GLP-1 increases heart rate by a direct action on the sinus node.

AIMS: Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) are increasingly used to treat type 2 diabetes and obesity. Albeit cardiovascular outcomes generally improve, treatment with GLP-1 RAs is associated with increased heart rate, the mechanism of which is unclear. METHODS AND RESULTS: We employed a large animal model, the female landrace pig, and used multiple in-vivo and ex-vivo approaches including pharmacological challenges, electrophysiology and high-resolution mass spectrometry to explore how GLP-1 elicits an increase in heart rate. In anaesthetized pigs, neither cervical vagotomy, adrenergic blockers (alpha, beta or combined alpha-beta blockade), ganglionic blockade (hexamethonium) nor inhibition of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (ivabradine) abolished the marked chronotropic effect of GLP-1. GLP-1 administration to isolated perfused pig hearts also increased heart rate, which was abolished by GLP-1 receptor blockade. Electrophysiological characterization of GLP-1 effects in vivo and in isolated perfused hearts localized electrical modulation to the atria and conduction system. In isolated sinus nodes, GLP-1 administration shortened action potential cycle length of pacemaker cells and shifted the site of earliest activation. The effect was independent of HCN blockade. Collectively, these data support a direct effect of GLP-1 on GLP-1 receptors within the heart. Consistently, single nucleus RNA sequencing (snRNAseq) showed GLP-1 receptor expression in porcine pacemaker cells. Quantitative phosphoproteomics analyses of sinus node samples revealed that GLP-1 administration leads to phosphorylation changes of calcium cycling proteins of the sarcoplasmic reticulum, known to regulate heart rate. CONCLUSION: GLP-1 has direct chronotropic effects on the heart mediated by GLP-1 receptors in pacemaker cells of the sinus node, inducing changes in action potential morphology and the leading pacemaker site through a calcium signaling response characterized by PKA-dependent phosphorylation of Ca2+ cycling proteins involved in pace making. Targeting the pacemaker calcium clock may be a strategy to lower heart rate in GLP-1 RA recipients.

Novel customized age-dependent corneal membranes and interactions with biodegradable nanoparticles loaded with dexibuprofen.

Ocular inflammation is one of the most prevalent diseases in ophthalmology and it is currently treated using eye drops of nonsteroidal antiinflammatory drugs such as dexibuprofen (DXI). However, their bioavailability is low and therefore, PLGA nanoparticles constitute a suitable approach to be administered as eyedrops. Therefore, DXI has been encapsulated into PLGA nanoparticles (DXI-NPs). Although the eye, and specifically the cornea, suffers from age-related changes in its composition, current medications are not focused on these variations. Therefore, to elucidate the interaction mechanism of DXI-NPs with the cornea in relation with age, two different corneal membrane models have been developed (corresponding to adult and elder population) using lipid monolayers, large and giant unilamellar vesicles. Interactions of both DXI and DXI-NPs were studied with these models by means of Langmuir balance technique, dipole potential, anisotropy and confocal microscopy. In addition, fluorescently labelled nanoparticles were administered to mice in order to corroborate these data obtained in vitro. It was observed that DXI-NPs interact with lipid membranes through an adhesion process, mainly in the rigid regions and afterwards DXI-NPs are internalized by a wrapping process. Furthermore, differences on the dipole potential caused by DXI-NPs in each corneal membrane have been obtained due to the increase of membrane rigidity on the ECMM. Additionally, it can be confirmed that DXI-NPs adhere to Lo phase and also inside the lipid membrane. Finally, in vitro and in vivo results corroborate that DXI-NPs are adhered to the more ordered phase. Finally, differences between interactions of DXI-NPs with the elder and adult corneal tissue were observed.

Loss of Nuclear Envelope Integrity and Increased Oxidant Production Cause DNA Damage in Adult Hearts Deficient in PKP2: A Molecular Substrate of ARVC.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by high propensity to life-threatening arrhythmias and progressive loss of heart muscle. More than 40% of reported genetic variants linked to ARVC reside in the PKP2 gene, which encodes the PKP2 protein (plakophilin-2). METHODS: We describe a comprehensive characterization of the ARVC molecular landscape as determined by high-resolution mass spectrometry, RNA sequencing, and transmission electron microscopy of right ventricular biopsy samples obtained from patients with ARVC with PKP2 mutations and left ventricular ejection fraction >45%. Samples from healthy relatives served as controls. The observations led to experimental work using multiple imaging and biochemical techniques in mice with a cardiac-specific deletion of Pkp2 studied at a time of preserved left ventricular ejection fraction and in human induced pluripotent stem cell-derived PKP2-deficient myocytes. RESULTS: Samples from patients with ARVC present a loss of nuclear envelope integrity, molecular signatures indicative of increased DNA damage, and a deficit in transcripts coding for proteins in the electron transport chain. Mice with a cardiac-specific deletion of Pkp2 also present a loss of nuclear envelope integrity, which leads to DNA damage and subsequent excess oxidant production (O2.- and H2O2), the latter increased further under mechanical stress (isoproterenol or exercise). Increased oxidant production and DNA damage is recapitulated in human induced pluripotent stem cell-derived PKP2-deficient myocytes. Furthermore, PKP2-deficient cells release H2O2 into the extracellular environment, causing DNA damage and increased oxidant production in neighboring myocytes in a paracrine manner. Treatment with honokiol increases SIRT3 (mitochondrial nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin-3) activity, reduces oxidant levels and DNA damage in vitro and in vivo, reduces collagen abundance in the right ventricular free wall, and has a protective effect on right ventricular function. CONCLUSIONS: Loss of nuclear envelope integrity and subsequent DNA damage is a key substrate in the molecular pathology of ARVC. We show transcriptional downregulation of proteins of the electron transcript chain as an early event in the molecular pathophysiology of the disease (before loss of left ventricular ejection fraction <45%), which associates with increased oxidant production (O2.- and H2O2). We propose therapies that limit oxidant formation as a possible intervention to restrict DNA damage in ARVC.

Spatial-proteomics reveals phospho-signaling dynamics at subcellular resolution.

Dynamic change in subcellular localization of signaling proteins is a general concept that eukaryotic cells evolved for eliciting a coordinated response to stimuli. Mass spectrometry-based proteomics in combination with subcellular fractionation can provide comprehensive maps of spatio-temporal regulation of protein networks in cells, but involves laborious workflows that does not cover the phospho-proteome level. Here we present a high-throughput workflow based on sequential cell fractionation to profile the global proteome and phospho-proteome dynamics across six distinct subcellular fractions. We benchmark the workflow by studying spatio-temporal EGFR phospho-signaling dynamics in vitro in HeLa cells and in vivo in mouse tissues. Finally, we investigate the spatio-temporal stress signaling, revealing cellular relocation of ribosomal proteins in response to hypertonicity and muscle contraction. Proteomics data generated in this study can be explored through https://SpatialProteoDynamics.github.io .

Netrin-1 receptor antibodies in thymoma-associated neuromyotonia with myasthenia gravis.

OBJECTIVE: To identify cell-surface antibodies in patients with neuromyotonia and to describe the main clinical implications. METHODS: Sera of 3 patients with thymoma-associated neuromyotonia and myasthenia gravis were used to immunoprecipitate and characterize neuronal cell-surface antigens using reported techniques. The clinical significance of antibodies against precipitated proteins was assessed with sera of 98 patients (neuromyotonia 46, myasthenia gravis 52, thymoma 42; 33 of them with overlapping syndromes) and 219 controls (other neurologic diseases, cancer, and healthy volunteers). RESULTS: Immunoprecipitation studies identified 3 targets, including the Netrin-1 receptors DCC (deleted in colorectal carcinoma) and UNC5A (uncoordinated-5A) as well as Caspr2 (contactin-associated protein-like 2). Cell-based assays with these antigens showed that among the indicated patients, 9 had antibodies against Netrin-1 receptors (7 with additional Caspr2 antibodies) and 5 had isolated Caspr2 antibodies. Only one of the 219 controls had isolated Caspr2 antibodies with relapsing myelitis episodes. Among patients with neuromyotonia and/or myasthenia gravis, the presence of Netrin-1 receptor or Caspr2 antibodies predicted thymoma (p < 0.05). Coexisting Caspr2 and Netrin-1 receptor antibodies were associated with concurrent thymoma, myasthenia gravis, and neuromyotonia, often with Morvan syndrome (p = 0.009). Expression of DCC, UNC5A, and Caspr2 proteins was demonstrated in paraffin-embedded thymoma samples (3) and normal thymus. CONCLUSIONS: Antibodies against Netrin-1 receptors (DCC and UNC5a) and Caspr2 often coexist and associate with thymoma in patients with neuromyotonia and myasthenia gravis. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that antibodies against Netrin-1 receptors can identify patients with thymoma (sensitivity 21.4%, specificity 100%).

Immunoproteomic studies on paediatric opsoclonus-myoclonus associated with neuroblastoma.

We aimed to identify new cell-membrane antigens implicated in opsoclonus-myoclonus with neuroblastoma. The sera of 3 out of 14 patients showed IgG electron-microscopy immunogold reactivity on SH-SY5Y neuroblastoma cells. Immunoprecipitation experiments using rat brain synaptosomes and SH-SY5Y cells led to the identification of: (1) thirty-one nuclear/cytoplasmic proteins (including antigens HuB, HuC); (2) seven neuronal membrane proteins, including the Shaw-potassium channel Kv3.3 (KCNC3), whose genetic disruption in mice causes ataxia and generalized muscle twitching. Although cell-based assays did not demonstrate direct antigenicity, our findings point to Shaw-related subfamily of the potassium voltage-gated channels complexed proteins as hypothetical antigenic targets.

Clinical and Immunological Features of Opsoclonus-Myoclonus Syndrome in the Era of Neuronal Cell Surface Antibodies.

IMPORTANCE: Most studies on opsoclonus-myoclonus syndrome (OMS) in adults are based on small case series before the era of neuronal cell surface antibody discovery. OBJECTIVE: To report the clinical and immunological features of idiopathic OMS (I-OMS) and paraneoplastic OMS (P-OMS), the occurrence of antibodies to cell surface antigens, and the discovery of a novel cell surface epitope. DESIGN, SETTING, AND PARTICIPANTS: Retrospective cohort study and laboratory investigations of 114 adult patients with OMS at a center for autoimmune neurological disorders done between January 2013 and September 2015. MAIN OUTCOMES AND MEASURES: Review of clinical records. Immunohistochemistry on rat brain and cultured neurons as well as cell-based assays were used to identify known autoantibodies. Immunoprecipitation and mass spectrometry were used to characterize novel antigens. RESULTS: Of the 114 patients (62 [54%] female; median age, 45 years; interquartile range, 32-60 years), 45 (39%) had P-OMS and 69 (61%) had I-OMS. In patients with P-OMS, the associated tumors included lung cancer (n = 19), breast cancer (n = 10), other cancers (n = 5), and ovarian teratoma (n = 8); 3 additional patients without detectable cancer were considered to have P-OMS because they had positive results for onconeuronal antibodies. Patients with I-OMS, compared with those who had P-OMS, were younger (median age, 38 [interquartile range, 31-50] vs 54 [interquartile range, 45-65] years; P < .001), presented more often with prodromal symptoms or active infection (33% vs 13%; P = .02), less frequently had encephalopathy (10% vs 29%; P = .01), and had better outcome (defined by a modified Rankin Scale score ≤ 2 at last visit; 84% vs 39%; P < .001) with fewer relapses (7% vs 24%; P= .04). Onconeuronal antibodies occurred in 13 patients (11%), mostly Ri/ANNA2 antibodies, which were detected in 7 of 10 patients (70%) with breast cancer. Neuronal surface antibodies were identified in 12 patients (11%), mainly glycine receptor antibodies (9 cases), which predominated in P-OMS with lung cancer (21% vs 5% in patients with OMS without lung cancer; P = .02); however, a similar frequency of glycine receptor antibodies was found in patients with lung cancer without OMS (13 of 65 patients [20%]). A novel cell surface epitope, human natural killer 1 (HNK-1), was the target of the antibodies in 3 patients with lung cancer and P-OMS. CONCLUSIONS AND RELEVANCE: Patients with I-OMS responded better to treatment and had fewer relapses than those with P-OMS. Older age and encephalopathy, significantly associated with P-OMS, are clinical clues suggesting an underlying tumor. Glycine receptor antibodies occur frequently in P-OMS with lung cancer, but the sensitivity and specificity are low. The HNK-1 epitope is a novel epitope in a subset of patients with P-OMS and lung cancer.

Conjugation of cell-penetrating peptides with poly(lactic-co-glycolic acid)-polyethylene glycol nanoparticles improves ocular drug delivery.

In this work, a peptide for ocular delivery (POD) and human immunodeficiency virus transactivator were conjugated with biodegradable poly(lactic-co-glycolic acid) (PGLA)-polyethylene glycol (PEG)-nanoparticles (NPs) in an attempt to improve ocular drug bioavailability. The NPs were prepared by the solvent displacement method following two different pathways. One involved preparation of PLGA NPs followed by PEG and peptide conjugation (PLGA-NPs-PEG-peptide); the other involved self-assembly of PLGA-PEG and the PLGA-PEG-peptide copolymer followed by NP formulation. The conjugation of the PEG and the peptide was confirmed by a colorimetric test and proton nuclear magnetic resonance spectroscopy. Flurbiprofen was used as an example of an anti-inflammatory drug. The physicochemical properties of the resulting NPs (morphology, in vitro release, cell viability, and ocular tolerance) were studied. In vivo anti-inflammatory efficacy was assessed in rabbit eyes after topical instillation of sodium arachidonate. Of the formulations developed, the PLGA-PEG-POD NPs were the smaller particles and exhibited greater entrapment efficiency and more sustained release. The positive charge on the surface of these NPs, due to the conjugation with the positively charged peptide, facilitated penetration into the corneal epithelium, resulting in more effective prevention of ocular inflammation. The in vitro toxicity of the NPs developed was very low; no ocular irritation in vitro (hen's egg test-chorioallantoic membrane assay) or in vivo (Draize test) was detected. Taken together, these data demonstrate that PLGA-PEG-POD NPs are promising vehicles for ocular drug delivery.

A novel non-rapid-eye movement and rapid-eye-movement parasomnia with sleep breathing disorder associated with antibodies to IgLON5: a case series, characterisation of the antigen, and post-mortem study.

BACKGROUND: Autoimmunity might be associated with or implicated in sleep and neurodegenerative disorders. We aimed to describe the features of a novel neurological syndrome associated with prominent sleep dysfunction and antibodies to a neuronal antigen. METHODS: In this observational study, we used clinical and video polysomnography to identify a novel sleep disorder in three patients referred to the Sleep Unit of Hospital Clinic, University of Barcelona, Spain, for abnormal sleep behaviours and obstructive sleep apnoea. These patients had antibodies against a neuronal surface antigen, which were also present in five additional patients referred to our laboratory for antibody studies. These five patients had been assessed with polysomnography, which was done in our sleep unit in one patient and the recording reviewed in a second patient. Two patients underwent post-mortem brain examination. Immunoprecipitation and mass spectrometry were used to characterise the antigen and develop an assay for antibody testing. Serum or CSF from 298 patients with neurodegenerative, sleep, or autoimmune disorders served as control samples. FINDINGS: All eight patients (five women; median age at disease onset 59 years [range 52-76]) had abnormal sleep movements and behaviours and obstructive sleep apnoea, as confirmed by polysomnography. Six patients had chronic progression with a median duration from symptom onset to death or last visit of 5 years (range 2-12); in four the sleep disorder was the initial and most prominent feature, and in two it was preceded by gait instability followed by dysarthria, dysphagia, ataxia, or chorea. Two patients had a rapid progression with disequilibrium, dysarthria, dysphagia, and central hypoventilation, and died 2 months and 6 months, respectively, after symptom onset. In five of five patients, video polysomnography showed features of obstructive sleep apnoea, stridor, and abnormal sleep architecture (undifferentiated non-rapid-eye-movement [non-REM] sleep or poorly structured stage N2, simple movements and finalistic behaviours, normalisation of non-REM sleep by the end of the night, and, in the four patients with REM sleep recorded, REM sleep behaviour disorder). Four of four patients had HLA-DRB1*1001 and HLA-DQB1*0501 alleles. All patients had antibodies (mainly IgG4) against IgLON5, a neuronal cell adhesion molecule. Only one of the 298 controls, who had progressive supranuclear palsy, had IgLON5 antibodies. Neuropathology showed neuronal loss and extensive deposits of hyperphosphorylated tau mainly involving the tegmentum of the brainstem and hypothalamus in the two patients studied. INTERPRETATION: IgLON5 antibodies identify a unique non-REM and REM parasomnia with sleep breathing dysfunction and pathological features suggesting a tauopathy. FUNDING: Fondo de Investigaciones Sanitarias, Centros de Investigación Biomédica en Red de enfermedades neurodegenerativas (CIBERNED) and Respiratorias (CIBERES), Ministerio de Economía y Competitividad, Fundació la Marató TV3, and the National Institutes of Health.

Longitudinally extensive transverse myelitis with AQP4 antibodies revealing ovarian teratoma.

Paraneoplastic myelitis is a rare inflammatory disorder most frequently associated with solid tumors or lymphoproliferative disorders. Patients often harbor onconeuronal antibodies and their prognosis is usually poor. Here we report a 42-year old woman with longitudinally extensive transverse myelitis and aquaporin-4 (AQP4) antibodies that led to the diagnosis of ovarian teratoma. After tumor removal and immune therapy (including corticosteroids, plasma exchange, intravenous immunoglobulins and rituximab) the patient progressively improved achieving complete recovery. Histological study of the teratoma demonstrated neural tissue containing AQP4 expressing cells and intense inflammatory infiltrates, providing evidence for a possible paraneoplastic link between both disorders.

Flurbiprofen PLGA-PEG nanospheres: role of hydroxy-ß-cyclodextrin on ex vivo human skin permeation and in vivo topical anti-inflammatory efficacy.

In this study, flurbiprofen (FB) loaded poly(d,l-lactide-co-glycolide) (PLGA) and PLGA with poly(ethylene glycol) (PLGA-PEG) nanospheres (NSs) with and without hydroxypropyl-ß-cyclodextrin (HPßCD) were developed as skin controlled delivery systems. X-ray diffraction was used to determine the physical state of the entrapped drug. Results showed that the drug in PLGA NSs was present in the form of a molecular dispersion (dissolved state) in the polymers, whereas in PLGA-PEG NSs, the drug was present in both molecular dispersion and crystalline forms. Furthermore, HPßCD provided solubilization of the free FB present on the surface of the PLGA-PEG NSs and a complete amorphosization of the drug was obtained. Optical analyses using TurbiscanLab(®) demonstrated that HPßCD provided an efficient steric stability of the NSs, preventing particle aggregation. The ex vivo permeation profiles of the NSs and conventional FB solution were evaluated using human skin. Results demonstrated that only PLGA-PEG NSs showed slight permeation improvement. However, after 24h, the FB retained in the skin was about 9-fold higher with NSs compared with the control solution, attributed to the reservoir effect of NSs acting as a depot, sustaining the drug and limiting its systemic absorption. In vivo performance of NSs was evaluated by assessing anti-inflammatory efficacy in TPA-induced mouse ear edema. Topically applied NSs significantly decreased in vivo inflammation compared to the control solution and the anti-inflammatory efficacy of HPßCD NSs was stronger than NSs without HPßCD. In vivo skin irritation evaluated by the in vivo Draize test showed no irritation of the formulations tested.

Role of hydroxypropyl-ß-cyclodextrin on freeze-dried and gamma-irradiated PLGA and PLGA-PEG diblock copolymer nanospheres for ophthalmic flurbiprofen delivery.

Poly(D,L-lactide-co-glycolide) and poly(D,L-lactide-co-glycolide) with poly(ethylene glycol) nanospheres (NSs) incorporating flurbiprofen (FB) were freeze-dried with several cryoprotective agents and sterilized by γ-irradiation. Only when 5.0% (w/v) hydroxypropyl-ß-cyclodextrin (HPßCD) was used, a complete resuspension by manual shaking and almost identical particle size of the NSs was obtained after freeze-drying. In vitro drug release and ex vivo corneal permeation of NSs with and without HPßCD were evaluated. The presence of HPßCD resulted in a reduction of burst effect, providing a more sustained release of the drug. A significant decrease in the FB transcorneal permeation of NSs containing HPßCD was obtained, related to the slower diffusion of FB observed in the in vitro results. The uptake mechanism of the NSs was examined by confocal microscopy, suggesting that NSs penetrate corneal epithelium through a transcellular pathway. Ocular tolerance was assessed in vitro and in vivo by the Eytex™ and Draize test, respectively. Long-term stability studies revealed that γ-irradiated NSs stored as freeze-dried powders maintained their initial characteristics. Stability studies of the resuspended NSs after 3 months of storage in the aqueous form showed that NSs were stable at 4°C, while formulations stored at 25°C and 40°C increased their initial particle size.

Particle sizes of colloids to be used in sentinel lymph node radiolocalization.

OBJECTIVES: To investigate particle size distribution and possible effects of colloid concentration and time after its preparation on the particle sizes of albumin nanocolloid (Nanocoll), rhenium sulfide nanocolloid (Nanocis) and tin colloid (Hepatate). METHODS: All three kits were prepared according to the manufacturers' instructions using the following volumes of decayed Tc generator eluate: 1.5 ml, 3.5 ml and 5.0 ml (albumin nanocolloid); 1.0 ml, 2.0 ml and 3.0 ml (rhenium sulfide nanocolloid); 3.0 ml, 5.0 ml and 9.0 ml (tin colloid). The particle sizes were determined by photon correlation spectroscopy inmediately after preparation, then at 30 min, 1 h, 2 h, 3 h and 4 h (all three kits) and at 6 h (Nanocoll and Hepatate). Each measurement was performed in three different preparations. RESULTS: The particle sizes of the three colloids did not change significantly after preparation throughout their period of validity. For Nanocoll, 95% of the particles were between 3 and 16 nm, approximately (mean around 8 nm). Small (< or =1 nm) but significant variations on the mean diameter were found depending on the volume of pertechnetate used in the preparation. For Nanocis, 95% of the particles were between 8 and 68 nm, approximately (mean around 23-25 nm). In this kit, there were no significant differences among colloidal suspensions prepared with different volumes of pertechnetate. For Hepatate, 95% of the particles were between 33 and 255 nm, approximately (mean, around 72-90 nm). The upper limit and the mean size increase with the volume of eluate used were from 190 nm and 73 nm (3 ml) to 255 nm and 85 nm (9 ml). CONCLUSIONS: The particle sizes of Nanocoll, Nanocis and Hepatate are very stable throughout their period of validity. For Nanocoll, 95% of particles have a diameter <16 nm, with a mean diameter around 8 nm. There are small (< or =1 nm) but significant variations on the mean diameter depending on the volume of pertechnetate used in its preparation. For Nanocis, 95% of particles have a diameter <70 nm, with a mean around 24 nm, and are independent of the volume of pertechnetate used in their preparation. For Hepatate, the upper size limit for 95% of particles is between 190 and 255 nm, with a mean between 72 and 88 nm, depending on the volume of pertechnetate used in its preparation. This tin colloid could be a good candidate for evaluating clinical suitability in sentinel node localization.