PLA and PBAT-Based Electrospun Fibers Functionalized with Antibacterial Bio-Based Polymers.

Antimicrobial fibers based on biodegradable polymers, poly(lactic acid) (PLA), and poly(butylene adipate-co-terephthalate) (PBAT) are prepared by electrospinning. For this purpose, a biodegradable/bio-based polyitaconate containing azoles groups (PTTI) is incorporated at 10 wt.% into the electrospinning formulations. The resulting fibers functionalized with azole moieties are uniform and free of beads. Then, the accessible azole groups are subjected to N-alkylation, treatment that provides cationic azolium groups with antibacterial activity at the surface of fibers. The positive charge density, roughness, and wettability of the cationic fibers are evaluated and compared with flat films. It is confirmed that these parameters exert an important effect on the antimicrobial properties, as well as the length of the alkylating agent and the hydrophobicity of the matrix. The quaternized PLA/PTTI fibers exhibit the highest efficiency against the tested bacteria, yielding a 4-Log reduction against S. aureus and 1.7-Log against MRSA. Then, biocompatibility and bioactivity of the fibers are evaluated in terms of adhesion, morphology and viability of fibroblasts. The results show no cytotoxic effect of the samples, however, a cytostatic effect is appreciated, which is ascribed to the strong electrostatic interactions between the positive charge at the fiber surface and the negative charge of the cell membranes.

Chondroprotective activity of N-acetyl phenylalanine glucosamine derivative on knee joint structure and inflammation in a murine model of osteoarthritis.

OBJECTIVE: Osteoarthritis (OA), the most common chronic degenerative joint disease, is characterized by joint structure changes and inflammation, both mediated by the IκB kinase (IKK) signalosome complex. The ability of N-acetyl phenylalanine derivative (NAPA) to increase cartilage matrix components and to reduce inflammatory cytokines, inhibiting IKKα kinase activity, has been observed in vitro. The present study aims to further clarify the effect of NAPA in counteracting OA progression, in an in vivo mouse model after destabilization of the medial meniscus (DMM). DESIGN: 26 mice were divided into three groups: (1) DMM surgery without treatment; (2) DMM surgery treated after 2 weeks with one intra-articular injection of NAPA (2.5 mM) and (3) no DMM surgery. At the end of experimental times, both knee joints of the animals were analyzed through histology, histomorphometry, immunohistochemistry and microhardness of subchondral bone (SB) tests. RESULTS: The injection of NAPA significantly improved cartilage thickness (CT) and reduced Chambers and Mankin modified scores and fibrillation index (FI), with weaker MMP13, ADAMTS5, MMP10 and IKKα staining. The microhardness measurements did not shown statistically significant differences between the different groups. CONCLUSIONS: NAPA markedly improved the physical structure of articular cartilage while reducing catabolic enzymes, extracellular matrix (ECM) remodeling and IKKα expression, showing to be able to exert a chondroprotective activity in vivo.

Effect of glucosamine and its peptidyl-derivative on the production of extracellular matrix components by human primary chondrocytes.

OBJECTIVE: Aim of this study is to investigate the effects of Glucosamine (GlcN) and its peptidyl-derivative, 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-ß-D-glucose (NAPA), on extracellular matrix (ECM) synthesis in human primary chondrocytes (HPCs). METHODS: Dose-dependent effect of GlcN and NAPA on Glycosaminoglycan (GAG), Collagen type II (Col2) and Small Leucine-Rich Proteoglycans (SLRPs) was examined by incubating HPCs, cultured in micromasses (3D), with various amounts of two molecules, administered as either GlcN alone or NAPA alone or GlcN plus NAPA (G + N). Immunohystochemical and immunofluorescent staining and biochemical analysis were used to determine the impact of the two molecules on ECM production. Gene expression analysis was performed by TaqMan Real-Time Polymerase Chain Reaction (PCR) assays. RESULTS: The lowest concentration to which GlcN and NAPA were able to affect ECM synthesis was 1 mM. Both molecules administered alone and as G + N stimulated GAGs and SLRPs synthesis at different extent, NAPA and mainly G + N stimulated Col2 production, whereas GlcN was not effective. Both molecules were able to induce Insulin Growth Factor-I (IGF-I) and to stimulate SOX-9, whereas NAPA and G + N were able to up-regulate both Hyaluronic Acid Synthase-2 and Hyaluronic acid. Very interesting is the synergistic effect observed when chondrocyte micromasses were treated with G + N. CONCLUSIONS: The observed anabolic effects and optimal concentrations of GlcN and NAPA, in addition to beneficial effects on other cellular pathways, previously reported, such as the inhibition of IKKα, could be useful to formulate new cartilage repair strategies.

Glucosamine and its N-acetyl-phenylalanine derivative prevent TNF-alpha-induced transcriptional activation in human chondrocytes.

OBJECTIVES: Glucosamine (GlcN) is used in the treatment of osteoarthritis as symptomatic slow-acting drug, but its mode of action is not completely known. We analyzed the influence of GlcN and its N-acetyl-phenylalanine derivative (NAPA) on mRNA transcription level of TNF-alpha-stimulated genes in cell culture. METHODS: Human immortalized chondrocyte cell line lbpva55 was stimulated with TNF-alpha and treated with GlcN and NAPA. mRNA transcription level of several genes, identified by complementary DNA microarray (cDNA microarray), was validated by Quantitative Real-Time Polymerase Chain Reaction (Q-RT-PCR). RESULTS: Several genes, whose mRNA level was increased by TNF-alpha treatment and significantly reduced by GlcN and NAPA in lbpva55 cells, were identified. These include cytokine receptors TNF-R1 and TNF-R2, their associated factor TRAF-6, signaling intermediates IGFB-6 and Rnd1, as well as cell cycle regulating proteins CUL-2 and G1S protein 1. Down- regulation of mRNA expression level of some of these genes is in accordance with inactivation of NF-kB transcription factor. Moreover, we found down-regulation of c-jun mRNA level, a component of AP-1 transcription factor. CONCLUSIONS: Our study suggests that GlcN and NAPA interfere with activation of NF-kB and AP-1 transcription factors, which are responsible for the expression of genes involved in diverse biological processes, such as cell growth and death, inflammatory and stress responses, accounting for the beneficial effects of GlcN in osteoarthritis.

Activity of quinupristin-dalfopristin in invasive isolates of Streptococcus pneumoniae from Italy.

Eighty-five recent isolates of Streptococcus pneumoniae from patients with invasive disease were examined for their susceptibility to erythromycin, clindamycin, penicillin and quinupristin-dalfopristin by E test. A novel duplex PCR assay was used to detect the presence of the erm(B) or mef(A) genes in all of the erythromycin-resistant isolates. All of the strains tested were susceptible to the combination quinupristin-dalfopristin, regardless of their susceptibility to penicillin or to erythromycin. By duplex PCR, two-thirds of the erythromycin-resistant strains harbored erm, and one-third harbored mef. The activity of quinupristin-dalfopristin was not influenced by the genetic determinant of erythromycin resistance. The in vitro susceptibility of S. pneumoniae to quinupristin-dalfopristin is promising for future use; however, it is important to monitor the possible emergence of resistance.

Molecular and biochemical characterization of the recombinant amidase from hyperthermophilic archaeon Sulfolobus solfataricus.

We have cloned, sequenced, and overexpressed in Escherichia coli the amidase gene from the hyperthermophilic archaeon Sulfolobus solfataricus (strain MT4). The recombinant thermophilic protein was expressed as a fusion protein with an N-terminus six-histidine-residue affinity tag. The enzyme, the first characterized archaeal amidase, is a monomer of 55,784 daltons, enantioselective, and active on 2- to 6-carbon aliphatic amides and on many aromatic amides, over the pH range 4-9 and at temperatures from 60 degrees to 95 degrees C. The S. solfataricus amidase belongs to the class of amidases that share a characteristic signature, GGSS(S/ G)GS, located in the central region of the protein, and which show remarkable variability in their individual substrate specificities, can hydrolyze aliphatic or aromatic substrates, and share a large invariance of their primary structure.

Crystallization and X-ray diffraction measurements of a thermophilic archaeal recombinant amidase from Sulfolobus solfataricus MT4.

Recombinant amidase is a 55.8 kDa enzyme from the thermophilic archaeon Sulfolobus solfataricus MT4 that catalyses the hydrolysis of aliphatic amides of 2-6 C atoms as well as many aromatic amides. Single crystals of purified amidase were obtained by the hanging-drop method at 294 K. Diffraction data for the native protein (2.55 A resolution) and a putative derivative (2.20 A) have been collected at low temperature using synchrotron radiation. The crystals belong to the rhombohedral space group R3. Structure determination by multiple isomorphous replacement is in progress. It is expected that structural information from this signatured thermostable amidase will increase our knowledge of the molecular mechanisms employed to maintain high-temperature stability in thermophilic proteins.

Production of a mouse antiserum to Bacteroides fragilis enterotoxin using a recombinant enterotoxin precursor.

The precursor of the Bacteroides fragilis metalloprotease enterotoxin was cloned and expressed in Escherichia coli, which was not able to process the precursor into the biologically active enterotoxin. Mouse antiserum elicited to the recombinant precursor reacted with the purified enterotoxin and with a crude enterotoxin preparation from an enterotoxigenic strain. The antiserum neutralized the cytotoxic activity of the enterotoxin in HT-29 cells.

The alleles of the bft gene are distributed differently among enterotoxigenic Bacteroides fragilis strains from human sources and can be present in double copies.

Enterotoxigenic Bacteroides fragilis (ETBF) strains are associated with diarrheal disease in children. These strains produce a zinc metalloprotease enterotoxin, or fragilysin, that can be detected by a cytotoxicity assay with HT-29 cells. Recently, three different isoforms or variants of the enterotoxin gene, designated bft-1, bft-2, and bft-3, have been identified and sequenced. We used restriction fragment length polymorphism analysis of the PCR-amplified enterotoxin gene to detect the isoforms bft-1 and bft-2 or bft-3 borne by ETBF. By sequencing the portion of the bft gene corresponding to the mature toxin in some strains and applying allele-specific PCR for strains categorized as bft-2 or bft-3, we found in our collection two strains harboring bft-3, a variant that had been described for isolates from East Asia. Analysis of 66 ETBF strains from different sources showed that bft-1 is the most frequent allele, being present in 65% of isolates; it is largely predominant in isolates from feces of adults, while bft-2 is present in isolates from feces of children. This association is statistically significant (P, 0.0064). Sixteen strains were examined by Southern hybridization using, as probes, the bft and second metalloprotease genes, both included in a pathogenicity islet. Five strains were found to harbor double copies of both genes, suggesting that the whole islet was duplicated. Four of these strains, harboring bft-1 (three strains) or bft-2 (one strain), were found to produce a large amount of biologically active toxin, as determined by a cytotoxicity assay with HT-29 cells. The strains harboring bft-3, either in a single copy or in double copies, produced the smallest amount of toxin in our collection.

The region 3' to Calpha1 gene of human IG heavy chain displays a polymorphic duplicated sequence and encodes an RNA associated with polysomes.

A highly spread polymorphism flanking the 3. Calpha1 human IG heavy chain gene was identified. This polymorphism allowed the detection of an internal duplication within the 3' flanking region of both Calpha1 and Calpha2. This region has a regulatory function with four enhancer structures also present at the 3' end of the human Calpha2 as well as in that of mouse and rat single Calpha genes. The 5682-bp sequence of clone lambdapl8 described here starts 3' of Calpha1 and presents three open reading frames; one of them contains part of the tandem repeats with the 20-bp consensus described previously that is expressed in a poly(A)+ RNA and found in three dbEST clones of the human tonsillar cDNA library. Here, we demonstrate that in the CLF1 B lymphoblastoid cell line, this transcript is associated with polysomes. We also discuss the possibility of the presence of a new regulatory gene that does not encode an immunoglobulin and maps in the human IG heavy chain gene cluster.

Association of responsiveness to the major pollen allergen of Parietaria officinalis with HLA-DRB1* alleles: a multicenter study.

Parietaria, a plant belonging to the family of Urticaceae, is a major source of allergenic pollen in Europe. In the context of a multinational study, we investigated whether in allergic subjects antibody response towards Par o 1, the major allergen from P. officinalis, was associated with defined HLA-DRB1* alleles. The study population consisted of 234 allergic patients: 65 from Bulgaria, 30 from Israel, 99 from Italy, and 40 from Spain. In the Italian study group, the prevalence of ST positivity to Parietaria was 77%. In Parietaria ST-positive subjects, the prevalences of IgG and IgE serum Ab towards Par o 1 were 91% and 75%, respectively. HLA-DRB1*1101 and/or 1104 were significantly positively associated with the presence of IgG Ab and with high levels of IgE Ab towards this allergen (p = 0.0007 and p = 0.012, respectively). In the Spanish study group, the positive association of DR1100 with responsiveness to Par o 1 was confirmed (p = 0.02, RR = 4, and p = 0.002, RR = 7, for IgG and IgE Ab, respectively). None of the Bulgarian patients had IgE Ab to Par o 1, whereas IgG Ab response was observed in 7 out of 65 subjects and was positively associated with DRB1*1101 and/or 1104 (p = 0.025). In the Israeli study group, responsiveness to Par o 1 was not associated with specific HLA-DRB1* alleles. In conclusion, this study shows that in allergic patients from three European populations antibody response to the major allergen from the pollen of Parietaria is associated with HLA-DRB1*1101 and/or 1104. Our data suggest that this association is stronger in subjects monosensitized to Parietaria.

Characterization of a dominant antigenic determinant of Par o I encoded by recombinant DNA.

BACKGROUND: The pollens from Parietaria judaica and Parietaria officinalis are a major cause of pollinosis in Europe. Par o I (13.5 kDa) and Par j I (12 kDa), the major allergens from these species, are highly crossreactive. METHODS: We have immunoscreened a P. judaica pollen cDNA expression library with a rabbit antiserum specific for Par j I and with a serum pool from allergic patients. An immunopositive clone containing a 26 bp insert was further characterized. The insert sequence was determined and the beta-galactosidase fusion protein was partially purified by electroelution from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. RESULTS: This fusion protein specifically and extensively inhibited Par o I and Par j I binding of a rabbit antiserum and of a serum pool obtained from allergic patients. The antifusion-protein antiserum obtained in a rabbit (anti 6a) specifically precipitated radioiodinated purified Par o I in the double antibody radioimmunoassay (DARIA) and competed with antibodies of sera from allergic patients for the binding to Parietaria pollen extract allergens by enzyme linked immunosorbent assay (ELISA). We investigated the prevalence of antibody response towards the 6a epitope in patients naturally sensitized to Parietaria. The presence of 6a specific IgE antibodies was assessed in the sera of 33 patients using inhibition assays. All sera had antibodies with this specificity: the extensive percentage of inhibition reached suggested that they dominated individual ab response. CONCLUSION: In conclusion, the antibody response induced by natural exposure to the pollen of Parietaria appears to be higly focused on a single linear antigenic determinant of the major allergens which may play a relevant role in the development of clinical allergy. This report is, to our knowledge, the first description of a dominant linear epitope of a major allergen.

Integration of an Epstein-Barr virus episome 3' into the gene encoding immunoglobulin heavy-chain alpha 1 in a lymphoblastoid cell line.

For the first time we have characterized an unoccupied site of Epstein-Barr (EBV) virus integration in a lymphoblastoid cell line, RGN1. The site of integration is about 1.5 kb downstream from the gene encoding the heavy chain constant alpha 1, specifying immunoglobulin A (IgA). Sequence and Southern analysis allowed us to hypothesize that integration occurred via a double exchange involving the viral latent origin of DNA replication (oriP) and the human DNA. The region involved in the integration is transcribed into poly(A)+ RNA in all the tested lymphoid lines, but not in the RGN1 line. We suggest a mechanism of integration primed by interactions between oriP and cell ori and its potential role in the establishment and/or evolution of EBV-carrying lines.

The search for a gene involved in the determination of limited duplicative capacity in human cells.

On the assumption that EBV integration into the genome of human B lymphocyte might lead to the inactivation of a hypothetical gene determining the limited duplicative capacity and consequently participate to the cell immortalization, a search for the human-virus junction was done. This led to the identification of a site of integration in the central part of the heavy chain of the immunoglobulin region. The coincidence of the involvement of the site in lymphomatogenesis with the first complete characterization of an integration site led to the speculation that the heavy chain gene itself might be an important controller of cell duplication in the B lymphocyte.

Purification and characterization of Par o I, major allergen of Parietaria officinalis pollen.

Par o I, a major allergen of Parietaria officinalis, was purified from the pollen extract. The purified allergen was obtained by ultrafiltration, Sephadex gel filtration and DE-52 ion exchange chromatography: the purified preparation yields a single band in polyacrylamide gel isoelectric focusing (PAG-IEF), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, a single immunoprecipitation arc in crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE) and a single peak in size exclusion high-performance liquid chromatography (HPLC). Par o I is a glycoprotein with a protein to carbohydrate ratio of 100:21. The molecular weight, determined by SDS-PAGE, Sephadex G-50 gel filtration and size exclusion HPLC, varied between 13.5 and 14.5 kDa according to the method employed. The isoelectric point was 4.6. The amino acid composition and the sequence of the first twelve N-terminal residues were determined. The allergenicity was assayed in vivo and in vitro. 29/29 Parietaria-allergic patients were skin positive to Par o I and possessed high level of specific serum IgE antibody as it determined by radioallergosorbent test (RAST). Par o I contained dominant epitopes for human IgE as inhibited to 85% the pollen extract RAST performed with a pool of sera of allergic patients. The RAST inhibitory activity was not abolished by deglycosylation.

Isolation and in vitro translation of mRNA from inflorescences of Parietaria judaica.

The aqueous extract of inflorescences of Parietaria judaica contains an allergen homologous to the major pollen allergen Par o I (14 kD), as shown by radio-allergosorbent test (RAST) inhibition and immunoblot analysis. Poly(A)+ RNA was obtained from inflorescences and was shown to be able to code in vitro for a protein homologous to Par o I with respect to sodium dodecylsulphate polyacrylamide gel electrophoretic mobility and to antigenic specificity as defined by the binding, in affinity chromatography, to solid-phase IgG of rabbit anti-Par o I antisera, and in RAST inhibition, to IgE antibodies of human reaginic serum pool.

Low molecular weight allergens of the pollen of Parietaria officinalis.

The allergenic composition of a low mol. wt fraction of the pollen extract of Parietaria officinalis (PO) was investigated. Fraction C, that was eluted after oxytocin (mol. wt 1040) when the pollen extract was gel filtered on Sephadex or on Biogel, was cross-reactive in the RAST with the major allergen P015 and was capable of eliciting histamine release from leukocytes of sensitive donors. RAST inhibition (RAST I) analysis of the eluate of gel filtration on Sephadex G-10 revealed several peaks of IgE binding activity. Analysis of fine specificity of response of individual patients carried out by skin-prick tests and by RAST I, revealed individual patterns of reactivity, indicating that allergens contained in fraction C were minor allergens.