Prevalence and pattern of cardiovascular magnetic resonance late gadolinium enhancement in highly trained endurance athletes.

BACKGROUND: Intensive endurance exercise may induce a broad spectrum of right ventricular (RV) adaptation/remodelling patterns. Late gadolinium enhancement (LGE) has also been described in cardiovascular magnetic resonance (CMR) of some endurance athletes and its clinical meaning remains controversial. Our aim was to characterize the features of contrast CMR and the observed patterns of the LGE distribution in a cohort of highly trained endurance athletes. METHODS: Ninety-three highly trained endurance athletes (> 12 h training/week at least during the last 5 years; 36 ± 6 years old; 53% male) and 72 age and gender-matched controls underwent a resting contrast CMR. In a subgroup of 28 athletes, T1 mapping was also performed. RESULTS: High endurance training load was associated with larger bi-ventricular and bi-atrial sizes and a slight reduction of biventricular ejection fraction, as compared to controls in both genders (p < 0.05). Focal LGE was significantly more prevalent in athletes than in healthy subjects (37.6% vs 2.8%; p < 0.001), with a typical pattern in the RV insertion points. In T1 mapping, those athletes who had focal LGE had higher extracellular volume (ECV) at the remote myocardium than those without (27 ± 2.2% vs 25.2 ± 2.1%; p < 0.05). CONCLUSIONS: Highly trained endurance athletes showed a ten-fold increase in the prevalence of focal LGE as compared to control subjects, always confined to the hinge points. Additionally, those athletes with focal LGE demonstrated globally higher myocardial ECV values. This matrix remodelling and potential presence of myocardial fibrosis may be another feature of the athlete's heart, of which the clinical and prognostic significance remains to be determined.

Three-dimensional regional bi-ventricular shape remodeling is associated with exercise capacity in endurance athletes.

AIMS: Endurance athletes develop cardiac remodeling to cope with increased cardiac output during exercise. This remodeling is both anatomical and functional and shows large interindividual variability. In this study, we quantify local geometric ventricular remodeling related to long-standing endurance training and assess its relationship with cardiovascular performance during exercise. METHODS: We extracted 3D models of the biventricular shape from end-diastolic cine magnetic resonance images acquired from a cohort of 89 triathlon athletes and 77 healthy sedentary subjects. Additionally, the athletes underwent cardio-pulmonary exercise testing, together with an echocardiographic study at baseline and few minutes after maximal exercise. We used statistical shape analysis to identify regional bi-ventricular shape differences between athletes and non-athletes. RESULTS: The ventricular shape was significantly different between athletes and controls (p < 1e-6). The observed regional remodeling in the right heart was mainly a shift of the right ventricle (RV) volume distribution towards the right ventricular infundibulum, increasing the overall right ventricular volume. In the left heart, there was an increment of left ventricular mass and a dilation of the left ventricle. Within athletes, the amount of such remodeling was independently associated to higher peak oxygen pulse (p < 0.001) and weakly with greater post-exercise RV free wall longitudinal strain (p = 0.03). CONCLUSIONS: We were able to identify specific bi-ventricular regional remodeling induced by long-lasting endurance training. The amount of remodeling was associated with better cardiopulmonary performance during an exercise test.

High-frequency burst vagal nerve simulation therapy in a natural primate model of genetic generalized epilepsy.

PURPOSE: Since the approval of Vagal Nerve Stimulation (VNS) Therapy for medically refractory focal epilepsies in 1997, it has been also reported to be effective for a wide range of generalized seizures types and epilepsy syndromes. Instead of conventional VNS Therapy delivered at 20-30Hz signal frequencies, this study evaluates efficacy and tolerability of high-frequency burst VNS in a natural animal model for genetic generalized epilepsy (GGE), the epileptic baboon. METHODS: Two female baboons (B1 P.h. Hamadryas and B2 P.h. Anubis x Cynocephalus) were selected because of frequently witnessed generalized tonic-clonic seizures (GTCS) for VNS implantation. High-frequency burst VNS Therapy was initiated after a 4-5 week baseline; different VNS settings (0.25, 2 or 2.5mA, 300Hz, 4 vs 7 pulses, 0.5-2.5s interburst interval, and intermittent stimulation for 1-2 vs for 24h per day) were tested over the subsequent 19 weeks, which included a 4-6 week wash-out period. GTCS frequencies were quantified for each setting, while seizure duration and postictal recovery times were compared to baseline. Scalp EEG studies were performed at almost every setting, including intermittent light stimulation (ILS) to evaluate photosensitivity. Pre-ILS ictal and interictal discharge rates, as well as ILS responses were compared between trials. The Novel Object test was used to assess potential treatment effects on behavior. RESULTS: High-frequency burst VNS Therapy reduced GTCS frequencies at all treatment settings in both baboons, except when output currents were reduced (0.25mA) or intermittent stimulation was restricted (to 1-2h/day). Seizure duration and postictal recovery times were unchanged. Scalp EEG studies did not demonstrate treatment-related decrease of ictal or interictal epileptic discharges or photosensitivity, but continuous treatment for 120-180s during ILS appeared to reduce photoparoxysmal responses. High-frequency burst VNS Therapy was well-tolerated by both baboons, without cardiac or behavioral changes. Repetitive muscle contractions involving the neck and left shoulder girdle were observed intermittently, most commonly at 0.5 interburst intervals, but these were transient, resolving with a few cycles of stimulation and not noted in wakefulness. CONCLUSIONS: This preclinical pilot study demonstrates efficacy and tolerability of high-frequency burst VNS Therapy in the baboon model of GGE. The muscle contractions may be due to aberrant propagation of the stimulus along the vagal nerve or to the ansa cervicalis, but can be reduced by minimal adjustment of current output or stimulus duration.

Multiple pulmonary embolisms in a male marathon athlete: Is intense endurance exercise a real thrombogenic risk?

Intense endurance exercise could be associated with multiple thrombogenic risk factors, including dehydration, hemoconcentration, inflammation, and injuries. Despite an increasing number of reported cases of deep venous thrombosis (DVT) in athletes that is raising awareness in the medical community, the prevalence is not well established and evidenced-based guidelines for management of this clinical scenario are lacking. We present an episode of unprovoked DVT and multiple pulmonary embolisms with severe right ventricular dysfunction in a male runner. We highlight the challenge of diagnosing DVT and pulmonary embolism in athletes due to frequently atypical symptomatology and the emergent need for longitudinal studies to evaluate their thrombogenic risk and develop specific guidelines in this field.

Haemophilus influenzae lysate induces aspects of the chronic obstructive pulmonary disease phenotype.

Nontypeable Haemophilus influenzae (NTHi) commonly colonizes the lower airways of patients with chronic obstructive pulmonary disease (COPD). Whether it contributes to COPD progression is unknown. Here, we determined which aspects of the COPD phenotype can be induced by repetitive exposure to NTHi products. Mice were exposed weekly to an aerosolized NTHi lysate, and inflammation was evaluated by measurement of cells and cytokines in bronchoalveolar lavage fluid (BALF) and immunohistochemical staining; structural changes were evaluated histochemically by periodic acid fluorescent Schiff's reagent, Masson's trichrome, and Picrosirius red staining; mucin gene expression was measured by quantitative RT-PCR; and the role of TNF-alpha was examined by transgenic airway overexpression and use of an inhibitory antibody. NTHi lysate induced rapid activation of NF-kappaB in airway cells and increases of inflammatory cytokines and neutrophils in BALF. Repetitive exposure induced infiltration of macrophages, CD8+ T cells, and B cells around airways and blood vessels, and collagen deposition in airway and alveolar walls, but airway mucin staining and gel-forming mucin transcripts were not increased. Transgenic overexpression of TNF-alpha caused BALF neutrophilia and inflammatory cell infiltration around airways, but not fibrosis, and TNF-alpha neutralization did not reduce BALF neutrophilia in response to NTHi lysate. In conclusion, NTHi products elicit airway inflammation in mice with a cellular and cytokine profile similar to that in COPD, and cause airway wall fibrosis but not mucous metaplasia. TNF-alpha is neither required for inflammatory cell recruitment nor sufficient for airway fibrosis. Colonization by NTHi may contribute to the pathogenesis of small airways disease in patients with COPD.

Subcellular fractions of Brucella ovis distinctively induce the production of interleukin-2, interleukin-4, and interferon-gamma in mice.

The aim of this study was to evaluate the effect of 3 Brucella ovis subcellular protein fractions: Outer membrane (OMP), inner membrane (IMP), and cytoplasm (CP), on cellular immune response by in vitro production of interleukin (IL)-2, IL-4, and interferon (IFN)-gamma. Each fraction was inoculated 3 times into Balb/c mice, primary cultures of mice spleen cells were done, and these were then stimulated with the fractions. Culture supernatants were collected at 24, 48, 72, 96, and 120 h postinoculation. Cytokine concentration was measured by Duoset-enzyme-linked immunosorbent assay (ELISA). The OMP fraction induced highest cellular immune response of 1000 pg/mL of IL-2 at 24 h, which decreased to < 100 pg/mL by 96 h. The IL-2 response for the IMP fraction was low at 24 h, but exceeded that of the OMP fraction at 72, 96, and 120 h. The CP showed a poor IL response. Regarding the IFN-gamma production, OMP and IMP induced a high response at 120 h. These results open the possibility for the use of B. ovis outer and inner membrane proteins as a subcellular vaccine.

The amoebicidal aqueous extract from Castela texana possesses antigenotoxic and antimutagenic properties.

Due to long-term treatment toxicity and clinical resistance to drugs commonly used against E. histolytica, new drugs against amoebiasis are urgently needed. Castela texana ("chaparro amargo") is a shrub taken traditionally in teas and capsules of dry plant to treat intestinal amoebic infections. An aqueous extract was prepared and its mutagenic, genotoxic and cytotoxicity properties were evaluated in prokaryotic and eukaryotic systems. This extract was neither mutagenic when evaluated with the Ames test in Salmonella typhimurium strains TA98, TA100 and TA102, nor genotoxic in unscheduled DNA synthesis in hepatocyte cultures, even at the highest concentrations tested. In fact, C. texana extract showed antimutagenic activity on S. typhimurium strains TA98 and TA100 in the Ames test. Furthermore, it was capable of protecting liver cell cultures against unscheduled DNA synthesis induced by 2-acetylaminofluorene at a concentration of 6.77 microg/ml. A free-radical scavenging test was used in order to explore the antioxidant capacity of C. texana extract with S. typhimurium strain TA102 pretreated with norfloxacin, a free radical producer. This extract showed a free radical withdrawal effect. The effective chemoprotective activity of this extract could be due to the antioxidant capacity of the C. texana extract components. In this paper it is shown that the antiamoebic natural product, C. texana, is also antimutagenic and protects against induction of preneoplastic lesions in rat liver. These results justify further studies to extend it use to human beings.

Antigenotoxic, antimutagenic and ROS scavenging activities of a Rhoeo discolor ethanolic crude extract.

Rhoeo discolor is a legendary plant used for treatment of superficial mycoses in Mexican traditional medicine. Despite its extended use, it is not known whether it has side-effects. An ethanolic crude extract from Rhoeo discolor was prepared, its mutagenic capacity was investigated by the Ames test, and its genotoxic activity in primary liver cell cultures using the unscheduled DNA synthesis assay. This extract was not mutagenic when tested with Salmonella typhimurium strains TA97, TA98 and TA100, and it did not elicit unscheduled DNA synthesis in hepatocyte cultures. In addition, we explored the antimutagenic and antigenotoxic activities of the extract and its ROS scavenger behaviour. Our results show that Rhoeo extract is antimutagenic for S. typhimurium strain TA102 pretreated with ROS-generating mutagen norfloxacin in the Ames test, and protects liver cell cultures against diethylnitrosamine induction of unscheduled DNA synthesis even at 1.9 ng per dish, which was the lowest dose tested. A free radical scavenging test was used in order to explore the antioxidant capacity of Rhoeo extract, as compared with three commercial well-known antioxidants quercetin, ascorbic acid and tocopherol. Rhoeo extract showed less radical scavenging effect than quercetin, but similar to that of alpha-tocopherol and more than ascorbic acid. It is important to note that this extract was neither mutagenic in S. typhimurium nor genotoxic in liver cell culture, even at concentrations as high as four- and 166-fold of those needed for maximal antimutagenic or chemoprotective activities, respectively.

Expressed CYP4A4 metabolism of prostaglandin E(1) and arachidonic acid.

Cytochrome P4504A4 (CYP4A4) is a hormonally induced pulmonary cytochrome P450 which metabolizes prostaglandins and arachidonic acid (AA) to their omega-hydroxylated products. Although the physiological function of this enzyme is unknown, prostaglandins play an important role in the regulation of reproductive, vascular, intestinal, and inflammatory systems and 20-hydroxyeicosatetraenoic acid, the omega-hydroxylated product of arachidonate, is a potent vasoconstrictor. Therefore, it is important to obtain sufficient quantities of the protein for kinetic and biophysical characterization. A CYP4A4 construct was prepared and expressed in Escherichia coli. The enzyme was purified, and its activity with substrates prostaglandin E(1) (PGE(1)) and AA was examined in the presence and absence of cytochrome b(5) (cyt b(5)) and with a heme-depleted form of cyt b(5) (apo b(5)). The stimulatory role played by cyt b(5) in this system is not dependent on electron transfer from cyt b(5) to the CYP4A4 as similar stimulation was observed with apo b(5). Rapid kinetic measurement of CYP4A4 electron transfer rates confirmed this result. Both flavin and heme reduction rates were constant in the absence and presence of cyt b(5) or apo b(5). CD spectroscopy demonstrated that a conformational change occurred in CYP4A4 protein upon binding of cyt b(5) or apo b(5). Finally, acetylenic fatty acid inhibitors 17-octadecynoic acid, 12-hydroxy-16-heptadecynoic acid, 15-hexadecynoic acid, and 10-undecynoic acid (10-UDYA) were used to probe the substrate-binding pocket of CYP4A4. The short-chain fatty acid inhibitor 10-UDYA was unable to inhibit either PGE(1) or AA metabolism. All but 10-UDYA were effective inhibitors of CYP4A4.

Entamoeba histolytica: transferrin binding proteins.

Entamoeba histolytica trophozoites depend on iron for their growth; thus, they must use some host iron-containing molecules to fulfill this requirement. In this work we report that amoebas are able to utilize human holo-Tf as iron source and to recognize it through transferrin binding proteins. By use of an anti-human transferrin antiserum in an immunoblotting assay, two main polypeptides with apparent molecular masses of 70 and 140 kDa were found in total extract of trophozoites cultured in vitro. However, when a monoclonal anti-human transferrin receptor antibody was used, only one band with molecular mass of 140 kDa was observed. Both the human transferrin and the monoclonal antibody recognized a protein on the amoebic surface, demonstrated by confocal microscopy. Furthermore, the complex transferrin-transferrin binding protein was internalized by an endocytic process and probably dissociated inside the cell. This mechanism could be one manner in which E. histolytica acquires iron from the human host transferrin.

Membrane vesicles released by Actinobacillus pleuropneumoniae contain proteases and Apx toxins.

Actinobacillus pleuropneumoniae serotype 1 releases vesicles containing proteases and Apx toxins into the culture medium. Vesicles were concentrated by ultracentrifugation and analyzed by electron microscopy and electrophoresis; their size ranged from 20 to 200 nm. A polyclonal antiserum raised against a purified high molecular mass secreted protease of serotype 1 recognized this protease on the surface of the vesicles by immunogold electron microscopy. Higher molecular mass polypeptides from vesicle extracts were recognized by the antiserum by Western immunoblot, indicating that the protease could form oligomers. However, these oligomers were not active against gelatin until secreted. Additionally, Apx toxins were also present in vesicles, and were recognized by Western immunoblot by an anti-serotype 1 toxins polyclonal serum. A. pleuropneumoniae antigens in vesicles were recognized by convalescent-phase pig sera from animals infected with serotype 1 or 5. The release of vesicles containing virulence factors could be a tissue damage mechanism in swine pleuropneumonia.

The C termini of constitutive nitric-oxide synthases control electron flow through the flavin and heme domains and affect modulation by calmodulin.

The sequences of nitric-oxide synthase flavin domains closely resemble that of NADPH-cytochrome P450 reductase (CPR). However, all nitric-oxide synthase (NOS) isoforms are 20-40 residues longer in the C terminus, forming a "tail" that is absent in CPR. To investigate its function, we removed the 33 and 42 residue C termini from neuronal NOS (nNOS) and endothelial NOS (eNOS), respectively. Both truncated enzymes exhibited cytochrome c reductase activities without calmodulin that were 7-21-fold higher than the nontruncated forms. With calmodulin, the truncated and wild-type enzymes reduced cytochrome c at approximately equal rates. Therefore, calmodulin functioned as a nonessential activator of the wild-type enzymes and a partial noncompetitive inhibitor of the truncated mutants. Truncated nNOS and eNOS plus calmodulin catalyzed NO formation at rates that were 45 and 33%, respectively, those of their intact forms. Without calmodulin, truncated nNOS and eNOS synthesized NO at rates 14 and 20%, respectively, those with calmodulin. By using stopped-flow spectrophotometry, we demonstrated that electron transfer into and between the two flavins is faster in the absence of the C terminus. Although both CPR and intact NOS can exist in a stable, one-electron-reduced semiquinone form, neither of the truncated enzymes do so. We propose negative modulation of FAD-FMN interaction by the C termini of both constitutive NOSs.

A 24-kDa cloned zinc metalloprotease from Actinobacillus pleuropneumoniae is common to all serotypes and cleaves actin in vitro.

Actinobacillus pleuropneumoniae causes pleuropneumonia in swine. This bacterium secretes proteases that degrade porcine hemoglobin and IgA in vitro. To further characterize A. pleuropneumoniae proteases, we constructed a genomic library expressed in Escherichia coli DH5alpha, and selected a clone that showed proteolytic activity. The recombinant plasmid carries an 800-base pair A. pleuropneumoniae gene sequence that.codes for a 24-kDa polypeptide. A 350-base pair PstI fragment from the sequence hybridized at high stringency with DNA from 12 serotypes of A. pleuropneumoniae, but not with DNA from Actinobacillus suis, Haemophilus parasuis, Pasteurella haemolytica, Pasteurella multocida A or D, or E. coli DH5alpha, thus showing specificity for A. pleuropneumoniae. The expressed polypeptide was recognized as an antigen by convalescent-phase pig sera. Furthermore, a polyclonal antiserum developed against the purified polypeptide recognized an A. pleuropneumoniae oligomeric protein in both crude-extract and cell-free culture media. This recombinant polypeptide cleaved azocoll, gelatin, and actin. Inhibition of the proteolytic activity by diethylpyrocarbonate suggests that this polypeptide is a zinc metalloprotease.

The C terminus of mouse macrophage inducible nitric-oxide synthase attenuates electron flow through the flavin domain.

The sequences of nitric-oxide synthase (NOS) flavin domains closely resemble that of NADPH-cytochrome P450 reductase (CPR), with the exception of a few regions. One such region is the C terminus; all NOS isoforms are 20-40 amino acids longer than CPR, forming a "tail" that is absent in CPR. To investigate its function, we removed the 21-amino acid C-terminal tail from murine macrophage inducible NOS (iNOS) holoenzyme and from a flavin domain construct. Both the truncated holoenzyme and reductase domain exhibited cytochrome c reductase activities that were 7-10-fold higher than the nontruncated forms. The truncated holoenzyme catalyzed NO formation approximately 20% faster than the intact form. Using stopped-flow spectrophotometry, we demonstrated that electron transfer into and between the two flavins and from the flavin to the heme domain is 2-5-fold faster in the absence of the C-terminal tail. The heme-nitrosyl complex, formed in all NOS isoforms during NO catalysis, is 5-fold less stable in truncated iNOS. Although both CPR and intact NOS can exist in a stable, one electron-reduced semiquinone form, neither the truncated holoenzyme nor the truncated flavin domain demonstrate such a form. We propose that this C-terminal tail curls back to interact with the flavin domain in such a way as to modulate the interaction between the two flavin moieties.

The clinically isolated FIZ15 bacteriophage causes lysogenic conversion in Pseudomonas aeruginosa PAO1.

FIZ15 bacteriophage, from a human clinical isolate of Pseudomonas aeruginosa, causes lysogenic conversion in the P. aeruginosa strain PAO1. The prophage-conferred phenotypes are: (1) increased resistance to phagocytosis by mouse peritoneal macrophages; (2) increased resistance to killing by normal human serum, and (3) increased adhesion to human buccal epithelial cells. These phenotypes are related to the prophage-induced change at the level of its own bacterial receptor, which appears to be the O-antigen.

Secretion of proteases from Pasteurella multocida isolates.

The capability of Pasteurella multocida to secrete proteases to the culture medium and their characterization were studied in five animal isolates (bovine, chicken, sheep, and two from pig). All the isolates produced proteases in a wide range of molecular mass. It is suggested that they are neutral metalloproteases, since they were optimally active between pH 6 and 7, inhibited by chelating agents but not by other protease inhibitors, and reactivated by calcium. Proteases from isolates were able to degrade IgG. Several proteins from supernatants of cultures precipitated with 70% (NH4)2SO4 of all the P. multocida isolates were recognized by a polyclonal antiserum raised against a purified protease from Actinobacillus pleuropneumoniae. Protease production might play an important role during tissue colonization and in P. multocida diseases.

Purification and characterization of a protease from Actinobacillus pleuropneumoniae serotype 1, an antigen common to all the serotypes.

A high molecular-mass proteolytic enzyme of Actinobacillus pleuropneumoniae serotype 1, was purified from culture supernatants (CSN) by using DEAE-cellulose and sepharose-4B-gelatin chromatography. In 10% SDS-polyacrylamide gels copolymerized with porcine gelatin, the protease showed a single band of activity of > 200 kDa. However, minor molecular-mass proteolytic bands were observed when the protease was electrophoresed in the presence of either 5% beta-mercaptoethanol, 50 mM dithiothreitol, or 0.25 M urea. Furthermore, when the > 200-kDa purified protein was passed through a sucrose gradient, several bands with proteolytic activity were found: 62, 90, 190, and 540 kDa. The proteolytic activity was increased in the presence of calcium or zinc and was not affected after being heated at 90 degrees C for 5 min. Proteolytic activities were also observed in CSN from all A. pleuropneumoniae serotypes and biotypes. The purified protease hydrolyzed porcine IgA and IgG in vitro. In addition, by immunoblot the protease was recognized by serum of naturally infected pigs with serotypes 1 and 5, and by serum of pigs experimentally infected with serotypes 1, 2, 8, or 9. Serum of a pig vaccinated with CSN of a serotype 3 strain also recognized the protease, but not sera of pigs vaccinated with a bacterin (serotype 1). Proteins from CSN of all the serotypes, which were precipitated with 70% (NH4)2SO4, were recognized by a polyclonal antibody raised against the purified protease. Taken together these results indicate that an antigenic protease is produced in vivo by all the serotypes of A. pleuropneumoniae. The results indicate that proteases could have a role in the disease and in the immune response of pigs infected with A. pleuropneumoniae.

Entamoeba histolytica HM1:IMSS: hemoglobin-degrading neutral cysteine proteases.

Entamoeba histolytica HMI:IMSS trophozoites were able to utilize human hemoglobin but not hemin as a sole iron source to grow in vitro. Proteases from crude extracts of E. histolytica degraded human, porcine, and bovine hemoglobins at pH 7.0. These proteolytic activities were found by electrophoresis in SDS-polyacrylamide gels copolymerized with hemoglobin, with apparent molecular weights of 116, 82, and 21 kDa, the 82-kDa protein being the most active protease against this substrate. The proteases were classified in the cysteine group since the activities were inhibited by l-trans-epoxysuccinylleucylamido(4-guanidino)butane, p-hydroxymercuribenzoate, iodoacetate, and N-ethylmaleimide and activated with dithiothreitol. Other pathogenic strains of E. histolytica showed the same pattern of hemoglobinases. These hemoglobin-degrading proteases could be playing an important role in iron acquisition by E. histolytica.

Entamoeba histolytica: proteinase secretion induced by collagen type I is dependent on cytoskeleton integrity.

Proteolytic activities of the protozoan parasite Entamoeba histolytica strain HM1:IMSS and the cytochalasin D-resistant mutant BG-3 were analyzed following stimulation with collagen type I and Ca2+, which induces electron-dense associated collagenase secretion. The mutant BG-3 had a protease activity of 73 kDa and secretion of total protease activity was not stimulated by collagen type I and Ca2+, which produced, in contrast, a 2-fold increase in protease secretion by the parental strain. This collagen-stimulated protease secretion was inhibited by cytochalasin D at a concentration of 1 microgram/ml. Cytochalasin D did not have any effect on the protease activity released by the mutant BG-3. These findings suggest that cytoskeleton integrity is necessary for collagen-induced protease secretion.