Characterisation of granulocyte stimulation by thionins from European mistletoe and from wheat

Thionins are small basic peptides found in different plant species, which are known to exert cytotoxic properties. In addition, previous data indicated an activation of human granulocytes by thionins from European mistletoe (viscotoxins, VT). To extend these latter findings, we investigated the influence of VT and from thionins from wheat flour (purothionin) on human granulocytes by flow cytometry and tried to characterise the involved molecular structures and mechanisms. Phagocytosis was determined by incorporation of FITC-labelled Escherichia coli and respiratory burst by oxidation of dihydrorhodamine 123 to rhodamine 123. VT and purothionin significantly enhanced E. coli-stimulated phagocytosis and respiratory burst at 25 and 250 microgram/ml. Phagocytosis of damaged lymphocytes by granulocytes was detected by electron microscopy in the VT-stimulated (100 microgram/ml) but not in the control cultures. The poly-cationic structure of the intact molecule seems to be crucial, as evidenced by comparison of the burst and phagocytosis-enhancing effects induced by other poly-cationic (protamine sulphate, histone, poly-l-arginine, poly-l-lysine) and poly-anionic (poly-l-glutamic acid) peptides, while pore forming due to amphipathic properties seems to be less important. Ca2+ and Mg2+ could not inhibit VT-enhanced phagocytosis and, thus, could not inhibit binding of VT to granulocytes. In addition, verapamil at low concentrations inhibited VT activity, suggesting the involvement of Ca2+ channels for granulocyte activation by the VT. Similarly, thionins and histones in contrast to protamine sulphate induced cell death of granulocytes at 250 microgram/ml as demonstrated by an enhanced release of reactive oxygen intermediates in unstimulated granulocytes. From these data one may suggest that activity of VT is induced by strong unspecific ionic binding, probably followed by specific receptor binding, and thionins exhibit stimulatory and cytotoxic effects on immune cells, which have to be further characterised.     

Scorpion toxins and defensins

Advances in CT, MR imaging, and catheter angiography provide the radiologist and neurosurgeon with a variety of imaging options for screening, diagnosis, presurgical evaluation, and postoperative monitoring of patients with intracranial aneurysms. Noninvasive imaging techniques have not replaced conventional angiography for the comprehensive evaluation o aneurysms but are effective in screening patients suspected to have an unruptured aneurysm or for preoperative planning in emergency situations that preclude catheter angiography. CT, CT angiography, MR imaging, and MR angiography can all complement the information obtained with catheter angiography in presurgical planning, and the choice of supplemental studies should be individualized. Rotational and intraoperative angiography are problem-solving options used for selected cases at our institution. Continuous improvements in techniques for CT and MR angiography may someday reach the point where surgery can be undertaken on the basis on noninvasive imaging alone, with catheter angiography reserved for endovascular therapy planning and guidance.     

Comparison of secretory responses as measured by membrane capacitance and by amperometry

We have compared capacitance and amperometric measurements in bovine chromaffin cells when secretion was elicited by flash photolysis of caged-calcium or step depolarizations. Total amperometric charge depended linearly on the amount of capacitance increase in both types of experiments. Furthermore, the properties of resolvable amperometric spikes after flashes were comparable to those observed after depolarizations, and their timing was compatible with the rate of capacitance increase. For a more detailed comparison, we used Monte Carlo simulations of multiple amperometric events occurring randomly over the surface of a sphere and summing together, to generate a reference amperometric signal for a given measured capacitance increase. Even after correction for endocytotic processes, the time courses of the integrated experimental records lagged behind the integrated Monte Carlo records by approximately 50 ms in flash and depolarization experiments. This delay was larger by approximately 40 ms than what can be expected from the "pre-foot delay" or the foot duration. Possible sources for the remaining delay could be diffusional barriers like the patch-pipette and the chamber bottom, which are not taken into account in the model. We also applied a novel type of fluctuation analysis to estimate the relative quantum size of an amperometric event. On average the estimates from experimental amperometric traces, in both flash and depolarization experiments, were 3-5 times smaller than estimates from simulated ones. This discrepancy can be due to contributions to the amperometric current from small vesicles, preferred release from cellular regions orientated toward the chamber bottom, or abundance of "foot-only" events. In conclusion, amperometric signals in flash and depolarization experiments displayed similar delayed average time courses and a lower estimate for the relative quantum size compared to the modeled amperometric signals. However, individual amperometric spikes were in agreement with expectations derived from capacitance signals.     

Protein conformational distortions induced by membrane interfacial interactions

It is clear that considerable conformational distortions may occur in peripheral proteins on interaction with anionic lipid bilayers. Specific lipid interactions do occur at least in the case of cytochrome c, and each perturbation and interaction may well take place before insertion into, or translocation across, a biomembrane.     

Fungal plasma membrane proton pumps as promising new antifungal targets

Fungi are widely dispersed in nature and frequently appear as pathogens in the animal and plant kingdoms. The incidence of opportunistic fungal infections in humans has increased due to the human immunodeficiency virus and the application of modern medical approaches that subvert natural protective barriers to infection. Also, fungal blights continue to threaten crops worldwide. As a result, new antifungal agents are needed to address these critical problems. Existing antifungals can be used to effectively treat most cases of topical infection caused by the opportunistic pathogen Candida albicans, which is the principal agent of nosocomially acquired fungal infections. However, life-threatening, disseminated Candida infections are treated with more modest success. Existing antifungals can be toxic or ineffective because of natural resistance or even induced resistance. This limited efficacy largely reflects the restricted range of cellular targets considered during the development of current antifungals. The advancement of highly selective fungicidal reagents requires the recognition of new essential cellular targets. The fungal plasma-membrane proton pump is a high-abundance essential enzyme with a number of well-understood molecular properties that should facilitate the development of new antifungals. The proton pump is important for intracellular pH regulation and the maintenance of electrochemical proton gradients needed for nutrient uptake. It is a member of the P-type class of ion-transport enzymes, which are present in nearly all external cellular membranes. Typical P-type enzymes such as the Na+,K(+)-ATPase and H+,K(+)-ATPase are well established as specific targets for surface-active cardiac glycosides and anti-ulcer therapeutics. The development of new classes of selective antifungals targeted to the proton pump will require exploitation of the well-characterized genetic, kinetic, topological, regulatory, and drug-interaction features of the fungal enzyme that discriminate it from related host P-type enzymes. New antifungal drugs of this type should be relevant to the control of fungal pathogens of medical and agricultural importance and may be applicable to the control of intracellular parasites that also depend on closely related proton pumps for survival.     

Modulation of plant plasma membrane H+-ATPase by phytotoxic lipodepsipeptides produced by the plant pathogen Pseudomonas fuscovaginae

OBJECTIVE: Dienogest, a synthetic steroid with progestational activity, is used as a component of oral contraceptives and is currently being evaluated clinically for the treatment of endometriosis. The present study was conducted to confirm the effects of dienogest on experimental endometriosis in rats and to elucidate its mechanism of action. DESIGN: Experimental endometriosis induced by autotransplantation of endometrium in rats. METHODS: Endometrial implants, immune system, and bone mineral were investigated after 3 weeks of medication. RESULTS: Dienogest (0.1-1 mg/kg per day, p.o.) reduced the endometrial implant volume to the same extent as danazol (100 mg/kg per day, p.o.). Simultaneously, dienogest ameliorated the endometrial implant-induced alterations of the immune system: i.e. it increased the natural killer activity of peritoneal fluid cells and splenic cells, decreased the number of peritoneal fluid cells, and decreased interleukin-1beta production by peritoneal macrophages. In contrast, danazol (100 mg/kg per day, p.o.) and buserelin (30 microg/kg per day, s.c.) had none of these immunologic effects. Additionally, combined administration of dienogest (0.1 mg/kg per day) plus buserelin (0.3 microg/kg per day) suppressed the bone mineral loss induced by buserelin alone, with no reduction of the effect on endometrial implants. In vitro studies on dienogest revealed an antiproliferative effect on rat endometrial cells due to inhibition of protein kinase C activity plus a partial progestational effect. CONCLUSIONS: Dienogest appears to be a potent agent with mechanisms of action different from those of danazol and GnRH agonists currently available for the treatment of endometriosis.     

Mechanisms for induction of acquired host immunity by neutrophil peptide defensins

Human neutrophil peptide (HNP) defensins were studied to determine their potential effects on adaptive mucosal immunity. Intranasal delivery of HNPs plus ovalbumin (OVA) enhanced OVA-specific serum IgG antibody (Ab) responses. However, OVA-specific IgA Abs were not induced in mucosal secretions or in serum. CD4(+) T cells of intranasally immunized mice displayed higher OVA-specific proliferative responses and elevated production of interferon gamma, interleukin (IL) 5, IL-6, and IL-10 when compared with control groups receiving OVA alone. In vitro, HNPs also enhanced both proliferative responses and T helper (Th) cytokine secretion profiles of CD3epsilon-stimulated spleen- and Peyer's patch-derived naive CD4(+) T cells. HNPs modulated the expression of costimulatory molecules by lipopolysaccharide- or CD3epsilon-stimulated splenic and Peyer's patch B or T cell populations, respectively. These studies show that defensins enhance systemic IgG, but not IgA, Ab responses through help provided by CD4(+) Th1- and Th2-type cytokines and foster B and T cell interactions to link innate immunity with the adaptive immune system.     

Causal beliefs and conditioned responses: Retrospective revaluation induced by experience and by instruction.

The author tested causal beliefs and conditioned responses in a task involving retrospective revaluation of the causal status of a target cue with respect to electric shock. Successful revaluation was observed on both self-report shock expectancy and skin conductance, whether the training trials were directly experienced, described, or partly experienced and partly described. The results contradict models that link anticipatory conditioned responses to a separate or earlier process from that underlying explicit causal knowledge. They suggest instead that a single learning process gives rise to propositional knowledge that (a) drives anticipatory responding, (b) forms the basis for self-reported causal beliefs, and (c) can be combined with other knowledge, provided either by experience or symbolically, to generate inferences such as retrospective revaluation. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Antitumor responses induced by transgenic expression of CD40 ligand

Because CD40 ligand (CD40L) is a co-stimulator molecule for multiple components of the immune response, we wanted to determine whether transgenic expression of the molecule would increase immune responses against a weakly immunogenic murine tumor, neuro-2a. Tumor cells were transduced with a retroviral construct containing the CD40L gene and co-injected with variable numbers of non-CD40L transduced cells into syngeneic mice. Mice injected with cells that expressed CD40L had a significant reduction in average tumor size as compared to controls (p < 0.0001). In addition, survival of the neuro-2a/CD40L mice was 48 days versus 34 days for the neuro-2a/neo controls (p < 0.02). Expression of CD40L by less than 1.5% of neuro-2a cells was sufficient for significant antitumor effects (p < 0.001). These antitumor effects protected mice from subsequent challenge with parental neuro-2a cells. The protective effects of CD40L were associated with systemic immunomodulation. In vivo depletion of CD8+ cells abrogated the CD40L-mediated antitumor effects. Analysis of spleens from CD40L-protected animals showed increased numbers of CD4+ and CD8+ cells, the majority of which co-expressed the activation marker CD25. In addition, an increased number of antigen-presenting cells (APCs) expressed the co-stimulatory molecule CD86. These experiments illustrate that transducing even a small percentage of tumor cells with CD40 ligand can create a long-lasting systemic immune response capable of impeding growth of unmodified neuroblastoma cells.     

Ultrastructures of the glial epiretinal membrane induced by activated macrophages

A rabbit model of glial epiretinal membrane was established following the injection of activated macrophages into the vitreous. The membrane was composed entirely of cells with glial characteristics, ie, abundant intermediate filaments, microvilli, junctional complexes and basement membranes. The extracellular matrix of the mature membranes contained collagen fibrils of 10 to 15 and 20 to 25 nm in diameter. Fusiform densities were seen adjacent to the cell membrane and cells with indented nuclei were found in thick membranes. These observations demonstrate that glial cells in epiretinal membranes may synthesize collagen and possess myofibroblast-like properties.     

Constitutive and induced cytokine production by human placenta and amniotic membrane at term

Results of our previous study on the immunity of human placenta and amniotic membranes revealed that in majority of cases these organs present constitutive non-specific antiviral immunity in the organ culture (OC) system. It is possible that interferons (IFNs), tumour necrosis factors (TNFs) and interleukin 6 (IL-6) may be responsible for the antiviral effect. Here, the constitutive and lipopolysaccharide (LPS)-induced production of these cytokines and, additionally, interleukin 10 (IL-10) were determined in OC of chorionic villi, decidua and amniotic membranes. Significant amounts of constitutive TNF-alpha (2-64 U/ml), IL-6 (200-12,000 U/ml) and IL-10 (1-70 ng/ml) were detected in the maternal decidua and chorionic villi of placenta. Amniotic membranes produced lower concentrations of the cytokines. LPS increased the production of cytokines from two- to eightfold. In contrast, activity of IFN released spontaneously was found only in four of 50 placentae and amniotic membranes. LPS and Newcastle disease virus (NDV) induced IFN production in the OC system. However, the increase of IFN after induction was also very small (up to 32 U/ml). Individual differentiation in the cytokines production was observed among placentas and amniotic membranes. TNF was identified as type alpha with addition TNF-beta, IFN as type alpha, beta and gamma.     

Melanosomal pattern in phototoxic pigmentary responses induced by topical psoralens and other photosensitizers

High resolution proton magnetic resonance measurements provide evidence for the formation of hydrogen-bonded complexes between 9-ethyladenine and p-cresol used as a model of tyrosine side chain in CDCl3. We have calculated the sum of the association constants corresponding to the three existing 1:1 complexes: K=6.3+/-0.15. By methylation of the amino group of adenine, we were able to calculate the ratio of the two strongest equilibrium constants K7/K1=1.6+/-0.3. Theoretical computations by the complete neglect of differential overlap (CNDO/2) method indicate that several hydrogen-bonded planar complexes can form between 9-methyladenine and phenol. The computed energy of the complexes with 6-dimethylamino adenine removes some ambiguity concerning the computed ratio of the association constants. Comparison of the calculated energies with free energies experimentally determined in organic solvent shows that despite the competition with CDCl3, which associates with both solute molecules, the preferential order of association is conserved. The small variations of charge density of adenine carbon atoms when complexed with phenol are in agreement with very small chemical shifts observed by 13C-nuclear magnetic resonance.     

Quantitative assessment of auditory cortex responses induced by imager acoustic noise

A clustered volume acquisition functional MRI pulse sequence was modified to assess the response to the acoustic noise of echo-planar imaging in the auditory cortex and to determine whether it is possible to obtain data which is relatively free of acoustic contamination. The spatial location and strength (percent signal change) of cortical responses to the imager noise were examined by introducing extra gradient readouts, without slice excitation, to provide acoustic stimulation immediately prior to acquisition of a cerebral volume. The duration of acoustic stimulation was controlled by varying the number of extra gradient readouts. Slice acquisitions were clustered at the end of the repetition time (TR) period to prevent a response from being induced by the volume acquisition itself ("Intra-Acquisition Response"). The cerebral volumes were acquired using a long TR in order to limit the integration of the cortical response across volume acquisitions ("Inter-Acquisition Response"). Cortical responses were observed to be largest and most significant on the medial two-thirds of Heschl's gyrus, the location of primary auditory cortex. Mean signal changes induced by the imager noise were observed to be as high as 0.95%. A 2 sec delay prior to onset of the BOLD response was empirically determined. These results demonstrate that clustered volume acquisitions may be utilized for up to 2 sec of volume acquisition without inducing an appreciable Intra-Acquisition Response and can be used, with a sufficiently long TR, to provide data which are similarly free of any Inter-Acquisition Response.     

Radiogenic responses of normal cells induced by fractionated irradiation--a simulation study. Part II. Late responses

AIM: Based on controlled theory, a computed simulation model has been constructed which describes the time course of slowly responding normal cells after irradiation exposure. Subsequently, different clinical irradiation schemes are compared in regard to their delayed radiogenic responses referred to as late effects in radiological terminology. METHOD: A cybernetic model of a parenchymal tissue consisting of dominantly resting functional cells has been developed and transferred into a computer model. The radiation effects are considered by characteristic cell parameters as well as by the linear-quadratic model. RESULTS: Three kinds of tissue (brain and lung parenchyma of the mouse, liver parenchyma of rat) have been irradiated in the model according to standard-, super-, hyperfractionation and a single high dose per week. The simulation studies indicate that the late reaction of brain parenchyma to hyperfractionation (3 x 1.5 Gy per day) and of lung parenchyma tissue with regard to all fractionation schemes applied is particularly severe. In contrast to these observations the behavior of liver parenchyma is not unique: If Dtotal amounts to 60 Gy there is no evidence for compensation of radiation damages, but if Dtotal is restricted to 30 Gy the corresponding evidence can be expected for all schemes. In the case of a high single dose of 6 Gy a reduction of the recovery time from 1 week to 2...2 days yields also an indication of a severe damage, even if Dtotal amounts only to 30 Gy. CONCLUSIONS: A comparison of the simulation results basing to the survival of cell numbers with clinical experience and practice shows that the clinical reality can qualitatively be represented by the model. This opens the door for connecting side effects to normal tissue with the corresponding tumor efficacy (discussed in previous papers). The model is open to further refinement and to discussions referring to the phenomenon of late effects.     

Effect of nitric oxide donors on neutrophil responses induced by immune complexes

The present study characterizes the effect of two nitric oxide (NO) donors, S-nitrosoglutathione (GSNO) and sodium nitroprusside (SNP), on the ability of neutrophils to perform different responses triggered by immune complexes (IC). Pretreatment of neutrophils with either GSNO or SNP exerted a biphasic action on antibody-dependent cellular cytotoxicity (ADCC) performed against erythrocytes (E) coated with IgG antibodies (IgG-E), depending on the amount of IgG employed. While with high amounts of antibodies ADCC was markedly inhibited, at low amounts of antibodies it was significantly increased. Both effects were prevented by haemoglobin, a NO scavenger. Moreover, these effects were reproduced by the cell-permeable analogue of cGMP, dibutyryl cGMP (Bt2cGMP). Other neutrophil functions triggered by IgG-E were also examined. It was found that NO donors did not affect either the phagocytosis of IgG-E or the emission of chemiluminescence (CL). Finally, neutrophil functions triggered by soluble IC (sIC) and precipitating IC (pIC) were analysed. It was observed that NO donors did not modify either cytotoxicity performed towards non-sensitized target cells or CL emission. The significance of these results is discussed.     

Cytokine and adhesion molecule requirements for lung injury induced by anti-glomerular basement membrane antibody

Acute hemorrhagic lung injury occurs in humans with anti-GBM antibody (Goodpasture's syndrome), however, the mechanism of this injury is still largely unknown. To date, treatment has been confined to steroids and plasmaphoresis. Infusion of anti-GBM antibody into rats caused lung injury with intra-alveolar hemorrhage and intrapulmonary accumulation of neutrophils. Lung injury was dependent on the presence of neutrophils and complement and required both TNF alpha and IL-1. Experiments employing blocking antibodies to adhesion molecules demonstrated requirements for the beta 1 integrin VLA-4, beta 2 integrins LFA-1 and Mac-1, and L-selection. The endothelial cell adhesion molecules, E-selectin and ICAM-1, were also required for the full development of lung injury. Inhibition of TNF alpha or IL-1 or adhesion molecules reduced both lung injury and tissue neutrophil accumulation. Thus, this study underscores cytokine and adhesion molecule requirements for neutrophil mediated injury in lung and kidney caused by anti-GBM, suggesting potential targets for the treatment of Goodpasture's syndrome in humans.     

KATP channel modulators and myocardial damages induced by ischemia-reperfusion: membrane lipids injury and arrhythmias

Although KATP channels have been proposed as playing a role in most types of myocardial damage associated with ischemia/reperfusion, the potential benefits of KATP channel modulators against the biochemical and electrical disturbances observed during ischemia remain unclear. We have thus studied the effects of glibenclamide and cromakalim, KATP channel blocker and opener respectively, on membrane lipid injury and arrhythmias, in a model of ischemic-reperfused guinea-pig myocardium. Ventricular strips were prelabeled with [3H] arachidonic acid, then subjected to normal conditions (Time-related Control) or to simulated ischemic-reperfused conditions in absence of drug (Control) or in presence of glibenclamide 10 microM or cromakalim 10 microM. The release of radioactive compounds was counted by liquid scintillation spectrometry, while action potentials (AP) were recorded with intracellular microelectrodes. Reperfusion induced a significant increase of arachidonic acid release (P < 0.05 versus Time-related Control). Glibenclamide inhibited the reperfusion-induced arachidonic acid release while cromakalim only delayed it (respectively 483 +/- 87 dpm/g, P < 0.05 and 790 +/- 143 dpm/g. NS versus 838 +/- 80 dpm/g for Control, after 30 min of reperfusion). Unlike glibenclamide, cromakalim was proarrhythmic during reperfusion (in 100% of preparations versus 33% in Control or in presence of glibenclamide, P < 0.05). This in vitro study shows that glibenclamide prevented the reperfusion-induced membrane arachidonic acid release, without proarrhythmic effect, whereas cromakalim, associated with proarrhythmicity, was unable to protect myocardium from cell lipid damage.