Diagnostic potential of NETosis-derived products for disease activity, atherosclerosis and therapeutic effectiveness in Rheumatoid Arthritis patients.

OBJECTIVES: 1) To assess the association of NETosis and NETosis-derived products with the activity of the disease and the development of cardiovascular disease in RA; 2) To evaluate the involvement of NETosis on the effects of biologic therapies such as anti-TNF alpha (Infliximab) and anti-IL6R drugs (Tocilizumab). METHODS: One hundred and six RA patients and 40 healthy donors were evaluated for the occurrence of NETosis. Carotid-intimae media thickness was analyzed as early atherosclerosis marker. Inflammatory and oxidative stress mediators were quantified in plasma and neutrophils. Two additional cohorts of 75 RA patients, treated either with Infliximab (n = 55) or Tocilizumab (n = 20) for six months, were evaluated. RESULTS: NETosis was found increased in RA patients, beside myeloperoxidase and neutrophil elastase protein levels. Cell-free nucleosomes plasma levels were elevated, and strongly correlated with the activity of the disease and the positivity for autoantibodies, alongside inflammatory and oxidative profiles in plasma and neutrophils. Moreover, ROC analyses showed that cell-free nucleosomes levels could identify RA patients showing early atherosclerosis with high specificity. RA patients treated either with IFX or TCZ for six months exhibited decreased generation of NETs. Concomitantly, clinical parameters and serum markers of inflammation were found reduced. Mechanistic in vitro analyses showed that inhibition of NETs extrusion by either DNase, IFX or TCZ, further abridged the endothelial dysfunction and the activation of immune cells, thus influencing the global activity of the vascular system. CONCLUSIONS: NETosis-derived products may have diagnostic potential for disease activity and atherosclerosis, as well as for the assessment of therapeutic effectiveness in RA.

Effect of dietary coenzyme Q and fatty acids on the antioxidant status of rat tissues.

Wistar rats were fed with different diets with or without supplement coenzyme Q(10) (CoQ(10)) and with oil of different sources (sunflower or virgin olive oil) for six or twelve months. Ubiquinone contents (CoQ(9) and CoQ(10)) were quantified in homogenates of livers and brains from rats fed with the four diets. In the brain, younger rats showed a 3-fold higher amount of ubiquinone than older ones for all diets. In the liver, however, CoQ(10) supplementation increased the amount of CoQ(9) and CoQ(10) in both total homogenates and plasma membranes. Rats fed with sunflower oil as fat source showed higher amounts of ubiquinone content than those fed with olive oil, in total liver homogenates, but the total ubiquinone content in plasma membranes was similar with both fat sources. Older rats showed a higher amount of ubiquinone after diets supplemented with CoQ(10). Two ubiquinone-dependent antioxidant enzyme activities were measured. NADH-ferricyanide reductase activity in hepatocyte plasma membranes was unaltered by ubiquinone accumulation, but this activity increased slightly with age. Both cytosolic and membrane-bound dicumarol-sensitive NAD(P)H:(quinone acceptor) oxidoreductase (DT-diaphorase, EC 1.6.99.2) activities were decreased by diets supplemented with CoQ(10). Animals fed with olive oil presented lower DT-diaphorase activity than those fed with sunflower oil, suggesting that the CoQ(10) antioxidant protection is strengthened by olive oil as fat source.

Redox-related peroxidative responses evoked by methyl-jasmonate in axenically cultured aeroponic sunflower (Helianthus annuus L.) seedling roots.

Methyl-jasmonate (MeJA) has been proposed to be involved in the evocation of defense reactions, as the oxidative burst in plants, substituting the elicitors or enhancing their effect. 48 h dark- and sterilely cultured (axenic) aeroponic sunflower seedling roots excised and treated with different concentrations of MeJA showed a strong and quick depression of the H(+) efflux rate, 1.80 microM MeJA totally stopping it for approximately 90 min and then reinitiating it again at a lower rate than controls. These results were wholly similar to those obtained with nonsterilely cultured roots and have been interpreted as mainly based on H(+) consumption for O(2)(*-) dismutation to H(2)O(2). Also K(+) influx was strongly depressed by MeJA, even transitorily reverting to K(+) efflux. These results were consistent with those associated to the oxidative burst in plants. MeJA induced massive H(2)O(2) accumulation in the middle lamella and intercellular spaces of both the root cap cells and the inside tissues of the roots. The native acidic extracellular peroxidase activity of the intact (nonexcised) seedling roots showed a sudden enhancement (by about 52%) after 5 min of MeJA addition, maintained for approximately 15 min and then decaying again to control rates. O(2) uptake by roots gave similar results. These and other results for additions of H(2)O(2) or horseradish peroxidase, diphenylene iodonium, and sodium diethyldithiocarbamate trihydrate to the reaction mixture with roots were all consistent with the hypothesis that MeJA induced an oxidative burst, with the generation of H(2)O(2) being necessary for peroxidase activity. Results with peroxidase activity of the apoplastic fluid were in accordance with those of the whole root. Finally, MeJA enhanced NADH oxidation and inhibited hexacyanoferrate(III) reduction by axenic roots, and diphenylene iodonium cancelled out these effects. Redox activities by CN(-)- preincubated roots were also studied. All these results are consistent with the hypothesis that MeJA enhanced the NAD(P)H oxidase of a redox chain linked to the oxidative burst, so enhancing the generation of O(2)(*-) and H(2)O(2), O(2) uptake, and peroxidase activity by roots.

Cloning, expression and immunolocalization pattern of a cinnamyl alcohol dehydrogenase gene from strawberry (Fragaria x ananassa cv. Chandler).

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) catalyses the conversion of p-hydroxy-cinnamaldehydes to the corresponding alcohols and is considered a key enzyme in lignin biosynthesis. By a differential screening of a strawberry (Fragariax ananassa cv. Chandler) fruit specific subtractive cDNA library, a full-length clone corresponding to a cad gene was isolated (Fxacad1). Northern blot and quantitative real time PCR studies indicated that the strawberry Fxacad1 gene is expressed in fruits, runners, leaves, and flowers but not in roots. In addition, the gene presented a differential expression in fruits along the ripening process. Moreover, by screening of a strawberry genomic library a cad gene was isolated (Fxacad2). Similar to that found in other cad genes from higher plants, this strawberry cad gene is structured in five exons and four introns. Southern blot analyses suggest that, probably, a small cad gene family exists in strawberry. RT-PCR studies indicated that only the Fxacad1 gene was expressed in all the fruit ripening stages and vegetative tissues analysed. The Fxacad1 cDNA was expressed in E. coli cells and the corresponding protein was used to raise antibodies against the strawberry CAD polypeptide. The antibodies obtained were used for immunolocalization studies. The results showed that the CAD polypeptide was localized in lignifying cells of all the tissues examined (achenes, fruit receptacles, runners, leaves, pedicels, and flowers). Additionally, the cDNA was also expressed in yeast (Pichia pastoris) as an extracellular protein. The recombinant protein showed activity with the characteristic substrates of CAD enzymes from angiosperms, indicating that the gene cloned corresponds to a CAD protein.

Role of Apoplastic and Cell-Wall Peroxidases on the Stimulation of Root Elongation by Ascorbate.

Elongation of onion (Allium cepa L.) roots was highly stimulated by ascorbate (ASC) and its natural precursor I-galactone-[gamma]-lactone (GL). When incubation media were supplemented with lycorine (Lyc), an inhibitor of the ASC biosynthesis, root growth was negligible even in the presence of ASC or GL. ASC completely inhibited in vitro guaiacol peroxidase activities that were isolated from both the apoplast and the cell wall. However, ferulic-acid-dependent peroxidase from the cell wall was partially inhibited by ASC, whereas ferulic acid peroxidase activity from the apoplastic fluid was completely inhibited by ASC as long as ASC was present in the assay medium. ASC content in cells was increased by preincubations with ASC or GL, whereas Lyc reduced it. On the other hand, ASC or GL treatments decreased both apoplast and cell-wall-bound peroxidase activities, whereas Lyc had a slight stimulating effect. These results are discussed on the basis of a possible control of root elongation by ASC via its action on peroxidases that are involved in the regulation of cell-wall extensibility.

The pH Requirement for in Vivo Activity of the Iron-Deficiency-Induced "Turbo" Ferric Chelate Reductase (A Comparison of the Iron-Deficiency-Induced Iron Reductase Activities of Intact Plants and Isolated Plasma Membrane Fractions in Sugar Beet).

The characteristics of the Fe reduction mechanisms induced by Fe deficiency have been studied in intact plants of Beta vulgaris and in purified plasma membrane vesicles from the same plants. In Fe-deficient plants the in vivo Fe(III)-ethylenediaminetetraacetic complex [Fe(III)-EDTA] reductase activity increased over the control values 10 to 20 times when assayed at a pH of 6.0 or below ("turbo" reductase) but increased only 2 to 4 times when assayed at a pH of 6.5 or above. The Fe(III)-EDTA reductase activity of root plasma membrane preparations increased 2 and 3.5 times over the controls, irrespective of the assay pH. The Km for Fe(III)-EDTA of the in vivo ferric chelate reductase in Fe-deficient plants was approximately 510 and 240 [mu]M in the pH ranges 4.5 to 6.0 and 6.5 to 8.0, respectively. The Km for Fe(III)-EDTA of the ferric chelate reductase in intact control plants and in plasma membrane preparations isolated from Fe-deficient and control plants was approximately 200 to 240 [mu]M. Therefore, the turbo ferric chelate reductase activity of Fe-deficient plants at low pH appears to be different from the constitutive ferric chelate reductase.

Topography of the 27- and 31-kDa electron transport proteins in the onion root plasma membrane.

Plasma membranes purified from onion roots contain two distinct NAD(P)H-dehydrogenases of 27 and 31 kDa that differ in their physicochemical properties, substrate specificities and inhibitors sensitivities. The 27-kDa enzyme used both NADH and NADPH as electron donors. The 31-kDa enzyme was fully specific for NADH and accounted for the bulk of NADH-ferricyanide oxidoreductase. We have used NADPH- and NADH-ferricyanide oxidoreductase activities as markers for investigating the orientation of the 27- and 31-kDa enzymes at the plasma membrane, respectively. These activities were assayed in right-side-out vesicles isolated by two-phase partition, inside-out vesicles obtained by treatment with the detergent Brij 58 and membranes permeabilized with Triton X-100. Upon addition of Brij 58 to right-side-out plasma membrane vesicles, both NADPH- and NADH-ferricyanide oxidoreductases were activated to the same degree as the plasma membrane H(+)-ATPase. Redox activities were similar when measured in the presence of either Brij 58 or Triton X-100. Our results demonstrate that both enzymes expose their catalytic sites toward the cytoplasmic side of the plasma membrane.

Purification and Characterization of Two Distinct NAD(P)H Dehydrogenases from Onion (Allium cepa L.) Root Plasma Membrane.

Highly purified plasma membrane fractions were obtained from onion (Allium cepa L.) roots and used as a source for purification of redox proteins. Plasma membranes solubilized with Triton X-100 contained two distinct polypeptides showing NAD(P)H-dependent dehydrogenase activities. Dehydrogenase I was purified by gel filtration in Sephacryl S-300 HR, ion-exchange chromatography in DEAE-Sepharose CL-6B, and dye-ligand affinity chromatography in Blue-Sepharose CL-6B after biospecific elution with NADH. Dehydrogenase I consisted of a single polypeptide of about 27 kD and an isoelectric point of about 6. Dehydrogenase II was purified from the DEAE-unbound fraction by chromatography in Blue-Sepharose CL-6B and affinity elution with NADH. Dehydrogenase II consisted of a single polypeptide of about 31 kD and an isoelectric point of about 8. Purified dehydrogenase I oxidized both NADPH and NADH, although higher rates of electron transfer were obtained with NADPH. Maximal activity was achieved with NADPH as donor and juglone or coenzyme Q as acceptor. Dehydrogenase II was specific for NADH and exhibited maximal activity with ferricyanide. Optimal pH for both dehydrogenases was about 6. Dehydrogenase I was moderately inhibited by dicumarol, thenoyltrifluoroacetone, and the thiol reagent N-ethyl-maleimide. A strong inhibition of dehydrogenase II was obtained with dicumarol, thenoyltrifluoroacetone, and the thiol reagent p-hydroxymercuribenzoate.

Ascorbate and plant cell growth.

Ascorbate and related enzymes are involved in the control of several plant growth processes. Ascorbate modulates cell growth by controlling (i) the biosynthesis of hydroxyproline-rich proteins required for the progression of G1 and G2 phases of the cell cycle, (ii) the cross-linking of cell wall glycoproteins and other polymers, and (iii) redox reactions at the plasma membrane involved in elongation mechanisms. The effect of ascorbate on onion root elongation is reviewed here. The ascorbate free radical induces a high vacuolization responsible for elongation. This effect may be dependent on the activity of the redox system linked to the plasma membrane. Current data are discussed on the basis of the modulation of the plasma membrane energetic state derived from the ascorbate-induced hyperpolarization and the activity of an intrinsic transplasmalemma ascorbate-regenerating enzyme.

Two distinct NAD(P)H-dependent redox enzymes isolated from onion root plasma membranes.

Plasma membranes purified by two-phase partition from onion roots catalyzed the NAD(P)H-dependent reduction of a variety of electron acceptor such as ferricyanide, quinones, dyes and ascorbate free radical. Among these, NAD(P)H-ferricyanide and -quinone oxidoreductase activities were effectively solubilized by Triton X-100. Both oxidoreductase activities were bound to an affinity column of Blue-Sepharose CL 6B. NADH eluted a redox enzyme showing more juglone than ferricyanide-dependent activity. Ulterior unspecific elution with salt allowed us to the partial purification of a different redox enzyme of about 31 kDa that reduced better ferricyanide than quinones and constituted the bulk of solubilized redox activity.

Nutrient Uptake Changes in Ascorbate Free Radical-Stimulated Onion Roots.

Long-term treatments with ascorbate free radical-stimulated glucose, fucose, sucrose, and nitrate uptake in Allium cepa roots. Glucose and fucose showed saturation kinetics in untreated roots, but after treatment with the ascorbate free radical, uptake was linear with time. Although the rates of nitrate and sucrose uptake increased after treatment with ascorbate free radical, the kinetics were similar to those observed in the controls. Ascorbate and dehydroascorbate inhibited nutrient uptake. The uptake rates for all nutrients increased throughout the 48-h period of pretreatment with ascorbate free radical. During the treatment an increase in the vacuole volume and tonoplast surface area also occurred. These results show the relationship between an increase in vacuolar volume and stimulated nutrient uptake from ascorbate-free radical, resulting in enhanced root elongation. These results suggest that activation of a transplasma membrane redox system by ascorbate-free radical is involved in these responses.

A quantitative ultrastructural and cytochemical study of TPA-induced differentiation in HL-60 cells.

The effects of the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate on morphometric and stereological parameters have been studied using the HL-60 cell line as a differentiation model for the monocytic pathway. Evaluation of the differentiation was carried out by quantification of endoplasmic reticulum, Golgi apparatus, mitochondria and cytoplasmic granules. Changes in both nuclear and cytoplasmic volumes during TPA-induced differentiation led to a decrease of the nucleus-cytoplasmic ratio after 3 days of treatment. Plasma membrane glycoprotein pattern was also determined. The major change in cell surface was the presence of high amounts of glycoproteins containing N-acetyl glucosamine residues that make wheatgerm agglutinin lectin a valuable marker of the monocytic differentiation pathway in HL-60 cells.

Effects of hypothyroidism on the ultrastructure of rat pancreatic acinar cells: a stereological analysis.

The morphological and stereological characteristics of the exocrine pancreas subcellular organelles from healthy and thyroidectomized rats have been studied. The acinar tissue from hypothyroid rats showed an interstitial edema and evidence of degenerative processes. Stereological parameters of zymogen granules were significantly reduced in thyroidectomized rats. The hypothyroidism induced degenerative changes in the pancreatic acinar cells as well as a decrease in the number and size of the zymogen granules. These modifications probably cause functional alterations.

Differential morphometric values induced in Golgi apparatus of higher plant cells by aldehyde and permanganate fixation.

In order to determine the best conditions to carry out quantitative ultrastructural studies in plant specimens, five different fixation techniques, including some of the most reported electron microscopy fixatives (glutaraldehyde-paraformaldehyde, osmium tetroxide, potassium permanganate), were assayed in onion root meristems to check their ability to induce morphometric changes in Golgi apparatus ultrastructure. Although the parameters evaluated showed in all cases the same tendency, values obtained after permanganate fixation were always higher than those found after aldehyde techniques (especially aldehyde-osmium). Aldehyde followed by osmium fixation appears as the most indicated fixation method when accurate quantitative ultrastructural studies are to be developed.

Growth kinetics of the Golgi apparatus during the cell cycle in onion root meristems.

In onion root meristems, the number of dictyosomes per cell shows a kinetics of growth strongly related to the cell cycle. During the interphase of steady-state proliferative cells, the volume density and numerical density of the Golgi apparatus decrease to reach minimum values in late-interphase cells, characterized by their greatest length. This pattern is also found in the total volume occupied by Golgi apparatus. Once in mitosis, the above-mentioned parameters begin to increase reaching maximum mean values in telophase. After the experimental uncoupling of chromosome and growth cycles by presynchronization with hydroxyurea, we found a similar behaviour pattern in the Golgi apparatus: decreasing values during interphase and a triggering of Golgi-apparatus growth in prophase independently of the bigger cell sizes reached in mitosis as an effect of pretreatment with hydroxyurea. These results indicate a cyclic kinetics of this subcellular component in higher-plant meristems, coupled with early mitotic events.

Subcellular responses in frog pituitary gonadotrophic cells to constant environmental conditions: a morphometric study.

An ultrastructural study of the pars distalis of the pituitary in Rana ridibunda specimens subjected to constant environmental conditions revealed significant structural differences which indicate a short-time increase in the activity of gonadotrophic cells. Planimetry was used for morphometric and stereological analysis of the rough endoplasmic reticulum, Golgi complex, secretory granules and lysosomes. The presence of a vesicular endoplasmic reticulum throughout the cytoplasm of gonadotrophic cells was observed in the pituitary gland of the intact frog. After 7 days of constant environmental conditions, there was an increase in the volume density of the endoplasmic reticulum vesicles, while on the 11th day there was a slight reversal in the vesicle content that was complete the 15th day. In the hypertrophy of the endoplasmic reticulum, the larger vesicles (bigger than 0,52 micron2) play a more important role than the smaller ones (similar to the endoplasmic reticulum observed in control cells). However, the small vacuoles were also hypertrophied in relation to the control. Degranulation was also observed after 7 days of constant environmental conditions. On the 15th days, there was an increase in the numerical density of secretory granules and lysosomes compared to the control while their volume density was similar to the control.

An electron microscopic study of stellate cells and cavities in the pars distalis of frog pituitary.

Stellate cells in the pars distalis of adult Rana ridibunda were observed electron microscopically under normal and experimental conditions (TRH injection). The stellate cells have lengthy processes extending into the intercellular spaces between the secretory cells and scanty cytoplasm surrounding the nucleus. Occasional desmosomes link stellate cells to adjacent secretory cells. In the pars distalis of animals injected with thyrotropic-releasing hormone (TRH), the stellate cells form large cavities (2-6 mum) filled with heterogeneous material. Their cytoplasm contains well-developed Golgi complexes and some lysosomes; these are the principal morphological alterations as compared to those observed in control animals. It is suggested that stellate cells could play an active role in addition to providing a structural framework for the pars distalis.