Cell Surface Protein Disulfide Isomerase Regulates Natriuretic Peptide Generation of Cyclic Guanosine Monophosphate

Rationale

The family of natriuretic peptides (NPs), including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), exert important and diverse actions for cardiovascular and renal homeostasis. The autocrine and paracrine functions of the NPs are primarily mediated through the cellular membrane bound guanylyl cyclase-linked receptors GC-A (NPR-A) and GC-B (NPR-B). As the ligands and receptors each contain disulfide bonds, a regulatory role for the cell surface protein disulfide isomerase (PDI) was investigated.

Objective

We utilized complementary in vitro and in vivo models to determine the potential role of PDI in regulating the ability of the NPs to generate its second messenger, cyclic guanosine monophosphate.

Methods and Results

Inhibition of PDI attenuated the ability of ANP, BNP and CNP to generate cGMP in human mesangial cells (HMCs), human umbilical vein endothelial cells (HUVECs), and human aortic smooth muscle cells (HASMCs), each of which were shown to express PDI. In LLC-PK1 cells, where PDI expression was undetectable by immunoblotting, PDI inhibition had a minimal effect on cGMP generation. Addition of PDI to cultured LLC-PK1 cells increased intracellular cGMP generation mediated by ANP. Inhibition of PDI in vivo attenuated NP-mediated generation of cGMP by ANP. Surface Plasmon Resonance demonstrated modest and differential binding of the natriuretic peptides with immobilized PDI in a cell free system. However, PDI was shown to co-localize on the surface of cells with GC-A and GC-B by co-immunoprecpitation and immunohistochemistry.

Conclusion

These data demonstrate for the first time that cell surface PDI expression and function regulate the capacity of natriuretic peptides to generate cGMP through interaction with their receptors.     

Nitroxyl (HNO) stimulates soluble guanylyl cyclase to suppress cardiomyocyte hypertrophy and superoxide generation

Background

New therapeutic targets for cardiac hypertrophy, an independent risk factor for heart failure and death, are essential. HNO is a novel redox sibling of NO• attracting considerable attention for the treatment of cardiovascular disorders, eliciting cGMP-dependent vasodilatation yet cGMP-independent positive inotropy. The impact of HNO on cardiac hypertrophy (which is negatively regulated by cGMP) however has not been investigated.

Methods

Neonatal rat cardiomyocytes were incubated with angiotensin II (Ang II) in the presence and absence of the HNO donor Angeli''s salt (sodium trioxodinitrate) or B-type natriuretic peptide, BNP (all 1 µmol/L). Hypertrophic responses and its triggers, as well as cGMP signaling, were determined.

Results

We now demonstrate that Angeli''s salt inhibits Ang II-induced hypertrophic responses in cardiomyocytes, including increases in cardiomyocyte size, de novo protein synthesis and β-myosin heavy chain expression. Angeli''s salt also suppresses Ang II induction of key triggers of the cardiomyocyte hypertrophic response, including NADPH oxidase (on both Nox2 expression and superoxide generation), as well as p38 mitogen-activated protein kinase (p38MAPK). The antihypertrophic, superoxide-suppressing and cGMP-elevating effects of Angeli''s salt were mimicked by BNP. We also demonstrate that the effects of Angeli''s salt are specifically mediated by HNO (with no role for NO• or nitrite), with subsequent activation of cardiomyocyte soluble guanylyl cyclase (sGC) and cGMP signaling (on both cGMP-dependent protein kinase, cGK-I and phosphorylation of vasodilator-stimulated phosphoprotein, VASP).

Conclusions

Our results demonstrate that HNO prevents cardiomyocyte hypertrophy, and that cGMP-dependent NADPH oxidase suppression contributes to these antihypertrophic actions. HNO donors may thus represent innovative pharmacotherapy for cardiac hypertrophy.     

A novel small molecule target in human airway smooth muscle for potential treatment of obstructive lung diseases: a staged high-throughput biophysical screening

Background

A newly identified mechanism of smooth muscle relaxation is the interaction between the small heat shock protein 20 (HSP20) and 14-3-3 proteins. Focusing upon this class of interactions, we describe here a novel drug target screening approach for treating airflow obstruction in asthma.

Methods

Using a high-throughput fluorescence polarization (FP) assay, we screened a library of compounds that could act as small molecule modulators of HSP20 signals. We then applied two quantitative, cell-based biophysical methods to assess the functional efficacy of these molecules and rank-ordered their abilities to relax isolated human airway smooth muscle (ASM). Scaling up to the level of an intact tissue, we confirmed in a concentration-responsive manner the potency of the cell-based hit compounds.

Results

Among 58,019 compound tested, 268 compounds caused 20% or more reduction of the polarized emission in the FP assay. A small subset of these primary screen hits, belonging to two scaffolds, caused relaxation of isolated ASM cell in vitro and attenuated active force development of intact tissue ex vivo.

Conclusions

This staged biophysical screening paradigm provides proof-of-principle for high-throughput and cost-effective discovery of new small molecule therapeutic agents for obstructive lung diseases.     

The Arabidopsis Wall Associated Kinase-Like 10 Gene Encodes a Functional Guanylyl Cyclase and Is Co-Expressed with Pathogen Defense Related Genes

Background

Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, guanosine 3′,5′-cyclic monophosphate (cGMP), has long been known to be an essential signaling molecule in many different physiological processes in higher plants, including biotic stress responses. To date, however, the guanylyl cyclase (GC) enzymes that catalyze the formation of cGMP from GTP have largely remained elusive in higher plants.

Principal Findings

We have identified an Arabidopsis receptor type wall associated kinase–like molecule (AtWAKL10) as a candidate GC and provide experimental evidence to show that the intracellular domain of AtWAKL10_431–700 can generate cGMP in vitro. Further, we also demonstrate that the molecule has kinase activity indicating that AtWAKL10 is a twin-domain catalytic protein. A co-expression and stimulus-specific expression analysis revealed that AtWAKL10 is consistently co-expressed with well characterized pathogen defense related genes and along with these genes is induced early and sharply in response to a range of pathogens and their elicitors.

Conclusions

We demonstrate that AtWAKL10 is a twin-domain, kinase-GC signaling molecule that may function in biotic stress responses that are critically dependent on the second messenger cGMP.     

Increased Light Exposure Alleviates One Form of Photoreceptor Degeneration Marked by Elevated Calcium in the Dark

Background

In one group of gene mutations that cause photoreceptor degeneration in human patients, guanylyl cyclase is overactive in the dark. The ensuing excess opening of cGMP-gated cation channels causes intracellular calcium to rise to toxic levels. The Y99C mutation in guanylate cyclase-activating protein 1 (GCAP1) has been shown to act this way. We determined whether prolonged light exposure, which lowers cGMP levels through activation of phototransduction, might protect photoreceptors in a line of transgenic mice carrying the GCAP1-Y99C.

Methodology/Principal Findings

We reared cohorts of GCAP1-Y99C transgenic mice under standard cyclic, constant dark and constant light conditions. Mouse eyes were analyzed by histology and by immunofluorescence for GFAP upregulation, a non-specific marker for photoreceptor degeneration. Full-field electroretinograms (ERGs) were recorded to assess retinal function. Consistent with our hypothesis, constant darkness accelerated disease, while continuous lighting arrested photoreceptor degeneration.

Conclusions/Significance

In contrast to most forms of retinal degeneration, which are exacerbated by increased exposure to ambient light, a subset with mutations that cause overly active guanylyl cyclase and high intracellular calcium benefitted from prolonged light exposure. These findings may have therapeutic implications for patients with these types of genetic defects.     

Bioactivity Guided Fractionation of Leaves Extract of Nyctanthes arbor tristis (Harshringar) against P falciparum

Background

Nyctanthes arbor-tristis (Harshringar, Night Jasmine) has been traditionally used in Ayurveda, Unani and other systems of medicine in India. The juice of its leaves has been used by various tribal populations of India in treatment of fevers resembling malaria.

Aim of the study

This work reports the antiplasmodial activity guided fractionation of Harshringar leaves extract.

Methodology

Crude ethanolic Harshringar leaves extract and its RPHPLC purified fractions were studied for antiplasmodial potency against 3D7 (CQ sensitive) and Dd2 (CQ resistant) strains of P.falciparum and subsequently subjected to bioassay guided fractionation using reverse phase chromatography to pursue the isolation of active fractions.

Principal Findings

Harshringar crude leaves extract and some of its RPHPLC purified fractions exhibited promising antiplasmodial potency against 3D7 and Dd2 strains of P.falciparum.

Conclusions

The present study has provided scientific validity to the traditional use of leaves extract of Harshringar against malaria leading to the conclusion that this plant holds promise with respect to antimalarial phytotherapy. This is the first scientific report of antiplasmodial activity of RPHPLC fractions of Harshringar leaves extract against P.falciparum strains.     

Direct Immunochemiluminescent Assay for proBNP and Total BNP in Human Plasma proBNP and Total BNP Levels in Normal and Heart Failure

Background

Recent studies have shown that in addition to brain (or B-type) natriuretic peptide (BNP) and the N-terminal proBNP fragment, levels of intact proBNP are also increased in heart failure. Moreover, present BNP immunoassays also measure proBNP, as the anti-BNP antibody cross-reacts with proBNP. It is important to know the exact levels of proBNP in heart failure, because elevation of the low-activity proBNP may be associated with the development of heart failure.

Methodology/Principal Findings

We therefore established a two-step immunochemiluminescent assay for total BNP (BNP+proBNP) and proBNP using monoclonal antibodies and glycosylated proBNP as a standard. The assay enables measurement of plasma total BNP and proBNP within only 7 h, without prior extraction of the plasma. The detection limit was 0.4 pmol/L for a 50-µl plasma sample. Within-run CVs ranged from 5.2%–8.0% in proBNP assay and from 7.0%–8.4% in total BNP assay, and between-run CVs ranged from 5.3–7.4% in proBNP assay and from 2.9%–9.5% in total BNP assay, respectively. The dilution curves for plasma samples showed good linearity (correlation coefficients = 0.998–1.00), and analytical recovery was 90–101%. The mean total BNP and proBNP in plasma from 116 healthy subjects were 1.4±1.2 pM and 1.0±0.7 pM, respectively, and were 80±129 pM and 42±70 pM in 32 heart failure patients. Plasma proBNP levels significantly correlate with age in normal subjects.

Conclusions/Significance

Our immunochemiluminescent assay is sufficiently rapid and precise for routine determination of total BNP and proBNP in human plasma.     

Drug discovery for Duchenne muscular dystrophy via utrophin promoter activation screening

Background

Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by mutations in dystrophin, a muscle cytoskeletal protein. Utrophin is a homologue of dystrophin that can functionally compensate for its absence when expressed at increased levels in the myofibre, as shown by studies in dystrophin-deficient mice. Utrophin upregulation is therefore a promising therapeutic approach for DMD. The use of a small, drug-like molecule to achieve utrophin upregulation offers obvious advantages in terms of delivery and bioavailability. Furthermore, much of the time and expense involved in the development of a new drug can be eliminated by screening molecules that are already approved for clinical use.

Methodology/Principal Findings

We developed and validated a cell-based, high-throughput screening assay for utrophin promoter activation, and used it to screen the Prestwick Chemical Library of marketed drugs and natural compounds. Initial screening produced 20 hit molecules, 14 of which exhibited dose-dependent activation of the utrophin promoter and were confirmed as hits. Independent validation demonstrated that one of these compounds, nabumetone, is able to upregulate endogenous utrophin mRNA and protein, in C2C12 muscle cells.

Conclusions/Significance

We have developed a cell-based, high-throughput screening utrophin promoter assay. Using this assay, we identified and validated a utrophin promoter-activating drug, nabumetone, for which pharmacokinetics and safety in humans are already well described, and which represents a lead compound for utrophin upregulation as a therapy for DMD.     

Inhibition of striatal soluble guanylyl cyclase-cGMP signaling reverses basal ganglia dysfunction and akinesia in experimental parkinsonism

Objective

There is clearly a necessity to identify novel non-dopaminergic mechanisms as new therapeutic targets for Parkinson''s disease (PD). Among these, the soluble guanylyl cyclase (sGC)-cGMP signaling cascade is emerging as a promising candidate for second messenger-based therapies for the amelioration of PD symptoms. In the present study, we examined the utility of the selective sGC inhibitor 1H-[1], [2], [4] oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) for reversing basal ganglia dysfunction and akinesia in animal models of PD.

Methods

The utility of the selective sGC inhibitor ODQ for reversing biochemical, electrophysiological, histochemical, and behavioral correlates of experimental PD was performed in 6-OHDA-lesioned rats and mice chronically treated with MPTP.

Results

We found that one systemic administration of ODQ is sufficient to reverse the characteristic elevations in striatal cGMP levels, striatal output neuron activity, and metabolic activity in the subthalamic nucleus observed in 6-OHDA-lesioned rats. The latter outcome was reproduced after intrastriatal infusion of ODQ. Systemic administration of ODQ was also effective in improving deficits in forelimb akinesia induced by 6-OHDA and MPTP.

Interpretation

Pharmacological inhibition of the sGC-cGMP signaling pathway is a promising non-dopaminergic treatment strategy for restoring basal ganglia dysfunction and attenuating motor symptoms associated with PD.     

Negative Interference by Rheumatoid Factor of Plasma B-Type Natriuretic Peptide in Chemiluminescent Microparticle Immunoassays

Background

The chemiluminescent microparticle immunoassay (CMIA) is widely used for the quantitative determination of B-type natriuretic peptide (BNP) in human ethylenediaminetetraacetic acid plasma. Rheumatoid factor (RF) is usually thought to result in a positive interference in immunoassays, but it is not clear whether its presence in plasma can lead to interferences in the CMIA of BNP.

Methods

The estimation of BNP recovery was carried out by diluting high-concentration BNP samples with RF-positive or RF-negative plasma at a ratio of 1∶9. The diluted samples were then tested using the ARCHITECT i2000 System and ARCHITECT BNP Reagent Kits and the recovery was then calculated.

Results

When the RF level ranged from 48 to 1420 IU/mL, the average recovery of BNP was 79.29% and 91.61% in the RF-positive and RF-negative plasma samples, respectively, and was thus significantly lower in the group of RF-positive plasma samples than in the group of RF-negative plasma samples. At a dilution of 1∶16, the measured BNP level increased by >36% in six of the seven RF-positive plasma samples. The recovery of BNP increased significantly in the RF-positive plasma samples after pretreatment with IgG-sensitive latex particles. In addition, The BNP recovery was not significantly related to the plasma RF at concentrations ranging from 48 to 2720 IU/mL.

Conclusions

Measurement of BNP by CMIA is susceptible to interference from RF leading to predominantly (but not exclusively) lower results. Pretreatment of samples with blocking reagents is advisable prior to the initiation of denying patient''s necessary treatment.     

BNP but Not s-cTnln Is Associated with Cardioembolic Aetiology and Predicts Short and Long Term Prognosis after Cerebrovascular Events

Background

We analyzed the prognostic value of b-type natriuretic peptide (BNP) and sensitive cardiac Troponin (s-cTnI) in patients with ischemic stroke or transient ischemic attack (TIA) and their significance in predicting stroke aetiology.

Methods

In a prospectively enrolled cohort we measured BNP and s-cTnI levels upon admission. Primary endpoints were mortality, unfavorable functional outcome and stroke recurrence after 90 days and after 12 months. Secondary endpoint was cardioembolic aetiology.

Results

In 441 patients BNP but not s-cTnI remained an independent predictor for death with an adjusted HR of 1.2 (95% CI 1.1–1.4) after 90 days and 1.2 (95% CI 1.0–1.3) after one year. The comparison of the Area under Receiver Operating Characteristic (AUROC) of model A (age, NIHSS) and model B (age, NIHSS, BNP) showed an improvement in the prediction of mortality (0.85 (95% CI 0.79–0.90) vs. 0.86 (95% CI 0.81–0.92), Log Rank p = 0.004). Furthermore the category free net reclassification improvement (cfNRI) when adding BNP to the multivariate model was 57.5%, p<0.0001. For the prediction of functional outcome or stroke recurrence both markers provided no incremental value. Adding BNP to a model including age, atrial fibrillation and heart failure lead to a higher discriminatory accuracy for identification of cardioembolic stroke than the model without BNP (AUC 0.75 (95% CI 0.70–0.80) vs. AUC 0.79, (95% CI 0.75–0.84), p = 0.008).

Conclusion

BNP is an independent prognostic maker for overall mortality in patients with ischemic stroke or TIA and may improve the diagnostic accuracy to identify cardioembolic aetiology.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00390962","term_id":"NCT00390962"}}NCT00390962     

Evidence of Uncoupling between Renal Dysfunction and Injury in Cardiorenal Syndrome: Insights from the BIONICS Study

Objective

The objective of the study was to assess urinary biomarkers of renal injury for their individual or collective ability to predict Worsening renal function (WRF) in patients with acutely decompensated heart failure (ADHF).

Methods

In a prospective, blinded international study, 87 emergency department (ED) patients with ADHF were evaluated with biomarkers of cardiac stretch (B type natriuretic peptide [BNP] and its amino terminal equivalent [NT-proBNP], ST2), biomarkers of renal function (creatinine, estimated glomerular filtration rate [eGFR]) and biomarkers of renal injury (plasma neutrophil gelatinase associated lipocalin [pNGAL], urine kidney injury molecule-1 [KIM-1], urine N-acetyl-beta-D-glucosaminidase [NAG], urine Cystatin C, urine fibrinogen). The primary endpoint was WRF.

Results

26% developed WRF; baseline characteristics of subjects who developed WRF were generally comparable to those who did not. Biomarkers of renal function and urine biomarkers of renal injury were not correlated, while urine biomarkers of renal injury correlated between each other. Biomarker concentrations were similar between patients with and without WRF except for baseline BNP. Although plasma NGAL was associated with the combined endpoint, none of the biomarker showed predictive accuracy for WRF.

Conclusions

In ED patients with ADHF, urine biomarkers of renal injury did not predict WRF. Our data suggest that a weak association exists between renal dysfunction and renal injury in this setting (Clinicaltrials.gov NCT#0150153).     

Effect of Insulin Analogues on Insulin/IGF1 Hybrid Receptors: Increased Activation by Glargine but Not by Its Metabolites M1 and M2

Background

In diabetic patients, the pharmacokinetics of injected human insulin does not permit optimal control of glycemia. Fast and slow acting insulin analogues have been developed, but they may have adverse properties, such as increased mitogenic or anti-apoptotic signaling. Insulin/IGF1 hybrid receptors (IR/IGF1R), present in most tissues, have been proposed to transmit biological effects close to those of IGF1R. However, the study of hybrid receptors is difficult because of the presence of IR and IGF1R homodimers. Our objective was to perform the first study on the pharmacological properties of the five marketed insulin analogues towards IR/IGF1R hybrids.

Methodology

To study the effect of insulin analogues on IR/IGF1R hybrids, we used our previously developed Bioluminescence Resonance Energy Transfer (BRET) assay that permits specific analysis of the pharmacological properties of hybrid receptors. Moreover, we have developed a new, highly sensitive BRET-based assay to monitor phophatidylinositol-3 phosphate (PIP₃) production in living cells. Using this assay, we performed a detailed pharmacological analysis of PIP₃ production induced by IGF1, insulin and insulin analogues in living breast cancer-derived MCF-7 and MDA-MB231 cells.

Results

Among the five insulin analogues tested, only glargine stimulated IR/IGF1R hybrids with an EC50 that was significantly lower than insulin and close to that of IGF1. Glargine more efficiently stimulated PIP₃ production in MCF-7 cells but not in MDA-MB231 cells as compared to insulin. In contrast, glargine metabolites M1 and M2 showed lower potency for hybrid receptors stimulation, PIP₃ production, Akt and Erk1/2 phosphorylation and DNA synthesis in MCF-7 cells, compared to insulin.

Conclusion

Glargine, possibly acting through IR/IGF1R hybrids, displays higher potency, whereas its metabolites M1 and M2 display lower potency than insulin for the stimulation of proliferative/anti-apoptotic pathways in MCF-7 cells.     

Gene Expression of ANP,BNP and ET-1 in the Heart of Rats during Pulmonary Embolism

Aims

Atrial natriuretic petide (ANP), brain natriuretic peptide (BNP) and endothelin-1 (ET-1) may reflect the severity of right ventricular dysfunction (RVD) in patients with pulmonary embolism (PE). The exact nature and source of BNP, ANP and ET-1 expression and secretion following PE has not previously been studied.

Methods and Results

Polystyrene microparticles were injected to induce PE in rats. Gene expression of BNP, ANP and ET-1 were determined in the 4 cardiac chambers by quantitative real time polymerase chain reaction (QPCR). Plasma levels of ANP, BNP, ET-1 and cardiac troponin I (TNI) were measured in plasma. PE dose-dependently increased gene expression of ANP and BNP in the right ventricle (RV) and increased gene expression of ANP in the right atrium (RA). In contrast PE dose-dependently decreased BNP gene expression in both the left ventricle (LV) and the left atrium (LA). Plasma levels of BNP, TNI and ET-1 levels dose-dependently increased with the degree of PE.

Conclusion

We found a close correlation between PE degree and gene-expression of ANP, and BNP in the cardiac chambers with a selective increase in the right chambers of the heart.The present data supports the idea of natriuretic peptides as valuable biomarkers of RVD in PE.     

CSF concentrations of cAMP and cGMP are lower in patients with Creutzfeldt-Jakob disease but not Parkinson's disease and amyotrophic lateral sclerosis

Background

The cyclic nucleotides cyclic adenosine-3′,5′-monophosphate (cAMP) and cyclic guanosine-3′,5′-monophosphate (cGMP) are important second messengers and are potential biomarkers for Parkinson''s disease (PD), amyotrophic lateral sclerosis (ALS) and Creutzfeldt-Jakob disease (CJD).

Methodology/Principal Findings

Here, we investigated by liquid chromatography/tandem mass spectrometry (LC-MS/MS) the cerebrospinal fluid (CSF) concentrations of cAMP and cGMP of 82 patients and evaluated their diagnostic potency as biomarkers. For comparison with a well-accepted biomarker, we measured tau concentrations in CSF of CJD and control patients. CJD patients (n = 15) had lower cAMP (−70%) and cGMP (−55%) concentrations in CSF compared with controls (n = 11). There was no difference in PD, PD dementia (PDD) and ALS cases. Receiver operating characteristic (ROC) curve analyses confirmed cAMP and cGMP as valuable diagnostic markers for CJD indicated by the area under the curve (AUC) of 0.86 (cAMP) and 0.85 (cGMP). We calculated a sensitivity of 100% and specificity of 64% for cAMP and a sensitivity of 67% and specificity of 100% for cGMP. The combination of both nucleotides increased the sensitivity to 80% and specificity to 91% for the term cAMPxcGMP (AUC 0.92) and to 93% and 100% for the ratio tau/cAMP (AUC 0.99).

Conclusions/Significance

We conclude that the CSF determination of cAMP and cGMP may easily be included in the diagnosis of CJD and could be helpful in monitoring disease progression as well as in therapy control.     

Increased Cardiac Myocyte PDE5 Levels in Human and Murine Pressure Overload Hypertrophy Contribute to Adverse LV Remodeling

Background

The intracellular second messenger cGMP protects the heart under pathological conditions. We examined expression of phosphodiesterase 5 (PDE5), an enzyme that hydrolyzes cGMP, in human and mouse hearts subjected to sustained left ventricular (LV) pressure overload. We also determined the role of cardiac myocyte-specific PDE5 expression in adverse LV remodeling in mice after transverse aortic constriction (TAC).

Methodology/Principal Findings

In patients with severe aortic stenosis (AS) undergoing valve replacement, we detected greater myocardial PDE5 expression than in control hearts. We observed robust expression in scattered cardiac myocytes of those AS patients with higher LV filling pressures and BNP serum levels. Following TAC, we detected similar, focal PDE5 expression in cardiac myocytes of C57BL/6NTac mice exhibiting the most pronounced LV remodeling. To examine the effect of cell-specific PDE5 expression, we subjected transgenic mice with cardiac myocyte-specific PDE5 overexpression (PDE5-TG) to TAC. LV hypertrophy and fibrosis were similar as in WT, but PDE5-TG had increased cardiac dimensions, and decreased dP/dt_max and dP/dt_min with prolonged tau (P<0.05 for all). Greater cardiac dysfunction in PDE5-TG was associated with reduced myocardial cGMP and SERCA2 levels, and higher passive force in cardiac myocytes in vitro.

Conclusions/Significance

Myocardial PDE5 expression is increased in the hearts of humans and mice with chronic pressure overload. Increased cardiac myocyte-specific PDE5 expression is a molecular hallmark in hypertrophic hearts with contractile failure, and represents an important therapeutic target.     

Apparent B-type natriuretic peptide selectivity in the kidney due to differential processing

Two natriuretic peptides, atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), are found principally in the heart. In preliminary experiments with mouse kidney cells or slices, we found mouse BNP1-45 much more potent than ANP1-28 in causing elevations of cGMP (>50-fold). The guanylyl cyclase-A (GC-A) receptor has been suggested to represent the primary means by which both peptides signal. In cultured cells overexpressing GC-A, BNP and ANP were almost equivalent in potency, suggesting that a receptor unique for BNP exists in the kidney. However, in mice lacking the GC-A gene, neither BNP nor ANP significantly elevated cGMP in kidney slices. Phosphoramidon, a neutral endopeptidase inhibitor, shifted the apparent potency of ANP to values equivalent to that of BNP, suggesting these kidney cell/slices rapidly degrade ANP but not BNP. Mass spectroscopic analysis confirmed that ANP is rapidly cleaved at the first cysteine of the disulfide ring, whereas BNP is particularly stable to such cleavage. Other tissues (heart, aorta) failed to significantly degrade ANP or BNP, and therefore the kidney-specific degradation of ANP provides a mechanism for preferential regulation of kidney function by BNP independent of peripheral ANP concentration.     

Development of a Rapid Agglutination Latex Test for Diagnosis of Enteropathogenic and Enterohemorrhagic Escherichia coli Infection in Developing World: Defining the Biomarker,Antibody and Method

Background

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC/EHEC) are human intestinal pathogens responsible for diarrhea in both developing and industrialized countries. In research laboratories, EPEC and EHEC are defined on the basis of their pathogenic features; nevertheless, their identification in routine laboratories is expensive and laborious. Therefore, the aim of the present work was to develop a rapid and simple assay for EPEC/EHEC detection. Accordingly, the EPEC/EHEC-secreted proteins EspA and EspB were chosen as target antigens.

Methodology

First, we investigated the ideal conditions for EspA/EspB production/secretion by ELISA in a collection of EPEC/EHEC strains after cultivating bacterial isolates in Dulbecco’s modified Eagle’s medium (DMEM) or DMEM containing 1% tryptone or HEp-2 cells-preconditioned DMEM, employing either anti-EspA/anti-EspB polyclonal or monoclonal antibodies developed and characterized herein. Subsequently, a rapid agglutination latex test (RALT) was developed and tested with the same collection of bacterial isolates.

Principal findings

EspB was defined as a biomarker and its corresponding monoclonal antibody as the tool for EPEC/EHEC diagnosis; the production of EspB was better in DMEM medium. RALT assay has the sensitivity and specificity required for high-impact diagnosis of neglected diseases in the developing world.

Conclusion

RALT assay described herein can be considered an alternative assay for diarrhea diagnosis in low-income countries since it achieved 97% sensitivity, 98% specificity and 97% efficiency.     

Do Optimal Prognostic Thresholds in Continuous Physiological Variables Really Exist? Analysis of Origin of Apparent Thresholds,with Systematic Review for Peak Oxygen Consumption,Ejection Fraction and BNP

Background

Clinicians are sometimes advised to make decisions using thresholds in measured variables, derived from prognostic studies.

Objectives

We studied why there are conflicting apparently-optimal prognostic thresholds, for example in exercise peak oxygen uptake (pVO₂), ejection fraction (EF), and Brain Natriuretic Peptide (BNP) in heart failure (HF).

Data Sources and Eligibility Criteria

Studies testing pVO₂, EF or BNP prognostic thresholds in heart failure, published between 1990 and 2010, listed on Pubmed.

Methods

First, we examined studies testing pVO₂, EF or BNP prognostic thresholds. Second, we created repeated simulations of 1500 patients to identify whether an apparently-optimal prognostic threshold indicates step change in risk.

Results

33 studies (8946 patients) tested a pVO₂ threshold. 18 found it prognostically significant: the actual reported threshold ranged widely (10–18 ml/kg/min) but was overwhelmingly controlled by the individual study population''s mean pVO₂ (r = 0.86, p<0.00001). In contrast, the 15 negative publications were testing thresholds 199% further from their means (p = 0.0001). Likewise, of 35 EF studies (10220 patients), the thresholds in the 22 positive reports were strongly determined by study means (r = 0.90, p<0.0001). Similarly, in the 19 positives of 20 BNP studies (9725 patients): r = 0.86 (p<0.0001).Second, survival simulations always discovered a “most significant” threshold, even when there was definitely no step change in mortality. With linear increase in risk, the apparently-optimal threshold was always near the sample mean (r = 0.99, p<0.001).

Limitations

This study cannot report the best threshold for any of these variables; instead it explains how common clinical research procedures routinely produce false thresholds.

Key Findings

First, shifting (and/or disappearance) of an apparently-optimal prognostic threshold is strongly determined by studies'' average pVO₂, EF or BNP. Second, apparently-optimal thresholds always appear, even with no step in prognosis.

Conclusions

Emphatic therapeutic guidance based on thresholds from observational studies may be ill-founded. We should not assume that optimal thresholds, or any thresholds, exist.