Italian consensus recommendations for a biomarker-based aetiological diagnosis in mild cognitive impairment patients.

BACKGROUND AND PURPOSE: Biomarkers support the aetiological diagnosis of neurocognitive disorders in vivo. Incomplete evidence is available to drive clinical decisions; available diagnostic algorithms are generic and not very helpful in clinical practice. The aim was to develop a biomarker-based diagnostic algorithm for mild cognitive impairment patients, leveraging on knowledge from recognized national experts. METHODS: With a Delphi procedure, experienced clinicians making variable use of biomarkers in clinical practice and representing five Italian scientific societies (neurology - Società Italiana di Neurologia per le Demenze; neuroradiology - Associazione Italiana di Neuroradiologia; biochemistry - Società Italiana di Biochimica Clinica; psychogeriatrics - Associazione Italiana di Psicogeriatria; nuclear medicine - Associazione Italiana di Medicina Nucleare) defined the theoretical framework, relevant literature, the diagnostic issues to be addressed and the diagnostic algorithm. An N-1 majority defined consensus achievement. RESULTS: The panellists chose the 2011 National Institute on Aging and Alzheimer's Association diagnostic criteria as the reference theoretical framework and defined the algorithm in seven Delphi rounds. The algorithm includes baseline clinical and cognitive assessment, blood examination, and magnetic resonance imaging with exclusionary and inclusionary roles; dopamine transporter single-photon emission computed tomography (if no/unclear parkinsonism) or metaiodobenzylguanidine cardiac scintigraphy for suspected dementia with Lewy bodies with clear parkinsonism (round VII, votes (yes-no-abstained): 3-1-1); 18 F-fluorodeoxyglucose positron emission tomography for suspected frontotemporal lobar degeneration and low diagnostic confidence of Alzheimer's disease (round VII, 4-0-1); cerebrospinal fluid for suspected Alzheimer's disease (round IV, 4-1-0); and amyloid positron emission tomography if cerebrospinal fluid was not possible/accepted (round V, 4-1-0) or inconclusive (round VI, 5-0-0). CONCLUSIONS: These consensus recommendations can guide clinicians in the biomarker-based aetiological diagnosis of mild cognitive impairment, whilst guidelines cannot be defined with evidence-to-decision procedures due to incomplete evidence.

Pore-forming bioinks to enable spatio-temporally defined gene delivery in bioprinted tissues.

The regeneration of complex tissues and organs remains a major clinical challenge. With a view towards bioprinting such tissues, we developed a new class of pore-forming bioink to spatially and temporally control the presentation of therapeutic genes within bioprinted tissues. By blending sacrificial and stable hydrogels, we were able to produce bioinks whose porosity increased with time following printing. When combined with amphipathic peptide-based plasmid DNA delivery, these bioinks supported enhanced non-viral gene transfer to stem cells in vitro. By modulating the porosity of these bioinks, it was possible to direct either rapid and transient (pore-forming bioinks), or slower and more sustained (solid bioinks) transfection of host or transplanted cells in vivo. To demonstrate the utility of these bioinks for the bioprinting of spatially complex tissues, they were next used to zonally position stem cells and plasmids encoding for either osteogenic (BMP2) or chondrogenic (combination of TGF-ß3, BMP2 and SOX9) genes within networks of 3D printed thermoplastic fibers to produce mechanically reinforced, gene activated constructs. In vivo, these bioprinted tissues supported the development of a vascularised, bony tissue overlaid by a layer of stable cartilage. When combined with multiple-tool biofabrication strategies, these gene activated bioinks can enable the bioprinting of a wide range of spatially complex tissues.

Mesenchymal stem cell fate following non-viral gene transfection strongly depends on the choice of delivery vector.

Controlling the phenotype of mesenchymal stem cells (MSCs) through the delivery of regulatory genes is a promising strategy in tissue engineering (TE). Essential to effective gene delivery is the choice of gene carrier. Non-viral delivery vectors have been extensively used in TE, however their intrinsic effects on MSC differentiation remain poorly understood. The objective of this study was to investigate the influence of three different classes of non-viral gene delivery vectors: (1) cationic polymers (polyethylenimine, PEI), (2) inorganic nanoparticles (nanohydroxyapatite, nHA) and (3) amphipathic peptides (RALA peptide) on modulating stem cell fate after reporter and therapeutic gene delivery. Despite facilitating similar reporter gene transfection efficiencies, these nanoparticle-based vectors had dramatically different effects on MSC viability, cytoskeletal morphology and differentiation. After reporter gene delivery (pGFP or pLUC), the nHA and RALA vectors supported an elongated MSC morphology, actin stress fibre formation and the development of mature focal adhesions, while cells appeared rounded and less tense following PEI transfection. These changes in MSC morphology correlated with enhanced osteogenesis following nHA and RALA transfection and adipogenesis following PEI transfection. When therapeutic genes encoding for transforming growth factor beta 3 (TGF-ß3) and/or bone morphogenic protein 2 (BMP2) were delivered to MSCs, nHA promoted osteogenesis in 2D culture and the development of an endochondral phenotype in 3D culture, while RALA was less osteogenic and appeared to promote a more stable hyaline cartilage-like phenotype. In contrast, PEI failed to induce robust osteogenesis or chondrogenesis of MSCs, despite effective therapeutic protein production. Taken together, these results demonstrate that the differentiation of MSCs through the application of non-viral gene delivery strategies depends not only on the gene delivered, but also on the gene carrier itself. STATEMENT OF SIGNIFICANCE: Nanoparticle-based non-viral gene delivery vectors have been extensively used in regenerative medicine, however their intrinsic effects on mesenchymal stem cell (MSC) differentiation remain poorly understood. This paper demonstrates that different classes of commonly used non-viral vectors are not inert and they have a strong effect on cell morphology, stress fiber formation and gene transcription in MSCs, which in turn modulates their capacity to differentiate towards osteogenic, adipogenic and chondrogenic lineages. These results also point to the need for careful and tissue-specific selection of nanoparticle-based delivery vectors to prevent undesired phenotypic changes and off-target effects when delivering therapeutic genes to damaged or diseased tissues.

Long-chain amine-templated synthesis of gallium sulfide and gallium selenide nanotubes.

We describe the soft chemistry synthesis of amine-templated gallium chalcogenide nanotubes through the reaction of gallium(iii) acetylacetonate and the chalcogen (sulfur, selenium) using a mixture of long-chain amines (hexadecylamine and dodecylamine) as a solvent. Beyond their role as solvent, the amines also act as a template, directing the growth of discrete units with a one-dimensional multilayer tubular nanostructure. These new materials, which broaden the family of amine-stabilized gallium chalcogenides, can be tentatively classified as direct large band gap semiconductors. Their preliminary performance as active material for electrodes in lithium ion batteries has also been tested, demonstrating great potential in energy storage field even without optimization.

Efficient fluorescence quenching in electrochemically exfoliated graphene decorated with gold nanoparticles.

High surface area graphene sheets were obtained by electrochemical exfoliation of graphite in an acid medium under constant potential conditions. Filtration and centrifugation processes played an important role in order to obtain stable dispersions in water. Scanning electron microscopy and transmission electron microscopy imaging revealed highly exfoliated crystalline samples of ∼5 µm. Raman, Fourier transform infrared and x-ray photoelectron spectroscopy further confirmed the high quality of the exfoliated material. The electrochemically exfoliated graphene (EEG) was decorated with gold nanoparticles (AuNPs) using sodium cholate as a buffer layer. This approach allowed for a non-covalent functionalization without altering the desirable electronic properties of the EEG. The AuNP-EEG samples were characterized with various techniques including absorbance and fluorescence spectroscopy. These samples displayed a fluorescence signal using an excitation wavelength of 290 nm. The calculated quantum yield (Φ) for these samples was 40.04%, a high efficiency compared to previous studies using solution processable graphene.

Graphene resist interlacing process for versatile fabrication of free-standing graphene.

We present a graphene resist interlacing process (GRIP) to sandwich graphene between polymer lines in a cloth-like fashion, making it more accessible for experiments and applications. We demonstrate the handling of large-area graphene in this way. Here, GRIP is used to fabricate supports for transmission electron microscopy. These supports improve the imaging quality of nanoparticles, as we show by comparison to imaging on standard lacey carbon supports.

Antibiotic susceptibility profiles of uncommon bacterial species causing severe infections in Italy.

This study presents the results of the italian "Severe infections project" involving bacteria that can be considered rare causes of disease. we isolated 30 uncommon human pathogens from a total of 60 strains (1.2% of all the isolates). The most frequent sources of uncommon human pathogens were primary bloodstream infections (48.3%) and pneumonia (20%). Species such as Comamonas testosteroni, Enterococcus hirae, Kluyvera ascorbata, Kluyvera cryocrescens, Leclercia adecarboxylata and Ochrobactrum anthropi were recovered from bacteremia patients. Clinically useful antimicrobial agents were tested against each isolate. Resistance to 4 or more antibiotics tested was found in Achromobacter xylosoxidans, O. anthropi, Pseudomonas stutzeri, Citrobacter braakii, Enterobacter sakazakii, K. ascorbata, Proteus penneri and Serratia plymuthica. About 16% of the Gram-negative species were resistant to third-generation cephalosporins and 28.6% of the staphylococci were oxacillin-resistant. the results from this study offer indications for empirical therapy for severe infections from uncommon human pathogens.

Comparative activity of cefodizime and ceftriaxone against respiratory pathogens in an in vitro pharmacodynamic model simulating concentration-time curves.

The duration of time that serum levels are above the minimum inhibitory concentration (MIC; T >MIC) seems to be an important pharmacodynamic parameter for beta-lactams. The aim of this study was to evaluate the bactericidal activity of cefodizime and ceftriaxone in a pharmacokinetic model mimicking the concentrations in bronchial mucus and in serum (total and free) obtained at 2, 4, 8, 12 and 24 h, after 1 g i.m. administration once daily. The species investigated were respiratory pathogens (1 strain of Staphylococcus aureus, 2 strains of Streptococcus pneumoniae, 1 strain b-lactamase negative and 1 strain beta-lactamase positive of Haemophilus influenzae, 1 strain of Escherichia coli and 1 strain of Klebsiella pneumoniae); MIC50s of the chosen strains were reported. In this in vitro model the concentrations (serum and bronchial mucus) for both antibiotics are generally at or above the MIC values of the tested strains until 24 hours. The killing curve showed rapid killing for both antibiotics: 99.9% killing (a 3-log reduction in growth) within 6 to 8 h, depending upon the microorganism tested. There was no significant difference in the log kill between cefodizime and ceftriaxone. These data confirm that T >MIC for beta-lactams is the pharmacodynamic parameter which best correlates with bactericidal efficacy. On the basis of the killing curve determined for cefodizime versus ceftriaxone at concentrations that these antibiotics can reach during therapy with 1 g i.m. once daily we expect reasonable clinical efficacy with monoadministration of cefodizime as well as for ceftriaxone in respiratory tract infections.

Correlation of trimethoprim and brodimoprim physicochemical and lipid membrane interaction properties with their accumulation in human neutrophils.

Dipalmitoylphosphatidylcholine vesicles were used as a biological membrane model to investigate the interaction and the permeation properties of trimethoprim and brodimoprim as a function of drug protonation. The drug-membrane interaction was studied by differential scanning calorimetry. Both drugs interacted with the hydrophilic phospholipid head groups when in a protonated form. An experiment on the permeation of the two drugs through dipalmitoylphosphatidylcholine biomembranes showed higher diffusion rate constants when the two drugs were in the uncharged form; lowering of the pH (formation of protonated species) caused a reduction of permeation. Drug uptake by human neutrophil cells was also investigated. Both drugs may accumulate within neutrophils; however, brodimoprim does so to a greater extent. This accumulation is probably due to a pH gradient driving force, which allows the two drugs to move easily from the extracellular medium (pH approximately 7.3) into the internal cell compartments (acid pH). Once protonated, both drugs are less able to permeate and can be trapped by the neutrophils. This investigation showed the importance of the physicochemical properties of brodimoprim and trimethoprim in determining drug accumulation and membrane permeation pathways.

Synergistic effect of deoxyspergualin (DSP) and cyclosporin A (CsA) in the prevention of spontaneous autoimmune diabetes in BB rats.

Dose-dependent side effects are frequently observed with immunosuppressive drugs of potential relevance for the immunotherapy of insulin-dependent diabetes mellitus (IDDM), such as CsA and DSP. If CsA and DSP acted synergistically in vivo, their combined use would allow using each compound at lower doses than those required when each drug is given in monotherapy. Consequently, dose-dependent side effects could be reduced and the therapeutic activity maintained or even enforced. Toward this end we studied the effects of combined treatment with CsA and DSP on the course of IDDM in the diabetes-prone (DP)-BB rat. The results show that two 'low' doses of CsA (2 mg/kg) and DSP (1 mg/kg) that are clinically ineffective in suppressing IDDM development in BB rats when administered alone under a prolonged prophylactic regimen (30-105 days old), may successfully prevent, but not cure, the disease when given contemporaneously under the same experimental conditions. The combined treatment was well tolerated, and no side effects were noticed. These data suggest that the combined use of CsA and DSP may deserve consideration for its possible application in the prevention/treatment of human IDDM and other autoimmune diseases.

In vitro activity of cefdinir against respiratory pathogens isolated in Sicily with reference to beta-lactamase production.

The in vitro activity of cefdinir (CI-983, FK-482), an orally absorbed aminothiazolyl cephalosporin, was evaluated against all 287 strains of Haemophilus influenzae, Haemophilus parainfluenzae, Moraxella catarrhalis, Streptococcus pneumoniae and Streptococcus pyogenes in comparison with cefaclor, cefuroxime, amoxicillin, amoxicillin-clavulanic acid, erythromycin and cotrimoxazole. The bactericidal activity of cefdinir, cotrimoxazole, amoxicillin-clavulanic acid and erythromycin was determined against H. influenzae, M. catarrhalis and S. pneumoniae. With the exception of one beta-lactamase negative ampicillin-resistant strain of H. influenzae (resistant to all antibiotics tested), no resistance to cefdinir was observed (MIC < or = 1 mg/l). Cefdinir was active against H. influenzae, H. parainfluenzae and M. catarrhalis regardless of whether or not they produced beta-lactamase. In general, the inhibitory concentrations of cefdinir against H. influenzae, H. parainfluenzae and M. catarrhalis were similar to those of amoxicillin/clavulanic acid, one or two dilutions lower than those of cefuroxime and four dilutions lower than those of cefaclor and cotrimoxazole. Against S. pneumoniae and S. pyogenes cefdinir had the same activity as cefuroxime and amoxicillin but was more effective than the other antibiotics tested. Kinetic studies showed that cefdinir was rapidly bactericidal at concentrations 2 and 4 times the minimum inhibitory concentration (MIC): a reduction of 99.9% in CFU values was generally observed after 6-8 h.

In vitro antimicrobial activity of Pistacia lentiscus L. extracts: preliminary report.

The in vitro antimicrobial activity of Pistacia lentiscus L. extracts was determined. Pistacia lentiscus L. extracts were tested on bacteria (Sarcina lutea, Staphylococcus aureus and Escherichia coli) and fungi (Candida albicans, Candida parapsilosis, Torulopsis glabrata and Cryptococcus neoformans). Of the different plant extractions, decoctions showed the best antibacterial activity, but the activity against fungal cells appears to be much more interesting.

Bactericidal kinetics and postantibiotic effect of sparfloxacin against selected species of respiratory pathogens.

We determined the bactericidal kinetics and postantibiotic effect (PAE) of sparfloxacin against Haemophilus influenzae, Moraxella (Branhamella) catarrhalis, Klebsiella pneumonia, Streptococcus pneumoniae, and Staphylococcus aureus. Time-kill studies were performed by using 1 x and 4 x the minimum inhibitory concentrations (MICs) of sparfloxacin, ciprofloxacin, co-trimoxazole, amoxicillin/clavulanic acid and erythromycin (inoculum 10(5) and 10(7) CFU/ml). The PAE was induced by exposing the strains to 1xMIC and 4xMIC of sparfloxacin and ciprofloxacin for 1 h. Sparfloxacin was the most bactericidal of all the antibiotics tested, being active against Gram-positive and Gram-negative isolates with a 99.9% reduction within 3 to 6 h of exposure, depending upon strain, inoculum and concentration. The PAE of sparfloxacin against all species tested ranged from 1.1 to 2.6 hours; the most notable PAE occurring with M. catarrhalis.

[Etiology and rational therapy of acute otitis media in adults]. / Eziologia e razionale terapeutico dell'otite media acuta nell'adulto.

Acute otitis media (AOM) is an infection frequently found in children, but tends to be less frequent with age and its frequency in adults is only about 1%. The etiology of AOM in children is prevalently bacterial; numerous studies have shown the most common etiological agents. But the etiology in adults has not been well studied. The authors examined 40 cases of AOM in adults, the pathologic material was obtained by needle aspiration; in 32 samples there was bacterial growth. In the majority of the cases (94%) the bacteria isolated were the same as those found in children: S. pneumoniae, H. influenzae and B. catarrhalis; much rarer were S. pyogenes and S. aureus. On the potential beta-lactamase producing strains, this activity was measured. From our findings we believe that there is the necessity to have a rational antibiotic therapy (due to the difficulty in obtaining pathologic material) with active drugs for the probable etiological agents of AOM.

Action of clindamycin-phosphate in foreign body infections due to Staphylococcus epidermidis in mice.

The virulence of Staphylococcus epidermidis strain slime producer was examined in an experimental model of foreign body infection in mice. In the course of this experimental infection the mice were injected with two antibiotics (clindamycin and cefazolin) active in vitro toward the Staphylococcus strain used. The results obtained after a week of antibiotic therapy show that clindamycin alone has a therapeutic action against the infection caused by S. epidermidis. Cefazolin showed a very poor therapeutic effect. The results are discussed on the basis of inflammatory reaction elicited from the foreign body and the characteristics of clindamycin in connection with the host's defense mechanisms.

In vitro study of dactimicin (ST 900) against methicillin-susceptible and -resistant staphylococci.

The current knowledge on the antimicrobial activity of dactimicin is poor and limited to the study of its activity against gram-negative bacilli. The new acquisition about the use of aminoglycoside antibiotics in infections due to Staphylococci, induced us to evaluate the behavior of this drug against methicillin-susceptible and -resistant Staphylococci, in comparison with fortimicin A and amikacin. Dactimicin (ST 900) shows good antibacterial activity and a strong bactericidal effect at MIC concentrations in all strains tested.

Enterococci and aminoglycosides: evaluation of susceptibility and synergism of their combination with piperacillin.

Some clinical isolates of enterococci were tested for susceptibility to gentamicin, tobramycin, amikacin and netilmicin. Five percent of Streptococcus faecalis tested demonstrated high level resistance (minimum inhibitory concentration (MIC) greater than 2024 micrograms/ml) to gentamicin, tobramycin and netilmicin, while amikacin had MICs greater than 128 micrograms/ml for all strains tested. Since a combination of a beta-lactam and streptomycin does not produce synergism against all strains of enterococci for an increase in the number of highly resistant strains, the effect of piperacillin plus gentamicin, tobramycin, and netilmicin was examined. The combination piperacillin + netilmicin seemed to be very effective against all enterococci tested.

The sensitivity of gram-negative and gram-positive bacteria to ofloxacin.

The in vitro antimicrobial activity of ofloxacin, a new fluorinated quinolone, was evaluated against 165 gram-negative rods, both fermentative and non-fermentative and against 57 gram-positive strains (coagulase-positive and -negative, staphylococci both methicillin-resistant and susceptible and Streptococcus faecalis). Minimal inhibitory concentrations were determined by using the macrodilution test and activity was compared with nalidixic acid, norfloxacin, ampicillin, piperacillin, ceftazidime and gentamicin for gram-negative rods, norfloxacin and gentamicin for Staphylococcus strains, and norfloxacin, ampicillin, piperacillin and gentamicin for enterococci. Ofloxacin inhibited all fermentative gram-negative bacteria tested, in a range of 0.05-3.12 micrograms/ml. It also had good antimicrobial activity against non-fermentative gram-negative strains: in fact it inhibited 90% of Acinetobacter and 80% of Pseudomonas aeruginosa tested at 3.12 micrograms/ml. Ofloxacin had good antimicrobial activity against Staphylococcus and Enterococcus tested.

The sensitivity of gram-negative and gram-positive bacteria to ofloxacin.

The in vitro antimicrobial activity of ofloxacin, a new fluorinated quinolone, was evaluated against 165 Gram-negative rods, both fermentative and non-fermentative, and against 57 Gram-positive strains (coagulase-positive and -negative staphylococci both methicillin-resistant and -susceptible, and Streptococcus faecalis). Minimal inhibitory concentrations were determined by using the macrodilution test and the activity was compared with nalidixic acid, norfloxacin, ampicillin, piperacillin, ceftazidime and gentamicin for Gram-negative rods; norfloxacin and gentamicin for Staphylococcus strains; and norfloxacin, ampicillin, piperacillin and gentamicin for enterococci. Ofloxacin inhibited all fermentative Gram-negative bacteria tested, in a range of 0.05-3.12 mcg/ml, and had good antimicrobial activity against non-fermentative Gram-negative strains: it inhibited 90% of Acinetobacter and 80% of P. aeruginosa tested, at 3.12 mcg/ml. Ofloxacin had a high antimicrobial activity against Staphylococcus and Enterococcus strains tested.