Characterization and predicted role of the microRNA expression profile in amnion from obese pregnant women.

Maternal obesity and nutrient excess in utero increase the risk of future metabolic diseases. The mechanisms underlying this process are poorly understood, but probably include genetic, epigenetic alterations and changes in fetal nutrient supply. We have studied the microRNA (miRNA) expression profile in amnion from obese and control women at delivery to investigate if a specific miRNA signature is associated with obesity. The expression profile of 365 human miRNAs was evaluated with the TaqMan Array in amnion from 10 obese and 5 control (prepregnancy body mass index (BMI) >30 and <25 kg m(-2), respectively) women at delivery. Target genes and miRNA-regulated pathways were predicted by bioinformatics. Anthropometric and biochemical parameters were also measured in mothers and newborns. Seven miRNAs were expressed only in obese women (miR-422b, miR-219, miR-575, miR-523, miR-579, miR-618 and miR-659), whereas 13 miRNAs were expressed at a higher level and 12 miRNAs at a lower level in obese women than in controls. MicroRNAs significantly downregulated the neurotrophin, cancer/ErbB, mammalian target of rapamycin, insulin, adipocytokine, actin cytoskeleton and mitogen-activated protein kinase signaling pathways. In conclusion, we show that the miRNA profile is altered in amnion during obesity and hypothesize that this could affect pathways important for placental growth and function, thereby contributing to an increase in the newborn's risk of future metabolic diseases.

Prenatal diagnosis of congenital rubella infection and ultrasonography: a preliminary study.

AIM: The aim of this study was to analyze the role of ultrasonography in the prenatal diagnosis of women with confirmed rubella infection in pregnancy. METHODS: We performed a retrospective, population-based study on 175 women referred to our Centre of Infectious Disease in Pregnancy of AOU Federico II for rubella infection, in the period between January 1999 and December 2009. In confirmed cases of infection we performed periodic ultrasonographic assessment of fetal anatomy looking for prenatal findings of rubeovirus infection. Neonatal outcomes were collected. RESULTS: Among 175 women, 48 (27.4%) were confirmed cases, in 83 (47.4%) cases rubella infection remain suspected. The remaining 44 (25.1%) women were excluded to having rubella infection in pregnancy. No defined probable cases were found. Among children born to mother with confirmed rubella infection, 9 (18.7%) resulted infected by rubella virus. In particular 6 newborns (66% of the affected) were asymptomatic, two babies showed incomplete congenital rubella syndrome (CRS), due to the presence of deafness in the absence of other congenital abnormalities without showing any USG findings. Only one baby showed a complete CRS: USG prenatal rubella infection findings were IUGR, polydramnios, cardiomegaly, defects of atrial septum, hepatosplenomegaly, ascites, echogenic bowel, placentomegaly. USG specificity and sensitivity was 100% and 11% respectively. CONCLUSION: USG has an important role in the detection rubella intrauterine infection in case of severe abnormalities. The obstetricians should understand the limitations of ultrasound in detection of deafness and minor abnormalities.

Mitochondrial DNA sequence variations in some Italian wild boar populations.

In order to investigate the relationships between Italian wild boar and major pig breeds, we studied the genetic variability of four wild boar populations in Italy (Arezzo, Pisa, Parma, Bergamo) using a 533-bp fragment of the mitochondrial control region. Sixty-nine wild boar samples were analysed, allowing the identification of 10 distinct haplotypes, which involve a total of 15 single nucleotide polymorphisms. Phylogenetic and network analyses were performed also considering several sequences of wild and domesticated forms available in the databases. The Bayesian phylogenetic tree and the Median-Joining network analyses show three main groups: the Italian (IT), European (EU) and Asian (AS) clades. The IT clade corresponds to the Maremma endemic wild boar population and also includes Sardinian individuals, while the EU and AS groups include wild boars as well as domestic pig breeds. Only two individuals from Pisa cluster in the IT group, whereas two haplotypes from Bergamo cluster in the AS group and all other samples cluster in the EU clade. These findings suggest that in Italy wild boar populations have a mixed origin, both EU and AS, and that an interbreeding between wild and domesticated strains has probably occurred. Eight of the 10 wild boars coming from the Migliarino-San Rossore-Massaciuccoli Regional Park (Pisa) belong to H2 and H3 haplotypes, and cluster into the EU clade, suggesting that this regional park is not anymore exclusive of the endemic Maremma wild boar.

Fetal borderline cerebral ventriculomegaly: clinical significance and management.

AIM: The aim of this study was to evaluate the clinical significance and the management of fetal borderline lateral cerebral ventriculomegaly. METHODS: Fetuses with a sonographic diagnosis of lateral cerebral ventriculomegaly isolated or associated to other fetal malformations were followed monthly and a review of the English-language literature was made. RESULTS: Of 86 fetuses analyzed, 30 suffered from hydrocephaly (monolateral, bilateral), 56 showed also several other malformations . Chromosomal aberrations is possible also in case of isolated hydrocephaly. Neurological sequelae, mostly a mild to moderate delay in cognitive and/or motor development, is present in 10% of the cases. CONCLUSIONS: In most cases, isolated borderline lateral cerebral ventriculomegaly has no consequence. However, the risk of cerebral maldevelopment, delayed neurological development and, possibly, chromosomal aberrations is increased. The optimum management of these cases remains uncertain.

[Retroperitoneal abscess due to asymptomatic duodenal perforation by foreign body. A case report]. / Ascesso retroperitoneale da corpo estraneo dopo perforazione duodenale asintomatica. Case report.

We present a case of retroperitoneal abscess due to asymptomatic duodenal perforation by foreign body. Patient has been admitted for lumbar pain and subocclusive crisis, with a medical history negative for acute symptomatology. After both clinical and radiologic evaluation, an abscess-like mass was detected in the context of right psoas muscle. Patient underwent surgical operation and a lumbar abscess has been found containing a foreign body (toothpick). There has been a transduodenal migration of the foreign body, without clinical signs of duodenal perforation.

Microparticle-embedded fibroin/alginate beads for prolonged local release of simvastatin hydroxyacid to mesenchymal stem cells.

In the present work, we propose silk fibroin/alginate (SF/Alg) beads embedding simvastatin-loaded biodegradable microparticles as a versatile platform capable of tuning SVA release and in so doing osteogenic effects. In a first part of the study, microparticles of poly(lactic-co-glycolic) acid incorporating simvastatin either as lactone (SVL) or as hydroxyacid form (SVA) were prepared by spray-drying. While SVA-loaded microparticles released the drug in three days, long-term release of SVA could be obtained from SVL-loaded microparticles. In this latter case, SVL was promptly transformed to the osteogenic active SVA during release. When tested on mesenchymal stem cells, a time- and dose-dependent effect of SVL-loaded microparticles on cell proliferation and alkaline phosphatase (ALP) activity was found. Thereafter, SVL-loaded microparticles were embedded in SF/Alg beads to limit the initial simvastatin burst and to achieve easier implantation as well. Microparticle-embedded beads showed no cytotoxicity while ALP activity increased. If correctly exploited, the developed system may be suitable as osteogenic polymer scaffolds releasing correct amount of the drug locally for long time-frames.

Nasal powders of thalidomide for local treatment of nose bleeding in persons affected by hereditary hemorrhagic telangiectasia.

In this work nasal powder formulations of thalidomide were designed and studied to be used by persons affected by hereditary hemorrhagic telangiectasia as a complementary anti-epistaxis therapy, with the goal of sustaining the effect obtained with thalidomide oral treatment after its discontinuation for adverse effects. Three nasal powders were prepared using as carriers ß-CD or its more hydrophilic derivatives such as hydropropyl-ß-CD and sulphobutylether-ß-CD and tested with respect to technological and biopharmaceutical features after emission with active and passive nasal powder devices. For all formulated powders, improved dissolution rate was found compared to that of the raw material, making thalidomide promptly available in the nasal environment at a concentration favouring an accumulation in the mucosa. The very limited transmucosal transport measured in vitro suggests a low likelihood of significant systemic absorption. The topical action on bleeding could benefit from the poor absorption and from the fact that about 2-3% of the thalidomide applied on the nasal mucosa was accumulated within the tissue, particularly with the ß-CD nasal powder.

Development of inhalable hyaluronan/mannitol composite dry powders for flucytosine repositioning in local therapy of lung infections.

Flucytosine (5-fluorocytosine, 5-FC) is a fluorinated analogue of cytosine currently approved for the systemic treatment of fungal infections, which has recently demonstrated a very promising antivirulence activity against the bacterial pathogen Pseudomonas aeruginosa. In this work, we propose novel inhalable hyaluronic acid (HA)/mannitol composite dry powders for repositioning 5-FC in the local treatment of lung infections, including those affecting cystic fibrosis (CF) patients. Different dry powders were produced in one-step by spray-drying. Powder composition and process conditions were selected after in depth formulation studies aimed at selecting the 5-FC/HA/mannitol formulation with convenient aerosolization properties and drug release profile in simulated lung fluids. The optimized 5-FC/HA/mannitol powder for inhalation (HyaMan_FC#3) was effectively delivered from different breath-activated dry powder inhalers (DPI) already available to CF patients. Nevertheless, the aerodynamic assessment of fine particles suggested that the developed formulation well fit with a low-resistance DPI. HyaMan_FC#3 inhibited the growth of the fungus Candida albicans and the production of the virulence factor pyoverdine by P. aeruginosa at 5-FC concentrations that did not affect the viability of both wild type (16HBE14o-) and CF (CFBE41o-) human bronchial epithelial cells. Finally, pharmacokinetics of HyaMan_FC#3 inhalation powder and 5-FC solution after intratracheal administration in rats were compared. In vivo results clearly demonstrated that, when formulated as dry powder, 5-FC levels in both bronchoalveolar lavage fluid and lung tissue were significantly higher and sustained over time as compared to those obtained with the 5-FC solution. Of note, when the same 5-FC amount was administered intravenously, no significant drug amount was found in the lung at each time point from the injection. To realize a 5-FC lung concentration similar to that obtained by using HyaMan_FC#3, a 6-fold higher dose of 5-FC should be administered intravenously. Taken together, our data demonstrate the feasibility to deliver 5-FC by the pulmonary route likely avoiding/reducing the well-known side effects associated to the high systemic 5-FC doses currently used in humans. Furthermore, our results highlight that an appropriate formulation design can improve the persistence of the drug at lungs, where microorganisms causing severe infections are located.

Prenatal diagnosis of placental chorioangioma: our experience.

Placental chorioangioma is the most common benign tumor of placenta. The relationship of vascularized chorioangiomas to adverse pregnancy outcome is well recognized. We report 3 cases of placental chorioangioma. Hypervascularization of the lesions in all patients and the immune hydrops with adverse fetal outcome in 2 cases are the complications of our mini-series. Ultrasonography and Doppler ultrasonography findings were useful in establishing the prenatal diagnosis and the prognosis.

Functional characterization of biodegradable nanoparticles as antigen delivery system.

BACKGROUND: Peptide based vaccines may suffer from limited stability and inefficient delivery to professional antigen-presenting cells (APCs), such as dendritic cells (DCs). In order to overcome such limitations, several types of biodegradable nanoparticles (NPs) have been developed as carrier system for antigens. The present study describes for the first time the extensive biological characterization of cationic NPs made of poly (D,L-lactide-co-glycolide) (PLGA) and polyethylenimine (PLGA/PEI) as delivery system for protein/peptide antigens, with potential in therapeutic cancer vaccine development. RESULTS: Flow cytometry as well as confocal laser scanning microscopy (CLSM) showed that PLGA/PEI NPs are more readily taken up than PLGA NPs by both human CD14(+) monocytes and mouse Hepa 1-6 hepatoma cell line. No signs of toxicity were observed in either cellular setting. Sequential image acquisition by TEM showed an intracellular apical localization for PLGA NPs and a perinuclear localization for PLGA/PEI NPs. Both NPs showed a clathrin-dependent as well as a caveolin-dependent internalization pathway and, once in the cells, they formed multivesicular endosomes (MVE). Finally, an ex vivo priming experiment showed that PLGA/PEI NPs are comparable to PLGA NPs in delivering a non-self antigen (i.e., ovalbumin - OVA) to immature dendritic cells (imDCs), which matured and induced autologous naïve CD4(+) T cells to differentiate to memory (i.e., central memory and effector memory) cells. Such a differentiation was associated with a Th1 phenotype suggesting a downstream activation and amplification of a CD8(+) T cell cytotoxic response. The same OVA antigen in a soluble form was unable to induce maturation of DCs, indicating that both NP formulations provided an intrinsic adjuvanting effect combined to efficient antigen delivery. CONCLUSIONS: Our study represents the first report on side-by-side comparison of PLGA and PLGA/PEI NPs as strategy for protein antigen delivery. PLGA/PEI NPs are superior for cellular uptake and antigen delivery as compared to PLGA NPs. Such an evidence suggests their great potential value for vaccine development, including therapeutic cancer vaccines.

Spray-by-spray in situ cross-linking alginate hydrogels delivering a tea tree oil microemulsion.

In this paper we propose an in situ forming ionically cross-linked alginate (Alg) hydrogel delivering a Tea Tree Oil microemulsion (MeTTO) and potentially useful as an advanced dressing for infected wounds. Alg hydrogels were prepared by a spray-by-spray deposition method with the aim to minimize the discomforts during application. From pseudoternary phase diagrams, it was found that proper combination of TTO, water, polysorbate 80 and ethanol gave stable spherical MeTTO with good antimicrobial activity. On this basis, MeTTO at 20% TTO was selected for further inclusion in an Alg hydrogel prepared by alternating sprays of Alg/MeTTO and calcium chloride solutions. Homogeneous dispersion of MeTTO inside cross-linked Alg was assessed by different macroscopic and microscopic methods demonstrating the superior propensity of MeTTO to be integrated in the water-based hydrogel as compared to TTO. Antimicrobial effect of Alg/MeTTO hydrogels on Escherichia Coli strains was remarkable, highlighting the potential of the system as bioactive wound dressing.

Alginate-hyaluronan composite hydrogels accelerate wound healing process.

In this paper we propose polysaccharide hydrogels combining alginate (ALG) and hyaluronan (HA) as biofunctional platform for dermal wound repair. Hydrogels produced by internal gelation were homogeneous and easy to handle. Rheological evaluation of gelation kinetics of ALG/HA mixtures at different ratios allowed understanding the HA effect on ALG cross-linking process. Disk-shaped hydrogels, at different ALG/HA ratio, were characterized for morphology, homogeneity and mechanical properties. Results suggest that, although the presence of HA does significantly slow down gelation kinetics, the concentration of cross-links reached at the end of gelation is scarcely affected. The in vitro activity of ALG/HA dressings was tested on adipose derived multipotent adult stem cells (Ad-MSC) and an immortalized keratinocyte cell line (HaCaT). Hydrogels did not interfere with cell viability in both cells lines, but significantly promoted gap closure in a scratch assay at early (1 day) and late (5 days) stages as compared to hydrogels made of ALG alone (p<0.01 and 0.001 for Ad-MSC and HaCaT, respectively). In vivo wound healing studies, conducted on a rat model of excised wound indicated that after 5 days ALG/HA hydrogels significantly promoted wound closure as compared to ALG ones (p<0.001). Overall results demonstrate that the integration of HA in a physically cross-linked ALG hydrogel can be a versatile strategy to promote wound healing that can be easily translated in a clinical setting.

Minimal morphological criteria for defining bone marrow dysplasia: a basis for clinical implementation of WHO classification of myelodysplastic syndromes.

The World Health Organization classification of myelodysplastic syndromes (MDS) is based on morphological evaluation of marrow dysplasia. We performed a systematic review of cytological and histological data from 1150 patients with peripheral blood cytopenia. We analyzed the frequency and discriminant power of single morphological abnormalities. A score to define minimal morphological criteria associated to the presence of marrow dysplasia was developed. This score showed high sensitivity/specificity (>90%), acceptable reproducibility and was independently validated. The severity of granulocytic and megakaryocytic dysplasia significantly affected survival. A close association was found between ring sideroblasts and SF3B1 mutations, and between severe granulocytic dysplasia and mutation of ASXL1, RUNX1, TP53 and SRSF2 genes. In myeloid neoplasms with fibrosis, multilineage dysplasia, hypolobulated/multinucleated megakaryocytes and increased CD34+ progenitors in the absence of JAK2, MPL and CALR gene mutations were significantly associated with a myelodysplastic phenotype. In myeloid disorders with marrow hypoplasia, granulocytic and/or megakaryocytic dysplasia, increased CD34+ progenitors and chromosomal abnormalities are consistent with a diagnosis of MDS. The proposed morphological score may be useful to evaluate the presence of dysplasia in cases without a clearly objective myelodysplastic phenotype. The integration of cytological and histological parameters improves the identification of MDS cases among myeloid disorders with fibrosis and hypocellularity.

Biodegradable microspheres of novel segmented poly(ether-ester-amide)s based on poly(epsilon-caprolactone) for the delivery of bioactive compounds.

A novel class of multiblock poly(epsilon-caprolactone)-based polymers containing hydrophilic trioxyethylene segments and potentially relevant to the delivery of drugs is described in this work. L-phenylalanine residues may also be inserted into the hydrophilic blocks to generate peptide bonds susceptible to enzymatic attack. The investigated polymers were poly(ether-ester-amide)s (PEEAs) obtained by a two-step polymerization procedure from OH-end capped low molecular weight poly(epsilon-caprolactone), sebacoyl chloride and either 4,7,10-trioxa-1,13-tridecanediamine (PEEA1) or 1,13-di(L-phenylalaninamido)-4,7,10-trioxatridecane (PEEA2). PEEAs were characterized by 1H-NMR spectroscopy, differential scanning calorimetry, gel permeation chromatography and were tested for their suitability in producing microspheres. Particles obtained by the single emulsion-solvent evaporation technique were regular and smooth (SEM analysis) showing a monomodal distribution of dimensions. To assess the potentiality of PEEAs in the oral delivery of drugs, three model compounds with different pKa and solubilities--diclofenac, nicardipine and dicumarol--were encapsulated within PEEA microspheres. For the sake of comparison, microspheres prepared from poly(epsilon-caprolactone) (PCL) with a molecular weight similar to PEEAs were also prepared and tested. The release of diclofenac from all the microspheres was very rapid (100% released within 2 h) whereas nicardipine release was slower and biphasic. The initial phase approximated a near zero-order release, being the fraction of nicardipine released after 8 h from PEEA microspheres higher with respect to PCL particles (about 70 vs. 30%). This result was ascribed to the lower crystallinity of PEEAs with respect to PCL which results in a facilitated access of water molecules through the polymer matrix. The lipophilic-unionizable dicumarol was released from PEEA microspheres at a very slow rate. Therefore, dicumarol-loaded PEEA2 microspheres allowed the study of the influence on the release rate of the insertion into the polymer chain of enzymatically degradable bonds. PEEA2 microspheres released dicumarol at the same rate in a medium with or without the proteolitic enzyme alpha-chymotrypsin. Although the insertion of an isolated amino acid was not sufficient to confer enzyme susceptibility to the polymer, the distinctive properties of PEEAs make their use very attractive in the field of controlled release.

Novel microparticulate system made of poly(methylidene malonate 2.1.2).

Formulation of PMM 2.1.2 microparticles entrapping ovalbumin as a model protein was achieved by using a double emulsion solvent evaporation method. Parameters such as the nature of the solvent, polymer concentration and polymer molecular weight were investigated. Preparation process led to the formation of spherical and smooth particles with a mean diameter of 5 microm, and an encapsulation efficiency and protein loading level of up to 16 and 2.9% w/w, respectively. After an initial burst of approximately 10%, the protein was released at a rate of less than 1% per day. This slow release kinetics of encapsulated ovalbumin in phosphate buffer indicates that most of the protein was encapsulated within the polymer matrix. Degradation of PMM 2.1.2 microparticles in the presence of esterases indicated that side chain hydrolysis of the polymer was the rate-determining step in bioerosion; cleavage of the ester side chain, which was further hydrolyzed to glycolic acid and ethanol, led to an acrylic acid and subsequent solubilization of the polymer. However, slow polymer backbone solubilization after degradation was observed.

Influence of the co-encapsulation of different non-ionic surfactants on the properties of PLGA insulin-loaded microspheres.

The aim of this work was to produce insulin-loaded microspheres allowing the preservation of peptide stability during both particle processing and insulin release. Our strategy was to combine the concepts of using surfactants to improve insulin stability while optimising overall microsphere characteristics such as size, morphology, peptide loading and release. Bovine insulin was encapsulated within poly(lactide-co-glycolide) (PLGA 50:50, Resomer RG504H) microspheres by the multiple emulsion-solvent evaporation technique. Microspheres were prepared by adding to the primary emulsion three non-ionic surfactants, poloxamer 188, polysorbate 20 and sorbitan monooleate 80, at different concentrations (1.5 and 3. 0% w/v). The presence of surfactants was found to decrease the mean diameter and to affect the morphology of the microspheres. Insulin encapsulation efficiency was reduced in the presence of surfactants and especially for sorbitan monooleate 80, in a concentration-dependent mode. The influence of the surfactants on the interactions between insulin and PLGA together with the primary emulsion stability were found to be the major determinants of insulin encapsulation. The release of insulin from microspheres was biphasic, showing an initial burst effect followed by a near zero-order release for all the batches prepared. The initial burst was related to the presence of insulin molecules located onto or near to the microsphere surface. In the presence of surfactants, a faster insulin release with respect to microspheres encapsulating insulin alone was observed. Insulin stability within microspheres after processing, storage and release was evaluated by reversed phase- and size-exclusion-HPLC. The analysis of microsphere content after processing and 6 months of storage showed that insulin did not undergo any chemical modification within microspheres. On the contrary, during the period of sustained release insulin was transformed in a high-molecular weight product, the amount of which was related to the surfactant used. In conclusion, polysorbate 20 at 3% w/v concentration was the most effective in giving regular shaped particles with both good insulin loading and slow release, and limiting insulin modification within microspheres.

Modulation of drug release from hydrogels by using cyclodextrins: the case of nicardipine/beta-cyclodextrin system in crosslinked polyethylenglycol.

A simple approach is presented to modulate drug delivery from swellable systems by using complexants. The effect of complexants has been interpreted by means of simple mass balances on diffusing species and the involved relevant parameters have been individuated. The application of this strategy to the release of nicardipine (NIC) from swellable systems by using beta-cyclodextrin (CD) as complexant has evidenced the potential of the approach to tailor drug release. Crosslinked polyethyleneglycol has been synthesized, characterized and used as the swellable matrix. Swelling kinetics, NIC and CD diffusivities in the swollen matrix and NIC/CD phase solubility studies have been performed. The polymer matrix has been loaded with pure NIC or with NIC and CD at different ratios and release kinetics evaluated. Release profiles have shown that the presence of CD significantly affected drug delivery by decreasing the effective diffusivity of NIC. The higher the CD/NIC ratio the slower is the release. This effect has been interpreted on the basis of the proposed model and physically sound assumptions.

Interactions of nonsteroidal antiinflammatory drugs with phospholipids: comparison between octanol/buffer partition coefficients and chromatographic indexes on immobilized artificial membranes.

A set of seventeen nonsteroidal antiinflammatory drugs (NSAIDs), consisting of structurally unrelated carboxylic acids and piroxicam, was examined by high-performance liquid chromatography (HPLC) on an immobilized artificial membrane (IAM) column that is a solid-phase model of fluid membranes. The chromatographic capacity factors extrapolated to 100% aqueous phase (log KWIAM) were compared with n-octanol/buffer lipophilicity parameters. The interactions with phospholipids were much better predicted from the intrinsic partition coefficient, log P, than from the apparent partition value, log D7.4, indicating that phospholipids can counteract the influence of electrically charged functions of analytes on lipophilic interactions. The log KWIAM and log P values for both NSAIDs and structurally unrelated neutral compounds result in unique scale if uniquely partition-based mechanisms take place. However, an electrostatic repulsion component was observed for the NSAIDs bearing the carboxylic function directly linked to the aromatic ring, and for ibuprofen. Hence, the IAM-derived scale is distinctive from the one obtained by lipophilic parameters. The IC50 values on cyclooxygenase 2 (COX-2) in intact cells determined by different authors have been successfully correlated with respective IAM parameters, whereas no correlation was found with COX-1 activity data. These results suggest that membrane affinity may represent an important prerequisite for the specific binding NSAIDs/COX-2.