Cytokine modulation in patients with idiopathic pulmonary fibrosis undergoing treatment with steroids, immunosuppressants, and IFN-γ 1b.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease of unknown etiology and pathogenic mechanisms. From an etiopathogenic point of view, alveolar macrophages play a key role in accumulation of fibroblasts and deposition of collagen and extracellular matrix by releasing specific cytokines and inflammatory mediators. IPF seems to be also associated with circulating fibrocytes, which might be involved with an abnormal pulmonary vascular repair and remodeling. Based on its hypothesized pathologic mechanisms, anti-inflammatory, anti-fibrotic and immunosuppressive therapies are often used. For these reasons, Interferon-g (IFN-g) has been used to exploit its activity on macrophages and fibroblasts. The aim of this study was to investigate the response to corticosteroids and/or IFN-g 1b treatments based on pulmonary function tests and on inflammatory cytokine patterns of expression on bronchoalveolar lavage (BAL), at baseline and during and after the therapies. Unlike previous studies, we analyzed a period of therapy longer than 1 year. Our results demonstrated the effectiveness of IFN-γ in a group of IPF patients in whom the treatment was prolonged for over a year. These data suggest a positive role of IFN-γ; treatment in patients in the initial stage of the disease.

Absorption correction and phase function shape effects on the closure of apparent optical properties.

We present a closure experiment between new inherent optical properties (IOPs: absorption a, scattering b, backscattering bb) and apparent optical properties (AOPs: remote-sensing reflectance Rrs, irradiance reflectance R, and anisotropic factor at nadir Qn) data of Ionian and Adriatic seawaters, from very clear to turbid waters, ranging across one order of magnitude in Rrs. The internal consistency of the IOP-AOP matchups was investigated though radiative transfer closure. Using the in situ IOPs, we predicted the AOPs with the commercial radiative transfer solver Hydrolight. Closure was limited by two unresolved issues, one regarding processing of in situ data and the other related to radiative transfer modeling. First, different correction methods of the absorption data measured by the Wetlabs ac-s produced high variations in simulated reflectances, reaching 40% for the highest reflectances in our dataset. Second, the lack of detailed volume scattering function measurements forces us to adopt analytical functions that are consistent with a given particle backscattering ratio. The analytical phase functions named Fournier-Forand and two-term Kopelevich presented reasonable angular shapes with respect to measurements at a few backward angles. Between these phase functions, induced changes were within 4% for Rrs, within 11% for R, and within 10% for Qn. Additionally, closure of Qn was generally not successful considering radiometric uncertainties. Simulated Qn overestimated low values and underestimated high values, especially at 665 nm, where Hydrolight was unable to predict measured Qn values greater than 6 sr. The physical nature of Qn makes this mismatch almost independent of the measured IOPs, thus precluding Qn tuning by varying the former. The non-closure of Qn might be caused by an inaccurate phase function and, to a lesser extent, by the modeling of the incoming radiance. For the future, this remains the task of accurate absorption and phase function measurements, especially at red wavelengths.

Evidence of Covid-19 lockdown effects on riverine dissolved organic matter dynamics provides a proof-of-concept for needed regulations of anthropogenic emissions.

The fast spread of SARS-CoV-2 virus in Italy resulted in a 3-months lockdown of the entire country. During this period, the effect of the relieved anthropogenic activities on the environment was plainly clear all over the country. Herein, we provide the first evidence of the lockdown effects on riverine dissolved organic matter (DOM) dynamics. The strong reduction in anthropogenic activities resulted in a marked decrease in dissolved organic carbon (DOC) concentration in the Arno River (-44%) and the coastal area affected by its input (-15%), compared to previous conditions. The DOM optical properties (absorption and fluorescence) showed a change in its quality, with a shift toward smaller and less aromatic molecules during the lockdown. The reduced human activity and the consequent change in DOM dynamics affected the abundance and annual dynamics of heterotrophic prokaryotes. The results of this study highlight the extent to which DOM dynamics in small rivers is affected by secondary and tertiary human activities as well as the quite short time scales to return to the impacted conditions. Our work also supports the importance of long-term research to disentangle the effects of casual events from the natural variability.

Genomic insight into the amino acid relations of the pea aphid, Acyrthosiphon pisum, with its symbiotic bacterium Buchnera aphidicola.

The pea aphid genome includes 66 genes contributing to amino acid biosynthesis and 93 genes to amino acid degradation. In several respects, the pea aphid gene inventory complements that of its symbiotic bacterium, Buchnera aphidicola (Buchnera APS). Unlike other insects with completely sequenced genomes, the pea aphid lacks the capacity to synthesize arginine, which is produced by Buchnera APS. However, consistent with other insects, it has genes coding for individual reactions in essential amino acid biosynthesis, including threonine dehydratase and branched-chain amino acid aminotransferase, which are not coded in the Buchnera APS genome. Overall the genome data suggest that the biosynthesis of certain essential amino acids is shared between the pea aphid and Buchnera APS, providing the opportunity for precise aphid control over Buchnera metabolism.

Fibronectin and vitronectin regulate the organization of their respective Arg-Gly-Asp adhesion receptors in cultured human endothelial cells.

Human umbilical vein endothelial cells (ECs) adhere in vitro to proteins of the extracellular matrix including fibronectin (fn) and vitronectin (vn). Specific receptors for fn and vn have been previously characterized. These receptors belong to a family of membrane glycoproteins characterized (a) by being a transmembrane complex of two noncovalently linked subunits and (b) by recognizing the tripeptide Arg-Gly-Asp on their respective ligands. In this paper we investigated how vn and fn control the organization of their respective receptors over the surface of ECs. It was found that the clustering of individual receptors and the organization thereafter of focal contacts occurred only when ECs were exposed to the specific ligand and did not occur on the opposite ligand. The shape of receptor clusters was slightly different and a colocalization of the two receptors was found when ECs were cultured on a mixed matrix of fn plus vn. Adhesion was selectively inhibited by vn or fn receptor antibodies on their respective substrates. The clustering of both receptors preceded the association of vinculin with focal contacts and stress fiber formation. Also, the vn receptor, in the absence of associated fn receptor, was capable of inducing the organization of the membrane-microfilament interaction complex. Overall, these results indicate that individual matrix ligands induce only the clustering of their respective membrane receptors. The clustering of only one receptor is capable of supporting the subsequent formation of focal contacts and the local assembly of related cytoskeletal proteins.

Fibrinogen induces adhesion, spreading, and microfilament organization of human endothelial cells in vitro.

Human umbilical vein endothelial cells (ECs) have been shown to attach to a substratum of fibrinogen (fg). Later, ECs undergo spreading, organization of thick microfilament bundles of the stress fiber type, and formation of focal contacts (adhesion plaques) that correspond to accumulation of vinculin at the cytoplasmic aspect of the ventral membrane. The rate of attachment to fg and the type of spreading is virtually identical to that obtained on substrata coated with fibronectin (FN). Antibodies to fg, but not to FN, prevent EC adhesion to fg; conversely, antibodies to FN, but not to fg, prevent adhesion of ECs to a FN-coated substratum. The removal of residual FN contamination from fg preparations by means of DEAE-cellulose chromatography does not result in any difference in EC adhesion on fg. Moreover, pretreatment of cells with inhibitors of synthesis and release of proteins does not impair their adhesion capacity on an fg-coated substratum. In contrast, human arterial smooth muscle cells do not adhere and spread on fg substrata but do so on FN. The synthetic peptides (Gly-Arg-Gly-Asp[GRGD] and Gly-Arg-Gly-Asp-Ser-Pro[GRGDSP]) containing the tripeptide Arg-Gly-Asp (RGD), originally found to be responsible for the cell binding activity of FN, have been found to inhibit EC spreading and the redistribution of their cytoskeleton, including the formation of stress fibers and the localization of vinculin either on fg or on FN. Conversely, the synthetic peptide Arg-Gly-Gly (RGG) was completely uneffective in inhibiting the adhesion and the sequence of events leading to spreading and cytoskeletal organization. These results indicate that ECs, but not smooth muscle cells, specifically adhere and spread on an fg substratum and this occurs by recognition mechanisms similar to those reported for FN.

Elucidating the effect of the lead iodide complexation degree behind the morphology and performance of perovskite solar cells.

The inclusion of iodide additives in hybrid perovskite precursor solutions has been successfully exploited to improve the solar cell efficiency but their impact on perovskite formation, morphology and photovoltaic performance is still not clear. Here an extensive analysis of the effect of iodide additives in the solution-phase and during the perovskite film formation, as well as their effect on device performance is provided. The results demonstrate that in the solution-phase the additives promote the formation of lead poly-iodide species resulting in the disaggregation of the inorganic lead iodide framework and in the formation of smaller nuclei inducing the growth of uniform and smooth perovskite films. Most importantly, the complexation capability of different iodide additives does not only directly affect film morphology but also influences the density of defect states by varying the stoichiometry of precursors. These findings demonstrate that the fine control of the interactions of the chemical species in the solution-phase is essential for the precise control of the morphology at the nanoscale and the growth of the perovskite films with a reduced density of defect states. Therefore, the in-depth understanding of all the processes involved in the solution-phase is the first step for the development of a facile and reproducible approach for the fabrication of hybrid perovskite solar cells with enhanced photovoltaic performance.

An unusual thoracic localizations of Erdheim- Chester disease: A case report.

Erdheim- Chester disease is a rare non- Langerhans cell histiocytosis that usually involves the bones, heart, central nervous system, retroperitoneum, eyes, kidneys, skin and adrenals. Lungs are affected in up to one-half cases; at CT scan various patterns are described: interstitial disease, consolidations, micronodules and microcysts, with or without pleural involvement. We presented a case of a 59 year-old man with unusual intrathoracic manifestation of Erdheim- Chester disease. Singularities of our report are the lonely thoracic involvement at the onset of the disease and a histiocytic lesion in the posterior mediastinum.

Gene expression profiling of low-grade diffuse astrocytomas by cDNA arrays.

Diffuse astrocytoma WHO grade II is a well-differentiated, slowly growing tumor that has an inherent tendency to progress to anaplastic astrocytoma (WHO grade III) and, eventually, to glioblastoma (WHO grade IV). Little is known about its molecular basis, except for p53 mutations that are found in >60% of cases. In a search for additional genetic alterations, we carried out gene expression profiling of 11 diffuse astrocytomas using cDNA expression arrays. Expression of six genes (TIMP3, c-myc, EGFR, DR-nm23, nm23-H4, and GDNPF) was detected in 64-100% of diffuse astrocytomas, but not in nontumorous brain tissue. Seven genes (AAD14, SPARC, LRP, PDGFR-alpha, 60S ribosomal protein L5, PTN, and hBAP) were found to be up-regulated more than 2-fold in 20-60% of cases, whereas 11 genes (IFI 9-27, protein kinase CLK, TDGF1, BIN1, GAB1, TYRO3, LDH-A, adducin 3, GUK1, CDC10, and KRT8) were down-regulated to less than 50% of normal levels in 64-100% of cases. Semiquantitative conventional reverse transcription-PCR was performed for 11 genes, 9 of which showed an expression profile similar to that obtained with cDNA expression arrays. Immunohistochemical staining for SPARC showed cytoplasmic immunoreactivity of neoplastic cells in all diffuse astrocytomas analyzed. These results indicate significant changes in gene expression in diffuse astrocytomas, but it remains to be shown which of these are causally related to the transformation of glial cells.

Endobronchial-ultrasound needle aspiration and endoscopic ultrasound-fine-needle aspiration in thoracic diseases.

EBUS-TBNA and EUS-FNA are minimally invasive techniques rapidly gaining ground in the non-surgical invasive diagnostic approach to thoracic diseases due to their high accuracy and low morbidity and mortality compared to surgical techniques. Moreover, in the diagnosis and staging of lung cancer the combination of the two techniques is superior to either test alone. In this review we focus on the role of EBUS-TBNA and EUS-FNA in both malignant and non-malignant thoracic diseases.

Prenatal diagnosis of 30 fetuses at risk for fragile X syndrome.

The results of 30 prenatal diagnoses for fragile X syndrome are reported. Amniotic fluid cells were examined in 1 case, fetal blood in 4, and chorionic villi samples in the others. Of the 5 fetuses analyzed by cytogenetic methods, 1 had showed 4% of fraXq27.3 expression sites and the pregnancy was terminated. For 1 diagnosis, linkage analysis was used: the female fetus turned out to be normal. In 24 fetuses, the direct analysis of the mutation by StB12.3 probe was performed: 6 female and 3 male fetuses were found to carry a full mutation and 1 female fetus was found to carry a premutation. In 3 cases, the diagnoses were verified on fetal blood samples. Several tissues of 2 aborted male fetuses were analyzed for the fragile X mutation. The results are reported and discussed.

Osteoprotegerin and RANKL in the pathogenesis of osteoporosis in patients with thalassaemia major.

Osteoporosis represents an important cause of morbidity in thalassaemia major patients; the etiopathogenesis is multifactorial and includes expansion of the bone marrow, endocrine disorders, iron overload and genetic factors. Two cytokines, osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL), have recently been identified as important mediators in the pathogenesis of osteoporosis. In this study, the possible role of the OPG-RANKL system in the pathogenesis of osteoporosis in thalassemia major is assessed, as well as any correlations between the serum levels of OPG and RANKL and bone mineral density (BMD), 17 beta-estradiol and free testosterone and the relationship between T-score of BMD and OPG/RANKL ratio. In 31 thalassaemia major patients and a control group, the serum values of OPG and RANKL were assayed and correlated with BMD, as well as with the sex hormones values. All the thalassemic patients had reduced BMD and 35.5% presented osteoporosis. The thalassemic patients had significantly higher serum levels of OPG than the controls, while their higher RANKL levels, were at the threshold of significance. The OPG/RANKL ratio showed higher level respect to the controls. No statistically significant correlation was observed between the T-score and RANKL neither between the T-score and OPG nor between T-score and OPG/RANKL ratio. Instead, a statistically significant correlation was found between the T-score and free testosterone and between the T-score and 17 beta-estradiol. There was no correlation between the sex hormones and OPG and RANKL. The increased OPG values in thalassemic patients could be considered to compensate the increased bone turnover. The authors confirm hypogonadism as a primary etiopathogenetic factor in the reduced BMD observed in thalassaemia major patients.

LRRTM1 on chromosome 2p12 is a maternally suppressed gene that is associated paternally with handedness and schizophrenia.

Left-right asymmetrical brain function underlies much of human cognition, behavior and emotion. Abnormalities of cerebral asymmetry are associated with schizophrenia and other neuropsychiatric disorders. The molecular, developmental and evolutionary origins of human brain asymmetry are unknown. We found significant association of a haplotype upstream of the gene LRRTM1 (Leucine-rich repeat transmembrane neuronal 1) with a quantitative measure of human handedness in a set of dyslexic siblings, when the haplotype was inherited paternally (P=0.00002). While we were unable to find this effect in an epidemiological set of twin-based sibships, we did find that the same haplotype is overtransmitted paternally to individuals with schizophrenia/schizoaffective disorder in a study of 1002 affected families (P=0.0014). We then found direct confirmatory evidence that LRRTM1 is an imprinted gene in humans that shows a variable pattern of maternal downregulation. We also showed that LRRTM1 is expressed during the development of specific forebrain structures, and thus could influence neuronal differentiation and connectivity. This is the first potential genetic influence on human handedness to be identified, and the first putative genetic effect on variability in human brain asymmetry. LRRTM1 is a candidate gene for involvement in several common neurodevelopmental disorders, and may have played a role in human cognitive and behavioral evolution.

Chemotactic activity of human recombinant granulocyte-macrophage colony-stimulating factor.

Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) seeded in the lower compartment of chemotaxis chambers induced migration of human peripheral blood monocytes and polymorphonuclear phagocytes. rhGM-CSF was active in inducing phagocyte migration at concentrations as low as 10 CML units/ml (corresponding to 0.002 microgram), and maximal activity was observed at 10(2)-10(4)/ml. Checkerboard analysis performed by seeding different concentrations of rhGM-CSF above and below the filter revealed that maximal induction of migration required a positive concentration gradient between the lower and upper compartment, and that rhGM-CSF elicited an actual chemotactic response in phagocytes. Human umbilical vein endothelial cells and blood large granular lymphocytes (LGL) responded poorly or not at all (endothelial cells) to rhGM-CSF under conditions in which appropriate reference chemoattractants were active. The chemotactic activity of rhGM-CSF may serve to recruit phagocytic cells from the blood compartment to amplify resistance against noxious agents.

Identical mutations in the CSB gene associated with either Cockayne syndrome or the DeSanctis-cacchione variant of xeroderma pigmentosum.

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are two hereditary disorders in which photosensitivity is associated with distinct clinical and cellular phenotypes and results from genetically different defects. We have identified the primary molecular alteration in two patients in whom clinical manifestations strongly reminiscent of a severe form of XP were unexpectedly associated with the CS cellular phenotype and with a defect in the CSB gene. Sequencing of the CSB -coding region in both cDNA and genomic DNA showed that these patients had identical alterations to those in a patient with the clinical features of the classical form of CS. These data, together with fluorescence in situ hybridization analysis, demonstrated that the two siblings with XP as well as the CS patient were homozygous for the same CSB mutated allele, containing a silent C2830T change and a nonsense mutation C2282T converting Arg735 to a stop codon. The finding that the same inactivating mutation underlies different pathological phenotypes indicates that there is no simple correlation between the molecular defect and the clinical features. Therefore, alterations in the CSB gene give rise to the same repair defect at the cellular level but other genetic and/or environmental factors determine the pathological phenotype.

Chemotactic activity of recombinant human granulocyte colony-stimulating factor.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) induced migration across polycarbonate filters of human polymorphonuclear leukocytes (PMN). rhG-CSF was active in inducing PMN migration at concentrations greater than or equal to 10 to 100 U/mL (7 to 70 ng/mL). rhG-CSF did not contain appreciable levels of endotoxin contamination as assessed by Limulus amebocyte assay, and Polymixin B did not affect the chemotactic activity of rhG-CSF. A monoclonal anti-G-CSF antibody blocked the induction of migration by G-CSF, thus establishing that the cytokine was responsible for the activity of the recombinant preparation. Checkerboard analysis was performed by seeding different concentrations of G-CSF above and/or below the filter and revealed that the migratory response to this cytokine was best observed in the presence of a positive concentration gradient between the lower and upper compartments of the chamber, thus indicating an actual chemotactic effect. When different migrating cells were examined, rhG-CSF was inactive on large granular lymphocytes and endothelial cells under conditions in which appropriate reference attractants were active. In contrast, rhG-CSF elicited a chemotactic response in monocytes inhibited by specific antibody. Thus, G-CSF is a chemotactic signal for phagocytes. This cytokine, when produced at inflammatory sites, may contribute to the recruitment of phagocytes from the blood compartment to amplify resistance against certain noxious agents.

The localization of a platelet GpIIb-IIIa-related protein in endothelial cell adhesion structures.

Previous studies have shown that human endothelial cells (ECs) adhere to fibrinogen (fg) and fibronectin (fn) and organize their cytoskeleton on these substrata. However, the mechanism governing this chain of events is poorly known. In ECs glycoproteins immunologically and biochemically similar to the platelet membrane GpIIb-IIIa complex have been described. The functional role of this complex in ECs remains to be established. In this study we show that the antigens recognized by polyclonal antibodies raised against human platelet GpIIb-IIIa and crossreacting with the EC form have a discrete and well-organized distribution at cell adhesion structures. Indeed these antigens are located at vinculin-rich focal contacts found at the membrane insertion of microfilament bundles of the stress fiber type. They are also found at cell-to-cell contacts and, with a diffuse pattern, at the dorsal surface of ECs. GpIIb-IIIa antibodies, added to EC suspensions prior to plating, inhibit EC spreading on fg and vitronectin (vn) substrata in a concentration-dependent way. In contrast, the antibodies are very poorly active when the cells are seeded on fn-coated glass. The same antibodies, added to adherent cells, disrupt cell-to-cell contacts and cause their rounding and detachment. Overall these results indicate that EC GpIIb-IIIa complex is involved in controlling the adhesion mechanism of these cells to extracellular matrix proteins.

Reduced expression of the Aalpha subunit of protein phosphatase 2A in human gliomas in the absence of mutations in the Aalpha and Abeta subunit genes.

Protein phosphatase 2A (PP2A) consists of 3 subunits: the catalytic subunit, C, and the regulatory subunits, A and B. The A and C subunits both exist as 2 isoforms (alpha and beta) and the B subunit as multiple forms subdivided into 3 families, B, B' and B". It has been reported that the genes encoding the Aalpha and Abeta subunits are mutated in various human cancers, suggesting that they may function as tumor suppressors. We investigated whether Aalpha and Abeta mutations occur in human gliomas. Using single strand conformational polymorphism analysis and DNA sequencing, 58 brain tumors were investigated, including 23 glioblastomas, 19 oligodendrogliomas and 16 anaplastic oligodendrogliomas. Only silent mutations were detected in the Aalpha gene and no mutations in the Abeta gene. However, in 43% of the tumors, the level of Aalpha was reduced at least 10-fold. By comparison, the levels of the Balpha and Calpha subunits were mostly normal. Our data indicate that these tumors contain very low levels of core and holoenzyme and high amounts of unregulated catalytic C subunit.

Mutation analysis of hBUB1, hBUBR1 and hBUB3 genes in glioblastomas.

Glioblastomas, the most malignant human brain tumors, are characterized by marked aneuploidy, suggesting chromosomal instability which may be caused by a defective mitotic spindle checkpoint. We screened 22 glioblastomas for mutations in the mitotic spindle check-point genes hBUB1, hBUBR1 and hBUB3. DNA sequencing revealed a silent mutation at codon 144 of hBUB1 (CAG-->CAA, Gln-->Gln) in one glioblastoma, a silent mutation at codon 952 of hBUBR1 (GAC-->GAT, Asp-->Asp) in another glioblastoma, and a silent mutation at codon 388 of the hBUBR1 gene (GCG-->GCA, Ala-->Ala) in 8 glioblastomas. We also observed a known polymorphism at hBUBR1 codon 349 (CAA/CGA, Gln/Arg), with an allelic frequency of 0.75 for Gln and 0.25 for Arg, which is similar to that among healthy Caucasian individuals (0.73 vs 0.27). The coding sequence of the hBUB3 gene did not contain any mutation, but in 4 glioblastomas (18%), a C-->T point mutation was detected at position -6 (6 nucleotides upstream of the ATG initiator codon). Analysis of blood DNA of these patients showed identical sequence alterations, indicating that this is a polymorphism. Again, the frequency in glioblastomas was similar to that in healthy Caucasians (15%). We further screened hBUB1 in 18 cases of giant cell glioblastoma, a variant characterized by a predominance of bizarre, multinucleated giant cells. There were no changes, except for a silent mutation at codon 144 in two cases. These results suggest that mutations in these mitotic spindle checkpoint genes do not play a significant role in the causation of chromosomal instability in glioblastomas.