Survey on the usage of therapeutic erythrocytapheresis in transfusion services in Italy for the treatment of polycythemia vera, secondary erythrocytosis and hemochromatosis.

INTRODUCTION: Erythrocytapheresis, an apheresis treatment which selectively removes red blood cells, is an alternative to therapeutic phlebotomy, over which it has several advantages. Actually there is a high degree of variability in the use of this treatment. This prompted SIdEM (Italian Society of Hemapheresis and Cell Manipulation) to conduct a survey on the use of erythrocytapheresis in the Italian Transfusion Services. The purpose is to monitor this activity in the treatment of Polycythemia Vera (pv), secondary erythrocytosis and hemochromatosis. MATERIALS AND METHODS: A data collection file was sent to the SIdEM regional delegates who, in turn, involved the Transfusion Centers in the areas they cover. The data collected were processed on a Microsoft Excel spreadsheet. RESULTS: 75 centers from 14 Italian regions responded to the Survey: 36 centers (48 %) use erythrocytapheresis (35 centers perform therapeutic apheresis and 1 center only donor apheresis), 39 centers (52 %) do not (15 centers perform therapeutic apheresis, 18 centers only donor apheresis and 6 centers do not perform either therapeutic apheresis or donor apheresis). Although most centers have a substantially uniform attitude concerning the indications for which erythrocytapheresis is used, the survey shows that there are still differences more evident in the treatment of secondary erythrocytosis than in the treatment of pv or hemochromatosis. CONCLUSIONS: This survey has been useful to document the current Italian reality and to raise awareness about the need for improvement in optimizing and standardizing the use of a therapy with a great potential to exploit properly.

CNS Hypomyelination Disrupts Axonal Conduction and Behavior in Larval Zebrafish.

Myelination is essential for central nervous system (CNS) formation, health and function. As a model organism, larval zebrafish have been extensively employed to investigate the molecular and cellular basis of CNS myelination, because of their genetic tractability and suitability for non-invasive live cell imaging. However, it has not been assessed to what extent CNS myelination affects neural circuit function in zebrafish larvae, prohibiting the integration of molecular and cellular analyses of myelination with concomitant network maturation. To test whether larval zebrafish might serve as a suitable platform with which to study the effects of CNS myelination and its dysregulation on circuit function, we generated zebrafish myelin regulatory factor (myrf) mutants with CNS-specific hypomyelination and investigated how this affected their axonal conduction properties and behavior. We found that myrf mutant larvae exhibited increased latency to perform startle responses following defined acoustic stimuli. Furthermore, we found that hypomyelinated animals often selected an impaired response to acoustic stimuli, exhibiting a bias toward reorientation behavior instead of the stimulus-appropriate startle response. To begin to study how myelination affected the underlying circuitry, we established electrophysiological protocols to assess various conduction properties along single axons. We found that the hypomyelinated myrf mutants exhibited reduced action potential conduction velocity and an impaired ability to sustain high-frequency action potential firing. This study indicates that larval zebrafish can be used to bridge molecular and cellular investigation of CNS myelination with multiscale assessment of neural circuit function.SIGNIFICANCE STATEMENT Myelination of CNS axons is essential for their health and function, and it is now clear that myelination is a dynamic life-long process subject to modulation by neuronal activity. However, it remains unclear precisely how changes to myelination affects animal behavior and underlying action potential conduction along axons in intact neural circuits. In recent years, zebrafish have been employed to study cellular and molecular mechanisms of myelination, because of their relatively simple, optically transparent, experimentally tractable vertebrate nervous system. Here we find that changes to myelination alter the behavior of young zebrafish and action potential conduction along individual axons, providing a platform to integrate molecular, cellular, and circuit level analyses of myelination using this model.

Surface characterization and interfacial activity of chitinase chi18-5 against chitosan in langmuir monolayers.

One of the challenges for producing active chitinase formulations relies on the gap between the laboratory tests and the biological scenarios where the enzyme will perform its function. In this work, we have employed different Langmuir monolayer arrays to evaluate the interfacial behavior of a recently purified recombinant chitinase, Chi18-5. We have demonstrated that two conformations exist for the chitinase at pH values close to its pI, showing very distinct structural properties at the air/aqueous interface. Enzyme activity was assessed by implementing different kinetic approaches and using a chitosan-1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) mixed film as organized substrate model membrane. Combining these strategies, we demonstrated that better catalytic efficiencies can be obtained for Chi18-5 at pH 5. Moreover, the chitinase activity at the air/aqueous interface can be tuned by introducing in situ pH modifications over the surrounding milieu. We also studied the changes in the topography at the mesoscale level using Brewster Angle Microscopy (BAM). We found that Chi18-5 segregated onto the chitosan domains of the membrane, showing differences in homogeneity depending on the pH imposed. Alternatively, pure Chi18-5 was tested for immobilization onto a hydrophilic activated solid support using the Langmuir-Blodgett technique. Atomic Force Microscopy (AFM) analyses showed successfully stabilization and preservation of molecular features attributed to the pH at which the enzyme deposition was performed.

The biocompatible polysaccharide chitosan enhances the oral tolerance to type II collagen.

Chitosan is a mucoadhesive polysaccharide that promotes the transmucosal absorption of peptides and proteins. At mucosal sites chitosan exhibits immunomodulatory activities and stimulates the release of regulatory cytokines. Herein we evaluated the effect of the co-administration of chitosan in the tolerance to type II collagen (CII) using an experimental model of arthritis. Rats were fed diluent (acetic acid), 1 mg CII, 1 mg chitosan or 1 mg CII + 1 mg chitosan during 5 days before immunization with CII in Freund's complete adjuvant. Systemic effects were evaluated in draining lymph nodes after antigenic challenge or during the clinical evolution of arthritis. Specific antibodies, proliferation against CII and the production of interferon (IFN)-gamma and interleukin-10 were assessed. Clinical signs were observed 13-15 days after primary immunization. The CII : chitosan group presented the lowest incidence and developed moderate arthritis, with reduced levels of immunoglobulin (Ig)G2a anti-CII, a limited proliferation in draining lymph nodes and a lower release of IFN-gamma after restimulation with CII. Our results demonstrate that chitosan enhances the tolerance to an articular antigen with a decrease in the inflammatory responses and, as a consequence, an improvement in clinical signs.

[Emergency, subjectivity, stress: on the side of the helper]. / Emergenza, soggettività, stress: dalla parte del soccorritore.

Rescue and emergency teams are affected by different stress factors. We investigated 201 subjects from Red Cross Organization, firemen "regular" and volunteer, policemen, and health rescue team. Mental fatigue and psychological impact are more common critical factors. 55% of subjects show burnout symptoms, more among Red Cross volunteers, and firemen. Among policemen the emotional exaustion has more pathologic results; depersonalization among fireman and personal accomplishment among Red Cross and firemen volunteers. The normality of the data from other psychophysical health investigation confirm occupational origin of the complaints.

[Satisfaction and stress if the health care system: comparison of two different situations]. / Fattori di soddisfazione e stress in sanità: confronto tra due realtà differenti.

We examined two different health care settings to identify if some stress and satisfaction factors are common. In both neonatological ward and psychiatric residence the relation with the patient has been perceived as gratifying but in paediatric setting it is perceived through procedures and protocols, on the opposite site, in the psychiatric setting the daily life care is perceived as a low technical degree. Formation of workers is indispensable tools to understand the "technical" aspects of the work, without promoving a remoteness from the patient.

Effect of sulfatide and gangliosides on phospholipase C and phospholipase A2 activity. A monolayer study.

The effect of sulfatide and gangliosides GM1, GD1a and GT1b on the activity of phospholipase C from Clostridium perfringens on dilauroylphosphatidylcholine and of porcine pancreatic phospholipase A2 on dilauroylphosphatidic acid was studied in lipid monolayers containing different proportions of glycolipids under zero-order kinetics at various constant surface pressures. The presence of sulfatide in the monolayer increases the activity of phospholipase C at high surface pressures. Gangliosides shift the cut-off pressure to lower values and inhibit the action of phospholipase C. In mixed monolayers with dilauroylphosphatidic acid, sulfatide at a molar fraction of 0.5 increases the activity of phospholipase A2 at surface pressures below 18 mN/m and shows an inhibitory effect at higher pressures. Ganglioside GM1 at a molar fraction of 0.25 completely inhibits the enzyme above 20 mN/m and markedly reduces its activity at lower pressures. Gangliosides GD1a and GT1b abolish the enzyme activity at all pressures at molar fractions of 0.25 and 0.15, respectively. The modified velocity of the enzymatic reaction in the presence of glycosphingolipids is not due to an irreversible alteration of the catalytic activity.

Identification of two specific lysines responsible for the inhibition of phospholipase A2 by manoalide.

Manoalide, a natural product of sponge, irreversibly inhibits phospholipase A2 (PLA2) by reacting with lysine residues. Cobra venom PLA2 mutants were constructed in which four of the six lysine residues were independently replaced by arginine or methionine, which cannot react with manoalide. The mutants were overexpressed in Escherichia coli, renatured, and purified. The enzyme mutants lacking Lys-6 (K6R and K6M) or Lys-79 (K79R) were inhibited only 40% by manoalide while the native cobra venom PLA2 was inhibited 80% under the same conditions. This means that the manoalide modification of either Lys-6 or Lys-79 accounted for only half of the manoalide inhibition. The double mutant (K6R79R) was not inhibited by manoalide at all. Lys-56 (K56R) and Lys-65 (K65R) mutants were inhibited to the same extent as the native enzyme which indicates that these residues are not responsible for any of the inhibitory effects produced by manoalide. These results demonstrate that the reaction of manoalide with both Lys-6 and Lys-79 can account for all of its inhibition of cobra venom PLA2. The inhibition of PLA2 and its mutants with manoalide did not affect the activity of the enzyme toward monomeric substrate, which suggests that manoalide does not modify the catalytic site residues, that it does not block access to this site, and that its inhibition requires an interface. Furthermore, as with native PLA2, the activation of phosphatidylethanolamine hydrolysis by phosphorylcholine-containing compounds was exhibited by all of the mutants suggesting that none of the lysines examined are essential for this activation.

Regulation by gangliosides and sulfatides of phospholipase A2 activity against dipalmitoyl- and dilauroylphosphatidylcholine in small unilamellar bilayer vesicles and mixed monolayers.

The modulation by gangliosides GM1 and GD1a, and sulfatide (Sulf) of the activity of porcine pancreatic phospholipase A2 was studied with small unilamellar vesicles of dipalmitoylphosphatidylcholine (L-dpPC) and lipid monolayers of dilauroylphosphatidylcholine (L-dlPC). The presence of Sulf always led to an increase of the maximum rate of the enzymatic reaction, irrespective on whether the vesicles were above, in the range of, or below the bilayer transition temperature. Sulf did not modify the latency period for the reaction that is observed at the bilayer transition temperature. Gangliosides inhibited the maximum rate of enzymatic activity bilayer vesicles in the gel phase but the effect was complex. When the reaction was carried out at a temperature within the range of the bilayer phase transition, the gangliosides inhibited the maximal rate of the reaction in proportion to their content in the bilayer. However, at the same time the latency period observed with vesicles of pure phospholipid at this temperature was shortened in proportion to the mole fraction of gangliosides in the bilayer. At temperatures above the bilayer phase transition, gangliosides stimulated the activity of PLA2. Preincubation of the enzyme with Sulf or gangliosides did not affect the activity against bilayer vesicles of pure substrate. These glycosphingolipids did not modify the rate or extent of desorption of the enzyme from the interface, nor the pre-catalytic steps for the interfacial activation of PLA2, or the enzyme affinity for the phospholipid substrate. Also, the activity of the enzyme was not altered irreversibly by glycosphingolipids. Our results indicate that Sulf and gangliosides modulate the catalytic activity of PLA2 at the interface itself, beyond the initial steps of enzyme adsorption and activation, probably through modifications of the intermolecular organization and surface electrostatics of the phospholipid substrate.

Chitosan-induced phospholipase A2 activation and arachidonic acid mobilization in P388D1 macrophages.

We have found that chitosan, a polysaccharide present in fungal cell walls, is able to activate macrophages for enhanced mobilization of arachidonic acid in a dose- and time-dependent manner. Studies aimed at identifying the intracellular effector(s) implicated in chitosan-induced arachidonate release revealed the involvement of the cytosolic Group IV phospholipase A2 (PLA2), as judged by the inhibitory effect of methyl arachidonoyl fluorophosphonate but not of bromoenol lactone. Interestingly, priming of the macrophages with lipopolysaccharide renders the cells more sensitive to a subsequent stimulation with chitosan, and this enhancement is totally blocked by the secretory PLA2 inhibitor 3-(3-acetamide)-1-benzyl-2-ethylindolyl-5-oxy-propanesulfonic acid (LY311727). Collectively, the results of this work establish chitosan as a novel macrophage-activating factor that elicits AA mobilization in P388D1 macrophages by a mechanism involving the participation of two distinct phospholipases A2.

Modulation of the inflammatory response by corticotropin-releasing factor.

Peptides of the corticotropin-releasing factor (CRF) family have been shown to have either pro- or anti-inflammatory activities. CRF (10-30 micrograms/kg) administered subcutaneously or intravenously could inhibit edema and dye leakage in the rat paw produced by several injuries. These findings are opposed to some results suggesting a predominantly pro-inflammatory effect of CRF mainly in arthritic processes. The purpose of this work was to identify in vivo and in vitro the conditions for the pro- or anti-inflammatory actions of CRF in order to clarify its physiological and pharmacological function. Using the rat paw edema test we observed that only the highest doses of CRF employed (5 micrograms) induced a moderate and sustained swelling. Pre-treatment with low doses of CRF (0.5-5 ng) was able to inhibit the edema induced by Naja naja phospholipase A2, carrageenin or histamine. Higher doses (50 ng-5 micrograms) had no anti-inflammatory activity. When co-inhibited with Naja naja phospholipase A2 or histamine the peptide did not modify the swelling at doses up to 500 ng, showing at 5 micrograms an additive edema with Naja naja phospholipase A2. In vitro, CRF did not modify the release of histamine but slightly increased the release of arachidonic acid to the medium. Our findings show a clear dose dependence on the local effects of CRF in inflammatory responses. These results suggest that the mechanisms of the two dose-related phenomena may be distinct.

Anti-inflammatory effect of gangliosides in the rat hindpaw edema test.

The influence of total brain gangliosides on acute inflammation was investigated using the rat hind paw edema test. Total gangliosides (10 micrograms/paw) inhibited the edema produced by the injection of bee venom phospholipase A2 (5 micrograms/paw) when the lipids were co-injected or injected 15 min before the phospholipase A2. Sulphatide (10 micrograms/paw) did not inhibit the edema but potentiated it. Gangliosides (40 micrograms/paw) inhibited the edema induced by carrageenin 1% when they were injected 1 h before the agent. However, gangliosides (up to 200 micrograms/paw) failed to inhibit the dextran-induced edema. The edema test was also used to investigate the effect of gangliosides on the production of mediators of inflammation by peritoneal adherent macrophages. Gangliosides inhibited the production of mediators of inflammation only when they were incubated with these cells before the stimulation with phospholipase A2 or carrageenin. Gangliosides did not inhibit the production of mediators of inflammation when arachidonic acid was added to the cells. These results suggest that the anti-inflammatory effect observed with gangliosides is mediated by inhibition at or before endogenous phospholipase activity.

Inhibition of human platelet aggregation by gangliosides.

The content and composition of gangliosides is modified upon platelet stimulation, suggesting that these lipids may play functional roles in platelet physiology. Therefore, the effect of exogenously added gangliosides on human platelet aggregation was evaluated. The pretreatment of platelets with a mixture of total gangliosides from bovine brain and a series of purified mono-, di- and tri-sialogangliosides partially inhibit the collagen-induced aggregation process and ATP release and completely block the generation of the second aggregation wave when ADP is used as agonist. The inhibition was exerted at around 100 microM by G(TOT) as well as purified G(M1), G(M3), G(D1a), and G(T1b) gangliosides, whereas asialoG(M1) and sulphatide did not show a significant influence on platelet aggregation. Thrombin, Ca(2+) ionophores (A23187 and Ionomycin), arachidonic acid, and U46619 were unable to bypass the inhibitory effect exerted by gangliosides, suggesting that gangliosides inhibit platelet aggregation by inhibiting the synthesis or action of prostaglandins. Gangliosides inhibited U46619-induced aggregation, thus suggesting that they block the action of thromboxane A(2). Epinephrine induces a partial aggregation on gangliosides-treated platelets, similar to fluoroaluminate and phorbol myristate acetate, indicating that these platelets are still functional. To summarize, these results indicate that the major pathway(s), but not all, driving to the aggregation process following the interaction of ligand-receptor may be blocked by pretreatment of human platelets with gangliosides.

The lower susceptibility to Plasmodium falciparum malaria of Fulani of Burkina Faso (west Africa) is associated with low frequencies of classic malaria-resistance genes.

The gene frequencies in 1993-94 for haemoglobin S, haemoglobin C, alpha-3.7 deletional thalassaemia, G6PDA-, HLAB*5301 were estimated in Fulani, Mossi and Rimaibé ethnic groups of Burkina Faso, West Africa. The aim of the study was to verify whether the previously reported Fulani lower susceptibility to Plasmodium falciparum malaria was associated with any of these malaria-resistance genes. Similar frequencies for haemoglobin S were recorded in the 3 ethnic groups (0.024 +/- 0.008, 0.030 +/- 0.011, 0.022 +/- 0.013; in Mossi, Rimaibé and Fulani, respectively). The Mossi and Rimaibé showed higher frequencies when compared to Fulani for haemoglobin C (0.117 +/- 0.018, 0.127 +/- 0.020, 0.059 +/- 0.020), alpha-3.7 deletional thalassaemia (0.227 +/- 0.040, 0.134 +/- 0.032, 0.103 +/- 0.028), G6PDA- (0.196 +/- 0.025, 0.187 +/- 0.044, 0.069 +/- 0.025) and HLA B*5301 (0.189 +/- 0.038, 0.202 +/- 0.041, 0.061 +/- 0.024). Among Fulani the proportion of individuals not having any of these protective alleles was more than 3-fold greater than in the Mossi-Rimaibé group (56.8% vs 16.7%; P < 0.001). These findings exclude the involvement of these genetic factors of resistance to P. falciparum in the lower susceptibility to malaria of Fulani. This evidence, in association with the previously reported higher immune reactivity to malaria of Fulani, further supports the existence in this ethnic group of unknown genetic factor(s) of resistance to malaria probably involved in the regulation of humoral immune responses.

A new phospholipase A2 isoform isolated from Bothrops neuwiedii (Yarará chica) venom with novel kinetic and chromatographic properties.

A new phospholipase A2 isoform, called P-3, isolated from Bothrops neuwiedii (Yarará chica) venom, showed different chromatographic, enzymatic and cytotoxic properties compared to the previously purified isoforms P-1 and P-2 but it had a similar edema-inducing activity. In contrast to previously reported B. neuwiedii phospholipase A2 isoforms, P-3 did not interact with the oligosaccharide matrix of gel filtration columns (Superose, Superdex). Its molecular weight was 15,000 and its N-terminal 14 amino acid sequence was Asn-Leu-Val-Gln-Phe-Glu-Thr-Leu-Ile-Met-Lys-Ile-Ala-Gly. Amino acid analyse revealed the presence of an unique histidine, presumably located at the active site, because a full inhibition of enzymatic activity was observed after treatment with p-bromophenacyl bromide. The new isoform also differentiated in its surface pressure activity profile when assayed in lipid monolayers. P-3 had an optimum activity towards dilauroylphosphatidylcholine monolayers of 27 mN/m and a cut-off pressure of 30 mN/m, whereas P-1 and P-2 had an optimum of 13 mN/m with a cut-off of 22 mN/m. P-3 retained its edema-inducing activity in the absence of hydrolytic activity, suggesting that the inflammatory activity was not dependent on the enzymatic activity. Neither the enzymatic nor the edema-inducing activity was affected by heparin. The new isoform was not lethal when a single dose of 5 micrograms/g body weight was injected intraperitoneally into mice. All of the isoforms displayed cytotoxic activity in vitro on B16F10 melanoma cells evaluated by direct MTT assay, with an EC50 of 31 micrograms/ml for P-3 and of 15 micrograms/ml for P-1 and P-2. The cytotoxic activity of P-3 was inhibited by p-bromophenacyl bromide treatment of the enzyme (up to 170 micrograms/ml), whereas the same treatment on P-1 and P-2 changed their EC50 to 60 micrograms/ml. The difference observed with inhibited enzymes suggests a different mechanism for the cytotoxic action of P-3 with respect to P-1 and P-2.

Calcium dependency of arachidonic acid incorporation into cellular phospholipids of different cell types.

Ca2+ -independent phospholipase A2 (iPLA2) is involved in the incorporation of arachidonic acid (AA) into resting macrophages by the generation of the lysophospholipid acceptor. The role of iPLA2 in AA remodeling in different cells was evaluated by studying the Ca2+ dependency of AA uptake from the medium, the incorporation into cellular phospholipids, and the effect of the iPLA2 inhibitor bromoenol lactone on these events. Uptake and esterification of AA into phospholipids were not affected by Ca2+ depletion in human polymorphonuclear neutrophils and rat fibroblasts. The uptake was Ca2+ independent in chick embryo glial cells, but the incorporation into phospholipids was partially dependent on extracellular Ca2+. Both events were fully dependent on extra and intracellular Ca2+ in human platelets. In human polymorphonuclear neutrophils, the kinetics of incorporation in several isospecies of phospholipids was not affected by the absence of Ca2+ at short times (<30 min). The involvement of iPLA2 in the incorporation of AA from the medium was confirmed by the selective inhibition of this enzyme with bromoenol lactone, which reduced < or =50% of the incorporation of AA into phospholipids of human neutrophils. These data provide evidence that suggests iPLA2 plays a major role in regulating AA turnover in different cell types.

Gangliosides raise the intracellular Ca2+ level in different cell types.

Total gangliosides from bovine brain at micromolar concentration induce intracellular Ca2+ increments in a temperature, time and dose dependent manner when assayed with suspensions of rat macrophages, rat and chicken neurons, human erythrocytes and liposomes, loaded with the fluorescent Ca2+ indicator FURA 2. The effect was independent on the endogenous ganglioside composition of the cells and in the case of neurons it was also independent on the differentiation state. Gangliosides do not induce the release of Ca2+ from inner stores. These findings indicate that the reported inhibition of arachidonic acid release (Bressler, J., et al., (1994) Life Sci., 54, 49-60) and anti-inflammatory properties of gangliosides (Correa, S.G. et al., (1991) Eur. J. Pharmacol. 199, 93-98) are not due to impairments of Ca2+ flux. The results also suggest the possibility that the well-known neurotrophic effect produced by gangliosides on undifferentiated neurons in culture may be due to subtoxic cytosolic Ca2+ increments.

Psychotic symptoms in mood disorders: Evaluation of 159 inpatients.

The authors investigated the historical and clinical variables of 159 inpatients affected by mood disorders in order to identify variables which might differentiate psychotic from non-psychotic forms. The results showed that 32% of the patients had psychotic symptoms. Although no significant difference was detected with regard to the severity of depression, psychotic depressives were younger and had a lower age at onset, as well as a shorter episode length. These features suggest that depression may express itself with or without psychotic symptoms, according to the different individual and, perhaps, biological substrate. Taken together, our findings seem to indicate that psychotic depression should not be considered a separate clinical entity, but a subtype of mood disorders.

Improvement of HDL- and LDL-cholesterol levels in diabetic subjects by feeding bread containing chitosan.

In this work we evaluated the efficacy and safety of a bread formulation containing chitosan in dyslipidemic type 2 diabetic subjects. For this purpose a total of 18 patients were allowed to incorporate to their habitual diets 120 g/day of bread containing 2% (wt/wt) chitosan (chitosan group, n= 9) or standard bread (control group, n= 9). Before the study and after 12 weeks on the modified diet, the following parameters were evaluated: body weight, plasma cholesterol, high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol, triglyceride, and hemoglobin A(1c) (HbA(1c)). Compared with the control group, the patients receiving chitosan-containing bread decreased their mean levels of LDL-cholesterol and significantly increased their mean levels of HDL-cholesterol at the end of the study. There were no significant differences in the body weight, serum triglyceride, and HbA(1c). These results suggest that chitosan incorporated into bread formulations could improve the lipoprotein balance similar to typical biliary salts trappers, increasing the HDL- and lowering the LDL-cholesterol, without changing the triglyceride levels. These results warrant further studies over a longer period of time to evaluate if a persistent improvement in levels of lipoproteins can be attained with this strategy.