Como Planear um Ensaio Clínico em Cardiologia. Aspectos Fundamentais

A experiência clínica acumulada resultante da observação seriada de doentes introduz enviesamento na avaliação da eficácia dos fármacos. Apenas com um ensaio clínico, se adequadamente desenhado, é possível minimizar o enviesamento e avaliar essa eficácia. No entanto, o planeamento de um ensaio clínico é a área em que a experiência clínica pode ser útil. Os autores revêem os aspectos fundamentais da metodologia dos ensaios clínicos em Cardiologia, nomeadamente as várias fases da investigação clínica em humanos, a definição da população, as formas de limitar o enviesamento, os desenhos possíveis dos estudos, as opções de aleatorização, a dimensão da amostra, os tipos de resultados e as precauções a ter no seguimento

Percutaneous retrieval of foreign bodies from the cardiovascular system

We report our experience of seven patients referred to our hospital with foreign bodies embolized in the cardiovascular system, namely fragmented catheters and devices used in interventional techniques, which were retrieved by a percutaneous approach. The patients' ages ranged from 2 to 29 years, with a mean age of 17. The majority (57%) were male. The retrieval equipment used included a pigtail catheter, multipurpose catheter with hand-prepared snare, Amplatz gooseneck snare, nitinol multisnare set and basket catheter. The foreign bodies were successfully removed percutaneously in all cases, with no complications

Quantification of Urinary Mevalonic Acid as a Biomarker of HMG-CoA Reductase Activity by a Novel Translational LC-MS/MS Method

Background: Mevalonic acid (MVA), as a product of 3-hydroxy-3-methylglutaryl coenzyme A reductase, represents a potential multipurpose biomarker in health and disease. A translational urinary MVA quantification method was developed, validated and used to demonstrate the diurnal variation of urinary MVA excretion in rats and healthy children. Methods: Urinary MVA was converted to mevalonolactone at pH 2, extracted with ethyl acetate and quantified by reversed-phase liquid chromatography-tandem mass spectrometry. Results: The assay had a dynamic range of 0.0156-10 µg/ml with precision <15% CV, accuracy 85-115% and was transferred between laboratories. Urinary MVA excretion in rats and healthy children displayed a diurnal variation consistent with the known diurnal variation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. Conclusion: Urinary MVA can be quantified accurately over a wide dynamic range by a validated translational and transferable method with biomarker capability

Liposome-based dry powder vaccine immunization targeting the lungs induces broad protection against pneumococcus

Streptococcus pneumoniae is an important human pathogen. Currently used conjugate vaccines are effective against invasive disease, but protection is restricted to serotypes included in the formulation, leading to serotype replacement. Furthermore, protection against non-invasive disease is reported to be considerably lower. The development of a serotype-independent vaccine is thus important and Pneumococcal surface protein A (PspA) is a promising vaccine candidate. PspA shows some diversity and can be classified in 6 clades and 3 families, with families 1 and 2 being the most frequent in clinical isolates. The ideal vaccine should thus induce protection against the two most common families of PspA. The aim of this work was to develop a liposome-based vaccine containing PspAs from family 1 and 2 and to characterize its immune response. Liposomes (LP) composed of dipalmitoylphosphatidylcholine (DPPC) and 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol) with or without α-galactosylceramide (α-GalCer) were produced by microfluidics, encapsulating PspA from clade 1 (PspA1, family 1) and/or clade 4 (PspA4Pro, family 2) followed by spray-drying with trehalose to form nanocomposite microparticles carriers (NCMP). LP/NCMPs showed good stability and preservation of protein activity. LP/NCMPs containing PspA1 and/or PspA4Pro were used for immunization of mice targeting the lungs. High serum IgG antibody titers against both PspA1 and PspA4Pro were detected in animals immunized with LP/NCMPs containing α-GalCer, with a balance of IgG1 and IgG2a titers. IgG in sera from immunized mice bound to pneumococcal strains from different serotypes and expressing different PspA clades, indicating broad recognition. Mucosal IgG and IgA were also detected. Importantly, immunization with LP/NCMPs induced full protection against strains expressing PspAs from family 1 and 2. Furthermore, CD4+ resident memory T cells were detected in the lungs of the immunized animals that survived the challenge

Pneumococcal Surface Protein A-Hybrid Nanoparticles Protect Mice from Lethal Challenge after Mucosal Immunization Targeting the Lungs

Pneumococcal disease remains a global burden, with current conjugated vaccines offering protection against the common serotype strains. However, there are over 100 serotype strains, and serotype replacement is now being observed, which reduces the effectiveness of the current vaccines. Pneumococcal surface protein A (PspA) has been investigated as a candidate for new serotype-independent pneumococcal vaccines, but requires adjuvants and/or delivery systems to improve protection. Polymeric nanoparticles (NPs) are biocompatible and, besides the antigen, can incorporate mucoadhesive and adjuvant substances such as chitosans, which improve antigen presentation at mucosal surfaces. This work aimed to define the optimal NP formulation to deliver PspA into the lungs and protect mice against lethal challenge. We prepared poly(glycerol-adipate-co-ω-pentadecalactone) (PGA-co-PDL) and poly(lactic-co-glycolic acid) (PLGA) NPs using an emulsion/solvent evaporation method, incorporating chitosan hydrochloride (HCl-CS) or carboxymethyl chitosan (CM-CS) as hybrid NPs with encapsulated or adsorbed PspA. We investigated the physicochemical properties of NPs, together with the PspA integrity and biological activity. Furthermore, their ability to activate dendritic cells in vitro was evaluated, followed by mucosal immunization targeting mouse lungs. PGA-co-PDL/HCl-CS (291 nm) or CM-CS (281 nm) NPs produced smaller sizes compared to PLGA/HCl-CS (310 nm) or CM-CS (299 nm) NPs. Moreover, NPs formulated with HCl-CS possessed a positive charge (PGA-co-PDL +17 mV, PLGA + 13 mV) compared to those formulated with CM-CS (PGA-co-PDL −20 mV, PLGA −40 mV). PspA released from NPs formulated with HCl-CS preserved the integrity and biological activity, but CM-CS affected PspA binding to lactoferrin and antibody recognition. PspA adsorbed in PGA-co-PDL/HCl-CS NPs stimulated CD80+ and CD86+ cells, but this was lower compared to when PspA was encapsulated in PLGA/HCl-CS NPs, which also stimulated CD40+ and MHC II (I-A/I-E)+ cells. Despite no differences in IgG being observed between immunized animals, PGA-co-PDL/HCl-CS/adsorbed-PspA protected 83% of mice after lethal pneumococcal challenge, while 100% of mice immunized with PLGA/HCl-CS/encapsulated-PspA were protected. Therefore, this formulation is a promising vaccine strategy, which has beneficial properties for mucosal immunization and could potentially provide serotype-independent protection