Proposing Urothelial and Muscle In Vitro Cell Models as a Novel Approach for Assessment of Long-Term Toxicity of Nanoparticles.

Many studies evaluated the short-term in vitro toxicity of nanoparticles (NPs); however, long-term effects are still not adequately understood. Here, we investigated the potential toxic effects of biomedical (polyacrylic acid and polyethylenimine coated magnetic NPs) and two industrial (SiO2 and TiO2) NPs following different short-term and long-term exposure protocols on two physiologically different in vitro models that are able to differentiate: L6 rat skeletal muscle cell line and biomimetic normal porcine urothelial (NPU) cells. We show that L6 cells are more sensitive to NP exposure then NPU cells. Transmission electron microscopy revealed an uptake of NPs into L6 cells but not NPU cells. In L6 cells, we obtained a dose-dependent reduction in cell viability and increased reactive oxygen species (ROS) formation after 24 h. Following continuous exposure, more stable TiO2 and polyacrylic acid (PAA) NPs increased levels of nuclear factor Nrf2 mRNA, suggesting an oxidative damage-associated response. Furthermore, internalized magnetic PAA and TiO2 NPs hindered the differentiation of L6 cells. We propose the use of L6 skeletal muscle cells and NPU cells as a novel approach for assessment of the potential long-term toxicity of relevant NPs that are found in the blood and/or can be secreted into the urine.

Complete Assessment of Multilineage Differentiation Potential of Human Skeletal Muscle-Derived Mesenchymal Stem/Stromal Cells.

The minimal criteria for mesenchymal stem/stromal cell (MSC) identification set by the International Society for Cellular Therapy include plastic adherence, presence and absence of a set of surface antigens and in vitro multilineage differentiation. This differentiation is assessed through stimulation of MSCs with defined combination and concentration of growth factors towards specific lineages and histological confirmation of the presence of differentiated cells. Here we provide protocols for multilineage differentiation, namely, osteogenesis, adipogenesis, chondrogenesis and myogenesis. We also provide their respective histological analyses.

Novel insights into in-vivo diffusion tensor cardiovascular magnetic resonance using computational modeling and a histology-based virtual microstructure.

PURPOSE: To develop histology-informed simulations of diffusion tensor cardiovascular magnetic resonance (DT-CMR) for typical in-vivo pulse sequences and determine their sensitivity to changes in extra-cellular space (ECS) and other microstructural parameters. METHODS: We synthesised the DT-CMR signal from Monte Carlo random walk simulations. The virtual tissue was based on porcine histology. The cells were thickened and then shrunk to modify ECS. We also created idealised geometries using cuboids in regular arrangement, matching the extra-cellular volume fraction (ECV) of 16-40%. The simulated voxel size was 2.8 × 2.8 × 8.0 mm3 for pulse sequences covering short and long diffusion times: Stejskal-Tanner pulsed-gradient spin echo, second-order motion-compensated spin echo, and stimulated echo acquisition mode (STEAM), with clinically available gradient strengths. RESULTS: The primary diffusion tensor eigenvalue increases linearly with ECV at a similar rate for all simulated geometries. Mean diffusivity (MD) varies linearly, too, but is higher for the substrates with more uniformly distributed ECS. Fractional anisotropy (FA) for the histology-based geometry is higher than the idealised geometry with low sensitivity to ECV, except for the long mixing time of the STEAM sequence. Varying the intra-cellular diffusivity (DIC ) results in large changes of MD and FA. Varying extra-cellular diffusivity or using stronger gradients has minor effects on FA. Uncertainties of the primary eigenvector orientation are reduced using STEAM. CONCLUSIONS: We found that the distribution of ECS has a measurable impact on DT-CMR parameters. The observed sensitivity of MD and FA to ECV and DIC has potentially interesting applications for interpreting in-vivo DT-CMR parameters.

A genome-wide assessment of conserved SNP alleles reveals a panel of regulatory SNPs relevant to the peripheral nerve.

BACKGROUND: Identifying functional non-coding variation is critical for defining the genetic contributions to human disease. While single-nucleotide polymorphisms (SNPs) within cis-acting transcriptional regulatory elements have been implicated in disease pathogenesis, not all cell types have been assessed and functional validations have been limited. In particular, the cells of the peripheral nervous system have been excluded from genome-wide efforts to link non-coding SNPs to altered gene function. Addressing this gap is essential for defining the genetic architecture of diseases that affect the peripheral nerve. We developed a computational pipeline to identify SNPs that affect regulatory function (rSNPs) and evaluated our predictions on a set of 144 regions in Schwann cells, motor neurons, and muscle cells. RESULTS: We identified 28 regions that display regulatory activity in at least one cell type and 13 SNPs that affect regulatory function. We then tailored our pipeline to one peripheral nerve cell type by incorporating SOX10 ChIP-Seq data; SOX10 is essential for Schwann cells. We prioritized 22 putative SOX10 response elements harboring a SNP and rapidly validated two rSNPs. We then selected one of these elements for further characterization to assess the biological relevance of our approach. Deletion of the element from the genome of cultured Schwann cells-followed by differential gene expression studies-revealed Tubb2b as a candidate target gene. Studying the enhancer in developing mouse embryos revealed activity in SOX10-positive cells including the dorsal root ganglia and melanoblasts. CONCLUSIONS: Our efforts provide insight into the utility of employing strict conservation for rSNP discovery. This strategy, combined with functional analyses, can yield candidate target genes. In support of this, our efforts suggest that investigating the role of Tubb2b in SOX10-positive cells may reveal novel biology within these cell populations.

Mitochondrial efficiency and exercise economy following heat stress: a potential role of uncoupling protein 3.

Heat stress has been reported to reduce uncoupling proteins (UCP) expression, which in turn should improve mitochondrial efficiency. Such an improvement in efficiency may translate to the systemic level as greater exercise economy. However, neither the heat-induced improvement in mitochondrial efficiency (due to decrease in UCP), nor its potential to improve economy has been studied. Determine: (i) if heat stress in vitro lowers UCP3 thereby improving mitochondrial efficiency in C2C12 myocytes; (ii) whether heat acclimation (HA) in vivo improves exercise economy in trained individuals; and (iii) the potential improved economy during exercise at altitude. In vitro, myocytes were heat stressed for 24 h (40°C), followed by measurements of UCP3, mitochondrial uncoupling, and efficiency. In vivo, eight trained males completed: (i) pre-HA testing; (ii) 10 days of HA (40°C, 20% RH); and (iii) post-HA testing. Pre- and posttesting consisted of maximal exercise test and submaximal exercise at two intensities to assess exercise economy at 1600 m (Albuquerque, NM) and 4350 m. Heat-stressed myocytes displayed significantly reduced UCP3 mRNA expression and, mitochondrial uncoupling (77.1 ± 1.2%, P < 0.0001) and improved mitochondrial efficiency (62.9 ± 4.1%, P < 0.0001) compared to control. In humans, at both 1600 m and 4350 m, following HA, submaximal exercise economy did not change at low and moderate exercise intensities. Our findings indicate that while heat-induced reduction in UCP3 improves mitochondrial efficiency in vitro, this is not translated to in vivo improvement of exercise economy at 1600 m or 4350 m.

Consumption and expenditure on food prepared away from home among Mexican adults in 2006 / Consumo y gasto en alimentos preparados fuera de casa en adultos mexicanos en 2006

Objective. To describe food expenditure and consumption of foods prepared away from home among Mexican adults. Materials and methods. Data were from 45 241 adult participants in the National Health and Nutrition Survey 2006, a nationally-representative, cross-sectional survey of Mexican households. Descriptive statistics and multivariable linear and logistic regression were used to assess the relationship between location of residence, educational attainment, socioeconomic status and the following: 1) expenditure on all food and at restaurants, and 2) frequency of consumption of comida corrida or restaurant food and street food. Results. Food expenditure and consumption of food prepared away from home were positively associated with socioeconomic status, educational attainment, and urban vs. rural residence (p<0.001 for all relationships in bivariate analyses). Conclusions. Consumption of food prepared outside home may be an important part of the diet among urban Mexican adults and those with high socioeconomic status and educational attainment.

Objetivo. Describir los gastos en alimentos y el consumo de alimentos preparados fuera de casa en población mexicana. Material y métodos. Los datos fueron de 45 241 adultos mexicanos en la Encuesta Nacional de Salud y Nutrición de 2006, representativa al nivel nacional. Se utilizaron estadísticas descriptivas y regresión linear y logística para estimar la relación entre el lugar de residencia, el nivel educativo y el nivel socioeconómico, con el gasto en todos los alimentos y en restaurantes, y con la frecuencia de consumo de comida corrida, en restaurantes y de la calle. Resultados. El gasto en alimentos y el consumo de alimentos preparados se asociaron positivamente con el nivel socioeconómico, el nivel educativo y la residencia rural (p<0,001 para todas las relaciones). Conclusiones. El consumo de alimentos preparados puede ser una parte importante de la dieta de los adultos urbanos y de aquéllos con altos niveles socioeconómicos y educativos.

Mechanisms and assessment of statin-related muscular adverse effects.

Statin-associated muscular adverse effects cover a wide range of symptoms, including asymptomatic increase of creatine kinase serum activity and life-threatening rhabdomyolysis. Different underlying pathomechanisms have been proposed. However, a unifying concept of the pathogenesis of statin-related muscular adverse effects has not emerged so far. In this review, we attempt to categorize these mechanisms along three levels. Firstly, among pharmacokinetic factors, it has been shown for some statins that inhibition of cytochrome P450-mediated hepatic biotransformation and hepatic uptake by transporter proteins contribute to an increase of systemic statin concentrations. Secondly, at the myocyte membrane level, cell membrane uptake transporters affect intracellular statin concentrations. Thirdly, at the intracellular level, inhibition of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase results in decreased intracellular concentrations of downstream metabolites (e.g. selenoproteins, ubiquinone, cholesterol) and alteration of gene expression (e.g. ryanodine receptor 3, glycine amidinotransferase). We also review current recommendations for prescribers.

Methods for the assessment of ssODN-mediated gene correction frequencies in muscle cells.

The past decade has seen the development of new technologies capable of editing the genome that have naturally led to exploring their therapeutic application for the treatment of many disorders. Among those, Duchenne muscular dystrophy (DMD) represents an ideal candidate for gene editing primarily due to the large size of dystrophin, the gene responsible for the disease, which limits the use of gene replacement approaches. Critical in the evaluation of the efficacy of the treatment is the development of a method that can accurately quantitate the frequencies of gene repair obtained in the dystrophin gene at both the genomic level as well as the mRNA level. The mdx (5cv) mouse model of DMD offers an ideal system to precisely determine the frequencies of gene repair. Here we describe the methods used for determining those frequencies and the limitations associated with the use of gene correction for the treatment of DMD. Clinical approaches to muscle disorders using ssODNs will heavily rely on the optimization of the technology and will have to take into consideration the safety, efficacy and cost of the procedure in vision of systemic delivery of the therapeutic treatment.

The use of biomarkers in the patient with heart failure.

Heart failure is a major burden to the health care system in terms of not only cost, but also morbidity and mortality. Appropriate use of biomarkers is critically important to allow rapid identification and optimal risk stratification and management of patients with both acute and chronic heart failure. This review will discuss the biomarkers that have the most diagnostic, prognostic, and therapeutic value in patients with heart failure. We will discuss established biomarkers such as natriuretic peptides as well as emerging biomarkers reflective of myocyte stress, myocyte injury, extracellular matrix injury, and both neurohormonal and cardio-renal physiology.

Bioluminescence imaging for assessment of immune responses following implantation of engineered heart tissue (EHT).

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; ). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Synthesis and biological evaluation of novel leonurine-SPRC conjugate as cardioprotective agents.

The synthesis and biological evaluation of novel leonurine-SPRC conjugate, 3,5-dimethoxy-4-(2-amino-3-prop-2-ynylsulfanyl-propionyl)-benzoic acid 4-guanidino-butyl ester (1) is reported in this Letter. It is designed to improve the pharmacology efficiency by combining leonurine with S-propargyl-L-cysteine (SPRC), a cysteine analog, via a phenolic hydroxyl ester bond, which could be readily hydrolyzed to release bioactive leonurine and SPRC. Pharmacological evaluation has shown that 1 possesses potent cardioprotective effect against hypoxia-induced neonatal rat ventricular myocytes damage at lower molar concentration (10-fold less than leonurine required and 100-fold less than SPRC required). The mechanism is in partial related to improve hydrogen sulfide production, anti-oxidative stress and anti-apoptosis.

Multi-image colocalization and its statistical significance.

Accurately localizing molecules within the cell is one of main tasks of modern biology, and colocalization analysis is one of its principal and most often used tools. Despite this popularity, interpretation is often uncertain because colocalization between two or more images is rarely analyzed to determine whether the observed values could have occurred by chance. To address this, we have developed a robust methodology, based on Monte Carlo randomization, to measure the statistical significance of a colocalization. The method works with voxel-based, intensity-based, object-based, and nearest-neighbor metrics. We extend all of these to measure colocalization in images with three colors. We also introduce three new metrics; blob colocalization, where the blob consists of a local maximum surrounded by a three-dimensional group of voxels; cluster diameter, to measure the clustering of fluorophores in three or more images; and the intercluster distance to measure the distance between these clusters. The robustness of these metrics was tested by varying the image thresholds over a broad range, which produced no change in the statistical significance of the colocalizations. A comparison of blob colocalization with voxel and Manders colocalization metrics shows that the different measures produce consistent results with similar values for significance and nonsignificance. Using our methodology, we are able to determine not only whether the labeled molecules colocalize with a probability greater than chance, but also whether they are sequestrated into different compartments. The program, written in C++, is freely available as source, as well as in a Linux version.

Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation.

While bile acids (BAs) have long been known to be essential in dietary lipid absorption and cholesterol catabolism, in recent years an important role for BAs as signalling molecules has emerged. BAs activate mitogen-activated protein kinase pathways, are ligands for the G-protein-coupled receptor (GPCR) TGR5 and activate nuclear hormone receptors such as farnesoid X receptor alpha (FXR-alpha; NR1H4). FXR-alpha regulates the enterohepatic recycling and biosynthesis of BAs by controlling the expression of genes such as the short heterodimer partner (SHP; NR0B2) that inhibits the activity of other nuclear receptors. The FXR-alpha-mediated SHP induction also underlies the downregulation of the hepatic fatty acid and triglyceride biosynthesis and very-low-density lipoprotein production mediated by sterol-regulatory-element-binding protein 1c. This indicates that BAs might be able to function beyond the control of BA homeostasis as general metabolic integrators. Here we show that the administration of BAs to mice increases energy expenditure in brown adipose tissue, preventing obesity and resistance to insulin. This novel metabolic effect of BAs is critically dependent on induction of the cyclic-AMP-dependent thyroid hormone activating enzyme type 2 iodothyronine deiodinase (D2) because it is lost in D2-/- mice. Treatment of brown adipocytes and human skeletal myocytes with BA increases D2 activity and oxygen consumption. These effects are independent of FXR-alpha, and instead are mediated by increased cAMP production that stems from the binding of BAs with the G-protein-coupled receptor TGR5. In both rodents and humans, the most thermogenically important tissues are specifically targeted by this mechanism because they coexpress D2 and TGR5. The BA-TGR5-cAMP-D2 signalling pathway is therefore a crucial mechanism for fine-tuning energy homeostasis that can be targeted to improve metabolic control.

Mechanisms of excitation-contraction coupling in an integrative model of the cardiac ventricular myocyte.

It is now well established that characteristic properties of excitation-contraction (EC) coupling in cardiac myocytes, such as high gain and graded Ca(2+) release, arise from the interactions that occur between L-type Ca(2+) channels (LCCs) and nearby ryanodine-sensitive Ca(2+) release channels (RyRs) in localized microdomains. Descriptions of Ca(2+)-induced Ca(2+) release (CICR) that account for these local mechanisms are lacking from many previous models of the cardiac action potential, and those that do include local control of CICR are able to reconstruct properties of EC coupling, but require computationally demanding stochastic simulations of approximately 10(5) individual ion channels. In this study, we generalize a recently developed analytical approach for deriving simplified mechanistic models of CICR to formulate an integrative model of the canine cardiac myocyte which is computationally efficient. The resulting model faithfully reproduces experimentally measured properties of EC coupling and whole cell phenomena. The model is used to study the role of local redundancy in L-type Ca(2+) channel gating and the role of dyad configuration on EC coupling. Simulations suggest that the characteristic steep rise in EC coupling gain observed at hyperpolarized potentials is a result of increased functional coupling between LCCs and RyRs. We also demonstrate mechanisms by which alterations in the early repolarization phase of the action potential, resulting from reduction of the transient outward potassium current, alters properties of EC coupling.

Caveolin-3 is adjacent to a group of extradyadic ryanodine receptors.

Caveolae are present in almost all cells and concentrate a wide variety of signaling molecules, receptors, transporters, and ion pumps. We have investigated the distribution of the ryanodine receptor, the Na(+)/Ca(2+) exchanger, the predominant Na(+) channel isoform rH1, and the L-type calcium channel, Ca(v)1.2, relative to the muscle-specific caveolin isoform, caveolin-3, in adult rat ventricular myocytes. Three-dimensional immunofluorescence images were deconvolved and analyzed. Caveolin-3 colocalizes with all of these molecules at the surface of the cell, but there is no significant colocalization between caveolin-3 and either the Na(+)/Ca(2+) exchanger or the Na(+) channel in the cell interior. The distribution of the surface colocalization indicates that the caveolae that colocalize with each molecule form distinct populations. This organization indicates that there are multiple populations of caveolae separable by location and occupants. In the interior of the cell, caveolin-3 shows a marked colocalization with a population of ryanodine receptors that are separate from those within the dyad. Because of their location, the signaling molecules contained within these caveolae may have preferred access to the neighboring nondyadic ryanodine receptors.

The role of stochastic and modal gating of cardiac L-type Ca2+ channels on early after-depolarizations.

Certain signaling events that promote L-type Ca2+ channel (LCC) phosphorylation, such as beta-adrenergic stimulation or an increased expression of Ca(2+)/calmodulin-dependent protein kinase II, promote mode 2 gating of LCCs. Experimental data suggest the hypothesis that these events increase the likelihood of early after-depolarizations (EADs). We test this hypothesis using an ionic model of the canine ventricular myocyte incorporating stochastic gating of LCCs and ryanodine-sensitive calcium release channels. The model is extended to describe myocyte responses to the beta-adrenergic agonist isoproterenol. Results demonstrate that in the presence of isoproterenol the random opening of a small number of LCCs gating in mode 2 during the plateau phase of the action potential (AP) can trigger EADs. EADs occur randomly, where the likelihood of these events increases as a function of the fraction of LCCs gating in mode 2. Fluctuations of the L-type Ca2+ current during the AP plateau lead to variability in AP duration. Consequently, prolonged APs are occasionally observed and exhibit an increased likelihood of EAD formation. These results suggest a novel stochastic mechanism, whereby phosphorylation-induced changes in LCC gating properties contribute to EAD generation.

Assessment of the influence of fatty acids on indices of insulin sensitivity and myocellular lipid content by use of magnetic resonance spectroscopy in cats.

OBJECTIVE: To determine whether dietary fatty acids affect indicators of insulin sensitivity, plasma insulin and lipid concentrations, and lipid accumulation in muscle cells in lean and obese cats. ANIMALS: 28 neutered adult cats. PROCEDURE: IV glucose tolerance tests and magnetic resonance imaging were performed before (lean phase) and after 21 weeks of ad libitum intake of either a diet high in omega-3 polyunsaturated fatty acids (3-PUFAs; n = 14) or high in saturated fatty acids (SFAs; 14). RESULTS: Compared with the lean phase, ad libitum food intake resulted in increased weight, body mass index, girth, and percentage fat in both groups. Baseline plasma glucose or insulin concentrations and glucose area under the curve (AUC) were unaffected by diet. Insulin AUC values for obese and lean cats fed 3-PUFAs did not differ, but values were higher in obese cats fed SFAs, compared with values for lean cats fed SFAs and obese cats fed 3-PUFAs. Nineteen cats that became glucose intolerant when obese had altered insulin secretion and decreased glucose clearance when lean. Plasma cholesterol, triglyceride, and non-esterified fatty acid concentrations were unaffected by diet. Ad libitum intake of either diet resulted in an increase in both intra- and extramyocellular lipid. Obese cats fed SFAs had higher glycosylated hemoglobin concentration than obese cats fed 3-PUFAs. CONCLUSION AND CLINICAL RELEVANCE: In obese cats, a diet high in 3-PUFAs appeared to improve long-term glucose control and decrease plasma insulin concentration. Obesity resulted in intra- and extramyocellular lipid accumulations (regardless of diet) that likely modulate insulin sensitivity.

Assessment of O2 uptake dynamics in isolated single skeletal myocytes.

The purpose of this research was to develop a technique for rapid measurement of O(2) uptake (Vo(2)) kinetics in single isolated skeletal muscle cells. Previous attempts to measure single cell Vo(2) have utilized polarographic-style electrodes, thereby mandating large fluid volumes and relatively poor sensitivity. Thus our laboratory has developed an approximately 100-microl, well-stirred chamber for the measurement of Vo(2) in isolated Xenopus laevis myocytes using a phosphorescence quenching technique [Ringer solution with 0.05 mM Pd-meso-tetra(4-carboxyphenyl)porphine] to monitor the fall in extracellular Po(2) (which is proportional to cellular Vo(2) within the sealed chamber). Vo(2) in single living myocytes dissected from Xenopus lumbrical muscles was measured from rest across a bout of repetitive tetanic contractions (0.33 Hz) and in response to a ramp protocol utilizing an increasing contraction frequency. In response to the square-wave contraction bout, the increase in Vo(2) to steady state (SS) was 16.7 +/- 1.3 ml x 100 g(-1) x min(-1) (range 13.0-21.9 ml x 100 g(-1) x min(-1); n = 6). The rise in Vo(2) at contractions onset (n = 6) was fit with a time delay (2.1 +/- 1.2 s, range 0.0-7.7 s) plus monoexponential rise to SS (time constant = 9.4 +/- 1.5 s, range 5.2-14.9 s). Furthermore, in two additional myocytes, Vo(2) increased progressively as contraction frequency increased (ramp protocol). This technique for measuring Vo(2) in isolated, single skeletal myocytes represents a novel and powerful investigative tool for gaining mechanistic insight into mitochondrial function and Vo(2) dynamics without potential complications of the circulation and other myocytes.

[Assessment of Tc-99m MIBI uptake mechanism in Mycobacterium tuberculosis and normal cells (fibroblasts and myocytes) cultures]. / Studiul mecanismului de captare a Tc-99m MIBI pe culturi de Mycobacterium tuberculosis si de celule normale (fibroblaste si miocite).

One of the factors which can explain the scintigraphic Tc-99m MIBI positives images in pulmonary tuberculosis could be the Mycobacterium tuberculosis radiotracer uptake; this can be investigated in vitro, on cell culture, in comparison with normal cell types known to have high (myocites) or low (fibroblasts) uptake. Mycobacterium tuberculosis cultures were realised on Löwenstein Jensen medium, by standard methodology. Myocites and fibroblasts cultures were realized from neonatal rat hearts. Monolayer cells culture were incubated with a same 1.85 kBq/?l Tc-99m MIBI concentration, at 37 degrees C for 15, 60 and 90 minutes. The kinetic was stopped by rapidly washing the cells, with a 4 degrees C physiological saline solution and than scrapped cells were counted for the uptaken radioactivity. The results show that radiotracer cellular uptake (reported at the protein concentration) in myocites was maximum at 60 minutes. Cellular uptake in fibroblasts was very low at all the intervals. In Mycobacterium tuberculosis a peak was observed after 15 minutes, the uptake being similar to that of 60 minutes incubated myocites, considered 100%. After 90 minutes the Mycobacterium tuberculosis uptake was smaller than the 15 minutes value (65.82%). In conclusion, Mycobacterium tuberculosis in vitro uptake results could explain the more positives scintigraphic images in BK positive patients obtain at 15 minutes, in comparison with the delayed images.