Developmental regulated mechanisms affect the ability of a fungal pathogen to infect and colonize tobacco leaves.

During tobacco development, a transition state from susceptibility to resistance to fungal pathogen infection is observed. Leaves acquire resistance to Phytophthora parasistica when the plant becomes committed to flowering. The ability to develop resistance does not imply pathogen-induced defence responses as for the onset of systemic acquired resistance (SAR). Throughout flowering growth, fungal establishment is restrained at two levels. The first level is the control of infection effectiveness. Using the salicylic acid non-accumulating NahG plants, we demonstrate that this control does not require salicylic acid accumulation. The intercellular fluids (IFs) from tobacco leaves committed to flowering exhibit a cytotoxic activity on fungal zoospore cells based on in vitro germination assays. Its accumulation is correlated to the control of infection effectiveness that occurs during flowering growth. The expression of this activity appears to constitute a developmental regulated mechanism that inhibits early steps of fungal pathogen installation. A second level of fungal growth control is the restriction of fungal hyphae expansion. In contrast to infection initiation, fungal hyphae spreading appears to be restricted by similar mechanisms induced during SAR as it is attested by the requirement of salicylic acid accumulation and by the correlating apoplastic accumulation of PR1 proteins. These results provide evidence for the activation of a set of at least two regulatory pathways during flowering growth. This activation leads to the induction of mechanisms which control fungal development by affecting the ability of the fungus to both infect and colonise plant tissues.

[Cardiac tamponade after withdrawal of a peripheral access central catheter]. / Taponamiento cardíaco tras movilización de catéter central de inserción periférica.

Central venous catheterization is a very common technique, although its complications can be multiple and sometimes fatal. A case is presented of cardiac tamponade by parenteral nutrition a few hours after moving a central venous catheter peripherally inserted a few days before. The diagnosis was made by echocardiography, and an emergency pericardiocentesis was performed, achieving complete recovery of the patient. Peripherally inserted central venous catheters are more likely to change their position secondary to the movements of the patient's arm, thus it is important to use soft catheters, make sure the tip lies above the carina to avoid perforation of the pericardial reflexion, and fix it well to the skin. Diagnosis must be made as soon as possible, given the high mortality rate of this complication, and the essential diagnostic tool is echocardiography. Elective treatment consists of early catheter withdrawal and emergency pericardiocentesis.

On the prediction of Hodgkin lymphoma treatment response.

PURPOSE: The cure rate in Hodgkin lymphoma is high, but the response along with treatment is still unpredictable and highly variable among patients. Detecting those patients who do not respond to treatment at early stages could bring improvements in their treatment. This research tries to identify the main biological prognostic variables currently gathered at diagnosis and design a simple machine learning methodology to help physicians improve the treatment response assessment. METHODS: We carried out a retrospective analysis of the response to treatment of a cohort of 263 Caucasians who were diagnosed with Hodgkin lymphoma in Asturias (Spain). For that purpose, we used a list of 35 clinical and biological variables that are currently measured at diagnosis before any treatment begins. To establish the list of most discriminatory prognostic variables for treatment response, we designed a machine learning approach based on two different feature selection methods (Fisher's ratio and maximum percentile distance) and backwards recursive feature elimination using a nearest-neighbor classifier (k-NN). The weights of the k-NN classifier were optimized using different terms of the confusion matrix (true- and false-positive rates) to minimize risk in the decisions. RESULTS AND CONCLUSIONS: We found that the optimum strategy to predict treatment response in Hodgkin lymphoma consists in solving two different binary classification problems, discriminating first if the patient is in progressive disease; if not, then discerning among complete and partial remission. Serum ferritin turned to be the most discriminatory variable in predicting treatment response, followed by alanine aminotransferase and alkaline phosphatase. The importance of these prognostic variables suggests a close relationship between inflammation, iron overload, liver damage and the extension of the disease.

[Anesthesia techniques in epidermolysis bullosa]. / Técnicas anestésicas en la epidermólisis ampollosa.

Epidermolysis bullosa encompasses a group of rare clinical profiles marked by the formation of bullae on the skin and mucosa as the result of slight mechanical trauma. The anesthesiologist must take certain safety measures to monitor the airway and must expect difficult venous access in patients with this disease. We report our experience in providing anesthesia by various techniques for plastic and maxillofacial surgery. Most anesthetic techniques can be considered safe if they are performed with care and attention to detail.

Establishment of 'normal' nervous cell lines after transfer of polyoma virus and adenovirus early genes into murine brain cells.

Brain cells from murine embryos were transfected with the polyoma virus large T or the adenovirus 5 EIA gene and, simultaneously, with the phosphotransferase coding NeoR gene. The efficiently transfected cells were selected for their resistance to Geneticin (G418) and their ability to clone at low cell density. Subsequently, most of the selected cells could be sub-cloned and continuously grown for 6-18 months so far. Their doubling time varied between 18 and 72 h. From independent transfections, more than one hundred cell lines were established. They did not exhibit a transformed phenotype, but subsequent transfection with the polyoma middle T gene induced their oncogenic transformation. The maintenance and expression of the transferred genes were verified. Most of the analyzed cell lines retained glial properties. These results suggest that the lines obtained as well as a further extension of this in vitro system should be of interest for the study of nervous cell interactions, differentiation and functions.

Immortalization of bipotential glial progenitors and generation of permanent "blue" cell lines.

The transfer of the adenovirus 5 E1A gene into brain cells from rat embryos led to the establishment of phenotypically untransformed clonal glial cell lines. Some lines displayed properties of oligodendrocyte-astrocyte progenitors. Their differentiation involved several steps that were dependent on culture conditions and cell interactions. Subsequently, a few lines were cotransfected with a vector carrying a modified dihydrofolate reductase gene and with the Escherichia coli lacZ gene. After selection for resistance to methotrexate, cell lines were derived that stably expressed the lacZ gene. These cells were individually detectable by histochemical staining for beta-galactosidase activity, even in the presence of other cells. These results suggest that this type of cell line could be of interest for further in vitro, and possibly transplant, studies of the differentiation and interactions of glial cells.

A permanent glial precursor cell line, immortalized with the adenovirus E1A gene, undergoes apoptosis in restrictive growth conditions.

We have previously described some differentiation properties of the ERD.1.1 cell line, obtained after transfer and integration of the adenovirus-5 E1A gene. Depending on the growth conditions, these cells differentiate towards the astrocyte or early oligodendrocyte differentiation pathway. However, in growth restrictive conditions, we observed dying cells that detached from the monolayer constituted of differentiating cells. This led us to examine the characteristics of the dying cells. The study of the low-molecular-weight DNA and the ultrastructural examination of chromatin showed that these cells were undergoing apoptosis. The results also suggest that the expression of an integrated polyoma middle T gene can partly save the cells from apoptosis.

Symptomatic gastroparesis in a patient with achalasia.

We present a case of a patient with achalasia who developed symptomatic gastroparesis after botulinum toxin injection therapy. Symptoms responded to prokinetics. Pathophysiology of gastric motility disturbances in patients with achalasia is discussed.

Identification of a neural-specific cDNA, NPDC-1, able to down-regulate cell proliferation and to suppress transformation.

Immortalized neural precursor cell lines carrying the polyoma large tumor (T) gene have been shown previously to retain a clear-cut contact inhibition of growth and to differentiate in vitro. In the present study, we have identified and isolated cDNA clones corresponding to RNA expressed preferentially when these cells reach confluence. One of them, NPDC-1, is expressed specifically in the nervous system. The transfection of dividing cells with a NPDC-1 expression vector results in the inhibition of cell proliferation. In addition, the stable introduction of NPDC-1 into transformed cells, even of nonneural origin, leads to the suppression of transformed characteristics.

Immortalization of different precursors of glial cells with a targeted and temperature-sensitive oncogene.

Different types of glial precursor cell lines were obtained after stable transfection of brain cells with the pJC-SVLTtsA vector carrying the tsA58 simian virus 40 large T (SVLT) gene driven by the promoter of a gliotropic strain of JC papovavirus. The immortalized cells were conditional for growth: they expressed the SVLT antigen and proliferated at 34 degrees C, but their growth was either reduced or arrested when they were shifted to 39 degrees C. The differentiation characteristics of four representative lines were more extensively studied. The CR15 and CM8 lines displayed properties of bipotential glial progenitors: they were able to express oligodendrocyte markers at both temperatures, but could differentiate into astrocytes only at 39 degrees C. In contrast, the CR19 and CM3r lines corresponded to more committed oligodendrocyte precursors: they expressed various oligodendroglial markers but they could not synthesize the glial fibrillary acidic protein. More particularly, the CM3r mouse cells displayed a typical oligodendrocyte progenitor morphology and expressed the proteolipid protein mRNA.

Immortalization of bipotential and plastic glio-neuronal precursor cells.

Permanent clonal cell lines from newborn mouse striatum have been established after transfer of the simian virus 40 large tumor oncogene by means of a retroviral vector. Some of the lines obtained displayed properties of bipotential and plastic glio-neuronal precursors. Depending on the culture conditions, these cells express either the glial fibrillary acidic protein or neurofilaments. In addition, the cells can display adrenergic, D1 and D2 dopaminergic, muscarinic, and 5-hydroxytryptamine type 2 serotoninergic receptors, which are coupled either to the adenylate cyclase or to the phosphatidylinositol signaling pathways. The panel of receptors for neurotransmitters exhibited by these lines closely resembles that of primary striatal neurons. Results suggest that plastic common precursors of astrocytes and neurons persist in the striatum at a late developmental stage. As these permanent cell lines constitute an unlimited source of homogenous cell material, we suggest that they should be useful for molecular and pharmacological studies on the mechanisms and regulation of signal transduction as well as the commitment, plasticity, and differentiation of neural cells.

Proliferation and differentiation properties of bipotent glial progenitor cell lines immortalized with the adenovirus E1A gene.

Bipotent glial progenitors have been immortalized by the transfer of the adenovirus E1A gene into primary cultured cells from embryonic rat brain. The lines obtained are phenotypically untransformed, retain growth contact-inhibition, and are able to differentiate, unless they are surtransfected with transforming oncogenes. Depending on the growth conditions, these immortalized cells express differentially either oligodendrocyte or astrocyte-specific markers and genes. After being seeded in serum-free medium, they display gangliosides recognized by A2B5 monoclonal antibody, and then they express sequentially O4 epitopes, galactocerebroside, and the myelin protein DM20. When grown in serum-supplemented medium, the cells express at first A2B5 epitopes, and then transiently O4 and galactocerebroside; after reaching confluence, O4 and galactocerebroside become undetectable, whereas the cells begin to coexpress glial fibrillary acidic protein and glutamine synthetase. These results indicate that the cell lines can undergo a differentiation reminiscent both of O-2A progenitors and of plastic process-bearing glial subpopulations. The cells were also genetically marked by the stable introduction of the nlslacZ reporter gene. Thus, the lines could be useful for studying direct interactions in vitro, or for post-grafting investigations. They should also provide a model for studying the mechanisms involved in the commitment and in the control of proliferation and differentiation of this cell lineage. This suggestion is consistent with the data indicating a growth arrest-dependent differential expression of a novel gene encoding a protein with a helix-loop-helix domain.

RNase activity prevents the growth of a fungal pathogen in tobacco leaves and increases upon induction of systemic acquired resistance with elicitin.

The hypersensitive response and systemic acquired resistance (SAR) can be induced in tobacco (Nicotiana tabacum L.) plants by cryptogein, an elicitin secreted by Phytophthora cryptogea. Stem application of cryptogein leads to the establishment of acquired resistance to subsequent leaf infection with Phytophthora parasitica var nicotianae, the agent of the tobacco black shank disease. We have studied early events that occur after the infection and show here that a tobacco gene encoding the extracellular S-like RNase NE is expressed in response to inoculation with the pathogenic fungus. Upon induction of SAR with cryptogein, the accumulation of NE transcripts coincided with a rapid induction of RNase activity and with the increase in the activity of at least two different extracellular RNases. Moreover, exogenous application of RNase activity in the extracellular space of leaves led to a reduction of the fungus development by up to 90%, independently of any cryptogein treatment and in the absence of apparent necrosis. These results indicate that the up-regulation of apoplastic RNase activity after inoculation could contribute to the control of fungal invasion in plants induced to SAR with cryptogein.

Establishment of permanent astroglial cell lines, able to differentiate in vitro, from transgenic mice carrying the polyoma virus large T gene: an alternative approach to brain cell immortalization.

Permanent untransformed cell lines have been established from the cerebral cortex of transgenic mice that carry the polyoma virus large T gene. The immortalized cells described here synthesize laminin and neural cell adhesion molecules and induce primary neurons to develop neuritic processes. As shown by immunofluorescence and immunoblotting assays, they begin to synthesize the glial fibrillary acidic protein (GFAP) after confluence. Double labelling experiments indicated that GFAP expression is reversibly correlated with the arrest of cell division. The present cells also display adrenergic, serotoninergic, and high levels of muscarinic receptors coupled to the phosphatidylinositol signalling pathway. Taken together, our data show that these cell lines constitute homogeneous cell material that has retained the main differentiative, functional, and growth properties of normal astrocytes. Therefore, such clonal untransformed cell lines should be useful for further molecular studies, addressing terminal differentiation of glial cells, glioneuronal interactions, and astroglial expression of receptors for neurotransmitters. Furthermore, we suggest that this approach of cell immortalization by the use of transgenic mice carrying a non-transforming oncogene might be extended to a variety of cell types.

Neurological disorder in transgenic mice that express the large T antigen of polyoma virus in the nervous system.

Among a series of 44 transgenic families established after microinjection into fertilized eggs of a plasmid DNA where the structural gene for the large T antigen of polyoma virus is located downstream from the viral early promoter-enhancer region, one family with a hereditary neurological disorder was observed. At about three weeks of age, these animals developed a syndrome of constant tremor with recurrent seizures. Histological and ultra-structural examination revealed extensive dysmyelination in the white matter of the brain stem, cerebellum and spinal cord, as well as of peripheral nerves. This phenotype is reminiscent of that of the mouse "twitcher" (twi) mutant and of the human hereditary leukodystrophies. Expression of the viral sequences, assayed by Northern analysis and immunolabeling of T antigen, occurred predominantly in cells of the central nervous system. Integration of the transgene was mapped by in situ hybridization on metaphasic plaques in region B-C1 of chromosome 12 (where the twi locus was previously localized). Long-term cultures of cells with neural characteristics could be established readily from the brain of the transgenic mice.