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Background: Beta thalassemia major (BT) is an inherited blood disorder caused by reduced or absent synthesis of the hemoglobin beta chains, associated with profound anemia, jaundice, splenomegaly, expanded bone marrow volume, siderosis and cardiomegaly. Because of repeated blood transfusions, BT patients are subjected to peroxidative tissue injury due to secondary iron overload., Objectives: The aim of the study was to analyze: 1) the total antioxidant capacity (TAC) value in BT patients (study group) and their healthy controls (control group) from Greece (Central Macedonia) and Italy (Sardinia); correlations between 2) the TAC and ferritin levels of BT patients, and 3) the TAC and ferritin values in BT patients with different chelation therapies., Material and Methods: The studied group consisted of 60 subjects diagnosed with BT (41 female, mean age: 41.5 ± 9.5 years) and 40 healthy controls matched with age and sex (31 female, mean age: 38.5 ± 3.7 years). Desferrioxamine (DFO) was the basic previous chelation regimen for all BT patients. Antioxidant activity was assayed spectrophotometrically, using a TAC Kit (Total Antioxidant Capacity Colorimetric assay kit, produced by Cayman Chemical Co.), and ferritin was assayed by immunoturbidimetry., Results: Lower levels of TAC were observed in BT patients of both countries when compared with controls (1.83 mmol/L vs 2.7 mmol/L in the Italian study group and controls and 2.42 mmol/L vs 3.2 mmol/L in the Greek study group and controls). There were no significant correlations between plasmatic TAC and ferritin. Furthermore, deferasirox was the only chelation treatment in which TAC showed a correlation in both regions., Conclusions: Our results potentially suggest that the reduced levels of TAC detectable in BT patients could demonstrate their reduced antioxidant defensive mechanisms.

Purpose: We examined different approaches aimed to deal with the signal fluctuation of pancreatic T2* values due to fat infiltration in order to obtain accurate estimates of iron overload., Methods: Pancreatic T2* values were assessed in 20 patients (13 females, 37.24 ± 9.12 years) enrolled in the Myocardial Iron Overload in Thalassemia network without and with the application of fat suppression-FS (T2*-NoFS and T2*-FS). T2* values were assessed in three different ways: (1) from the immediate fit (original T2*); (2) discarding the echoes until the achievement of a good visual concordance between the signal and the model (final&#95;vis T2*); (3) eliminating the echoes until the achievement of a fitting error (known) <5% (final&#95;thres T2*)., Results: For the T2*-NoFS sequence the original T2* values were significantly higher than the final&#95;vis T2* values (difference:4.8 ± 6.1 ms; P < 0.0001) and the final&#95;thres T2* values (difference:4.3 ± 6.1 ms; P = 0.006). For the T2*-FS sequence the original T2* values were comparable to final&#95;vis and final&#95;thres T2* values. The original T2*-FS values were significantly different from the original T2*-NoFS values. The final&#95;vis T2*-FS values were comparable to the final&#95;vis T2*-NoFS values and the final&#95;thresh T2*-FS values were comparable to the final&#95;thresh T2*-NoFS values. For both T2*-FS and T2*-NoFS sequences, the final&#95;thres T2* values were not significantly different from the final&#95;vis T2* values and no bias was present., Conclusions: In the clinical practice, an accurate pancreatic iron overload assessment should be done by applying FS and, when needed, by discarding the TEs until the fitting error goes below 5%.

The use of biological self-assembling materials, plant virus nanoparticles in particular, appears very intriguing as it allows a great choice of symmetries and dimensions, easy chemical and biological engineering of both surface and/or internal cavity as well as safe and rapid production in plants. In this perspective, we present an initial evaluation of the safety profile of two structurally different plant viruses produced in Nicotiana benthamiana L. plants: the filamentous Potato virus X and the icosahedral Tomato bushy stunt virus. In vitro haemolysis assay was used to test the cytotoxic effects, which could arise by pVNPs interaction with cellular membranes, while early embryo assay was used to evaluate toxicity and teratogenicity in vivo. Data indicates that these structurally robust particles, still able to infect plants after incubation in serum up to 24h, have neither toxic nor teratogenic effects in vitro and in vivo. This work represents the first safety-focused characterization of pVNPs in view of their possible use as drug delivery carriers., (Copyright © 2015 Elsevier B.V. All rights reserved.)

T2* maps obtained by the processing of multiecho MR sequences can be useful in several clinical applications. T2* map generation procedures should join a processing time compatible with on-line image analysis with a good precision in the entire T2* range of clinical interest. Fast generation of T2* maps can be achieved by the estimation of the T2* values by the weighted linear fitting of the logarithm of the signal (WLSL) method. This approach fails if the signal decay diverges from a pure exponential decay, as happens at low T2* values where the rapid decay in the signal intensity leads to a plateau in the later echo times (TE). The proposed method implements the automatic truncation of the signal decay curves to be fitted in order to compensate for the signal collapse at low T2* values, allowing the extension of the WLSL method through the entire clinical range of T2* values. Validation was performed on synthetic images and on 60 thalassemia major patients with different levels of myocardial iron overload. Phantom experiments showed that a 5% fitting error threshold represented the best compromise between T2* value measurement precision and processing time. A good agreement was found between T2* map pixel-wise measurements and ROI-based measurements performed by expert readers (CoV=1.84% in global heart T2*, CoV=5.8% in segmental analysis). In conclusion, the developed procedure was effective in generating correct T2* maps for the entire T2* clinical range., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Purpose: The aim of the work was to assess the long-term stability and the safety of lipid nanoparticles intended for brain drug delivery., Methods: Lipid nanoparticles, prepared by high pressure homogenization, were stored at room temperature and 4°C and monitored for their mean hydrodynamic diameter and Gaussian distribution width over time. Cetylpalmitate and polysorbate(®) 80 chemical integrity were investigated by nuclear magnetic resonance on diagnostic signals. Nanoparticle toxicity was assessed in chicken embryos by chorioallantoic membrane assay and in rodents by brain histological evaluation., Results: Data showed nanoparticle stability at 4°C over a period of time of 4 years with only a limited particle size increase while at room temperature destabilization was observed after 9 months. Nuclear magnetic resonance investigation confirmed the absence (<5%) of chemical degradation of the lipid matrix and the surfactant after 4 years of storage at 4°C. Chorioallantoic membrane assay and rat brain histology showed the absence of acute toxicity corroborating previously published data., Conclusions: Cetylpalmitate nanoparticle long-term physical and chemical stability, together with the in vivo safety, corroborate the existing evidences of the high value of colloidal lipids as parenteral formulations and carriers for brain drug delivery., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The pulmonary route is one of the main strategies investigated to improve tuberculosis therapy. The aim of this study was to develop a simple and scalable method to produce capreomycin inhalable powders to use as supergeneric. In vitro antimycobacterial activity and in vivo acute toxicity were assessed using agar proportion susceptibility test on Mycobacterium tuberculosis and chicken chorioallantoic membrane assay, respectively. Capreomycin and three different hydrophobic counterions, namely oleate, linoleate, and linolenate, were combined in solution to obtain hydrophobic ion-pairs that were successively spray-dried. Ion-pairing efficiency was influenced by the spray-dryer employed to produce the powder. In the case of capreomycin oleate, both instruments, mini and nano spray-dryer, were suitable to maintain a high ion-paired content, while for capreomycin linoleate and linolenate, mini spray-dryer was the most appropriate instrument. The three formulations showed morphology and particle sizes potentially suitable for inhalation. Capreomycin oleate and linoleate showed the same efficacy of capreomycin sulfate against M. tuberculosis, while capreomycin linolenate showed a reduced efficacy, even though strain growth was inhibited at 10(-4) mycobacterial inoculum. In vivo acute toxicity studies evidenced the lowest toxic potential for capreomycin oleate when compared to the single components or the other two salts. Overall, capreomycin oleate seems to possess the most promising characteristics to be used as supergenerics in pulmonary tuberculosis treatment., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Erythroid carbonic anhydrase (CA) activity of chick embryos from the third day of incubation to the egg hatching has been determined. Five minor activity peaks with maxima at 3, 6, 9, 15 and 17 days of development and a major one with maximum at 19 days have been found. The correlation between the peak distribution and the timing of release into the blood stream of waves of newly produced erythroid cells has been demonstrated on the basis of the following observations: 1) a linear correlation exists between red cell maturation and increase of CA activity; 2) chick red cells undergo lysis in the "Ørskov" medium when their CA activity exceeds a threshold value (23±3 Units/10 9 red cells); and 3) the lysis kinetics of red cells in the Ørskov medium is proportional to their CA content. We have thus been able to distinguish the immature erythroid forms from the mature ones on the basis of their behaviour in the Ørskov medium. In the blood of developing chick embryos, we have found waves of newly produced red cells at about 2, 4, 7, 10, 16 and 18 days of development. The same experimental criteria allowed us to detect the waves of red cell production in the erythropoietic organs. One wave has been detected in the blood islands at about 2 days; four waves in the yolk sac at about 5, 6, 11 and 15 days; two waves in the spleen at about 18 and 20 days; two waves in the bone marrow at about 19 days of incubation and 1 day after hatching. Primitive erythroid cells are produced in the first two waves: that of blood islands at 2 days and that of yolk sac at 5 days. Definitive red cells are produced in the other waves with the exception of the second wave of spleen and of the second wave of bone marrow, which are constituted by red cells of adult type.

Erythroid carbonic anhydrase activity of chick embryos from the 3rd day of incubation to the egg hatching has been determined. Three minor activity peaks at 3, 9 and 15 days of development and a major one at 19 days were found. The enzyme molecular forms were purified by affinity chromatography from haemolysates of embryos at several stages of development. As has been found for the adult erythrocytes, only type II isozyme was detected in the embryo red cells. Isoelectrofocusing analysis demonstrated that two different molecular forms of this isozyme are synthesized by the red cells of developing embryos. Only the early form is present up to 5 days of development; the late form, which is indistinguishable from the adult isozyme, appears in the haemolysate at 6-7 days and quickly replaces the early form. Analysis of purified primitive and definitive erythroid lines from 7-days-old embryos showed a compartmentalization of the early and late forms into the primitive and definitive erythroid cells, respectively.

1. Three Koelliker haemoglobins, HbKE, HbKA and HbKH, derived from a post-translational loss of alpha-Arg-141, were isolated from red cells of chicken embryos. HbKE is typical of embryos up to 7 days of incubation, HbKA and HbKH are found in mature embryos. 2. All the precursor haemoglobins contain alpha A chains. HbKA derives from adult haemoglobin A whose globin composition is alpha A2 beta 2, HbKH from embryonic haemoglobin H with a globin composition alpha A2 beta H2 and HbKE from embryonic haemoglobin E with globin composition alpha A2 epsilon 2. 3. No Koelliker derivatives of haemoglobins with alpha-like chains other than alpha A were observed.

Three haemoglobins were isolated by ion-exchange chromatography from the haemolysates of embryonic duck erythrocytes up to 8 days of development. The component globins were characterized both by electrophoresis in dissociating conditions and by finger-printing analysis. The major haemoglobin fraction E1 appears to be an embryonic tetramer since its constituent globins are different from all the others synthesized during embryonic and adult life. The two minor fractions E2 and E3 show alpha-type subunits that are very similar to those of the two adult haemoglobins A1 and A2 respectively. They are present all through embryonic life, as demonstrated by chromatographic analysis. For these reasons they have been considered foetal. The two haemoglobins typical of the adult animal are found in the red cells of the embryo from 8 days of incubation. Their relative amounts change continuously during embryonic development and reach the adult value after hatching.

1. Adult chicken hemoglobins were analysed by ion exchange chromatography and isoelectric focusing and a minor hemoglobin fraction (HbK) was isolated. 2. Analysis of the constituent chains shows that HbK differs from the two major hemoglobins HbA and HbD in the alpha globin. 3. The amino acid composition, the tryptic peptide maps, the results of carboxypeptidase digestion and the functional properties show that the HbK alpha globin is quite similar to that of HbA except that the C-terminal amino acid Arg 141 is lacking. 4. HbK must then be considered a Koelliker-type hemoglobin.

The synthesis of globins in the chick embryo before the onset of circulation has been studied in situ by specific immunofluorescence labelling of embryonic sections and by labelling newly synthesized proteins in ovo and in vitro in embryonic explants with [3H]leucine. The presence of major primitive haemoglobins is observed by 28 h of incubation. The minor primitive haemoglobins become detectable by immunofluorescence after 40 h of development, shortly before the onset of circulation. 3H-labelling shows that one definitive alpha chain is synthesized, though in low concentration, from the initial globin detection. The other definitive alpha chain is observed in embryos of at least 40 h of development. The relative concentration of the two definitive alpha chains changes rapidly with development indicating a specific mechanism of regulation. An erythropoietic site is observed in the wall of the dorsal aorta in embryos of about 45-50 h of development. From the initial detection, those cells contain all four primitive embryonic haemoglobins, in contrast to what is observed for the cells of the blood islands.

1. Chick embryos with primary circulation, up to about 3 days of development, show no hemoglobin-mediated transport of oxygen. 2. In embryos with secondary circulation, between 3 and 6 days of incubation, the vascular area acts as the respiratory organ. Its efficiency in the oxygen uptake is less than that of the chorioallantois of later embryos. On the contrary, oxygen release to the tissues is highly efficient. 3. A full efficient hematic uptake of oxygen is reached at about the 6th incubation day, when chorioallantois acts as the embryonic respiratory organ. 4. The different respiratory mechanisms of developing chick embryo are closely related to the functional properties of the various hemoglobins which are produced during the embryonic life.