RESUMO
Transcatheter aortic valve replacement (TAVR) is performed to treat aortic stenosis and is increasingly being utilised in the low-to-intermediate-risk population. Currently, attention has shifted towards long-term outcomes, complications and lifelong maintenance of the bioprosthesis. Some patients with TAVR in-situ may develop significant coronary artery disease over time requiring invasive coronary angiography, which may be problematic with the TAVR bioprosthesis in close proximity to the coronary ostia. In addition, younger patients may require a second transcatheter heart valve (THV) to 'replace' their in-situ THV because of gradual structural valve degeneration. Implantation of a second THV carries a risk of coronary obstruction, thereby requiring comprehensive pre-procedural planning. Unlike in the pre-TAVR period, cardiac CT angiography in the post-TAVR period is not well established. However, post-TAVR cardiac CT is being increasingly utilised to evaluate mechanisms for structural valve degeneration and complications, including leaflet thrombosis. Post-TAVR CT is also expected to have a significant role in risk-stratifying and planning future invasive procedures including coronary angiography and valve-in-valve interventions. Overall, there is emerging evidence for post-TAVR CT to be eventually incorporated into long-term TAVR monitoring and lifelong planning.
Assuntos
Estenose da Valva Aórtica , Valva Aórtica , Angiografia por Tomografia Computadorizada , Angiografia Coronária , Próteses Valvulares Cardíacas , Valor Preditivo dos Testes , Substituição da Valva Aórtica Transcateter , Humanos , Substituição da Valva Aórtica Transcateter/efeitos adversos , Valva Aórtica/diagnóstico por imagem , Valva Aórtica/cirurgia , Valva Aórtica/fisiopatologia , Estenose da Valva Aórtica/diagnóstico por imagem , Estenose da Valva Aórtica/cirurgia , Estenose da Valva Aórtica/fisiopatologia , Resultado do Tratamento , Fatores de Risco , Desenho de Prótese , Bioprótese , Fatores de TempoRESUMO
To localize the unusual cardiac activities non-invasively, one has to build a prior forward model that relates the heart, torso, and detectors. This model has to be constructed to mathematically relate the geometrical and functional activities of the heart. Several methods are available to model the prior sources in the forward problem, which results in the lead field matrix generation. In the conventional technique, the lead field assumed the fixed prior sources, and the source vector orientations were presumed to be parallel to the detector plane with the unit strength in all directions. However, the anomalies cannot always be expected to occur in the same location and orientation, leading to misinterpretation and misdiagnosis. To overcome this, the work proposes a new forward model constructed using the VCG signals of the same subject. Furthermore, three transformation methods were used to extract VCG in constructing the time-varying lead fields to steer to the orientation of the source rather than just reconstructing its activities in the inverse problem. In addition, the unit VCG loop of the acute ischemia patient was extracted to observe the changes compared to the normal subject. The abnormality condition was achieved by delaying the depolarization time by 15ms. The results involving the unit vectors of VCG demonstrated the anisotropic nature of cardiac source orientations, providing information about the heart's electrical activity.
Assuntos
Eletrocardiografia , Coração , Humanos , Eletrocardiografia/métodos , Coração/fisiologia , Algoritmos , Modelos Cardiovasculares , Simulação por Computador , Isquemia Miocárdica/diagnóstico , Processamento de Sinais Assistido por ComputadorRESUMO
BACKGROUND AND AIMS: Telehealth plays an integral part in healthcare delivery. The impact of telehealth and the COVID-19 pandemic on medication prescribing and patient satisfaction with telehealth in cardiology clinics remains unknown. METHODS: A retrospective study of cardiology clinic patients at an Australian tertiary hospital was conducted; 630 patients seen before the COVID-19 pandemic (0.6% telehealth) and 678 during the pandemic (91.2% telehealth) were included. Medication changes, new prescriptions and time to obtaining prescriptions after clinic were compared. To evaluate patients' experiences, cardiology clinic patients reviewed during the pandemic were prospectively invited to participate in an electronic survey sent to their mobile phones. RESULTS: The overall rates of medication changes made in the clinic between the prepandemic and the pandemic periods did not differ significantly (26.9% vs 25.8%). Compared with prepandemic, new cardiac medication prescriptions during clinic were significantly less (9.3% vs 2.5%; P < 0.0001) and recommendations to general practitioners (GP) to initiate cardiac medications were significantly more (2.6% vs 9.1%; P < 0.0001). Time to obtaining new prescriptions was significantly longer in the pandemic cohort (median 0 days (range: 0-32) vs 10.5 days (range: 0-231); P < 0.0001). Two hundred forty-three (32.7%) patients participated in the survey; 50% reported that telehealth was at least as good as face-to-face consultations. Most patients (61.5%) were satisfied with telehealth and most (62.9%) wished to see telehealth continued postpandemic. CONCLUSION: Telehealth during the COVID-19 pandemic was associated with greater reliance on GP to prescribe cardiac medications and delays in obtaining prescriptions among cardiology clinic patients. Although most patients were satisfied with telehealth services, nearly half of the cardiac patients expressed preference towards traditional face-to-face consultations.
RESUMO
In this paper, the inverse problems of cardiac sources using analytical and probabilistic methods are solved and discussed. The standard Tikhonov regularization technique is solved initially to estimate the under-determined heart surface potentials from Magnetocardiographic (MCG) signals. The results of the deterministic method subjected to noise in the measurements are discussed and compared with the probabilistic models. Hierarchical Bayesian modeling with fixed Gaussian prior is employed to quantify the uncertainties in source reconstructions. A novel application of Variational Bayesian inference approach has been presented to estimate the heart sources. The reconstruction results of Variational Bayesian model with non-stationary priors are compared with solutions of simplistic Bayesian approach; and the performances are evaluated using Root Mean Square Error (RMSE) and correlation co-efficient metrics. The Bayesian solutions in the study are also extended to localize the MCG sources for two types of Myocardial infarction cases.
Assuntos
Magnetocardiografia , Infarto do Miocárdio , Humanos , Teorema de Bayes , Algoritmos , Coração , Infarto do Miocárdio/diagnósticoRESUMO
Recombinant adeno-associated virus (rAAV) vectors are often produced in HEK293 or Spodoptera frugiperda (Sf)-based cell lines. We compared expression profiles of "oversized" (â¼5,000 bp) and "standard-sized" (4,600 bp) rAAV5-human α1-antitrypsin (rAAV5-hA1AT) vectors manufactured in HEK293 or Sf cells and investigated molecular mechanisms mediating expression decline. C57BL/6 mice received 6 × 1013 vg/kg of vector, and blood and liver samples were collected through week 57. For all vectors, peak expression (weeks 12-24) declined by 50% to week 57. For Sf- and HEK293-produced oversized vectors, serum hA1AT was initially comparable, but in weeks 12-57, Sf vectors provided significantly higher expression. For HEK293 oversized vectors, liver genomes decreased continuously through week 57 and significantly correlated with A1AT protein. In RNA-sequencing analysis, HEK293 vector-treated mice had significantly higher inflammatory responses in liver at 12 weeks compared with Sf vector- and vehicle-treated mice. Thus, HEK293 vector genome loss led to decreased transgene protein. For Sf-produced vectors, genomes did not decrease from peak expression. Instead, vector genome accessibility significantly decreased from peak to week 57 and correlated with transgene RNA. Vector DNA interactions with active histone marks (H3K27ac/H3K4me3) were significantly reduced from peak to week 57, suggesting that epigenetic regulation impacts transgene expression of Sf-produced vectors.
Assuntos
Epigênese Genética , Insetos , Humanos , Camundongos , Animais , Células HEK293 , Camundongos Endogâmicos C57BL , RNA , MamíferosRESUMO
INTRODUCTION: Biomarkers that predict the risk of immune-mediated adverse events (irAEs) among patients with non-small cell lung cancer (NSCLC) may reduce morbidity and mortality associated with these treatments. METHODS: We carried out high resolution human leucocyte antigen (HLA)-I typing on 179 patients with NSCLC treated with anti-program death (PD)-1/program death ligand (PDL)-1. Toxicity data were collected and graded as per common terminology criteria for adverse event (CTCAE) v5.0. We used 14.8-week for landmark analysis to address lead-time bias to investigate the correlation between HLA-I/II zygosity, supertypes and alleles with irAE. Furthermore, we assessed the association for irAE with clinical benefit rate (CBR), progression-free survival (PFS) and overall survival (OS). RESULTS: Homozygosity at one or more HLA-I loci, but not HLA-II, was associated with a reduced risk of irAE (relative risk (RR) = 0.61, 95% CI 0.33-0.95, P = 0.035) especially pneumonitis or any grade 3 toxicity. Patients with HLA-A03 supertype had a higher risk of developing irAE (RR = 1.42, 95% CI 1.02-2.01, P = 0.039). The occurrence of any irAE was significantly associated with improved CBR (RR = 1.48, P < 0.0001), PFS (HR = 0.45, P = 0.0003) and OS (HR = 0.34, P < 0.0001). CONCLUSIONS: Homozygosity at one or more HLA-I loci may serve as biomarker to predict patients who are unlikely to experience severe irAEs among patients with NSCLC and treated with anti-PD1/PDL1, but less likely to derive clinical benefit. Patients with HLA-I homozygous might benefit from additional therapy.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Doenças do Sistema Imunitário , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Genótipo , Antígenos HLA/genética , Humanos , Doenças do Sistema Imunitário/epidemiologia , Fatores Imunológicos/uso terapêutico , Imunoterapia/efeitos adversos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Nivolumabe/efeitos adversos , Estudos RetrospectivosRESUMO
Quantifying the composition of viral vectors used in vaccine development and gene therapy is critical for assessing their functionality. Adeno-associated virus (AAV) vectors, which are the most widely used viral vectors for in vivo gene therapy, are typically characterized using PCR, ELISA, and analytical ultracentrifugation which require laborious protocols or hours of turnaround time. Emerging methods such as charge-detection mass spectroscopy, static light scattering, and mass photometry offer turnaround times of minutes for measuring AAV mass using optical or charge properties of AAV. Here, we demonstrate an orthogonal method where suspended nanomechanical resonators (SNR) are used to directly measure both AAV mass and aggregation from a few microliters of sample within minutes. We achieve a precision near 10 zeptograms which corresponds to 1% of the genome holding capacity of the AAV capsid. Our results show the potential of our method for providing real-time quality control of viral vectors during biomanufacturing.
Assuntos
Dependovirus , Vetores Genéticos , Capsídeo , DNA , Dependovirus/genética , Vetores Genéticos/genéticaRESUMO
Adeno associated virus (AAV) capsids are a leading modality for in vivo gene delivery. Complete and precise characterization of capsid particles, including capsid and vector genome concentration, is necessary to safely and efficaciously dose patients. Size exclusion chromatography (SEC) coupled to multiangle light scattering (MALS) offers a straightforward approach to comprehensively characterize AAV capsids. The current study demonstrates that this method provides detailed AAV characterization information, including but not limited to aggregation profile, size-distribution, capsid content, capsid molar mass, encapsidated DNA molar mass, and total capsid and vector genome titer. Currently, multiple techniques are required to generate this information, with varying accuracy and precision. In the current study, a new series of equations for SEC-MALS are used in tandem with intrinsic properties of the capsids and encapsidated DNA to quantify multiple physical AAV attributes in one 20-min run with minimal sample manipulation, high accuracy, and high precision. These novel applications designate this well-established method as a powerful tool for product development and process analytics in future gene therapy programs.
RESUMO
Control of bleeding with direct-acting oral anticoagulants (DOACs) remains an unmet clinical need. Activated superFactor V (superFVa) is an engineered activated protein C (APC)-resistant FVa variant with enhanced procoagulant activity resulting from an A2/A3 domain disulfide bond and was studied here for control of DOAC-induced bleeding. SuperFVa reversed bleeding induced by FXa inhibitors (rivaroxaban, apixaban), and the FIIa inhibitor dabigatran in BalbC mice. The blocking anti-protein C and APC [(A)PC] antibody SPC-54 also reduced FXa inhibitor induced bleeding similar to superFVa, whereas dabigatran-induced bleeding was not affected. This indicated that sufficient APC was generated to contribute to bleeding in the presence of FXa inhibitors, but not in the presence of dabigatran, suggesting that mechanisms contributing to bleeding differed for FXa and FIIa inhibitors. Despite different mechanisms contributing to bleeding, superFVa effectively reduced bleeding for all DOACs, indicating the versatility of superFVa's properties that contribute to its universal prohemostatic effects for DOAC associated bleeding. Supported by thrombin generation assays on endothelial cells in normal plasma spiked with DOACs and patient plasma anticoagulated with DOACs, 3 complementary mechanisms were identified by which superFVa achieved DOAC class-independent prohemostatic efficiency. These mechanisms are resistance to inactivation by APC, overcoming the FV activation threshold, and maximizing the efficiency of the prothrombinase complex when the available FXa is increased by FVIIa-based prohemostatics. In summary, it is this versatility of superFVa that delineates it from other prohemostatic agents as a promising class-independent rescue agent in bleeding situations associated with DOACs.
Assuntos
Fator Va , Inibidores do Fator Xa , Animais , Anticoagulantes , Células Endoteliais , Hemorragia/induzido quimicamente , Hemorragia/tratamento farmacológico , Hemorragia/prevenção & controle , Humanos , CamundongosRESUMO
The electrical impulses of the heart will generate a tiny magnetic field outside the thorax that is measured as Magnetocardiographic signals. The challenging study is to estimate the cardiac activities in terms of depolarisation and repolarization maps from the measured signals called as inverse problem. This is computed only if one has solved generic or subject- specific prior models using the anatomical structures of the myocardium, the torso and the detectors called as forward problem. In this study, the Discretised heart is priorily assumed as the dipolar sources forming a double layer. The thorax structure modelled with finite element meshes is considered in the forward study. The magnetocardiographic data are simulated using uniform double layer model representing transmembrane distribution on the epicardium and endocardium. Using this data, the activation maps are non-invasively imaged on the heart surface using Tikhonov's regularisation technique. The inverse study is extended to reconstruct the depolarisation sequences of the abnormal cases.
Assuntos
Coração/fisiologia , Magnetocardiografia , Modelos Cardiovasculares , Análise de Elementos Finitos , Humanos , Masculino , Infarto do Miocárdio/fisiopatologia , Tórax/fisiologiaRESUMO
Vascular remodelling is a prominent feature of haemophilic arthropathy (HA) that may underlie re-bleeding, yet the nature of vascular changes and underlying mechanisms remain largely unknown. Here, we aimed to characterize synovial vascular remodelling and vessel integrity after haemarthrosis, as well as temporal changes in inflammatory and tissue-reparative pathways. Thirty acutely painful joints in patients with haemophilia (PWH) were imaged by musculoskeletal ultrasound with Power Doppler (MSKUS/PD) to detect vascular abnormalities and bloody effusions. Nineteen out of 30 painful joint episodes in PWH were associated with haemarthrosis, and abnormal vascular perfusion was unique to bleeding joints. A model of induced haemarthrosis in factor VIII (FVIII)-deficient mice was used for histological assessment of vascular remodelling (α-smooth muscle actin [αSMA] expression), and monitoring of in vivo vascular perfusion and permeability by MSKUS/PD and albumin extravasation, respectively. Inflammatory (M1) and reparative (M2) macrophage markers were quantified in murine synovium over a 10-week time course by real-time polymerase chain reaction. The abnormal vascular perfusion observed in PWH was recapitulated in FVIII-deficient mice after induced haemarthrosis. Neovascularization and increased vessel permeability were apparent 2 weeks post-bleed in FVIII-deficient mice, after a transient elevation of inflammatory macrophage M1 markers. These vascular changes subsided by week 4, while vascular remodelling, evidenced by architectural changes and pronounced αSMA expression, persisted alongside a reparative macrophage M2 response. In conclusion, haemarthrosis leads to transient inflammation coupled with neovascularization and associated vascular permeability, while subsequent tissue repair mechanisms coincide with vascular remodelling. Together, these vascular changes may promote re-bleeding and HA progression.
Assuntos
Permeabilidade Capilar/fisiologia , Fator VIII/genética , Hemartrose/fisiopatologia , Hemofilia A/fisiopatologia , Articulação do Joelho/diagnóstico por imagem , Macrófagos/imunologia , Remodelação Vascular/fisiologia , Actinas/metabolismo , Adulto , Animais , Modelos Animais de Doenças , Feminino , Humanos , Inflamação , Articulação do Joelho/irrigação sanguínea , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Neovascularização Patológica , CicatrizaçãoRESUMO
PURPOSE: Activated (super)Factor V ((super)FVa) is a novel engineered FV with excellent prohemostatic efficacy. (Super)FVa has three APC cleavage site mutations and an interdomain disulfide bond. Stability, pharmacokinetics, and immunogenic and thrombogenic potential are reported here. METHODS: Stability and circulating half-life were determined after incubation in buffer and human plasma, and after injection into FVIII-deficient mice. Immunogenicity potential was assessed by B- and T-cell specific epitope prediction and structural analysis using surface area and atomic depth computation. Thrombogenic potential was determined by quantification of lung fibrin deposition in wild-type mice after intravenous injection of (super)FVa (200 U/kg), recombinant human (rh) Tissue Factor (0.4-16 pmol/kg), rhFVIIa (3 mg/kg) or saline. RESULTS: FVa retained full activity over 30 h in buffer, the functional half-life in human plasma was 4.9 h, and circulating half-life in FVIII-deficient mice was ~30 min. Predicted immunogenicity was not increased compared to human FV. While rh Tissue Factor, the positive control, resulted in pronounced lung fibrin depositions (mean 121 µg/mL), (super)FVa did not (6.7 µg/mL), and results were comparable to fibrin depositions with rhFVIIa (7.6 µg/mL) or saline (5.6 µg/mL). CONCLUSION: FVa has an appropriate safety and stability profile for further preclinical development as a prohemostatic against severe bleeding.
Assuntos
Fator Va/farmacocinética , Hemofilia A/tratamento farmacológico , Hemostáticos/farmacocinética , Engenharia de Proteínas/métodos , Proteínas Recombinantes/farmacocinética , Animais , Modelos Animais de Doenças , Estabilidade de Medicamentos , Fator VIII/genética , Fator VIII/metabolismo , Fator Va/administração & dosagem , Fator Va/genética , Fator Va/toxicidade , Feminino , Fibrina/metabolismo , Meia-Vida , Hemofilia A/sangue , Hemofilia A/genética , Hemostáticos/administração & dosagem , Hemostáticos/toxicidade , Humanos , Injeções Intravenosas , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Camundongos Knockout , Mutagênese Sítio-Dirigida , Mutação , Estabilidade Proteica , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidade , Índice de Gravidade de Doença , Trombina/metabolismoRESUMO
BACKGROUND: Xenin-25 is a K-cell derived gut peptide with insulin-releasing activity which is rapidly degraded following release into the circulation. We hypothesized that substitution of all naturally-occurring Lys and Arg residues with Gln would lead to prolonged enzyme resistance and enhanced biological efficacy. METHODS: Peptide stability was assessed using murine plasma, in vitro insulin-releasing actions evaluated in BRIN-BD11 cells and acute glucose-lowering and insulin-releasing actions examined in high fat fed mice. For sub-chronic studies, a range of metabolic parameters and pancreatic histology were assessed in high fat fed mice which had received saline vehicle or xenin-25(gln) twice-daily for 21 days. RESULTS: In contrast to native xenin-25, xenin-25(gln) was resistant to plasma-mediated degradation and significantly stimulated insulin secretion in BRIN-BD11 cells. Acute administration of xenin-25(gln) in high fat fed mice significantly reduced blood glucose and increased plasma insulin concentrations. Twice-daily administration of xenin-25(gln) in high fat fed mice did not affect food intake, body weight or circulating insulin concentrations but significantly decreased blood glucose from day 9 onwards. Furthermore, glucose tolerance, glucose-mediated insulin secretion, insulin sensitivity and GIP-stimulated insulin-release were significantly enhanced in xenin-25(gln)-treated mice. Pancreatic immunohistochemistry revealed decreased alpha cell area with increased beta cell area and beta-to-alpha cell ratio in xenin-25(gln)-treated mice. In addition, xenin-25(gln) exerted similar beneficial actions in ob/ob mice as demonstrated by reduced blood glucose, superior glycaemic response and glucose-mediated insulin release. CONCLUSIONS: Xenin-25(gln) is resistant to plasma-mediated degradation and exerts sustained and beneficial metabolic actions in high fat fed and ob/ob mice. GENERAL SIGNIFICANCE: Glutamine (gln)-modified analogues of xenin may represent an attractive therapeutic approach for type 2 diabetes.
Assuntos
Glicemia/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Insulina/sangue , Neurotensina/farmacologia , Neurotensina/farmacocinética , Animais , Linhagem Celular , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Preparações de Ação Retardada/farmacologia , Diabetes Mellitus Tipo 2/sangue , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/efeitos adversos , Camundongos , Neurotensina/químicaRESUMO
Bypassing inhibitors in haemophilia patients is limited to activated (a) Factor(F)VII products. We introduced "FVa activity augmentation" as another bypassing strategy and studied effects of an engineered FVa variant designated superFVa. Procoagulant and clot stabilising properties of superFVa and recombinant human (rh)FVIIa, either alone or in combination, were studied in thrombin generation and clot lysis assays in normal human plasma (NHP) with or without anti-FVIII inhibitors, in haemophilia plasma, and in FVIII-deficient mice or in wild-type mice with anti-FVIII inhibitors. SuperFVa was as effective as rhFVIIa to improve thrombin generation or clot lysis. Furthermore, procoagulant effects were significantly enhanced when these compounds were combined. RhFVIIa at 40 nM (a therapeutic concentration) improved thrombin generation mildly, but markedly improved thrombin generation when combined with a low concentration (e. g. 3 nM) of superFVa. In clot lysis studies, the concentration of rhFVIIa to normalise clot lysis times could be reduced by 100-fold (e. g. from 40 nM to 0.4 nM) when combined with a low concentration (0.37 nM) of superFVa. In haemostasis studies of FVIII-deficient mice, blood loss was dose-dependently reduced by either superFVa or rhFVIIa. SuperFVa (200 U/kg) corrected mean blood loss indistinguishably from rhFVIII. Blood loss correction by rhFVIIa was greatly improved when combined with superFVa. Similar blood loss correction results were observed for therapies in wild-type mice after infusion with anti-FVIII inhibitors. Thus, superFVa may be an effective procoagulant agent in the setting of haemophilia with inhibitors and it merits further evaluation for new bypassing strategies.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Coagulantes/administração & dosagem , Fator VIIa/administração & dosagem , Fator Va/administração & dosagem , Hemofilia A/imunologia , Animais , Anticorpos/química , Relação Dose-Resposta a Droga , Fator VIII/antagonistas & inibidores , Feminino , Hemofilia A/sangue , Hemostasia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/administração & dosagem , Trombina/metabolismoRESUMO
Hemophilic arthropathy is a debilitating condition that can develop as a consequence of frequent joint bleeding despite adequate clotting factor replacement. The mechanisms leading to repeated spontaneous bleeding are unknown. We investigated synovial, vascular, stromal, and cartilage changes in response to a single induced hemarthrosis in the FVIII-deficient mouse. We found soft-tissue hyperproliferation with marked induction of neoangiogenesis and evolving abnormal vascular architecture. While soft-tissue changes were rapidly reversible, abnormal vascularity persisted for months and, surprisingly, was also seen in uninjured joints. Vascular changes in FVIII-deficient mice involved pronounced remodeling with expression of α-Smooth Muscle Actin (SMA), Endoglin (CD105), and vascular endothelial growth factor, as well as alterations of joint perfusion as determined by in vivo imaging. Vascular architecture changes and pronounced expression of α-SMA appeared unique to hemophilia, as these were not found in joint tissue obtained from mouse models of rheumatoid arthritis and osteoarthritis and from patients with the same conditions. Evidence that vascular changes in hemophilia were significantly associated with bleeding and joint deterioration was obtained prospectively by dynamic in vivo imaging with musculoskeletal ultrasound and power Doppler of 156 joints (elbows, knees, and ankles) in a cohort of 26 patients with hemophilia at baseline and during painful episodes. These observations support the hypothesis that vascular remodeling contributes significantly to bleed propagation and development of hemophilic arthropathy. Based on these findings, the development of molecular targets for angiogenesis inhibition may be considered in this disease.
Assuntos
Fator VIII/genética , Hemartrose/patologia , Hemofilia A/patologia , Neovascularização Patológica/patologia , Remodelação Vascular , Actinas/genética , Actinas/metabolismo , Animais , Tornozelo/irrigação sanguínea , Tornozelo/patologia , Modelos Animais de Doenças , Articulação do Cotovelo/irrigação sanguínea , Articulação do Cotovelo/metabolismo , Articulação do Cotovelo/patologia , Endoglina , Fator VIII/metabolismo , Expressão Gênica , Hemartrose/genética , Hemartrose/metabolismo , Hemofilia A/genética , Hemofilia A/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Articulação do Joelho/irrigação sanguínea , Articulação do Joelho/metabolismo , Articulação do Joelho/patologia , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Vaccination with tumor-associated antigens can induce cancer-specific CD8+ T cells. A recent improvement has been the targeting of antigen to dendritic cells (DC) using antibodies that bind DC surface molecules. This study explored the use of multi-trimers of CD40L to target the gp100 melanoma tumor antigen to DC. The spontaneously-multimerizing gene Surfactant Protein D (SPD) was used to fuse gp100 tumor antigen and CD40L, creating the recombinant protein SPD-gp100-CD40L. This "third generation" DC-targeting vaccine was designed to both target antigen to DC and optimally activate dendritic cells by aggregating CD40 trimers on the DC membrane surface. SPD-gp100-CD40L expressed as a 110kDa protein. Analytical light scattering analysis gave elution data corresponding to 4-trimer and multi-trimer SPD-gp100-CD40L oligomers. The protein was biologically active on dendritic cells and induced CD40-mediated NF-κB signaling. DNA vaccination with SPD-gp100-CD40L plasmid, together with plasmids encoding IL-12p70 and GM-CSF, significantly enhanced survival and inhibited tumor growth in a B16-F10 melanoma model. Expression of gp100 and SPD-CD40L as separate molecules did not enhance survival, highlighting the requirement to encode gp100 within SPD-CD40L for optimal vaccine activity. These data support a model where DNA vaccination with SPD-gp100-CD40L targets gp100 to DC in situ, induces activation of these DC, and generates a protective anti-tumor response when given in combination with IL-12p70 and GM-CSF plasmids.
Assuntos
Adjuvantes Imunológicos/metabolismo , Ligante de CD40/metabolismo , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Melanoma/terapia , Vacinas de DNA/imunologia , Antígeno gp100 de Melanoma/imunologia , Adjuvantes Imunológicos/genética , Animais , Ligante de CD40/genética , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/genética , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos C57BL , Proteína D Associada a Surfactante Pulmonar/genética , Proteína D Associada a Surfactante Pulmonar/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Análise de Sobrevida , Resultado do Tratamento , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Antígeno gp100 de Melanoma/genéticaRESUMO
Crude venom from three venomous snakes, Crotalus adamanteus, Crotalus vegrandis and Bitis nasicornis was fractionated by gel filtration chromatography, and selected fractions screened for in-vitro insulinotropic activity using clonal pancreatic BRIN-BD11 cells. Nineteen fractions stimulated insulin secretion and the structural identity of bioactive compounds responsible was probed using MALDI-ToF MS and N-terminal Edman degradation sequencing. Partial N-terminal sequences were determined and their homology to existing sequences identified using BLAST searching. The main insulinotropic peptide families identified were made up of snake venom serine proteinases, phospholipases A2 (PLA2) and disintegrins. Snake venom constituents may have therapeutic potential for diabetes, with each of the three viper venoms tested providing insulinotropic compounds from a range of different toxin families.
Assuntos
Venenos de Crotalídeos/farmacologia , Crotalus/metabolismo , Insulina/metabolismo , Venenos de Víboras/farmacologia , Viperidae/metabolismo , Animais , Linhagem Celular , Cromatografia em Gel , Venenos de Crotalídeos/química , Crotalus/classificação , Desintegrinas/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Fosfolipases A2/metabolismo , Ratos , Serina Proteases/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Venenos de Víboras/química , Viperidae/classificaçãoRESUMO
While being devoid of the ability to recognize ligands itself, the WW2 domain is believed to aid ligand binding to the WW1 domain in the context of a WW1-WW2 tandem module of WW domain-containing oxidoreductase (WWOX) tumor suppressor. In an effort to test the generality of this hypothesis, we have undertaken here a detailed biophysical analysis of the binding of WW domains of WWOX alone and in the context of the WW1-WW2 tandem module to an array of putative proline-proline-x-tyrosine (PPXY) ligands. Our data show that while the WW1 domain of WWOX binds to all ligands in a physiologically relevant manner, the WW2 domain does not. Moreover, ligand binding to the WW1 domain in the context of the WW1-WW2 tandem module is two-to-three-fold stronger than when treated alone. We also provide evidence that the WW domains within the WW1-WW2 tandem module physically associate so as to adopt a fixed spatial orientation relative to each other. Of particular note is the observation that the physical association of the WW2 domain with WW1 blocks access to ligands. Consequently, ligand binding to the WW1 domain not only results in the displacement of the WW2 lid but also disrupts the physical association of WW domains in the liganded conformation. Taken together, our study underscores a key role of allosteric communication in the ability of the WW2 orphan domain to chaperone physiological action of the WW1 domain within the context of the WW1-WW2 tandem module of WWOX.