Diagnostic role of internal mammary lymph node involvement in tuberculous pleurisy: a multicenter study.

BACKGROUND AND OBJECTIVES: Diagnosis of tuberculous pleurisy (TP) may be challenging and it often requires pleural biopsy. A tool able to increase pre-test probability of TP may be helpful to guide diagnostic work-up and enlargement of internal mammary lymph node (IMLN) has been suggested to play a potential role. The aim of the present investigation was to assess role of IMLN involvement in TP in a multi-centric case-control study, by comparing its prevalence and test performance to those observed in patients with infectious, non-tuberculous pleurisy (NTIP), and in controls free from respiratory diseases (CP). METHODS: A total of 419 patients, from 14 Pulmonology Units across Italy were enrolled (127 patients affected by TP, 163 affected by NTIP and 129 CP). Prevalence, accuracy and predictive values of ipsilateral IMLN involvement between cases and control groups were assessed, as well as concordance between chest computed tomography (CT scan) and thoracic ultrasound (TUS) measurements. RESULTS: The prevalence of ipsilateral IMLN involvement in TP was significantly higher than that observed in NTIP and CP groups (respectively 77.2%, 39.3% and 14.7%). Results on test performance, stratified by age, revealed a high positive predictive value in patients aged ≤50 years, while a high negative predictive value in patients aged >50 years. The comparison between CT scan and ultrasound showed moderate agreement (Kappa=0.502). CONCLUSIONS: Evaluation of IMLN involvement plays a relevant role in assessing the pre-test probability of TP. Considering the increasing global prevalence of mycobacterial infections, a tool able to guide diagnostic work-up of suspected TP is crucial, especially where local sources are limited.

Novel complications of the tunnelled indwelling pleural catheter.

Tunnelled indwelling pleural catheters (TIPC) are a modality of treatment for malignant pleural effusions. Though relatively easy, safe and efficacious, they are associated with a small risk of complications. We describe newer complications of the TIPC including the retention of the polyester plug and the blockage of the catheter with thick organised material consisting of malignant cells taking the shape of the catheter.

Diagnosing allergic sensitizations in the third millennium: why clinicians should know allergen molecule structures.

Diagnostic tests to detect allergic sensitization were introduced at the end of the nineteenth century but only in the late 1990s did the advent of molecular allergology revolutionize the approach to the allergic patient. Personalized Medicine, a medical procedure that separates patients into different groups with different medical decisions, practices and interventions has sanctioned this change. In fact, in the last few years molecular allergology and the observation that not every patient has the same allergic profile, even when allergic to the same allergenic source, has originated the concept "one size does not fit all". This new approach requires the identification of still unknown allergens, but also the more detailed investigation of those already known. In depth studies of the structure-function relationships in allergenic molecules can reveal the structural determinants involved in the IgE-binding. Then, the knowledge of the epitope profile of each allergen and of the environmental/experimental conditions affecting the exposure of IgE-binding epitopes can provide important contributions to the understanding of cross-reaction processes and to the improvement of diagnosis, immunotherapy and the overall patient treatment. The evolution of diagnostic systems cannot ignore these new needs in this field.

The kiwi fruit peptide kissper displays anti-inflammatory and anti-oxidant effects in in-vitro and ex-vivo human intestinal models.

Literature reports describe kiwi fruit as a food with significant effects on human health, including anti-oxidant and anti-inflammatory activity. Fresh fruit or raw kiwi fruit extracts have been used so far to investigate these effects, but the molecule(s) responsible for these health-promoting activities have not yet been identified. Kissper is a kiwi fruit peptide displaying pore-forming activity in synthetic lipid bilayers, the composition of which is similar to that found in intestinal cells. The objective of this study was to investigate the kissper influence on intestinal inflammation using cultured cells and ex-vivo tissues from healthy subjects and Crohn's disease (CD) patients. The anti-oxidant and anti-inflammatory properties of kissper were tested on Caco-2 cells and on the colonic mucosa from 23 patients with CD, by challenging with the lipopolysaccharide from Escherichia coli (EC-LPS) and monitoring the appropriate markers by Western blot and immunofluorescence. EC-LPS challenge determined an increase in the intracellular concentration of calcium and reactive oxygen species (ROS). The peptide kissper was highly effective in preventing the increase of LPS-induced ROS levels in both the Caco-2 cells and CD colonic mucosa. Moreover, it controls the calcium increase, p65-nuclear factor (NF)-kB induction and transglutaminase 2 (TG2) activation inflammatory response in Caco-2 cells and CD colonic mucosa. Kissper efficiently counteracts the oxidative stress and inflammatory response in valuable model systems consisting of intestinal cells and CD colonic mucosa. This study reports the first evidence supporting a possible correlation between some beneficial effects of kiwi fruit and a specific protein molecule rather than generic nutrients.

Models for institutional and professional accreditation of haemophilia centres in Italy.

The Health Commission of the Conference between the Italian State and Regions recognized the need to establish an institutional accreditation model for Haemophilia Centres (HCs) to be implemented by 21 Regions in order to provide patients with haemophilia and allied inherited coagulations disorders with high and uniform standards of care. The Italian National Blood Centre, on behalf of the Commission, convened a panel of clinicians, patients, experts, representatives from Regions and Ministry of Health. The agreed methodology included: systematic literature review and best practice collection, analysis of provisions and regulations of currently available services, priority setting, definition of principles and criteria for the development of recommendations on the optimal requirements for HCs. The result was the formulation of two recommendations sets. Two sets of recommendations were produced. The first concerns regional policy planning, in which the following aspects of comprehensive haemophilia care should be considered for implementation: monitoring and auditing, multidisciplinary approach to clinical care, protocols for emergency management, home treatment and its monitoring, patient registries, drug availability and procurement, recruitment and training of health care professionals. The second set concerns the accreditation process and lists 23 organizational requirements for level 1 HCs and 4 additional requirements for level 2 HCs. These recommendations help to provide Italian Regional Health Authorities with an organizational framework for the provision of comprehensive care to patients with inherited coagulation disorders based on current scientific evidence.

Peamaclein--a new peach allergenic protein: similarities, differences and misleading features compared to Pru p 3.

BACKGROUND: Among the peach-derived allergens which are already known, the lipid transfer protein (Pru p 3) seems to be the one to exert severe allergic reactions. OBJECTIVE: To identify and characterize a new peach allergen causing a clinical picture similar to that of Pru p 3. METHODS: Patients were selected on the basis of their severe clinical reactivity and negative results to a panel of peach allergens available on the ISAC103 microarray. Several in-house and commercial preparations were compared. Several methods were used to characterize the newly identified molecule. Specific IgE and inhibition assays were performed using the Allergen micro-Beads Array (ABA) assay. RESULTS: Negative ISAC results to Pru p 3 were confirmed by additional testing in contrast with the positive results obtained by commercial Pru p 3-enriched peach peel extracts. The analyses of one of these preparations led to the identification of Peamaclein, a new allergenic protein. It is a small, basic, cysteine-rich, heat-stable, digestion-resistant protein, homologous to a potato antimicrobial peptide. Peamaclein was able to trigger positive skin test reactions and to bind IgE in the ABA assay. It displays an electrophoretic mobility and chromatographic behaviour similar to that of Pru p 3; therefore, it can be hidden in Pru p 3 preparations. In fact, Pru p 3-enriched peach peel extracts were found to contain both Pru p 3 and Peamaclein by means of comparative in vivo testing, and by biochemical and immunochemical assays. Commercially available anti-Pru p 3 polyclonal antibodies were found to have a double specificity for the two molecules. CONCLUSIONS AND CLINICAL RELEVANCE: A new allergen from peach belonging to a new family of allergenic proteins has been identified and characterized. This knowledge on Peamaclein will improve our understanding on the clinical aspects of the peach allergy and the quality of diagnostic reagents.

Physico-chemical features of the environment affect the protein conformation and the immunoglobulin E reactivity of kiwellin (Act d 5).

BACKGROUND: Allergy diagnostic systems sometimes give false positive or negative results. In this respect, the influence of protein conformational changes on the allergen-IgE interaction sites is worthy to be investigated. OBJECTIVE: To investigate the influence of different experimental conditions on the structural properties and IgE reactivity of kiwellin (Act d 5) as a model system. METHODS: Act d 5 was purified from the natural source. To study its conformational features, experiments of circular dichroism (CD) in different media were performed. The IgE reactivity was investigated by skin testing, immunoblotting and ISAC microarray system, in a population of kiwifruit allergic subjects. RESULTS: CD experiments indicated that Act d 5 has a mainly helical structure and the conformation is strongly affected by the experimental conditions. The protein is more structured in low polarity media and at acidic pH values, similar to those of the natural source. Eleven subjects of 29 (38%) allergic to kiwifruit were positive to purified natural Act d 5 by skin test. Among them, three patients (10%) showed a reaction only to Act d 5 at pH 4.5, and three (10%) showed a reaction only to the allergen in standard neutral conditions. No one of the 11 subjects with positive skin test recognized Act d 5 immobilized on the ISAC system. Eight of nine subjects detected Act d 5 by IgE immunoblotting. One subject did not recognize the sequence epitopes of Act d 5 in IgE immunoblotting experiments and reacted to the skin test only when the allergen was in acidic conditions. CONCLUSIONS AND CLINICAL RELEVANCE: The conformation and IgE reactivity of Act d 5 are affected by the physico-chemical characteristics of the solvent. These findings suggest that the assay conditions influence the results of the diagnostic systems by modulating the pattern of exposed antigenic epitopes.

Biochemical, immunological and clinical characterization of a cross-reactive nonspecific lipid transfer protein 1 from mulberry.

BACKGROUND: Mulberry (Morus spp.) is a genus comprising several species of deciduous trees whose fruits are commonly eaten in southern Europe. Subjects with severe systemic reaction have been described. The aim of this study was to isolate the allergens of this species. METHODS: A nonspecific lipid transfer protein 1 (ns-LTP1) was purified from black mulberry by ion exchange and reverse phase high-performance liquid chromatography, and the primary structure was elucidated by direct protein sequencing. Its allergenic activity was evaluated in vivo by skin prick test and in vitro by Western Blot, CD203c basophil activation assay and high throughput multiplex inhibition method on immunosolid-phase allergen chip (ISAC). RESULTS: Mulberry ns-LTP (Mor n 3) comprises 91 amino acids producing a molecular mass of 9246 Da. This protein shows high sequence identity with several allergenic ns-LTP1. Immunoblot analysis and CD203c activation assay demonstrated its allergenic activity in symptomatic subjects and in ns-LTP allergic patients who are not mulberry consumers. Immunological co-recognition was studied in vivo on a selected group of well-characterized ns-LTP allergic patients showing a high percentage of nMor n 3(+) subjects (88.46%) even in patients who have never eaten mulberry before. IgE inhibition on ISAC micro-array demonstrated an almost complete cross-reactivity to nArt v 3, rCor a 8 and a very high percentage of inhibition to nPru p 3. CONCLUSIONS: Mor n 3 is the first allergen isolated in black mulberry and immunologically characterized. It displayed allergenic activity among symptomatic and nonconsumer patients and a pattern of cross-reactivity to other plant-derived LTPs.

Pectin methylesterase inhibitor.

Pectin methylesterase (PME) is the first enzyme acting on pectin, a major component of plant cell wall. PME action produces pectin with different structural and functional properties, having an important role in plant physiology. Regulation of plant PME activity is obtained by the differential expression of several isoforms in different tissues and developmental stages and by subtle modifications of cell wall local pH. Inhibitory activities from various plant sources have also been reported. A proteinaceous inhibitor of PME (PMEI) has been purified from kiwi fruit. The kiwi PMEI is active against plant PMEs, forming a 1:1 non-covalent complex. The polypeptide chain comprises 152 amino acid residues and contains five Cys residues, four of which are connected by disulfide bridges, first to second and third to fourth. The sequence shows significant similarity with the N-terminal pro-peptides of plant PME, and with plant invertase inhibitors. In particular, the four Cys residues involved in disulfide bridges are conserved. On the basis of amino acid sequence similarity and Cys residues conservation, a large protein family including PMEI, invertase inhibitors and related proteins of unknown function has been identified. The presence of at least two sequences in the Arabidopsis genome having high similarity with kiwi PMEI suggests the ubiquitous presence of this inhibitor. PMEI has an interest in food industry as inhibitor of endogenous PME, responsible for phase separation and cloud loss in fruit juice manufacturing. Affinity chromatography on resin-bound PMEI can also be used to concentrate and detect residual PME activity in fruit and vegetable products.

Molecular dynamics analysis of a second phosphate site in the hemoglobins of the seabird, south polar skua. Is there a site-site migratory mechanism along the central cavity?

Hemoglobin function is modulated by several non-heme ligands; among these effectors, organic phosphates generally bind to heterotropic sites with a one-to-one stoichiometry. The phosphate binding site of human hemoglobin is located at the interface between the two beta chains. An additional binding site for polyanions has been studied at the molecular level (Tamburrini, M., A. Riccio, M. Romano, B. Giardina, and G. di Prisco. 2000. Eur. J. Biochem. 267:6089-6098) in the hemoglobins of the south polar skua (Catharacta maccormicki). It is formed by a cluster of six positive charges of both alpha chains (Val-1, Lys-99, Arg-141); the two Lys-99alpha have an essential role in the site structure. The present investigation, carried out on skua deoxyhemoglobins by using a molecular dynamics approach, confirms the structural feasibility of the additional site, possibly having the role of an entry-leaving site, and leads to the proposal of a novel migration pathway for phosphate along the central cavity of hemoglobin from one binding site to the other, occurring according to the hypothesis of a site-site migratory mechanism, which may assign a functional role to the central cavity. The role of Lys-99alpha was further confirmed by molecular dynamics experiments on the mutant Lys-99alpha-->Ala in which, at the end of the simulation, the phosphate was external to the additional site.

The hemoglobin system of the brown moray Gymnothorax unicolor: structure/function relationships.

The Gymnothorax unicolor hemoglobin system is characterized by two components, called cathodic and anodic on the basis of their isoelectric point, which were separated by ion-exchange chromatography. The oxygen-binding properties of the purified components were studied in the absence and presence of chloride and/or GTP or ATP in the pH range 6.5-8.0. Stripped cathodic hemoglobin showed a small reverse Bohr effect, high oxygen affinity, and low co-operativity; the addition of chloride only caused a small decrease in oxygen affinity. In the presence of GTP or ATP, the oxygen affinity was dramatically reduced, the co-operativity increased, and the reverse Bohr effect abolished. Stripped anodic hemoglobin is characterized by both low oxygen affinity and co-operativity, and displayed a normal Bohr effect; the addition of chloride increased co-operativity, whereas ATP and GTP significantly modulated oxygen affinity at acidic pH values, enhancing the Bohr effect and giving rise to the Root effect. The complete amino-acid sequences of the alpha and beta chains of both hemoglobins were established; the molecular basis of the functional properties of the hemoglobins is discussed in the light of the primary structure and compared with those of other fish hemoglobins.

Structural and functional analysis of the two haemoglobins of the antarctic seabird Catharacta maccormicki characterization of an additional phosphate binding site by molecular modelling.

The amino-acid sequence and the oxygen-binding properties of the two haemoglobins of the Antarctic seabird south polar skua have been investigated. The two haemoglobins showed peculiar functional features, which were probably acquired to meet special needs in relation to the extreme environmental conditions. Both haemoglobins showed a weak alkaline Bohr effect which, during prolonged flight, may protect against sudden and uncontrolled stripping of oxygen in response to acidosis. We suggest that a weak Bohr effect in birds may reflect adaptation to extreme life conditions. The values of heat of oxygenation suggest different functional roles of the two haemoglobins. The experimental evidence suggests that both haemoglobins may bind phosphate at two distinct binding sites. In fact, analysis of the molecular models revealed that an additional phosphate binding site, formed by residues NA1alpha, G6alpha and HC3alpha, is located between the two alpha chains. This additional site may act as an entry/leaving site, thus increasing the probability of capturing phosphate and transferring it to the main binding site located between the two beta chains by means of a site-site migratory mechanism, thereby favouring the release of oxygen. It is suggested that most haemoglobins possess an additional phosphate binding site, having such a role in oxygen transport.

Soliton electro-optic effects in paraelectrics.

The combination of charge separation induced by the formation of a single photorefractive screening soliton and an applied external bias field in a paraelectric is shown to lead to a family of useful electro-optic guiding patterns and properties.

The myoglobin of Emperor penguin (Aptenodytes forsteri): amino acid sequence and functional adaptation to extreme conditions.

In the framework of a study on molecular adaptations of the oxygen-transport and storage systems to extreme conditions in Antarctic marine organisms, we have investigated the structure/function relationship in Emperor penguin (Aptenodytes forsteri) myoglobin, in search of correlation with the bird life style. In contrast with previous reports, the revised amino acid sequence contains one additional residue and 15 differences. The oxygen-binding parameters seem well adapted to the diving behaviour of the penguin and to the environmental conditions of the Antarctic habitat. Addition of lactate has no major effect on myoglobin oxygenation over a large temperature range. Therefore, metabolic acidosis does not impair myoglobin function under conditions of prolonged physical effort, such as diving.

The hemoglobins of the antarctic fishes Atedidraco orianae and Pogonophryne scotti. Amino acid sequence, lack of cooperativity, and ligand binding properties.

The oxygen-transport system of two species of Antarctic fishes belonging to the family Artedidraconidae, Artedidraco orianae and Pogonophryne scotti, was thoroughly investigated. The complete amino acid sequence of the alpha and beta chains of the single hemoglobins of the two species was established. The oxygen-binding properties were also investigated, and were found not to differ significantly from those shown by blood, intact erythrocytes, and unstripped hemolysates. Both hemoglobins have unusually high oxygen affinity and display a relatively small Bohr effect; the Root effect is elicited only by organophosphates and is also reduced. Remarkably, the Hill coefficient is close to one in the whole pH range, indicating absence of cooperative oxygen binding which, in A. orianae hemoglobin, could be ascribed to the subunit heterogeneity shown upon oxygen dissociation. In comparison with the other families of the suborder Notothenioidei, the oxygen-transport system of these two species of Artedidraconidae has unique characteristics, which raise interesting questions on the mode of function of a multisubunit molecule and the relationship with cold adaptation.

One-dimensional steady-state photorefractive spatial solitons in centrosymmetric paraelectric potassium lithium tantalate niobate.

We report the first observation of spatial one-dimensional photorefractive screening solitons in centrosymmetric media and compare the experimental results with recent theoretical predictions. We find good qualitative agreement with theory.

Molecular evolution of hemoglobins of Antarctic fishes (Notothenioidei).

Amino acid sequences of alpha- and beta-chains of human hemoglobin and of hemoglobins of coelacanth and 24 teleost fish species, including 11 antarctic and two temperate Notothenioidei, were analyzed using maximum parsimony. Trees were derived for the alpha- and beta-chains separately and for tandemly arranged sequences, using the human and coelacanth sequences as outgroups in all analyses. The topologies of the trees of the alpha- and beta-chains are highly congruent and indicate a specific pattern of gene duplications and gene expression of teleost hemoglobins which has not yet been investigated into more detail. The Notothenioid fish generally contain a single major hemoglobin and often a second minor component. The alpha- and beta-chains of the major components form a monophyletic group in all investigated trees, with the nonantarctic Pseudaphritis as their sister taxon. The minor chains also are a monophyletic group and form an unresolved cluster with the major chains and the hemoglobins of tuna and red gurnard. The Notothenioid families Nototheniidae and Bathydraconidae appear to be paraphyletic.

The hemoglobin system of Pleuragramma antarcticum: correlation of hematological and biochemical adaptations with life style.

The hematological properties and the oxygen-transport system of the antarctic fish Pleuragramma antarcticum were investigated. Most blood parameters are at the lower end of the range of values known for red-blooded antarctic fish, suggesting a link with the sluggish mode of life of this species. P. antarcticum is the only species of the family Nototheniidae and of the suborder Notothenioidei having three major hemoglobins, which were isolated and fully characterized. The complete amino acid sequence of the alpha- and beta-globin chains was determined. The three hemoglobins showed strong Bohr and Root effects, and their oxygen-binding properties were differently regulated by temperature. None of the three hemoglobins of P. antarcticum can be considered as evolutionary (or larval) remnants. Therefore, this oxygen-transport system is one of the most specialized ever found in fish. The data suggest a strong relationship between hematological/biochemical adaptation and life style.

The unique hemoglobin system of Pleuragramma antarcticum, an antarctic migratory teleost. Structure and function of the three components.

Pleuragramma antarcticum (suborder Notothenioidei, family Nototheniidae) is the most abundant fish in the antarctic shelf. This pelagic species has a circum-antarctic distribution and is characterized by spawning migration. This species displays the highest multiplicity of major hemoglobins (three); the other notothenioids have a single one (except one species, having two) with relatively low oxygen affinity regulated by pH and organophosphates. The hemoglobins of P. antarcticum display strong Bohr and Root effects; however, they reveal important functional differences in subunit cooperativity and organophosphate regulation and, above all, in the response of oxygenation to temperature. Despite the substitution ValbetaE11 --> Ile found in Hb 2, which decreases the affinity in human mutants, the hemoglobins have similar oxygen affinity, higher than that of the other notothenioids. Hb 1 has the alpha chain in common with Hb 2 and the beta in common with Hb 3. The amino acid sequence of all four chains has been established. Thus the hematological features of P. antarcticum differ remarkably from those of antarctic notothenioids. This unique and sophisticated oxygen transport system may adequately meet the requirements of the unusual mode of life of this fish.

Structure/function relationships in the hemoglobin components from moray (Muraena helena).

Concerning the number and type of the hemoglobin components, the moray Muraena helena is characterized by three different phenotypes whose frequencies are nearly identical. Thus, the cathodal component is present in all individuals, whereas one or both of two anodal components may be present in the same phenotype. These components have been separated by chromatography. The oxygen binding properties of the purified hemoglobin components have been studied in the absence and presence of saturating concentrations of ATP or GTP and as a function of pH. The cathodal component shows an intrinsic O2 affinity four times higher than that of both anodal components, a very small Bohr effect and a significant decrease in O2 affinity upon addition of ATP and GTP (three and four times respectively with respect to stripped conditions), the latter being more effective than the former over the entire pH range examined. The anodal components do not appear functionally distinguishable and show the presence of an enhanced Bohr effect (Root effect) that is under the strict control of nucleotide triphosphates ATP, GTP, which, unlike in the cathodic component, exert the same effect on oxygen affinity. The complete sequence of the beta chains of the cathodal and of one of the anodal components have been determined. The possible molecular basis of these different functional characteristics are discussed in the light of the globin sequence and of those amino acid residues which are known to be responsible of hemoglobin functional behaviour.