 1.43 to 4.05; p = 0.001; stroke OR: 0.50, 95% CI: 0.31 to 0.80; p = 0.004) approaches compared with CAS+CABG. The adjusted risk of death or stroke was similar in the 3 groups. Conclusions In patients with concomitant carotid and coronary disease undergoing combined revascularization, combined CEA+CABG is utilized most frequently, followed by staged CEA+CABG and staged CAS+CABG strategies. The staged CAS+CABG strategy was associated with lower risk of mortality, but higher risk of stroke. Future studies are needed to examine the risks/benefits of different carotid revascularization strategies for high-risk patients requiring concurrent CABG.

Coronary artery bypass grafting (CABG) decreases mortality in patients with significant left main (LM) coronary artery disease and for years remained the therapy of choice for patients with this ominous lesion. Advances in percutaneous coronary intervention (PCI) have enabled it to become an alternative to CABG. The results of observational registries and randomized comparisons have shown the safety and efficacy of PCI in appropriately selected patients with low or intermediate angiographic risk scores. Furthermore, the use of physiological measures of flow limitation and the use of intracoronary imaging techniques has added benefit and improved outcomes. The use of fractional flow reserve to more accurately evaluate the significance of intermediate lesions and guide the extent of revascularization has been an important refinement. Intravascular ultrasound and optical coherence tomography assessment of optimal stent deployment has led to reductions in restenosis. Newer generation stents, combined with improvements in specific techniques, especially at the LM bifurcation have extended PCI to more complex anatomic scenarios. The availability of left ventricular support devices in patients with complex coronary anatomy and severely depressed left ventricular function has added a margin of safety to LM and multivessel intervention. Randomized comparisons of CABG with PCI in carefully selected patients, using contemporaneous surgical and interventional techniques and optimal medical therapy, will further aid heart teams in the decision-making process. In conclusion, this review will give a concise overview of the management of unprotected LM disease.

Objectives The aim of this study was to compare the stroke/death rates between proximal embolic protection devices (P-EPDs) and distal filter embolic protection devices (F-EPDs) in elective carotid artery stenting (CAS). Background P-EPDs have theoretical advantages that may make them superior to F-EPDs for stroke prevention during CAS. Methods We examined 10,246 consecutive elective CAS procedures performed with embolic protection in the NCDR CARE registry between January 2009 and March 2013. We analyzed crude and propensity-matched rates of in-hospital combined death/stroke in patients treated with P-EPDs versus F-EPDs. Secondary analyses included 30-day adverse event rates and stroke rates by the involved cerebrovascular territory. Results P-EPDs were used in 590 of 10,246 cases (5.8%). Patients treated with P-EPDs had higher rates of symptomatic lesion status (46.8% vs. 39.7%, p Conclusions Use of a P-EPD during CAS was associated with low rates of in-hospital stroke/death similar to those with an F-EPD in the first comparative effectiveness study of the devices. An adequately powered randomized trial comparing clinical outcomes between these devices is unlikely to be feasible.

An 86-year-old woman with a medical history significant for hypertension, hyperlipidemia, dilated cardiomyopathy, remote history of coronary artery disease with a drug-eluting stent to the left anterior descending artery, permanent atrial fibrillation on warfarin, mitral and tricuspid valve repairs

Background— Given the controversy regarding whether carotid endarterectomy (CEA) or carotid artery stenting (CAS) may be superior for stroke prevention, it is uncertain how recent clinical evidence, guidelines, and reimbursement policies have influenced the volume and outcomes after these procedures. Methods and Results— We conducted a serial, cross-sectional study with time trends of patients undergoing CAS (n=124 265) and CEA (n=1 260 647) between 2001 and 2010 from the Nationwide Inpatient Sample database. During the 10-year period, the frequency of CEA declined, whereas CAS use slowly increased. After multivariate propensity score–matched analysis, CAS was associated with an increased risk of death (odds ratio [OR], 1.69; 95% confidence interval [CI], 1.40–2.04), stroke (OR, 1.43; 95% CI, 1.23–1.66), and major adverse events including death, stroke, and myocardial infarction (OR, 1.25; 95% CI, 1.13–1.39). In asymptomatic patients, there was no significant difference in major adverse events (OR, 1.08; 95% CI, 0.92–1.20; P =0.16 [ P P for trend=0.03] and major adverse events [OR, 0.75; 95% CI, 0.66–0.84; P for trend=0.05] comparing years 2010 versus 2001). Conclusions— In US hospitals between 2001 and 2010, CAS was associated with worse in-hospital outcomes, partly attributable to selection and ascertainment bias. Asymptomatic patients undergoing CAS versus CEA had similar adjusted rates of major adverse events. CAS outcomes improved significantly during the course of the decade likely attributable to improvements in patient selection, operator skills, and technological advancements.

Molecular identification of protein molecules surrounding nanoparticles (NPs) may provide useful information that influences NP clearance, biodistribution, and toxicity. Hence, nanoproteomics provides specific information about the environment that NPs interact with and can therefore report on the changes in protein distribution that occurs during tumorigenesis. Therefore, we hypothesized that characterization and identification of protein molecules that interact with 20 nm AuNPs from cancer and noncancer cells may provide mechanistic insights into the biology of tumor growth and metastasis and identify new therapeutic targets in ovarian cancer. Hence, in the present study, we systematically examined the interaction of the protein molecules with 20 nm AuNPs from cancer and noncancerous cell lysates. Time-resolved proteomic profiles of NP-protein complexes demonstrated electrostatic interaction to be the governing factor in the initial time-points which are dominated by further stabilization interaction at longer time-points as determined by ultraviolet–visible spectroscopy (UV–vis), dynamic light scattering (DLS), ζ-potential measurements, transmission electron microscopy (TEM), and tandem mass spectrometry (MS/MS). Reduction in size, charge, and number of bound proteins were observed as the protein-NP complex stabilized over time. Interestingly, proteins related to mRNA processing were overwhelmingly represented on the NP-protein complex at all times. More importantly, comparative proteomic analyses revealed enrichment of a number of cancer-specific proteins on the AuNP surface. Network analyses of these proteins highlighted important hub nodes that could potentially be targeted for maximal therapeutic advantage in the treatment of ovarian cancer. The importance of this methodology and the biological significance of the network proteins were validated by a functional study of three hubs that exhibited variable connectivity, namely, PPA1, SMNDC1, and PI15. Western blot analysis revealed overexpression of these proteins in ovarian cancer cells when compared to normal cells. Silencing of PPA1, SMNDC1, and PI15 by the siRNA approach significantly inhibited proliferation of ovarian cancer cells and the effect correlated with the connectivity pattern obtained from our network analyses.

Objectives This study sought to characterize usage and outcomes of carotid stenting platforms. Background A variety of stents and embolic protection devices (EPDs) are used for carotid artery stenting. Little is known about current usage patterns and differences in outcomes with these devices. Methods We analyzed 12,135 consecutive carotid stent procedures in the NCDR (National Cardiovascular Data Registry) CARE (Carotid Artery Revascularization and Endarterectomy) registry performed between January 1, 2007 and March 31, 2012. We compared baseline characteristics and crude and multivariable-adjusted rates of in-hospital combined death/stroke among patients treated with Acculink/Accunet (Abbott Laboratories, Abbott Park, Illinois), Xact/Emboshield (Abbott), and Precise/Angioguard (Cordis Corporation, Bridgewater, New Jersey) stent/EPD combinations. Results In 78.2% of cases, stents were used in conjunction with their specific, corresponding U.S. Food and Drug Administration–approved EPD. The Acculink/Accunet (n = 2,617, 21.6%), Xact/Emboshield (n = 3,507, 28.9%), and Precise/Angioguard (n = 2,696, 22.2%) stent/EPD combinations were used in 72.7% of all cases. The Protege/SpiderFx (ev3 Endovascular Inc., Plymouth, Minnesota) (n = 453, 3.7%) and Wallstent/Filterwire (Boston Scientific, Natick, Massachusetts) (n = 213, 1.8%) devices were used in a minority of cases. In unadjusted analyses, the Precise/Angioguard system was associated with higher rates of the primary outcome than were the Acculink/Accunet (2.5% vs. 1.8%; p = 0.058) and Xact/Emboshield (2.5% vs. 1.9%; p = 0.14) systems that were not statistically different. In adjusted analyses, differences between Precise/Angioguard and Accunet/Acculink (odds ratio [OR]: 1.48, 95% confidence interval [CI]: 0.89 to 2.47; p = 0.065), Precise/Angioguard and Xact/Emboshield (OR: 1.16, 95% CI: 0.77 to 1.76; p = 0.38), and Xact/Emboshield and Accunet/Acculink (OR: 1.28, 95% CI: 0.82 to 1.97; p = 0.18) remained nonsignificant. Conclusions In modern U.S. practice, the Acculink/Accunet, Xact/Emboshield, and Precise/Angioguard carotid stenting systems are used in most cases and are associated with similarly low rates of adverse events.

Urinary exosome-like vesicles (ELVs) are a heterogenous mixture (diameter 40–200nm) containing vesicles shed from all segments of the nephron including glomerular podocytes. Contamination with Tamm–Horsfall protein (THP) oligomers has hampered their isolation and proteomic analysis. Here we improved ELV isolation protocols employing density centrifugation to remove THP and albumin, and isolated a glomerular membranous vesicle (GMV)–enriched subfraction from 7 individuals identifying 1830 proteins and in 3 patients with glomerular disease identifying 5657 unique proteins. The GMV fraction was composed of podocin/podocalyxin-positive irregularly shaped membranous vesicles and podocin/podocalyxin-negative classical exosomes. Ingenuity pathway analysis identified integrin, actin cytoskeleton, and Rho GDI signaling in the top three canonical represented signaling pathways and 19 other proteins associated with inherited glomerular diseases. The GMVs are of podocyte origin and the density gradient technique allowed isolation in a reproducible manner. We show many nephrotic syndrome proteins, proteases, and complement proteins involved in glomerular disease are in GMVs and some were only shed in the disease state (nephrin, TRPC6, INF2 and phospholipase A 2 receptor). We calculated sample sizes required to identify new glomerular disease biomarkers, expand the ELV proteome, and provide a reference proteome in a database that may prove useful in the search for biomarkers of glomerular disease.

The diagnosis of brachiocephalic disease is often overlooked. Symptoms include arm claudication and vertebrobasilar insufficiency. In patients who have had the use of the internal mammary artery for coronary bypass surgery, the development of symptoms of myocardial ischemia should alert the clinician to the possibility of subclavian artery stenosis. Also, in patients who have had axillofemoral bypass, lower-extremity claudication may occur. Recognition involves physical examination and accurate noninvasive testing. Endovascular therapy has proven to be effective in alleviating symptoms in properly selected patients.

Thyroglobulin (Tg), a homodimer of 660 kD comprising 2748 amino acids, is the largest autoantigen known. The prevalence of autoimmune thyroid disease, including Hashimoto's thyroiditis and Graves' disease, has provided the impetus for identifying pathogenic T cell epitopes from human Tg over two decades. With no known dominant epitopes, the search has long been a challenge for investigators. After identifying HLA-DRB1∗03:01 (HLA-DR3) and H2E(b) as susceptibility alleles for Tg-induced experimental autoimmune thyroiditis in transgenic mouse strains, we searched for naturally processed T cell epitopes with MHC class II-binding motif anchors and tested the selected peptides for pathogenicity in these mice. The thyroiditogenicity of one peptide, hTg2079, was confirmed in DR3 transgenic mice and corroborated in clinical studies. In H2E(b)-expressing transgenic mice, we identified three T cell epitopes from mouse Tg, mTg179, mTg409 and mTg2342, based on homology to epitopes hTg179, hTg410 and hTg2344, respectively, which we and others have found stimulatory or pathogenic in both DR3- and H2E-expressing mice. The high homology among these peptides with shared presentation by DR3, H2E(b) and H2E(k) molecules led us to examine the binding pocket residues of these class II molecules. Their similar binding characteristics help explain the pathogenic capacity of these T cell epitopes. Our approach of using appropriate human and murine MHC class II transgenic mice, combined with the synthesis and testing of potential pathogenic Tg peptides predicted from computational models of MHC-binding motifs, should continue to provide insights into human autoimmune thyroid disease.

The EGFR/HER/ErbB family of receptor tyrosine kinases consists of EGFR/HER1, ErbB2/HER2/neu, ErbB3/HER3 and ErbB4/HER4. These receptors signal via both ligand-dependent (1) and -independent (2) pathways to regulate cell growth, differentiation, survival, migration, and adhesion, as well as oncogenic transformation. Consequently, it is not surprising that ErbB/HER overexpression and/or mutational activation play major roles in the etiology and progression of human cancer. In support of this concept, a number of HER receptor-targeted therapeutics have been FDA-approved (e.g., cetuximab, gefitinib, trastuzumab) for the treatment of adult solid malignancies, while many others are under clinical investigation (3). EGFR is the prototypic member of the ErbB/HER receptor family; it is a ~170-kDa glycoprotein consisting of an amino-terminal extracellular domain (ECD), a short transmembrane domain, and a carboxy-terminal intracellular domain (Figure 1). The ECD is divided into four subdomains, I–IV; subdomains I and III regulate ligand binding, while subdomains II and IV regulate receptor homo- or hetero-dimerization. In addition to full-length EGFR, we have reported the isolation of a cDNA clone encoding a soluble EGFR isoform that contains most of the ECD, but which lacks the transmembrane and intracellular domains. This EGFR isoform, sEGFR, arises from an alternate splice event, and incorporates a short region of novel coding sequence, including a termination codon and two polyadenylation sequences (4). Specifically, sEGFR contains sequences encompassing EGFR ECD subdomains I–III, most of subdomain IV, and the unique 78 amino acid carboxy-terminus (Figure 1) (5). This alternatively spliced sEGFR protein product is co-expressed with EGFR in diverse human tissues and also is the precursor for the major circulating isoform of EGFR in human blood, as discussed below. FIGURE 1 Schematic diagram illustrating the structure of EGFR compared to sEGFR. The extracellular domain is divided into four subdomains (I–IV); the transmembrane domain is indicated by the black box; the alternate C-terminal amino acid sequence of sEGFR ... While sEGFR does not contain a canonical transmembrane domain, it is, nevertheless, associated with the cell surface (6), and we recently have shown that cell surface sEGFR is released into the extracellular milieu via a complex process that is regulated by the extracellular matrix protein fibronectin (Wilken et al., unpublished experiments). Purification of anti-EGFR reactive protein(s) from human blood reveals one major protein species which contains peptides derived from the unique carboxy-terminus of sEGFR as determined by mass spectrometry (7). Taken together, these observations support the hypothesis that sEGFR released from the cell surface enters the bloodstream via the lymphatic system and thoracic duct. Translational studies have shown that low serum sEGFR concentrations are associated with the presence of ovarian cancer (7), poor overall survival in endometrial cancer patients (8), and responsiveness to aromatase inhibitor therapy in breast cancer patients (9). To develop further the clinical utility of sEGFR as a serum and tumor biomarker we have generated polyclonal antisera directed against a 31-mer peptide (residues 604–634) located within the unique carboxy-terminal sequence of sEGFR (Figure 1) (10). This affinity-purified antibody (anti-sEGFR) is specific for sEGFR, and does not detect the full-length EGFR, or any other HER receptor family member (10). Here, we report the further characterization of this anti-sEGFR antibody, including an apparent evolutionary convergence between the unique sEGFR carboxy-terminal sequence and a short bioactive sequence within the extracellular region of α5-integrin, i.e., the calf-1 domain, which coordinates ligand-dependent associations between the α and β subunits of the integrin heterodimer (11).

Use of iodinated contrast media (ICM) for angiography can result in contrast-induced nephropathy (CIN). Gadolinium-based contrast media (GCM) have been used in angiography with a goal to reduce the incidence of CIN. We performed a retrospective analysis involving 85 patients with renal insufficiency who underwent 97 carotid artery angiography and stenting (CAAS) procedures with a combination of GCM and ICM. The incidence of peri-procedural death, Q wave myocardial infarction (QWMI), stroke and CIN were recorded. Patients in GCM group had worse preprocedure renal function compared to ICM group. There were no peri-procedural deaths or QWMI in both groups. The incidence of stroke was 2.2% in GCM group and 0% in ICM group. The incidence of CIN were similar in GCM and ICM groups (8.5% vs. 10%, respectively, p NS). However, the predicted risk of CIN was 18.5% for GCM group and 10.4% for ICM group. Use of GCM and ICM combination for CAAS resulted in a 50% reduction in the incidence of predicted CIN risk compared to ICM.

Objectives Our aim was to design, synthesize and test in vivo and in vitro a new chimeric peptide that would combine the beneficial properties of 2 distinct natriuretic peptides with a biological profile that goes beyond native peptides. Background Studies have established the beneficial vascular and antiproliferative properties of C-type natriuretic peptide (CNP). While lacking renal actions, CNP is less hypotensive than the cardiac peptides atrial natriuretic peptide and B-type natriuretic peptide but unloads the heart due to venodilation. Dendroaspis natriuretic peptide is a potent natriuretic and diuretic peptide that is markedly hypotensive and functions via a separate guanylyl cyclase receptor compared with CNP. Methods Here we engineered a novel chimeric peptide CD-NP that represents the fusion of the 22-amino acid peptide CNP together with the 15-amino acid linear C-terminus of Dendroaspis natriuretic peptide. We also determined in vitro in cardiac fibroblasts cyclic guanosine monophosphate-activating and antiproliferative properties of CD-NP. Results Our studies demonstrate in vivo that CD-NP is natriuretic and diuretic, glomerular filtration rate enhancing, cardiac unloading, and renin inhibiting. CD-NP also demonstrates less hypotensive properties when compared with B-type natriuretic peptide. In addition, CD-NP in vitro activates cyclic guanosine monophosphate and inhibits cardiac fibroblast proliferation. Conclusions The current findings advance an innovative design strategy in natriuretic peptide drug discovery and development to create therapeutic peptides with favorable properties that may be preferable to those associated with native natriuretic peptides.

The phosphaturic activity of intact, full-length, fibroblast growth factor-23 (FGF-23) is well documented. FGF-23 circulates as the intact protein, and as fragments generated as the result of proteolysis of the full-length protein. To assess whether short fragments of FGF-23 are phosphaturic, we compared the effect of acute, equimolar infusions of full-length FGF-23 and various FGF-23 fragments carboxyl-terminal to amino acid 176. In rats, intravenous infusions of full-length FGF-23, and FGF-23 176-251, significantly and equivalently increased fractional phosphate excretion (FE Pi)) from 14±3 to 32±5% and 15±2 to 33±2% (p

MANP is a novel natriuretic peptide developed at the Mayo Clinic following its discovery from a familial frameshift mutation of the atrial natriuretic peptide (ANP) gene (NPPA) [1,2]. MANP possesses the 28 amino acid (AA) core structure of ANP with a novel 12 AA extended C-terminus resulting in a 40 AA peptide. MANP activates the particulate guanylyl cyclase A (pGC-A) receptor with equal potency to ANP and is highly resistant to degradation by neprilysin. In vivo, MANP has greater and longer lasting blood pressure (BP) lowering, natriuretic and aldosterone suppressing actions than ANP [3]. In animal models of hypertension, MANP potently reduces BP and enhances renal function while in experimental hypertensive heart failure, MANP has more potent renal and aldosterone suppressing actions than the cyclic guanylyl monophosphate (cGMP)-activating therapeutic agent nitroglycerin [4]. In vivo administered subcutaneously, MANP activates plasma cGMP 10-fold greater than ANP with a 4-fold greater half-life [3,4]. Based upon its potent cGMP mediated cardiorenal actions and studies which suggest that human hypertension may represent a relative natriuretic peptide deficiency state, MANP is being developed for treatment of severe resistant hypertension.

Aims Cenderitide is a novel dual natriuretic peptide (NP) receptor chimeric peptide activator, which targets the particulate guanylyl cyclase B (pGC-B) receptor and pGC-A unlike native NPs. Cenderitide was engineered to retain the anti-fibrotic properties of C-type natriuretic peptide (CNP)/pGC-B with renal-enhancing actions facilitated by fusion to the carboxyl terminus of Dendroaspis NP (DNP), a pGC-A agonist, to CNP. Here, we address significance of the DNP carboxyl terminus in dual pGC receptor activation and actions of cenderitide compared with CNP on renal function and cyclic guanosine monophosphate (cGMP) in vivo and ex vivo in normal canines. Methods and results In vitro , only cenderitide and not CNP or three CNP-based variants was a potent dual pGC-A/pGC-B activator of cGMP production (from 5 to 237 pmol/mL) in human embryonic kidney (HEK) 293 cells overexpressing human pGC-A while in pGC-B overexpressing cells cenderitide increased cGMP production (from 4 to 321 pmol/mL) while the three CNP-based variants were weak agonists. Based upon our finding that the DNP carboxyl terminus is a key structural requirement for dual pGC-A/pGC-B activation, we defined in vivo the renal-enhancing actions of cenderitide compared with CNP. Cenderitide increased urinary cGMP excretion (from 989 to 5977 pmol/mL), net generation of renal cGMP (821–4124 pmol/min), natriuresis (12–242 μEq/min), and glomerular filtration rate (GFR) (37–51 mL/min) while CNP did not. We then demonstrated the transformation of CNP ex vivo into a renal cGMP-activating peptide which increased cGMP in freshly isolated glomeruli eight-fold greater than CNP. Conclusion The current study establishes that dual pGC-A and pGC-B activation with CNP requires the specific carboxyl terminus of DNP. In normal canines in vivo and in glomeruli ex vivo , the carboxyl terminus of DNP transforms CNP into a natriuretic and GFR-enhancing peptide. Future studies of cenderitide are warranted in cardiorenal disease states to explore its efficacy in overall cardiorenal homeostasis.

Importance The Lariat device has received US Food and Drug Administration (FDA) 510(k) clearance for soft-tissue approximation and is being widely used off-label for left atrial appendage (LAA) exclusion. A comprehensive analysis of safety and effectiveness has not been reported. Objectives To perform a systematic review of published literature to assess safety and procedural success, defined as successful closure of the LAA during the index procedure, of the Lariat device. We performed a formal analytic review of the FDA MAUDE (Manufacturer and User Facility Device Experience) database to compile adverse event reports from real-world practice with the Lariat. Data Sources For the systematic review, PubMed, EMBASE, CINAHL, and the Cochrane Library were searched from January 2007 through August 2014 to identify all studies reporting use of the Lariat device in 3 or more patients. The FDA MAUDE database was queried for adverse events reports related to Lariat use. Data Extractions and Synthesis Data were abstracted in duplicate by 2 physician reviewers. Events from published literature were pooled using a generic inverse variance weighting with a random effects model. Cumulative and individual adverse events were also reported using the FDA MAUDE data set. Main Outcomes and Measures Procedural adverse events and procedural success. Results In the systematic review, 5 reports of Lariat device use in 309 participants were identified. Specific complications weighted for inverse of variance of individual studies were urgent need for cardiac surgery (2.3%; 7 of 309 procedures) and death (0.3%; 1 of 309 procedures). Procedural success was 90.3% (279 of 309 procedures). In the FDA MAUDE database, there were 35 unique reports of adverse events with use of the Lariat device. Among these, we identified 5 adverse event reports that noted pericardial effusion and death and an additional 23 reported urgent cardiac surgery without mention of death. Conclusions and Relevance This review of published reports and case reports identified risks of adverse events with off-label use of the Lariat device for LAA exclusion. Formal, controlled investigations into the safety and efficacy of the device for this indication are warranted.

Patients with previous percutaneous coronary intervention (PCI) are often excluded from clinical trials. As a result, limited data are available on the long-term outcome of such patients undergoing repeat PCI. In this study, we assessed the impact of previous PCI on outcomes in patients undergoing repeat PCI. We compared the baseline features and outcomes of 7,056 patients without previous PCI (group I) with those of 1,281 patients with previous PCI of the original target lesion (group II) and 1,408 patients with previous PCI of a nontarget lesion (group III) undergoing PCI in the Prevention of Restenosis with Tranilast and its Outcomes (PRESTO) trial. Compared with patients in group I, patients in groups II and III were more likely to have diabetes (25% and 24% vs 21%, p

Thyroid hormone-binding (THB) Abs are frequently detected in autoimmune thyroid disorders but it is unknown whether they can exert immunoregulatory effects. We report that a THB mAb recognizing the 5′ iodine atom of the outer phenolic ring of thyroxine (T4) can block T cell recognition of the pathogenic thyroglobulin (Tg) peptide (2549–2560) that contains T4 at aa position 2553 (T4(2553)). Following peptide binding to the MHC groove, the THB mAb inhibited activation of the Ak-restricted, T4(2553)-specific, mouse T cell hybridoma clone 3.47, which does not recognize other T4-containing epitopes or noniodinated peptide analogues. Addition of the same THB mAb to T4(2553)-pulsed splenocytes largely inhibited specific activation of T4(2553)-primed lymph node cells and significantly reduced their capacity to adoptively transfer thyroiditis to naive CBA/J mice. These data demonstrate that some THB Abs can block recognition of iodine-containing Tg epitopes by autoaggressive T cells and support the view that such Abs may influence the development or maintenance of thyroid disease.

A novel chemical-enzymatic approach was developed to facilitate identification of phosphorylation sites in isolated phosphoproteins. ESI-TOF mass spectrometry was used to characterize products from the chemical-enzymatic cleavage of specific phosphorylation sites in bovine alpha-S1 casein and synthetic phosphopeptides containing substitutions at a single phosphorylation site. Further refinements to this approach for identification of protein phosphorylation sites and its utility for the quantification of phosphopeptides by isotope-dilution mass spectrometry are presented.

To identify pathogenic epitopes on human thyroglobulin (hTg), a homodimer of 660kDa, we have applied a computer-based algorithm to predict potential HLA-DR3-binding peptides and have tested them in DR3-transgenic mice. Of the 39 peptides selected, four stimulated a proliferative response from hTg-primed cells of DR3+ mice, but not DQ8+ mice. Of the four peptides, one, hTg2079, was consistently pathogenic. Thyroiditis was not only produced by adoptive transfer of hTg-primed, hTg2079-activated cells but also by direct immunization with the peptide. These results demonstrate the utility of using this computer-based algorithm with synthetic peptides to help identify pathogenic T cell epitopes on hTg.

Molecular imaging is an important new direction in medical diagnosis; however, its success is dependent upon molecular probes that demonstrate selective tissue targeting. We report the design and chemical synthesis of a derivative of human amyloid-beta (Abeta) peptide that is capable of selectively targeting individual amyloid plaques in the brain of Alzheimer's disease transgenic mice after being intravenously injected. This derivative is based on the sequence of the first 30 amino acid residues of Abeta with asparagyl/glutamyl-4-aminobutane residues (N-4ab/Q-4ab) substituted at unique Asp and Glu positions and with Gd-DTPA-aminohexanoic acid covalently attached at the N-terminal Asp. The Gd[N-4ab/Q-4ab]Abeta30 peptide was homogeneous as shown by high-resolution analytical techniques with a mass of +/-4385 Da determined by electrospray ionization mass spectrometry. This diamine- and gadolinium-substituted derivative of Abeta is shown to have enhanced in vitro binding to Alzheimer's disease (AD) amyloid plaques and increased in vivo permeability at the blood-brain barrier because of the unique Asp/Glu substitutions. In addition, specific in vivo targeting to AD amyloid plaques is demonstrated throughout the brain of an APP, PS1 transgenic mouse after intravenous injection. Because of the magnetic resonance (MR) imaging contrast enhancement provided by gadolinium, this derivative should enable the in vivo MR imaging of individual amyloid plaques in the brains of AD animals or patients to allow for early diagnosis and also provide a direct measure of the efficacy of anti-amyloid therapies currently being developed.

Recent advances in technology have engendered a renewed enthusiasm in the use of intracardiac echocardiography (ICE) to guide and assess cardiac interventions. AcuNav is a phased-array sector imaging probe equipped with color and spectral Doppler capabilities. Previous-generation imaging catheters yielded unfamiliar limited-depth radial images with no flow information. Current imaging technology such as the AcuNav has not only consolidated the role of ICE but opened newer applications in the interventional laboratory. ICE has clear advantages over transesophageal echocardiography as the imaging modality of choice in the cardiac catheterization and electrophysiological laboratories. We review the technical evolution of ICE and describe the expanded utility of the AcuNav imaging catheter during cardiac interventions.

A 65-year-old woman with a history of pulmonary embolism and venous stasis presented to her doctor’s office reporting leg edema. In 1969, she developed a massive spontaneous pulmonary embolism that was treated with surgical clot removal. She was unable to be anticoagulated because of warfarin

In the twenty-six years since Gruntzig introduced a simple balloon angioplasty technique, percutaneous coronary intervention has undergone extraordinary growth and has now surpassed bypass surgery in frequency of performance. Several critical breakthrough technologies account for this remarkable progress: intracoronary stents have increased success rates and reduced restenosis, adjunctive antiplatelet therapy has reduced periprocedural complications, and restenosis after stent placement has been effectively treated with local radiation. Most recently, drug-eluting stents coated with cell-cycle inhibitors have shown great promise for further reducing restenosis, possibly to negligible levels.

The authors present an unusual case of a young male patient with a large left common carotid artery pseudoaneurysm in a shape similar to that of a bowtie treated with a covered Wallgraft. The Wallgraft is a covered stent originally designed to be used in the treatment of tracheobronchial fistula and peripheral arterial applications. The favorable out-come of this case illustrates its endovascular application in nonsurgical traumatic injuries of the carotid artery.

Background The posterior inferior cerebellar arteries (PICA) arise from the intracranial segments of the vertebral artery (VA). We report a case where a nondominant isolated vertebral artery, which terminated in PICA, was stenotic. This resulted in brainstem-lower cerebellar ischemia, corrected with balloon angioplasty. Case description A 62-year-old male presented primarily with transient vertigo, syncope, and dizziness and was diagnosed with transient ischemic attack. Angiography of the left vertebral artery (VA) demonstrated a small-caliber vessel terminating in PICA with a 90% stenosis at the C6 level. Angioplasty of the left VA was performed with excellent resolution of the stenosis. Conclusions This case illustrates cerebellar insufficiency in a unique case where the PICA was isolated, supplied by a small- caliber VA. Correction of the stenosis improved the patient's symptomatology and prevented an inferior brainstem-cerebellar infarction.

NT69L, a neurotensin analog that crosses the blood–brain barrier, reduces body temperature, reverses apomorphine-induced climbing, haloperidol-induced catalepsy, and d -amphetamine- and cocaine-induced locomotor activity in rats. In this study we tested the development of tolerance to these effects of NT69L in rats. The blockade of apomorphine-induced climbing behavior and d -amphetamine- and cocaine-induced hyperactivity seen after a single acute injection did not show significant change with repeated daily injections of NT69L. Thus, for example, NT69L after five daily injections at a fixed dosage was as effective at reversing cocaine-induced hyperactivity as after the first injection. On the other hand, repeated daily injections of NT69L resulted in a diminished hypothermic response and a diminished anticataleptic effect against haloperidol. The effect of NT69L on blood glucose, cortisol, and thyroxine (T4) were all back to control levels after five daily injections. Thus, tolerance developed to NT69L after the first injection, when it was tested for causing hypothermia, blockade of haloperidol-induced catalepsy, and change in blood glucose, cortisol and T4 levels. Since tolerance did not develop to the effects of drugs acting as direct (apomorphine) or indirect ( d -amphetamine and cocaine) agonists at dopamine receptors over the course of 5 days, these findings suggest a selective role of neurotensin in the modulation of dopamine neurotransmission. Furthermore, due to the lack of development of tolerance, NT69L or similar analogs might be useful in modulating certain behavioral effects of psychostimulants or have potential use as an antipsychotic drug in humans.

Synovial fluid (SF) is a dynamic reservoir for proteins originating from serum, synovial tissue, and cartilage. The composition of the SF proteome may reflect the pathophysiological conditions affecting the circulatory system and cartilage. Our long-term goal is to identify reliable protein markers for osteoarthritis (OA) in SF. We first evaluated the pattern of SF proteins on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) as a function of protein loading, pH range for isoelectric focusing, and concentration of acrylamide in SDS-PAGE. Removal of albumin and γ- globulins from the samples did not improve the detection of protein spots on 2D-PAGE. The repeatability of protein spot intensity was tested by triplicate 2D-PAGE of a given sample; these experiments showed low intrasample variability (correlation coefficients 0.89–0.95). Differences between multiple samples were tested by comparing the 2D-PAGE of four samples. These experiments showed slightly greater variation between samples (correlation coefficients 0.85–0.93) and a number of differentially expressed proteins. The intensity of 18 protein spots differed more than fivefold, and the intensity of nine protein spots differed more than 100-fold. These results show that 2D-PAGE can be used under standard conditions to screen SF samples and identify a small subset of proteins in SF that are potential markers associated with OA.

Experimental autoimmune thyroiditis (EAT) can be induced in genetically susceptible mice by immunization with the self antigen, thyroglobulin (Tg). Since susceptibility is linked to H2 class II molecules, we have generated human leukocyte antigen (HLA) class II transgenic mice to study potential HLA associations with Hashimoto's thyroiditis. DR3 (HLA-DRA/DRB1*0301) and DQ8 (HLA-DQA1*0301/DQB1*0302) transgenes were introduced into class II-negative Ab(0)/B10 and Ab(0) nonobese diabetic (Ab(0)/NOD) mice. Previous work had shown that DR3 transgenic mice were susceptible to both mouse Tg and human Tg-induced EAT, whereas DQ8 transgenic mice were moderately susceptible only to human Tg induction. In this report, we examined the effect of DQ8 transgene on mouse Tg- and human Tg-induced EAT in double transgenic DR3/DQ8 mice. After mouse Tg induction, thyroiditis in DR3(+)DQ8(+) Ab(0)/B10 mice was significantly less severe than in DR3(+) mice but more severe than in DQ8(+) mice. No difference in thyroiditis was observed between DR3(+) and DR3(+)DQ8(+) mice in another background strain, Ab(0)/NOD. However, after immunization with human Tg, DQ8 coexpression downregulated thyroiditis severity, compared to DR3(+) mice, whereas thyroiditis was more extensive than in DQ8(+) mice. Thus, depending on the background strain and the Tg used to induce disease, the presence of the DQ8 transgene can reduce thyroiditis mediated by DR3 molecules.

The results of the VeGAS 2 trial further extend knowledge of the AngioJet device and demonstrate benefits in success, safety, and greater convenience to the patient and physician due to a single, combined procedure for thrombus removal and definitive intervention in this high-risk patient group. AngioJet should be regarded as the therapy of choice for treatment of thrombotic SVGs or native coronary arteries.

We report preliminary data for three previously unrecognized hemoglobin (Hb) variants due to mutations of the α1-globin gene, that were initially ascertained by either chromatographic, electrophore...

The aim of this study was to compare the stroke/death rates between proximal embolic protection devices (P-EPDs) and distal filter embolic protection devices (F-EPDs) in elective carotid artery stenting (CAS).P-EPDs have theoretical advantages that may make them superior to F-EPDs for stroke prevention during CAS.We examined 10,246 consecutive elective CAS procedures performed with embolic protection in the NCDR CARE registry between January 2009 and March 2013. We analyzed crude and propensity-matched rates of in-hospital combined death/stroke in patients treated with P-EPDs versus F-EPDs. Secondary analyses included 30-day adverse event rates and stroke rates by the involved cerebrovascular territory.P-EPDs were used in 590 of 10,246 cases (5.8%). Patients treated with P-EPDs had higher rates of symptomatic lesion status (46.8% vs. 39.7%, p0.001), atrial fibrillation/flutter (16.1% vs. 13.0%, p=0.03), and history of a neurological event (51.2% vs. 46.6%, p=0.03). In unadjusted and propensity-matched analyses, differences in in-hospital stroke/death between P-EPD and F-EPD cohorts were nonsignificant (1.5% vs. 2.4%, p=0.16 and 1.6% vs. 2.0%, p=0.56, respectively). For patients with available data (n=7,693, 75.1%), 30-day adverse events rates were similar for P-EPDs and F-EPDs before (2.5% vs. 4.2%, p=0.07) and after (2.7% vs. 4.0%, p=0.22) propensity matching.Use of a P-EPD during CAS was associated with low rates of in-hospital stroke/death similar to those with an F-EPD in the first comparative effectiveness study of the devices. An adequately powered randomized trial comparing clinical outcomes between these devices is unlikely to be feasible.

Post-myocardial infarction ventricular septal defect is a devastating complication of ST-elevation myocardial infarction. Although surgical intervention is considered the gold standard for treatment, it carries high morbidity and mortality rates. We present 2 cases that illustrate the application of percutaneous closure of a post-myocardial infarction ventricular septal defect: the first in a patient who had undergone prior surgical closure and then developed a new shunt, and the second as a bridge to definitive surgery in a critically ill patient.

Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of ESRD. Affected individuals inherit a defective copy of either PKD1 or PKD2, which encode polycystin-1 (PC1) or polycystin-2 (PC2), respectively. PC1 and PC2 are secreted on urinary exosome-like vesicles (ELVs) (100-nm diameter vesicles), in which PC1 is present in a cleaved form and may be complexed with PC2. Here, label-free quantitative proteomic studies of urine ELVs in an initial discovery cohort (13 individuals with PKD1 mutations and 18 normal controls) revealed that of 2008 ELV proteins, 9 (0.32%) were expressed at significantly different levels in samples from individuals with PKD1 mutations compared to controls (P