Mycobacterium abscessus Infected Neutrophils as an In Vitro Model for Bronchiectasis. Neutrophils Prevent Mycobacterial Aggregation

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Increased serum concentrations of estrogen-induced growth factors Midkine and FGF2 in NF1 patients with plexiform neurofibroma.

Neurofibromatosis type 1 (NF1) predisposes to the development of dermal and plexiform neurofibromas and serum of NF1 patients stimulates neurofibroma proliferation in vitro. This study aimed to determine whether, in NF1 patients, serum levels of midkine (MK) and fibroblast growth factor 2 (FGF2) were associated with the number and/or type of neurofibromas. In addition, their concentrations were correlated with serum levels of dehydroepiandrosterone sulfate (DHEAS), a neurosteroid secreted by the peripheral nervous system. We performed a case control-study and measured, by ELISA assay, serum concentrations of MK, FGF2, and DHEAS in 20 NF1 patients and 30 controls. We found increased serum levels of MK and FGF2 in NF1 patients between 30 and 50 years old. Their concentrations were significantly higher in NF1 patients with plexiform neurofibromas than in controls (P=0.003 for MK and P=0.008 for FGF2). As an underlying hormonal regulation was suspected, DHEAS serum levels were measured but no difference was observed between patients and controls. We also observed a strong association between MK and FGF2 levels (P=0.0001) in NF1 patients and controls. In conclusion, we point out MK and FGF2 as biomarkers for plexiform neurofibroma in NF1 patients. As both growth factors are estrogen-responsive genes and neurofibromin is a co-repressor of estrogen receptor alpha activity, we suggest that the increased serum levels of MK and FGF2 observed in NF1 patients might be due to estradiol hypersensitivity.

Mycobacterium avium complex infected cells promote growth of the pathogen Pseudomonas aeruginosa.

Bronchiectasis is considered a consequence of the neutrophilic inflammatory response to infection. Mycobacterial infections, mainly from the Mycobacterium avium complex and M. abscessus, have been inextricably linked to bronchiectasis development. The most important pathogen that infect patients with bronchiectasis is Pseudomonas aeruginosa, associated with an increased risk of death. Patients with bronchiectasis are often co-infected with P. aeruginosa and M. avium complex, and it was studied whether they interacted in immune cell cultures. Peripheral blood mononuclear cells from healthy volunteers were infected overnight with clinical isolates of mycobacteria, 18 h later co-infected with P. aeruginosa and Pseudomonas multiplication was quantified. Inoculated P. aeruginosa multiply faster when cells were previously infected in vitro with M. avium complex or M. tuberculosis, but not with M. kansasii or M. gordonae, mycobacteria not regularly isolated from patients with bronchiectasis. The interaction between mycobacteria and P. aeruginosa also takes place in the absence of cells, but to a lower degree. Growth of Staphylococcus aureus, less frequently co-isolated with mycobacteria, was not affected by previous infection with mycobacteria. Surprisingly, multiplication of P. aeruginosa in neutrophil cultures did not vary in the presence of mycobacteria. Nevertheless, co-infection of mycobacteria and P. aeruginosa induced the production of IL-1ß, a mediator of neutrophilic inflammation. P. aeruginosa stimulation by mycobacteria provides evidence for explaining their common clinical association. Strategies to control mycobacteria may be useful to impair P. aeruginosa colonization.

A strategy based on Amplified Fragment Length Polymorphism (AFLP) for routine genotyping of nontuberculous mycobacteria at the clinical laboratory.

The increasing worldwide incidence of mycobacteriosis and the need to achieve improved clinical management makes nontuberculous mycobacteria (NTM) genotyping a useful tool. However, because of technical difficulties, medium size microbiology laboratories do not attempt to compare the genetic patterns that each of their isolates present. We have aimed to optimize a genotyping method with a reduced hands-on experimental time and that requires few technical resources. A strategy based on the Amplified Fragment Length Polymorphism (AFLP) methodology was developed using two rare-cutters enzymes (SacI and BglII). One out of seven primers was sequentially used in each amplification reaction that was analyzed by agarose gel electrophoresis. This approach makes it possible the timely genotyping of a moderate number of strains and its characterization without the need of image analysis software. We have genotyped 28 Mycobacterium intracellulare and 4 M. abscessus. Clinical researchers are encouraged to routinely genotype their NTM isolates.

The unexplained increase of nontuberculous mycobacteriosis.

Epidemiological data show a worldwide increase in nontuberculous mycobacteriosis. Although it has been partially attributed to the improvement of microbiological methodologies that has allowed a better recovery and identification of nontuberculous mycobacteria (NTM), it is generally accepted that there is a genuine incidence augmentation. The reasons of the increase are likely multifactorial, depending on the nature of the pathogen, the host, and their interaction. Mycobacteria from the Mycobacterium tuberculosis complex has been regarded as pathogenic and NTM as opportunistic and nontransmissible. Nevertheless, few differences have been found in either their phenotypic or genotypic characteristics. The phenomenon of M. tuberculosis adaptation to the human host may be taking place again in NTM as a consequence of human environmental alterations that facilitate the interaction with the pathogen. The current worsening of the immunological status of increasing numbers of individuals, a result of factors such as malnutrition (obesity and diabetes), population aging or the widespread use of immunosuppressive medication, may be allowing the rapid evolution and person-to-person transmission of NTM. It is likely that mycobacteriosis incidence will keep escalating. New measures should be taken to deal with these diseases, including their reportability and the implementation of strain genotyping that would shed light on the NTM dissemination routes from the environment or human hosts.

Gametocytes from K13 Propeller Mutant Plasmodium falciparum Clinical Isolates Demonstrate Reduced Susceptibility to Dihydroartemisinin in the Male Gamete Exflagellation Inhibition Assay.

Mutations in the kelch propeller domain (K13 propeller) of Plasmodium falciparum parasites from Southeast Asia are associated with reduced susceptibility to artemisinin. We exposed in vitro-cultured stage V gametocytes from Cambodian K13 propeller mutant parasites to dihydroartemisinin and evaluated the inhibition of male gamete formation in an in vitro exflagellation inhibition assay (EIA). Gametocytes with the R539T and C580Y K13 propeller alleles were less susceptible to dihydroartemisinin and had significantly higher 50% inhibitory concentrations (IC50s) than did gametocytes with wild-type alleles.

Predicting transmission blocking potential of anti-malarial compounds in the Mosquito Feeding Assay using Plasmodium falciparum Male Gamete Inhibition Assay.

Plasmodium falciparum Standard Membrane Feeding Assay (PfSMFA) is the current gold standard mosquito based confirmatory transmission blocking (TrB) assay for human malaria. However, owing to its complexity only selected gametocytocidal molecules are progressed into SMFA. Predictive tools for evaluation of TrB behavior of compounds in SMFA would be extremely beneficial, but lack of substantially large data sets from many mosquito feeds preempts the ability to perform correlations between outcomes from in vitro assays and SMFA. Here, a total of 44 different anti-malarial compounds were screened for inhibitory effect on male gamete formation in exflagellation inhibition assay (EIA) and the same drug-treated parasites were fed to mosquitoes in SMFA. Regression analysis was performed between outcomes of the two assays and regression models were applied to a randomly selected validation set of four compounds indicating no overfitting and good predictive power. In addition, the pIC50 for 11 different compounds obtained in the EIA was also correlated with pIC50's in SMFA. Resulting regression models provided pIC50 predictions in SMFA with reasonably good accuracy thereby demonstrating the use of a simple in vitro assay to predict TrB of molecules in a complex mosquito based assay.

Estudio retrospectivo en el diagnóstico de Mycoplasma y Ureaplasma en muestra seminal de 89 pacientes en la Ciudad de México / Retrospective study on ureaplasma and mycoplasma detected in 89 patients semen

Resumen: Antecedentes: El Mycoplasma hominis, Mycoplasma genitalium y Ureaplasma urealyticum son microorganismos que se encuentran asociados a problemas reproductivos como la enfermedad pélvica inflamatoria en mujeres y la uretritis no gonocócica en hombres. Se han descrito alteraciones vinculadas a la infección por estos microorganismos, en parámetros reproductivos como la morfología espermática, el índice de infertilidad y la subfertilidad en el hombre. Objetivos: Determinar la incidencia de Mycoplasma spp. y Ureaplasma spp. en pacientes del Distrito Federal (México) y evaluar las posibles correlaciones con sus resultados en la espermatobioscopía. Material y método: Estudio retrospectivo de 89 pacientes positivos a Mycoplasma y Ureaplasma. Se analizaron los resultados de la espermatobioscopía y se correlacionaron estadísticamente. Resultados: Se obtuvo un aumento en el diagnóstico de estos 2 microorganismos en un 15% en el primer semestre del 2013 respecto al año 2012. Se encontraron diferencias significativas (P < 0.001) en la morfología espermática de pacientes positivos a alguno de los 2 agentes. Conclusiones: El Mycoplasma y el Ureaplasma se consideran como flora normal del tracto genitourinario, aun así, debe realizarse su diagnóstico en pacientes con historial de fertilidad y subfertilidad y debe ser considerado cuando en el espermograma informe de alteraciones morfológicas y aumento del recuento de leucocitos.

Abstract: Background: The Mycoplasma hominis, Mycoplasma genitalium and Ureaplasma urealyticum are microorganisms associated with reproductive problems such as pelvic inflammatory disease in women and non-gonococcal urethritis in men. Alterations have been described in reproductive parameters and sperm morphology, rate of infertility and subfertility in man. Objectives: The aim of this study was to determine the incidence of Mycoplasma spp. and Ureaplasma spp. in patients Federal District (Mexico) and to evaluate the possible correlations with the semen analysis results. Material and methods: Retrospective study of 89 patients positive for Mycoplasma and Ureaplasma, we analyzed the results of the semen analysis and correlated statistically. Results: We obtained an increase in the diagnosis of these two microorganisms, by 15% in the first half of 2013 compared with 2012. Significant differences (P <0.001) in sperm morphology in patients positive to one of the two agents, the most frequent was the Ureaplasma. Conclusions: Mycoplasma and Ureaplasma are considered normal flora of the genitourinary tract, even so, the diagnosis should be performed in patients with a history of fertility and subfertility and should be considered when reporting morphological abnormalities and increased leukocyte count.

Macrophages from elders are more permissive to intracellular multiplication of Mycobacterium tuberculosis.

The elderly account for a disproportionate share of all tuberculosis cases, and the population ageing may not fully explain this phenomenon. We have performed in vitro infection experiments to investigate whether there is an immunological basis for the apparent susceptibility of elders to tuberculosis. In our infection model, Mycobacterium tuberculosis induces a higher production of interleukin (IL)-6 and reactive oxygen species in macrophages from elders than from younger adults. This response did not prevent, however, an increased multiplication of M. tuberculosis in macrophages from elders as compared with the growth observed within cells from adults. By performing a factorial experiment, we have found that IFN-γ, but not IL-1ß, IL-6 or TNF-α, stimulate the macrophages to restrict the multiplication of the bacterium in macrophages from elders. Although monocytes from elders seem to be in a higher level of activation, we present evidences that protein tyrosine phosphorylation response induced by M. tuberculosis is stronger in monocytes from adults than from elders. Using a protein array that detects 71 tyrosine phosphorylated kinases, we identified Pyk2 as the only kinase that displayed a difference of intensity larger than 50 % in adults than in elders. Furthermore, monocytes from elders that were incubated in the presence of tyrosine kinase inhibitors (genistein and PP2) allowed a higher level of bacterial multiplication. These observations may help to explain the susceptibility of elders to tuberculosis. An unexpected result was that both genistein and its negative control, daidzein, abundant soy isoflavones, promoted intracellular mycobacterial growth.

Non-chemotactic influence of CXCL7 on human phagocytes. Modulation of antimicrobial activity against L. pneumophila.

We have investigated the role of CXCL7 in the immune response of human phagocytes against the intracellular bacteria Mycobacterium tuberculosis and Legionella pneumophila. We have observed that polymorphonuclear neutrophil (PMN) chemotaxis induced by the supernatants of infected monocyte derived macrophages (MDM) may be attributed to CXCL8 rather than CXCL7, although both chemokines are present in large quantities. We have also found that CXCL7 is present not only in the supernatants of MDM, but also in the supernatants of PMN of some, but not all, individuals. Western blot analysis revealed that, in both MDM and PMN supernatants appeared two bands with molecular weights consistent with the platelet basic protein (PBP) and the neutrophil activating protein-2 (NAP-2) sizes. Regarding the influence on infected cells, recombinant NAP-2 enhanced the antimicrobial activity of IFNγ activated MDM against L. pneumophila, but not against M. tuberculosis. In addition, U937 cells transfected with a NAP-2 construct inhibited the intracellular multiplication of L. pneumophila, supporting its role in the modulation of the antimicrobial activity. Finally, U937 cells transfected with the NAP-2 construct showed an adherence that was dramatically enhanced when the substrate was fibronectin. We conclude that human phagocytes produce CXCL7 variants that may have a significant influence on the immune response against bacterial pathogens.

Apoptosis and oxidative burst in neutrophils infected with Mycobacterium spp.

Two of the better characterized antimicrobial mechanisms displayed by human neutrophils are the reactive oxygen species (ROS) production and the induction of apoptosis. Their importance in mycobacterial infections is, however, controversial and we aimed to analyze them simultaneously in neutrophils infected with either Mycobacterium tuberculosis or the non-pathogenic M. gordonae. Neither species is eliminated by neutrophils but the pattern exhibited for both activities is completely different. M. tuberculosis induces ROS production and apoptosis but M. gordonae does not. Additional evidence was provided by an attenuated strain of M. gordonae that, although it has become susceptible to the antimicrobial activity of neutrophils, it still does not promote ROS production or apoptosis. Therefore no relationship could be established between any of these activities and the ability of neutrophils to kill mycobacteria. We have also observed that neutrophil concentration, a variable that is important in the antimicrobial activity against other pathogens, has no influence in the mycobacterial intracellular growth.

Iron porphyrinate Fe(TPPS) reduces brain cell damage in rats intrastriatally lesioned by quinolinate.

It has been recently demonstrated that the reactive nitrogen species (RNS) peroxynitrite (ONOO(-)) is involved in the neurotoxic pattern produced by quinolinic acid in the rat brain [V. Pérez-De La Cruz, C. González-Cortés, S. Galván-Arzate, O.N. Medina-Campos, F. Pérez-Severiano, S.F. Ali, J. Pedraza-Chaverrí, A. Santamaría, Excitotoxic brain damage involves early peroxynitrite formation in a model of Huntington's disease in rats: protective role of iron porphyrinate 5,10,15,20-tetrakis (4-sulfonatophenyl)porphyrinate iron (III), Neuroscience 135 (2005) 463-474.]. The aim of this work was to investigate whether ONOO(-) can also be responsible for morphological alterations and inflammatory events in the same paradigm. For this purpose, we evaluated the effect of a pre-treatment with the iron porphyrinate Fe(TPPS), a well-known ONOO(-) decomposition catalyst (10 mg/kg, i.p., 120 min before lesion), on the quinolinate-induced striatal cell damage and immunoreactivities to glial-fibrilar acidic protein (GFAP), interleukin 6 (IL-6) and inducible nitric oxide synthase (iNOS), one and seven days after the intrastriatal infusion of quinolinate (240 nmol/microl) to rats. The striatal tissue from animals lesioned by quinolinate showed a significant degree of damage and enhanced immunoreactivities to GFAP, IL-6 and iNOS, both at 1 and 7 days post-lesion. Pre-treatment of rats with Fe(TPPS) significantly attenuated or prevented all these markers at both post-lesion times tested, except for GFAP immunoreactivity at 7 days post-lesion and iNOS immunoreactivity at 1 day post-lesion. Altogether, our results suggest that ONOO(-) is actively participating in triggering inflammatory events and morphological alterations in the toxic model produced by quinolinate, since the use of agents affecting its formation, such as Fe(TPPS), are effective experimental tools to reduce the brain lesions associated to excitotoxic and oxidative damage.

Human phagocytes lack the ability to kill Mycobacterium gordonae, a non-pathogenic mycobacteria.

Non-pathogenic mycobacteria, like Mycobacterium gordonae, are rarely associated to disease. The analysis of the mechanisms which are successful against them in the human host may provide useful information to understand why they fail against the pathogenic M. tuberculosis. We have developed an infection model to test the ability of human phagocytes to kill two strains of M. gordonae, HL184G and an attenuated variety, HL184Gat. As controls we included a strain of M. tuberculosis (HL186T) and another one of L. pneumophila (ATCC13151). We observed that human phagocytes lack the intrinsic ability to eliminate either M. gordonae or M. tuberculosis, but they can kill the attenuated strain. We found a relationship between pathogenicity and the pattern of cytokine production. Thus, both the pathogenic M. tuberculosis and Legionella pneumophila, but not the non-pathogenic M. gordonae, induced the production of significantly different levels of IL-1beta, IL-6 and TNF-alpha in monocytes and IL-8 in neutrophils. Although both monocytes and neutrophils killed HL184Gat, but not HL184G, the patterns of cytokine production induced by either strain were identical. Addition of INF-gamma and/or TNF-alpha did not enhance the antimycobacterial activity of phagocytes.

Protective effect of S-allylcysteine on 3-nitropropionic acid-induced lipid peroxidation and mitochondrial dysfunction in rat brain synaptosomes.

3-Nitropropionic acid is a neurotoxin that irreversibly inhibits succinate dehydrogenase, a relevant enzyme constituting the complex II of the respiratory chain during mitochondrial electron transport. 3-Nitropropionic acid is known to produce oxidative/nitrosative stress and evokes an experimental model of Huntington's disease. In this work we evaluated the effects of the antioxidant compound and major organosulfur garlic derivative, S-allylcysteine, on lipid peroxidation and mitochondrial dysfunction induced by 3-nitropropionic acid in synaptosomal fractions from rat brain. 3-Nitropropionic acid, at concentrations ranging 0.75-2.5 mM, produced enhanced levels of lipid peroxidation, while increasing concentrations of S-allylcysteine (0.1-2 mM) decreased the peroxidative action of 3-nitropropionic acid (1 mM) in synaptosomal fractions in a concentration-dependent manner. S-Allylcysteine (0.75 mM) also prevented the 3-nitropropionic acid (1mM)-induced mitochondrial dysfunction. These findings suggest that the protective actions that S-allylcysteine exert on the in vitro neurotoxicity induced by 3-nitropropionic acid are mediated by its antioxidant properties.

Selenium reduces the proapoptotic signaling associated to NF-kappaB pathway and stimulates glutathione peroxidase activity during excitotoxic damage produced by quinolinate in rat corpus striatum.

Quinolinate (QUIN) neurotoxicity has been attributed to degenerative events in nerve tissue produced by sustained activation of N-methyl-D-aspartate receptor (NMDAr) and oxidative stress. We have recently described the protective effects that selenium (Se), an antioxidant, produces on different markers of QUIN-induced neurotoxicity (Santamaría et al., 2003, J Neurochem 86:479-488.). However, the mechanisms by which Se exerts its protective actions remain unclear. Since some of these events are thought to be related with inhibition of deadly molecular cascades through the activation of antioxidant selenoproteins, in this study we investigated the effects of Se on QUIN-induced cell damage elicited by the nuclear factor kappaB (NF-kappaB) pathway, as well as the time-course response of striatal glutathione peroxidase (GPx) activity. Se (sodium selenite, 0.625 mg/kg/day, i.p.) was administered to rats for 5 days, and 120 min after the last administration, animals received a single striatal injection of QUIN (240 nmol/mul). Twenty-four hours later, their striata were tested for the expression of IkappaB-alpha (the NF-kappaB cytosolic binding protein), the immunohistochemical expression of NF-kappaB (evidenced as nuclear expression of P65), caspase-3-like activation, and DNA fragmentation. Additional groups were killed at 2, 6, and 24 h for measurement of GPx activity. Se reduced the QUIN-induced decrease in IkappaB-alpha expression, evidencing a reduction in its cytosolic degradation. Se also prevented the QUIN-induced increase in P65-immunoreactive cells, suggesting a reduction of NF-kappaB nuclear translocation. Caspase-3-like activation and DNA fragmentation produced by QUIN were also inhibited by Se. Striatal GPx activity was stimulated by Se at 2 and 6 h, but not at 24 h postlesion. Altogether, these data suggest that the protective effects exerted by Se on QUIN-induced neurotoxicity are partially mediated by the inhibition of proapoptotic events underlying IkappaB-alpha degradation, NF-kappaB nuclear translocation, and caspase-3-like activation in the rat striatum, probably involving the early activation of GPx.