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.

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.

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.

Reversible exposure of hydrophobic residues on albumin as a novel strategy for formulation of nanodelivery vehicles for taxanes.

BACKGROUND: We report herein a novel strategy for the preparation of protein-based nanodelivery vehicles for hydrophobic active pharmaceutical ingredients. METHODS: The procedure consisted of three steps, ie, exposure of hydrophobic residues of a protein to a pH-induced partial unfolding: interaction between hydrophobic residues on the protein and the hydrophobic active pharmaceutical ingredient, and a final step where the structure of the protein was reversed to a native-like state by returning to neutral pH. As proof of concept, the interaction of paclitaxel with partially unfolded states of human serum albumin was evaluated as a potential method for the preparation of water-soluble complexes of the taxane with albumin. RESULTS: We found that paclitaxel readily binds to pH-induced partially unfolded albumin, leading to the formation of optically clear water-soluble complexes. The complexes thus formed were more stable in solution when the albumin native state was at least partially restored by neutralization of the solution to a pH around 7. It was also observed that the hydrodynamic radius of human serum albumin was only slightly increased after the cycle of pH changes, remaining in a monomeric state with a size according to paclitaxel binding. Furthermore, paclitaxel binding did not affect the overall exposure of charged groups of human serum albumin, as evaluated by its interaction with an ionic exchange resin. CONCLUSION: The in vitro biological activity of the complexes formed was qualitatively equivalent to that of a Cremophor(®)-based formulation.

Polyisoprene matrix for progesterone release: in vitro and in vivo studies.

Latex, a polyisoprene (PI) hydrophobic elastomer, was evaluated in vitro and in vivo as a matrix for intravaginal steroid hormone delivery. Matrices containing hormone were prepared by swelling latex in chloroform that contained soluble progesterone (P4). In vitro studies demonstrate that P4 release from PI follows a zero order model during at least 100 h and depends on initial load up to 10 mg cm(-2). The release of P4 from a PI matrix was found to be two times faster than from a polydimethylsiloxane (PDMS) matrix. FT-IR and X-ray powder diffraction analysis of P4 polymorphs show that when nucleated in PDMS, the hormone crystallizes only in alpha-form while in latex, crystallizes as a mixture of alpha- and beta-form. In vivo studies show that devices with a PI matrix containing 0.5 g of P4 are effective to reach plasma levels above 1 ng ml(-1) that are needed to synchronize estrous in cattle. Altogether, the results show that PI, a vulcanized polymer with a carbon-carbon backbone, can be used as a new matrix for the intravaginal administration of progesterone with improved release profile than silicone and that the matrix can influence the crystalline state of the hormone.

Ability of the polysaccharide chitosan to inhibit proliferation of CD4+ lymphocytes from mucosal inductive sites, in vitro and in vivo.

OBJECTIVE: After oral administration of chitosan (a copolymer of glucosamine and N-acetylglucosamine), mesenteric lymph node (MLN) lymphocytes exhibited traits of anergy, a process coupled with inability of mature T cells to proliferate. We wondered whether biological activity of chitosan could be affecting division of lymphocytes at the mucosal inductive sites. MATERIALS AND METHODS: We studied the effect of chitosan on proliferation of carboxyfluorescein diacetate-labelled MLN lymphocytes stimulated with concanavalin A in vitro. We assessed expression of CD25 and CD71 activation markers and pro-apoptotic molecule CD95L. Moreover, we studied the effect of chitosan ex vivo, in carboxyfluorescein diacetate-labelled MLN cells isolated after feeding single or repetitive doses of the polysaccharide, and we evaluated cell cycle parameters. RESULTS: Chitosan suppressed cell proliferation and down-modulated expression of CD25 in these MLN CD4+ cells isolated from normal rats. After in vivo contact, chitosan inhibited proliferation of MLN cells and reduced secretion of interferon-gamma. Furthermore, sustained feeding produced reduction in percentage of CD4+ cells in S phase of the cell cycle. CONCLUSION: Here we demonstrate the ability of chitosan to suppress proliferation of CD4+ lymphocytes from mucosal inductive sites in vivo and in vitro This effect could be relevant in modulatory activity of chitosan in the intestinal microenvironment.

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.

Room temperature vulcanizing silicone sheaths on a reusable support for progesterone delivery in estrous synchronization treatments in cattle.

High temperature vulcanizing silicone elastomers have been widely used in controlled delivery systems of steroid hormones with the aim of controlling estrous cycle in livestock. This paper reports experiments conducted to evaluate the possibility of using room temperature vulcanizing (RTV) silicone elastomers for the intravaginal administration of progesterone to cattle. In vitro studies showed that RTV silicones and high-temperature vulcanizing silicone release progesterone at a similar rate. Y-shaped inserts made of different polymers were designed as supports of RTV silicone sheaths to test the in vivo release of progesterone. Field evaluation showed that RTV silicone sheaths containing 0.75 g of progesterone were at least as effective at estrous synchronization as commercially available intravaginal inserts.

[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.

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.

Treatment of rheumatoid arthritis by oral administration of bovine tracheal type II collagen.

We evaluated the efficacy and safety of orally administered bovine tracheal type II collagen (CGII) in the treatment of rheumatoid arthritis (RA). Twenty RA patients received 0.5 mg/day of CGII for 12 weeks. Eighteen of them had improvements in the clinical parameters studied (swollen and tender joint counts, 15-m walking time, duration of morning stiffness, and physician's global assessment of disease activity). Anti-CGII antibodies were positive in 57% and rheumatoid factor (RF) in 71% of the patients with a short history of RA ( < or =2 years), whereas only 23% of those with long histories (>2 years) presented autoantibodies to CGII and 38% had positive RF. After the treatment, four patients showed reduced RF levels and all those with detectable serum tumor necrosis factor alpha (TNF-alpha) experienced its return to normal or levels below those at study entry. Although a placebo effect cannot be discounted, the oral administration of bovine tracheal CGII induced clinical benefits in 90% of the patients, without the side effects usually associated with treatment. This is the first study showing that feeding CGII can induce reductions in RF and TNF-alpha. The data justify further controlled studies to assess the long-term efficacy of this treatment approach.

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.

Characterization of casein micelle precipitation by chitosans.

We have found that the addition of chitosan, a cationic polymer, on whole or skim milk produces destabilization and coagulation of casein micelles that takes place without changes in the milk pH or the stability of most whey proteins. The amount of lipids recovered in the chitosan-casein aggregates was similar or higher than that obtained with rennet or acid precipitation. Approximately 70% of milk Ca2+ (approximately 750 mg/L) was found in the chitosan-induced aggregates, which is 10 and 50% higher than the amounts observed with acid or rennet coagulations, respectively. Purified alpha, beta-, and kappa-caseins were extensively precipitated by different molecular weight chitosans at pH 6.8. The phosphate groups of caseins seem not to be relevant in this interaction because dephosphorylated alpha- and beta-caseins were equally precipitated with chitosans. Analysis by optical microscopy of the chitosan-casein complex reveals that the size of the aggregates increase as the molecular weight of chitosans increase. Hydrophobic and electrostatic interactions particpate in the association and coagulation of casein micelles with chitosans of different molecular weights. The phenomenon is observed over a broad range of temperature (4 to 70 degrees C) with a reduction in the concentration of chitosan needed to precipitate the caseins that parallels a reduction in the viscosity of the chitosan solutions. Taken together, the results indicate that the electrostatic interactions may contribute energetically to the association between the two biopolymers, but the hydrophobicity of the complex would be the key determinant in the overall energetics of the reaction.

Hydrolysis of a chitosan-induced milk aggregate by pepsin, trypsin and pancreatic lipase.

The addition of chitosan to whole milk results in dose dependent destabilization and coagulation of the casein micelles and milk fat. The present study evaluates how the presence of chitosan could affect the hydrolysis of this chitosan-induced aggregate by different gastrointestinal proteases (pepsin and trypsin) and by pancreatic lipase. The chitosan-milk aggregate was hydrolyzed by pepsin and trypsin, as evaluated by the UV absorbance of TCA-soluble peptides and by urea-PAGE. The kinetics and extent of hydrolysis were independent of the casein being soluble or aggregated. The release of soluble peptides from the aggregate was independent of the presence of chitosan. A progressive inhibition of pancreatic lipase was observed in proportion to the increase in molecular weight of the chitosan employed to induce the formation of the aggregate. Interestingly, the presence of chitosan not only affected the initial velocity of the reaction, but also reduced its extent. The results indicate that a milk aggregate induced by chitosan was very well digested by gastric and intestinal proteases independently of the molecular weight of the chitosan used, and that the aggregate could retain the lipid-lowering effect of chitosan.

Serum levels of erythropoietin and soluble transferrin receptor in the course of pregnancy in non beta thalassemic and beta thalassemic women.

BACKGROUND AND OBJECTIVES: In non-thalassaemic women serum erythropoietin (Epo) level increases during pregnancy, whereas that of soluble transferrin receptor (STFR) drops slightly in the first two trimesters to attain the original values in the third trimester. In this study the time-course of these two parameters was explored in b-thalassemic and non-b-thalassemic women, both pregnant and not. DESIGN AND METHODS: Two hundred and fifty-seven women were studied: 64 non-b-thalassemic, non-pregnant women made up the reference group, 89 were non-b-thalassemic pregnant women, and 104 were b-thalassemic pregnant or non-pregnant women. The full blood count, hemoglobin levels and iron status (serum iron and serum ferritin levels) were explored by traditional methods. Serum Epo and STFR levels were measured with standard commercial kits. RESULTS: In non-b-thalassemic women the mean non-pregnant Epo level (10.95 +/-4. 7 mU/mL) increased in the first trimester (17.12 +/-5.18 mU/mL), was stationary in the second, and increased again in the third (31. 43+/-14.13 mU/mL). STFR mean value dropped in early pregnancy from 2. 40+/-0.72 mg/L to 1.78 +/- 0.64 mg/L, and then returned to the original value (2.38 +/- 0.94 mg/L). In b-thalassemic women the mean non-pregnant Epo level (15.00 +/-6.56 mU/mL) was higher than in non-thal non-pregnant women. During pregnancy it progressively increased to 35.60+/-25.46 mU/mL. STFR (non-pregnant level 3.37+/-1. 07 mg/L) gradually increased throughout the whole gestation period and by the third trimester its level was markedly higher than that of non-b-thal women at the corresponding stage of gestation (9. 41+/-5.39 mg/L vs 2.40+/-0.72 mg/L). INTERPRETATION AND CONCLUSIONS: The STFR level changed to different extents in non-b-thal and b-thal women during their pregnancies. In the former STFR markedly decreased in early pregnancy; in the latter it showed no decrease in the first trimester, increased in the second and reached very high values in the third. This time course is likely to be the consequence of erythroid bone marrow hyperplasia and hyperactivity, which are usually present in all b-thalassemic patients and in heterozygous carriers as well.

Evidence of a role for galectin-1 in acute inflammation.

Galectin-1 (Gal-1), a member of a family of beta-galactoside-binding proteins, has been suggested to play key roles in immunological and inflammatory processes. The present study deals with the concept of an in vivo role for Gal-1 in acute inflammation by using the rat hind paw edema test. Local administration of Gal-1 (0.5, 2, 4 and 8 microg/ml) inhibited acute inflammation induced by bee venom phospholipase A(2) (PLA(2)) when it was injected 30 min before the enzyme or co-injected together with PLA(2). The anti-inflammatory effect was prevented by a specific antibody, but independent of its carbohydrate-binding properties. In contrast, Gal-1 failed to inhibit histamine-induced edema. Histopathological studies showed a clear reduction of the inflammatory process when Gal-1 was injected before PLA(2), evidenced by a diminished number of infiltrated polymorphonuclear neutrophils and scarce degranulated mast cells. The anti-inflammatory effect was also assessed in vitro, showing that Gal-1 treatment reduced prostaglandin E(2) secretion and arachidonic acid release from stimulated peritoneal macrophages. Results presented here provide the first evidence for a role of Gal-1 in acute inflammation and suggest that the anti-inflammatory effect involves the inhibition of both soluble and cellular mediators of the inflammatory response.