Effects of atmospheric carbon dioxide fertilization on biomass and secondary metabolites of some plant species with pharmacological interes under greenhouse conditions

RESUMEN El aumento del CO2 atmosférico debido al cambio global y/o a las prácticas hortícolas promueve efectos directos sobre crecimiento vegetal y el desarrollo. Estas respuestas pueden ocurrir en ecosistemas naturales, pero también se pueden utilizar para aumentar la producción de algunas plantas y de algunos compuestos secundarios. El actual trabajo intenta estudiar los efectos del enriquecimiento atmosférico del CO2 bajo condiciones de invernadero en el crecimiento y la concentración y la composición de metabolitos secundarios de Taxus bacatta, Hypericum perforatum y Echinacea purpurea en condiciones ambientales mediterráneas. La fertilización del CO2 muestra perspectivas interesantes para la mejorara y aplicabilidad de técnicas hortícolas para aumentar productividad de plantas medicinales, a pesar de diferencias claras entre la especie. En general esta técnica promueve aumentos importantes y significativos en producción primaria y, en algunos casos, también en compuestos secundarios. Esto tiene una gran importancia hortícola porque la productividad a nivel de cosecha total aumenta, directamente porque se aumenta la concentración e indirectamente porque se aumenta la biomasa. SUMMARY The increase of atmospheric CO2 due to global change and/or horticultural practices promotes direct effects on plant growth and development. These responses may occur in natural ecosystems, but also can be used to increase the production of some plants and some secondary compounds. Present work tries to study the effects of atmospheric CO2 enrichment under greenhouse conditions on growth and in the concentration and composition of secondary metabolites of Taxus bacatta, Hypericum perforatum and Echinacea purpurea under Mediterranean environmental conditions. CO2 fertilization shows interesting perspectives to increase and improve horticultural techniques in order to increase plant medicinal productivity, in spite of clear differences among the species. In general this technique promotes important and significant increases in primary productivity and, in some cases, also in secondary compounds. This has a great horticultural relevance because the total productivity of this kind of products increase at crop level, directly because concentration is increased and /or indirectly because biomass is increased. RESUM L'augment del CO2 atmosfèric a causa del canvi global i/o a les pràctiques hortícoles promou efectes directes sobre creixement vegetal i el desenvolupament. Aquestes respostes poden ocórrer en ecosistemes naturals, però també es poden utilitzar per a augmentar la producció d'algunes plantes i d'alguns compostos secundaris. L'actual treball intenta estudiar els efectes de l'enriquiment atmosfèric del CO2 sota condicions d'hivernacle en el creixement i la concentració i la composició de metabòlits secundaris de Taxus bacatta, Hypericum perforatum i Echinacea purpurea en condicions ambientals mediterrànies. La fertilització del CO2 mostra perspectives interessants per a la millora i aplicabilitat de tècniques hortícoles per a augmentar productivitat de plantes medicinals, a pesar de diferències clares entre l'espècie. En general aquesta tècnica promou augments importants i significatius en producció primària i, en alguns casos, també en compostos secundaris. Això té una gran importància hortícola perquè la productivitat a nivell de collita total augmenta, directament perquè s'augmenta la concentració i indirectament perquè s'augmenta la biomassa.

Liquid chromatography-tandem mass spectrometry (LC/APCI-MS/MS) methods for the quantification of captan and folpet phthalimide metabolites in human plasma and urine.

Captan and folpet are fungicides largely used in agriculture. They have similar chemical structures, except that folpet has an aromatic ring unlike captan. Their half-lives in blood are very short, given that they are readily broken down to tetrahydrophthalimide (THPI) and phthalimide (PI), respectively. Few authors measured these biomarkers in plasma or urine, and analysis was conducted either by gas chromatography coupled to mass spectrometry or liquid chromatography with UV detection. The objective of this study was thus to develop simple, sensitive and specific liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) methods to quantify both THPI and PI in human plasma and urine. Briefly, deuterated THPI was added as an internal standard and purification was performed by solid-phase extraction followed by LC/APCI-MS/MS analysis in negative ion mode for both compounds. Validation of the methods was conducted using spiked blank plasma and urine samples at concentrations ranging from 1 to 250 μg/L and 1 to 50 μg/L, respectively, along with samples of volunteers and workers exposed to captan or folpet. The methods showed a good linearity (R (2) > 0.99), recovery (on average 90% for THPI and 75% for PI), intra- and inter-day precision (RSD, <15%) and accuracy (<20%), and stability. The limit of detection was 0.58 μg/L in urine and 1.47 μg/L in plasma for THPI and 1.14 and 2.17 μg/L, respectively, for PI. The described methods proved to be accurate and suitable to determine the toxicokinetics of both metabolites in human plasma and urine.

Trapping of naive lymphocytes triggers rapid growth and remodeling of the fibroblast network in reactive murine lymph nodes.

Adaptive immunity is initiated in T-cell zones of secondary lymphoid organs. These zones are organized in a rigid 3D network of fibroblastic reticular cells (FRCs) that are a rich cytokine source. In response to lymph-borne antigens, draining lymph nodes (LNs) expand several folds in size, but the fate and role of the FRC network during immune response is not fully understood. Here we show that T-cell responses are accompanied by the rapid activation and growth of FRCs, leading to an expanded but similarly organized network of T-zone FRCs that maintains its vital function for lymphocyte trafficking and survival. In addition, new FRC-rich environments were observed in the expanded medullary cords. FRCs are activated within hours after the onset of inflammation in the periphery. Surprisingly, FRC expansion depends mainly on trapping of naïve lymphocytes that is induced by both migratory and resident dendritic cells. Inflammatory signals are not required as homeostatic T-cell proliferation was sufficient to trigger FRC expansion. Activated lymphocytes are also dispensable for this process, but can enhance the later growth phase. Thus, this study documents the surprising plasticity as well as the complex regulation of FRC networks allowing the rapid LN hyperplasia that is critical for mounting efficient adaptive immunity.

Combined clonotyping and gene signatures of individual tumor-reactive CD8 T-cells define their functional relevance following peptide vaccination in melanoma patients

Novel cancer vaccines are capableto efficiently induce and boost humantumor antigen specific T-cells. However,the properties of these CD8T-cells are only partially characterized.For in depth investigation ofT-cells following Melan-A/MART-1peptide vaccination in melanoma patients,we conducted a detailed prospectivestudy at the single cell level.We first sorted individual human naiveand effector CD8 T-cells from peripheralblood by flow cytometry, andtested a modified RT-PCR protocolincluding a global amplification ofexpressed mRNAs to obtain sufficientcDNAfromsingle cells.We successfullydetected the expression ofseveral specific genes of interest evendown to 106-fold dilution (equivalentto 10-5 cell). We then analyzed tumor-specific effector memory (EM)CD8T-cell subpopulations ex vivo, assingle cells from vaccinated melanomapatients. To elucidate the hallmarksof effective immunity the genesignatures were defined by a panel ofgenes related to effector functions(e.g. IFN-, granzyme B, perforin),and individual clonotypes were identifiedaccording to the expression ofdistinct T-cell receptors (TCR). Usingthis novel single cell analysis approach,we observed that T-cell differentiationis clonotype dependent,with a progressive restriction in TCRBV clonotype diversity from EMCD28pos to EMCD28neg subsets. However,the effector function gene imprintingis clonotype-independent,but dependent on differentiation,since it correlates with the subset oforigin (EMCD28pos or EMCD28neg). We also conducted a detailedcomparative analysis after vaccinationwith natural vs. analog Melan-Apeptide. We found that the peptideused for vaccination determines thefunctional outcome of individualT-cell clonotypes, with native peptideinducing more potent effector functions.Yet, selective clonotypic expansionwith differentiation was preservedregardless of the peptide usedfor vaccination. In summary, the exvivo single cell RT-PCR approach ishighly sensitive and efficient, andrepresents a reliable and powerfultool to refine our current view of molecularprocesses taking place duringT-cell differentiation.

Immuno-reactive proteins from Mycobacterium immunogenum useful for serodiagnosis of metalworking fluid hypersensitivity pneumonitis.

Metalworking fluid-associated hypersensitivity pneumonitis (MWF-HP) is a pulmonary disease caused by inhaling microorganisms present in the metalworking fluids used in the industrial sector. Mycobacterium immunogenum is the main etiological agent. Among the clinical, radiological and biological tools used for diagnosis, serological tests are important. The aim of this study was to identify immunogenic proteins in M. immunogenum and to use recombinant antigens for serological diagnosis of MWF-HP. Immunogenic proteins were detected by two-dimensional Western blot and candidate proteins were identified by mass spectrometry. Recombinant antigens were expressed in Escherichia coli and tested by enzyme-linked immunosorbent assay (ELISA) with the sera of 14 subjects with MWF-HP and 12 asymptomatic controls exposed to M. immunogenum. From the 350 spots visualized by two-dimensional gel electrophoresis with M. immunogenum extract, 6 immunogenic proteins were selected to be expressed as recombinant antigens. Acyl-CoA dehydrogenase antigen allowed for the best discrimination of MWF-HP cases against controls with an area under the receiver operating characteristics (ROC) curve of 0.930 (95% CI=0.820-1), a sensitivity of 100% and a specificity of 83% for the optimum threshold. Other recombinant antigens correspond to acyl-CoA dehydrogenase FadE, cytosol aminopeptidase, dihydrolipoyl dehydrogenase, serine hydroxymethyltransferase and superoxide dismutase. This is the first time that recombinant antigens have been used for the serodiagnosis of hypersensitivity pneumonitis. The availability of recombinant antigens makes it possible to develop standardized serological tests which in turn could simplify diagnosis, thus making it less invasive.

Reactive and proactive approaches for introspective CBR

This work investigates applying introspective reasoning to improve the performance of Case-Based Reasoning (CBR) systems, in both reactive and proactive fashion, by guiding learning to improve how a CBR system applies its cases and by identifying possible future system deficiencies. First we present our reactive approach, a new introspective reasoning model which enables CBR systems to autonomously learn to improve multiple facets of their reasoning processes in response to poor quality solutions. We illustrate our model’s benefits with experimental results from tests in an industrial design application. Then as for our proactive approach, we introduce a novel method for identifying regions in a case-base where the system gives low confidence solutions to possible future problems. Experimentation is provided for Zoology and Robo-Soccer domains and we argue how encountered regions of dubiosity help us to analyze the case-bases of a given CBR system.

Mitochondrial uncoupling prevents cold-induced oxidative stress: a case study using UCP1 knockout mice.

The relationship between metabolism and reactive oxygen species (ROS) production by the mitochondria has often been (wrongly) viewed as straightforward, with increased metabolism leading to higher generation of pro-oxidants. Insights into mitochondrial functioning show that oxygen consumption is principally coupled with either energy conversion as ATP or as heat, depending on whether the ATP-synthase or the mitochondrial uncoupling protein 1 (UCP1) is driving respiration. However, these two processes might greatly differ in terms of oxidative costs. We used a cold challenge to investigate the oxidative stress consequences of an increased metabolism achieved either by the activation of an uncoupled mechanism (i.e. UCP1 activity) in the brown adipose tissue (BAT) of wild-type mice or by ATP-dependent muscular shivering thermogenesis in mice deficient for UCP1. Although both mouse strains increased their metabolism by more than twofold when acclimatised for 4 weeks to moderate cold (12°C), only mice deficient for UCP1 suffered from elevated levels of oxidative stress. When exposed to cold, mice deficient for UCP1 showed an increase of 20.2% in plasmatic reactive oxygen metabolites, 81.8% in muscular oxidized glutathione and 47.1% in muscular protein carbonyls. In contrast, there was no evidence of elevated levels of oxidative stress in the plasma, muscles or BAT of wild-type mice exposed to cold despite a drastic increase in BAT activity. Our study demonstrates differing oxidative costs linked to the functioning of two highly metabolically active organs during thermogenesis, and advises careful consideration of mitochondrial functioning when investigating the links between metabolism and oxidative stress.

Sex difference and the role of leptin in the association between high-sensitivity C-reactive protein and adiposity in two different populations.

Elevated high-sensitivity C-reactive protein (hs-CRP) concentration is associated with an increased risk of cardiovascular disease but this association seems to be largely mediated via conventional cardiovascular risk factors. In particular, the association between hs-CRP and obesity has been extensively demonstrated and correlations are stronger in women than men. We used fractional polynomials-a method that allows flexible modeling of non linear relations-to investigate the dose/response mathematical relationship between hs-CRP and several indicators of adiposity in Caucasians (Switzerland) and Africans (Seychelles) surveyed in two population-based studies. This relationship was non-linear exhibiting a steeper slope for low levels of hs-CRP and a higher level in women. The observed sex difference in the relationship between hs-CRP and adiposity almost disappeared upon adjustment for leptin, suggesting that these sex differences might be partially mediated, by leptin. All these relationship were similar in Caucasians and Africans. This is the first report on a non-linear relation, stratified by gender, between hs-CRP and adiposity.

Simultaneous LC-MS/MS quantification of P-glycoprotein and cytochrome P450 probe substrates and their metabolites in DBS and plasma.

BACKGROUND: An LC-MS/MS method has been developed for the simultaneous quantification of P-glycoprotein (P-gp) and cytochrome P450 (CYP) probe substrates and their Phase I metabolites in DBS and plasma. P-gp (fexofenadine) and CYP-specific substrates (caffeine for CYP1A2, bupropion for CYP2B6, flurbiprofen for CYP2C9, omeprazole for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4) and their metabolites were extracted from DBS (10 µl) using methanol. Analytes were separated on a reversed-phase LC column followed by SRM detection within a 6 min run time. RESULTS: The method was fully validated over the expected clinical concentration range for all substances tested, in both DBS and plasma. The method has been successfully applied to a PK study where healthy male volunteers received a low dose cocktail of the here described P-gp and CYP probes. Good correlation was observed between capillary DBS and venous plasma drug concentrations. CONCLUSION: Due to its low-invasiveness, simple sample collection and minimal sample preparation, DBS represents a suitable method to simultaneously monitor in vivo activities of P-gp and CYP.

Fecal calprotectin more accurately reflects endoscopic activity of ulcerative colitis than the Lichtiger Index, C-reactive protein, platelets, hemoglobin, and blood leukocytes.

BACKGROUND: The correlation between noninvasive markers with endoscopic activity according to the modified Baron Index in patients with ulcerative colitis (UC) is unknown. We aimed to evaluate the correlation between endoscopic activity and fecal calprotectin (FC), C-reactive protein (CRP), hemoglobin, platelets, blood leukocytes, and the Lichtiger Index (clinical score). METHODS: UC patients undergoing complete colonoscopy were prospectively enrolled and scored clinically and endoscopically. Samples from feces and blood were analyzed in UC patients and controls. RESULTS: We enrolled 228 UC patients and 52 healthy controls. Endoscopic disease activity correlated best with FC (Spearman's rank correlation coefficient r = 0.821), followed by the Lichtiger Index (r = 0.682), CRP (r = 0.556), platelets (r = 0.488), blood leukocytes (r = 0.401), and hemoglobin (r = -0.388). FC was the only marker that could discriminate between different grades of endoscopic activity (grade 0, 16 [10-30] μg/g; grade 1, 35 [25-48] μg/g; grade 2, 102 [44-159] μg/g; grade 3, 235 [176-319] μg/g; grade 4, 611 [406-868] μg/g; P < 0.001 for discriminating the different grades). FC with a cutoff of 57 μg/g had a sensitivity of 91% and a specificity of 90% to detect endoscopically active disease (modified Baron Index ≥ 2). CONCLUSIONS: FC correlated better with endoscopic disease activity than clinical activity, CRP, platelets, hemoglobin, and blood leukocytes. The strong correlation with endoscopic disease activity suggests that FC represents a useful biomarker for noninvasive monitoring of disease activity in UC patients.

Desarrollo de biomarcadores del grado de exposición y activación para la evaluación de la neurotoxicidad de la MDMA en humanos

La 3,4-Metilendioximetanfetamina (MDMA, éxtasis) es un derivado anfetamínico sintético ampliamente usado como droga recreativa, que produce neurotoxicidad serotonérgica en animales y posiblemente también en humanos. El mecanismo subyacente de neurotoxicidad, incluye la formación de especies reactivas de oxigeno (ROS), pero la fuente de generación de estos es un punto de controversia. Se postula que la neurotoxicidad inducida por la MDMA es mediada por la formación de metabolitos bioreactivos. Específicamente, los metabolitos primarios de tipo catecol, la 3,4- dihidroximetanfetamina (HHMA) y la 3,4-dihidroxianfetamina (HHA), que luego dan lugar a la formación de conjugados con el glutatión y la N-acetilcisteína, y que conservan la capacidad de entrar en el ciclo redox y presentan neurotoxicidad serotonérgica en ratas. Aunque la presencia de dichos metabolitos se demostró recientemente en microdialisados de cerebros de ratas, su formación en humanos no se ha reportado aun. Este trabajo describe la detección de N-acetil-cisteína-HHMA (NAC-HHMA) y N-acetil-cisteína-HHA (NAC-HHA) en orina humana de 15 consumidores recreacionales de MDMA (1.5 mg/kg) en un entorno controlado. Los resultados revelan que en las primeras 4 horas después del consumo de MDMA aproximadamente el 0.002% de la dosis administrada es recuperada como aductos tioéter. Los polimorfismos genéticos en la expresión de las enzimas CYP2D6 y COMT, que en conjunto son las principales determinantes de los niveles estables de HHMA y HHA, posiblemente expliquen la variabilidad interindividual observada en la recuperación de la NAC-HHMA y la NAC-HHA en orina. Resumiendo, por primera vez se demuestra la formación de aductos tioéteres neurotóxicos de la MDMA en humanos. Estos resultados apoyan la hipótesis de que la bioactivación de la MDMA a metabolitos neurotóxicos es el mecanismo relevante para la generación de la neurotoxicidad en humanos.

A toxicokinetic model to assess exposure to folpet fungicide from urinary biomarkers

Folpet is one of the most widely employed fungicides in agriculture. It is typically used in the culture of vegetables, fruits and ornamental plants. Once absorbed in the human body, it has been found to be very reactive, especially in acid conditions. According to various in vitro and in vivo experiments in animals, Folpet is first fractioned at the N-S link when in contact with aqueous solutions and thiol groups. From this non-enzymatic process a phthalimide (PI) molecule is formed, which may be used as a biomarker of exposure, along with the short-lived thiophosgene. We have built a human toxicokinetic model to account for the biotransformation of Folpet into PI and its subsequent excretion while accounting for other non-monitored metabolites. The mathematical parameters of the model were determined accordingly from best-fits to the time courses of PI in blood and urine of five volunteers administered orally 1 mg/kg and dermally 10 mg/kg of Folpet. In both cases, the mean elimination half-life of PI from the body (either through faeces, urine or metabolism) was found to be 31.6 h. The average final fractions of administered dose recovered in urine as PI were 0.025% and 0.002%, for oral and dermal administration, respectively after 96 h. According to the model, when orally administered, PI rapidly hydrolyzes to phthalamic and phthalic acids such that only 0.04% of the PI found in the gastrointestinal tract is absorbed into the blood stream. Likewise, after dermal application, model predicts that only 7.4% of the applied Folpet dose crosses the epidermis. In the model, the PI initial metabolite of Folpet is formed in the dermis and further metabolized prior to reaching systemic circulation, such that only 0.125% of PI formed at the site-of-entry reaches systemic blood. Our mathematical model is in accordance with both measures of blood (R2=0.57 for dermal and R2=0.66 for oral) and urine (R2 =0.98 for dermal and R2=0.99 for oral).

Polypeptides reactive with antibodies eluted from the surface of Babesia bovis-infected erythrocytes

A technique was sought that would enable identification of surface-exposed parasite antigens on Babesia bovis-infected erythrocytes (BbIE) that are not detectable by surface-specific immunoprecipitations. Antibodies which bind to the surface of BbIE were recovered from intact cells using a low pH wash procedure. The eluted antibodies were then used in conventional immunoprecipitation assays to identify parasite-synthesized polypeptides carrying epitopes that are exposed on the surface or are cross reactive with shuch epitopes. The results of these experiments support our previous data, obtained using a surface-specific immunoprecipitation technique, in the identification of a repertoire of parasite-derived antigens on the surface of infected erythrocytes (Allred et al., 1991). In addition, two polypeptides of Mr 68,000 and 185,000 were identified wich react strongly with the eluted antibodies but wich are not detected by surface-immunoprecipitation. These data illustrate the potential of this approach for identification of parasite polypeptides wich carry epitopes exposed on, or cross-reactive with exposed epitopes of the infected erythrocyte surface.

Characterization of Melan-A reactive memory CD8+ T cells in a healthy donor.

Melan-A specific CD8+ T cells are thought to play an important role against the development of melanoma. Their in vivo expansion is often observed with advanced disease. In recent years, low levels of Melan-A reactive CD8+ T cells have also been found in HLA-A2 healthy donors, but these cells harbor naive characteristics and are thought to be mostly cross-reactive for the Melan-A antigen. Here, we report on a large population of CD8+ T cells reactive for the Melan-A antigen, identified in one donor with no evidence of melanoma. Interestingly, this population is oligoclonal and displays a clear memory phenotype. However, a detailed study of these cells indicated that they are unlikely to be directly specific for melanoma, so that their in vivo expansion may have been driven by an exogenous antigen. Screening of a Melan-A cross-reactive peptide library suggested that these cells may be specific for an epitope derived from a Mycobacterium protein, which would provide a further example of CD8+ T cell cross-reactivity between a pathogen antigen and a tumor antigen. Finally, we discuss potential perspectives regarding the role of such cells in heterologous immunity, by influencing the balance between protective immunity and pathology, e.g. in the case of melanoma development.