Prevalence of human Papillomavirus DNA in eyebrow hairs plucked from patients with psoriasis treated with TNF inhibitors.

BACKGROUND: Tumour necrosis factor alpha (TNF-α) inhibitors are associated with an increased risk of infections and with a still debatable risk of skin cancer. Furthermore, cutaneous human papillomavirus (HPV) infection may be involved in skin cancer. OBJECTIVES: Our primary objective was to assess the HPV DNA prevalence in psoriasis patients treated with TNF inhibitors and the secondary objective was to assess the same parameter before and during treatment. METHODS: Plucked eyebrow hairs were collected from 151 consecutive patients with moderate to severe chronic plaque psoriasis, including 48 patients treated with anti-TNF-α agents, 21 patients treated with methotrexate (MTX) and 82 patients with no previous systemic treatment. Among them, 38 patients were subsequently treated with either MTX or anti-TNF-α agents. HPV genotyping was performed using the HPV type-specific E7 PCR bead-based multiplex allowing the detection of 27 genus-α types, 25 genus-ß types, 16 genus-γ types and one single genus-µ type. Follow-up provided a total of 972.7 person-months of overall exposure for patients treated with TNF inhibitors and 326.9 person-months for patients treated with MTX. RESULTS: Our data confirm the high prevalence of ß-HPV infection in healthy skin of psoriasis patients (68.9%), with no significant difference between untreated patients (64.6%), patients treated with MTX (76.2%) and patients treated with anti-TNF-α agents (72.9%). The mean number of different HPV types and the distribution of HPV types were similar in different groups of patients. Moreover, in prospectively treated patients, we did not observe any change in the HPV DNA prevalence in the distribution of HPV types and the number of HPV types after a mean duration of treatment of 332 ± 39.8 days. CONCLUSION: Despite the small number of patients in our cohort, our results are quite encouraging in view of the increased use of anti-TNF-α agents in different auto inflammatory immune diseases.

Demencia semántica: presentación de dos casos y revisión de la literatura / Semantic dementia: report of two cases

Frontotemporal dementia is a neurodegenerative condition that presents with a number of distinct behavioral phenotypes. One of them is semantic dementia (SD), where exists a profound impairment for semantic knowledge related to atrophy of temporal poles. Pathologically, in most cases positive intraneuronal ubiquitin and tau negative inclusions are observed. SD is characterized by fluent, effortless, grammatical speech which lacks informational content, with limited and repetitive content, as well as semantic paraphasias. Also, patients may present with associative visual agnosia, surface dyslexia or dysgraphia, behavioral alterations. Both episodic and autobiographical memory are close to normality. Two female patients with fluent progressive aphasia are reported; they failed in a simple test of semantic association (to point to one of four objects with lesser relation to others). Autobiographical memory was fair. SD can be wrongly diagnosed as left-sided variant of Alzheimer's disease; absence of episodic amnesia and parietal defects may be useful for clinical diagnosis.

La demencia semántica es una variante de las atrofias lobares frontotemporales que se caracteriza por la degradación del conocimiento semántico, de los conceptos. En ella existe una atrofia predominante de los polos temporales, a veces asimétrica; la patología generalmente muestra neuronas con inclusiones ubiquitina (+) y tau (-). Se expresa como una afasia progresiva fluente, sin disartria ni agramatismo, con anomia y parafasias verbales o semánticas, poco informativa. Pueden existir agnosia visual asociativa y alteraciones conductuales; a veces existen alexia y/o agrafía de superficie. Hay respeto relativo de la memoria episódica y autobiográfica. Se presentan dos mujeres con afasia progresiva fluente, alteraciones conductuales y falla en una tarea semántica (4º excluido). A pesar de estos defectos la memoria autobiográfica y el desempeño en sus actividades cotidianas estaban dentro de lo normal. Se concluye que estos cuadros deben diferenciarse de los casos de enfermedad de Alzheimer de predominio izquierdo; el respeto de la memoria episódica y la falta de compromiso parietal descartarían clínicamente esta última patología.

A beta-1,3 glucan sulfate induces resistance in grapevine against Plasmopara viticola through priming of defense responses, including HR-like cell death.

Sulfated laminarin (PS3) has been shown previously to be an elicitor of plant defense reactions in tobacco and Arabidopsis and to induce protection against tobacco mosaic virus. Here, we have demonstrated the efficiency of PS3 in protecting a susceptible grapevine cultivar (Vitis vinifera cv. Marselan) against downy mildew (Plasmopara viticola) under glasshouse conditions. This induced resistance was associated with potentiated H2O2 production at the infection sites, upregulation of defense-related genes, callose and phenol depositions, and hypersensitive response-like cell death. Interestingly, similar responses were observed following P. viticola inoculation in a tolerant grapevine hybrid cultivar (Solaris). A pharmacological approach led us to conclude that both callose synthesis and jasmonic acid pathway contribute to PS3-induced resistance.

Estado actual de la terapia antiangiogénica en el manejo de gliomas de alto grado / Present state of the antiangiogenic therapy in the management of gliomas of high degree

High grade gliomas are lethal cancers. Despite recent advances in surgery, radiotherapy and chemotherapy, the overall survival is 15 months for glioblastoma. They are among the most vascular of human tumors, making them especially attractive targets for angiogenesis inhibitors. Most clinical trials of these agents as monotherapy have failed to demonstrate survival benefit in unselected high grade glioma patient populations. Several mechanisms of treatment failure have been postulated. In response, there are new intervention strategies on course: the combination of target therapy with classic chemotherapy, multitargeted kinase inhibitors and combinations of single-targeted kinase inhibitors and the identification of correlative biomarkers. These advances provide real opportunities for the development of effective therapies for high grade gliomas.

Nitric oxide: comparative synthesis and signaling in animal and plant cells.

Since its identification as an endothelium-derived relaxing factor in the 1980s, nitric oxide has become the source of intensive and exciting research in animals. Nitric oxide is now considered to be a widespread signaling molecule involved in the regulation of an impressive spectrum of mammalian cellular functions. Its diverse effects have been attributed to an ability to chemically react with dioxygen and its redox forms and with specific iron- and thiol-containing proteins. Moreover, the effects of nitric oxide are dependent on the dynamic regulation of its biosynthetic enzyme nitric oxide synthase. Recently, the role of nitric oxide in plants has received much attention. Plants not only respond to atmospheric nitric oxide, but also possess the capacity to produce nitric oxide enzymatically. Initial investigations into nitric oxide functions suggested that plants use nitric oxide as a signaling molecule via pathways remarkably similar to those found in mammals. These findings complement an emerging body of evidence indicating that many signal transduction pathways are shared between plants and animals.

Disruption of microtubular cytoskeleton induced by cryptogein, an elicitor of hypersensitive response in tobacco cells.

The dynamics of microtubular cytoskeleton were studied in tobacco (Nicotiana tabacum cv Xanthi) cells in response to two different plant defense elicitors: cryptogein, a protein secreted by Phytophthora cryptogea and oligogalacturonides (OGs), derived from the plant cell wall. In tobacco plants cryptogein triggers a hypersensitive-like response and induces systemic resistance against a broad spectrum of pathogens, whereas OGs induce defense responses, but fail to trigger cell death. The comparison of the microtubule (MT) dynamics in response to cryptogein and OGs in tobacco cells indicates that MTs appear unaffected in OG-treated cells, whereas cryptogein treatment caused a rapid and severe disruption of microtubular network. When hyperstabilized by the MT depolymerization inhibitor, taxol, the MT network was still disrupted by cryptogein treatment. On the other hand, the MT-depolymerizing agent oryzalin and cryptogein had different and complementary effects. In addition to MT destabilization, cryptogein induced the death of tobacco cells, whereas OG-treated cells did not die. We demonstrated that MT destabilization and cell death induced by cryptogein depend on calcium influx and that MT destabilization occurs independently of active oxygen species production. The molecular basis of cryptogein-induced MT disruption and its potential significance with respect to cell death are discussed.

Phosphoproteins involved in the signal transduction of cryptogein, an elicitor of defense reactions in tobacco.

We previously reported that the signal transduction of cryptogein, an elicitor of defense reactions in Nicotiana tabacum cells, involves upstream protein phosphorylation. In the present study, induction of these early physiological events was further investigated with inhibitors of protein phosphatase (PP), okadaic acid, and calyculin A. Calyculin A mimicked the effects of cryptogein, inducing an influx of calcium, an extracellular alkalinization, and the production of active oxygen species (AOS), suggesting that during cryptogein signal transduction the balance between specific protein kinase (PK) and PP activities was modified. To identify the phosphorylated proteins that could be involved early in the elicitor signaling pathway, we analyzed by 2-D electrophoresis the in vivo phosphorylation status of proteins after cryptogein, staurosporine, and calyculin A treatments of tobacco cells (5 min). Of about 100 phospho-labeled polypeptides, 19 showed increased 32P incorporation after 5 min of cryptogein treatment. Phosphorylation of 12 of the 19 polypeptides depended upon calcium influx. Staurosporine inhibited the phosphorylations induced by cryptogein whereas calyculin A activated the phosphorylation of 18 of these polypeptides. This study highlighted the role of PKs and/or constitutive active PPs whose activation and inhibition, respectively, resulted in an increased phosphorylation of proteins that may be involved in cryptogein signal transduction. Identification of the phosphoproteins is in progress and will increase our knowledge of signal transduction pathways implicated in plant defense responses.

Characterization of the cryptogein binding sites on plant plasma membranes.

Cryptogein is a 98-amino acid proteinaceous elicitor of tobacco defense reactions. Specific binding of cryptogein to high affinity binding sites on tobacco plasma membranes has been previously reported (K(d) = 2 nM; number of binding sites: 220 fmol/mg of protein). In this study, biochemical characterization of cryptogein binding sites reveals that they correspond to a plasma membrane glycoprotein(s) with an N-linked carbohydrate moiety, which is involved in cryptogein binding. Radiation inactivation experiments performed on tobacco plasma membrane preparations indicated that cryptogein bound specifically to a plasma membrane component with an apparent functional molecular mass of 193 kDa. Moreover, using the homobifunctional cross-linking reagent disuccinimidyl suberate and tobacco plasma membranes incubated with (125)I-cryptogein, we identified, after SDS-polyacrylamide gel electrophoresis and autoradiography, two (125)I-cryptogein linked N-glycoproteins of about 162 and 50 kDa. Similar results were obtained using Arabidopsis thaliana and Acer pseudoplatanus plasma membrane preparations, whereas cryptogein did not induce any effects on the corresponding cell suspensions. These results suggest that either cryptogein binds to nonfunctional binding sites, homologues to those present in tobacco plasma membranes, or that a protein involved in signal transduction after cryptogein recognition is absent or inactive in both A. pseudoplatanus and A. thaliana.

Involvement of plasma membrane proteins in plant defense responses. Analysis of the cryptogein signal transduction in tobacco.

Cryptogein, a 98 amino acid protein secreted by the fungus Phytophthora cryptogea, induces a hypersensitive response and systemic acquired resistance in tobacco plants (Nicotiana tabacum var Xanthi). The mode of action of cryptogein has been studied using tobacco cell suspensions. The recognition of this elicitor by a plasma membrane receptor leads to a cascade of events including protein phosphorylation, calcium influx, potassium and chloride effluxes, plasma membrane depolarization, activation of a NADPH oxidase responsible for active oxygen species (AOS) production and cytosol acidification, activation of the pentose phosphate pathway, and activation of two mitogen-activated protein kinase (MAPK) homologues. The organization of the cryptogein responses reveals that the earliest steps of the signal transduction pathway involve plasma membrane activities. Their activation generates a complex network of second messengers which triggers the specific physiological responses. This study may contribute to our understanding of plant signaling processes because elicitors and a variety of signals including hormones, Nod factors, light, gravity and stresses share some common transduction elements and pathways.

Comparison of binding properties and early biological effects of elicitins in tobacco cells

Elicitins are a family of small proteins secreted by Phytophthora species that have a high degree of homology and elicit defense reactions in tobacco (Nicotiana tabacum). They display acidic or basic characteristics, the acidic elicitins being less efficient in inducing plant necrosis. In this study we compared the binding properties of four elicitins (two basic and two acidic) and early-induced signal transduction events (Ca2+ influx, extracellular medium alkalinization, and active oxygen species production). The affinity for tobacco plasma membrane-binding sites and the number of binding sites were similar for all four elicitins. Furthermore, elicitins compete with one another for binding sites, suggesting that they interact with the same receptor. The four elicitins induced Ca2+ influx, extracellular medium alkalinization, and the production of active oxygen species in tobacco cell suspensions, but the intensity and kinetics of these effects were different from one elicitin to another. As a general observation the concentrations that induce similar levels of biological activities were lower for basic elicitins (with the exception of cinnamomin-induced Ca2+ uptake). The qualitative similarity of early events induced by elicitins indicates a common transduction scheme, whereas fine signal transduction tuning is different in each elicitin.

Activation of MAPK homologues by elicitors in tobacco cells.

Elicitors of plant defence reactions (such as cryptogein, an elicitin produced by Phytophthora cryptogea, or oligogalacturonides (OGs)), induced in tobacco cell suspensions (Nicotiana tabacum var Xanthi) a rapid and transient activation of two protein kinases (PKs) with apparent molecular masses of 50 and 46 kDa, respectively. These PKs activated and phosphorylated at tyrosine residues, phosphorylated myelin basic protein (MBP) at serine/threonine residues. Both are recognized by anti-MAPK antibodies. The two MBP kinases possessed the same kinetics of activation, and their activation depended, to the same extent, on different exogenously applied compounds (staurosporine, lanthanum, EGTA). We demonstrate here that the activation of the MBP kinases is calcium dependent and sensitive to staurosporine, a protein kinase inhibitor which annihilates all known responses of tobacco cells to cryptogein. The activation of MBP kinases appeared to be independent of the production of active oxygen species (AOS) and insensitive to calyculin A, a protein phosphatase type 1 and 2A inhibitor. The activation of MAPKs is discussed in relation to the early responses induced by cryptogein.

Cloning of two plant cDNAs encoding a beta-type proteasome subunit and a transformer-2-like SR-related protein: early induction of the corresponding genes in tobacco cells treated with cryptogein.

We report the successful combination of mRNA differential-display reverse-transcription PCR (DDRT-PCR) and 5'-rapid amplification of cDNA ends (5'-RACE) in order to isolate full-length cDNAs corresponding to genes activated in tobacco cells treated with cryptogein within 60 min. Cloning and sequencing of two cDNAs, called 'tcI 7' and 'tcI 14' (for tobacco cryptogein-induced), allowed the identification of open reading frames. Deduced amino-acid sequences of 'tcI 7' and 'tcI 14' showed significant homologies with a beta-type proteasome subunit and a transformer-2-like serine/arginine-rich (SR) ribonucleoprotein, respectively. The accumulation of mRNAs corresponding to 'tcI 7' started 30 min after the addition of cryptogein to tobacco cell suspensions and continued up to 180 min, whereas the accumulation of 'tcI 14' corresponding mRNAs was transitory between 30 and 60 min. These results indicated a transcriptional activation of the corresponding genes early after elicitation of tobacco cells by cryptogein. The biological significance of this activation remains to be elucidated.

Early Events Induced by the Elicitor Cryptogein in Tobacco Cells: Involvement of a Plasma Membrane NADPH Oxidase and Activation of Glycolysis and the Pentose Phosphate Pathway.

Application of the elicitor cryptogein to tobacco (cv Xanthi) is known to evoke external medium alkalinization, active oxygen species production, and phytoalexin synthesis. These are all dependent on an influx of calcium. We show here that cryptogein also induces calcium-dependent plasma membrane depolarization, chloride efflux, cytoplasm acidification, and NADPH oxidation without changes in NAD+ and ATP levels, indicating that the elicitor-activated redox system, responsible for active oxygen species production, uses NADPH in vivo. NADPH oxidation activates the functioning of the pentose phosphate pathway, leading to a decrease in glucose 6-phosphate and to the accumulation of glyceraldehyde 3-phosphate, 3- and 2-phosphoglyceric acid, and phosphoenolpyruvate. By inhibiting the pentose phosphate pathway, we demonstrate that the activation of the plasma membrane NADPH oxidase is responsible for active oxygen species production, external alkalinization, and acidification of the cytoplasm. A model is proposed for the organization of the cryptogein responses measured to date.

Cercospora beticola toxins. Part XVII. The role of the beticolin/Mg2+ complexes in their biological activity. Study of plasma membrane H(+)-ATPase, vacuolar H(+)-PPase, alkaline and acid phosphatases.

Beticolin-1 and beticolin-2, yellow toxins produced by the phytopathogenic fungus Cercospora beticola, inhibit the plasma membrane H(+)-ATPase. Firstly, since beticolins are able to form complexes with Mg2+, the role of the beticolin/Mg2+ complexes in the inhibition of the plasma membrane proton pump has been investigated. Calculations indicate that beticolins could exist under several forms, in the H(+)-ATPase assay mixture, both free or complexed with Mg2+. However, the percentage inhibition of the H(+)-ATPase activity is correlated to the concentration of one single form of beticolin, the dimeric neutral complex Mg2H2B2, which appears to be the active form involved in the H(+)-ATPase inhibition. Secondly, since previous data suggested that beticolins could also be active against other Mg2(+)-dependent enzymes, we tested beticolin-1 on the vacuolar H(+)-PPase, which requires Mg2+ as co-substrate, and on the alkaline and acid phosphatases, which do not use Mg2+ as co-substrate. Only vacuolar H(+)-PPase is sensitive to beticolin-1, which suggests that beticolins are specific to enzymes that use a complex of Mg2+ as the substrate. The same Mg2H2B2 complex which is responsible of the plasma membrane H(+)-ATPase inhibition appears to be also involved in the inhibition of the vacuolar H(+)-PPase.

Relationship between Active Oxygen Species, Lipid Peroxidation, Necrosis, and Phytoalexin Production Induced by Elicitins in Nicotiana.

Excised leaves of Nicotiana tabacum var Xanthi and Nicotiana rustica were treated with cryptogein and capsicein, basic and acidic elicitins, respectively. Both compounds induced leaf necrosis, the intensity of which depended on concentration and duration of treatment. N. tabacum var Xanthi was the most sensitive species and cryptogein was the most active elicitin. Lipid peroxidation in elicitin-treated Nicotiana leaves was closely correlated with the appearance of necrosis. Elicitin treatments induced a rapid and transient burst of active oxygen species (AOS) in cell cultures of both Nicotiana species, with the production by Xanthi cells being 6-fold greater than that by N. rustica. Similar maximum AOS production levels were observed with both elicitins, but capsicein required 10-fold higher concentrations than those of cryptogein. Phytoalexin production was lower in response to both elicitins in N. tabacum var Xanthi cells than in N. rustica cells, and capsicein was the most efficient elicitor of this response. In cryptogein-treated cell suspensions, phytoalexin synthesis was unaffected by diphenyleneiodonium, which inhibited AOS generation, nor was it affected by tiron or catalase, which suppressed AOS accumulation in the extracellular medium. These results suggest that AOS production, lipid peroxidation, and necrosis are directly related, whereas phytoalexin production depends on neither the presence nor the intensity of these responses.

Involvement of Free Calcium in Action of Cryptogein, a Proteinaceous Elicitor of Hypersensitive Reaction in Tobacco Cells.

Treatment of suspension-cultured tobacco (Nicotiana tabacum var Xanthi) cells with cryptogein, a proteinaceous elicitor from Phytophthora cryptogea, induced a great stimulation of Ca2+ influx within the first minutes. Ca2+ influx is essential for the initiation of cryptogein-induced responses, since ethyleneglycol-bis([beta]-amino-ethyl ether)-N,N[prime]-tetraacetic acid or La3+, which block Ca2+ entrance, suppress cryptogein-induced responses such as extracellular alkalinization, active oxygen species, and phytoalexin production. Moreover, once initiated, these responses require sustained Ca2+ influx within the 1st h. A Ca2+ ionophore (A23187) was able to trigger an extracellular alkalinization but not the formation of active oxygen species and phytoalexins, even in the presence of cryptogein. Staurosporine, a protein kinase inhibitor that was recently reported to suppress cryptogein-induced responses (M.-P. Viard, F. Martin, A. Pugin, P. Ricci, J.-P. Blein [1994] Plant Physiol 104: 1245-1249), inhibited Ca2+ influx induced by cryptogein in a dose-dependent manner. These results suggest that protein phosphorylation followed by Ca2+ influx might be involved in the initial steps of cryptogein signal transduction.

Evidence for specific, high-affinity binding sites for a proteinaceous elicitor in tobacco plasma membrane.

Binding of cryptogein, a proteinaceous elicitor, was studied on tobacco plasma membrane. The binding of the [125I]cryptogein was saturable, reversible and specific with an apparent Kd of 2 nM. A single class of cryptogein binding sites was found with a sharp optimum pH for binding at about pH 7.0. The high-affinity correlates with crytogein concentrations required for biological activity in vivo.

The Tonoplast H+-ATPase of Acer pseudoplatanus Is a Vacuolar-Type ATPase That Operates with a Phosphoenzyme Intermediate.

The tonoplast H+-ATPase of Acer pseudoplatanus has been purified from isolated vacuoles. After solubilization, the purification procedure included size-exclusion and ion-exchange chromatography. The H+-ATPase consists of at least eight subunits, of 95, 66, 56, 54, 40, 38, 31, and 16 kD, that did not cross-react with polyclonal antibodies raised to the plasmalemma ATPase of Arabidopsis thaliana. The 66-kD polypeptide cross-reacted with monoclonal antibodies raised to the 70-kD subunit of the vacuolar H+-ATPase of oat roots. The functional molecular size of the tonoplast H+-ATPase, analyzed in situ by radiation inactivation, was found to be around 400 kD. The 66-kD subunit of the tonoplast H+-ATPase was rapidly phosphorylated by [[gamma]-32P]ATP in vitro. The complete loss of radio-activity in the 66-kD subunit after a short pulse-chase experiment with unlabeled ATP reflected a rapid turnover, which characterizes a phosphorylated intermediate. Phosphoenzyme formed from ATP is an acylphosphate-type compound as shown by its sensitivity to hydroxylamine and alkaline pH. These results lead us to suggest that the tonoplast H+-ATPase of A. pseudoplatanus is a vacuolar-type ATPase that could operate with a plasmalemma-type ATPase catalytic mechanism.

Protein Phosphorylation Is Induced in Tobacco Cells by the Elicitor Cryptogein.

Changes in plasmalemma ion fluxes were observed when tobacco (Nicotiana tabacum) cells were treated with cryptogein, a proteinaceous elicitor from Phytophthora cryptogea. A strong alkalization of the culture medium, accompanied by a leakage of potassium, was induced within a few minutes of treatment. These effects reached a maximum after 30 to 40 min and lasted for several hours. This treatment also resulted in a rapid, but transient, production of activated oxygen species. All these physiological responses were fully sensitive to staurosporine, a known protein kinase inhibitor. Furthermore, a study of protein phosphorylation showed that cryptogein induced a staurosporine-sensitive phosphorylation of several polypeptides. These data suggest that phosphorylated proteins may be essential for the transduction of elicitor signals.