Synthetic Glycopeptide-Based Delivery Systems for Systemic Gene Targeting to Hepatocytes

Purpose. To design, synthesize, and test synthetic glycopeptide-baseddelivery systems for gene targeting to hepatocytes by systemicadministration.Methods. All peptides were synthesized by the solid phase methoddeveloped using Fmoc chemistry on a peptide synthesizer. The bindingof galactosylated peptides to HepG2 cells and accessibility of thegalactose residues on particle surface was demonstrated by acompetition assay using ¹²⁵I-labeleld asialoorosomucoid and RCA lectinagglutination assay, respectively. DNA plasmid encoding chloramphenicolacetyl transferase (CAT) gene was complexed with a tri-galactosylatedpeptide (GM245.3) or tri-galactosylated lipopeptide (GM246.3) in thepresence of an endosomolytic peptide (GM225.1) or endosomolyticlipopeptide (GM227.3) to obtain DNA particles of 100–150 nm insize. The plasmid/peptide complexes were added to HepG2 cell culturesor intravenously administered by tail vein injection into normal miceor rats. Plasmid uptake and expression was quantified by qPCR andELISA, respectively.Results. Multiple antennary glycopeptides that have the ability tocondense and deliver DNA plasmid to hepatocytes were synthesized andcomplexed with DNA plasmid to obtain colloidally stable DNA/peptidecomplexes. Addition of DNA/GM245.3/GM225.1 peptide complexes(1:3:1 (–/+/–)) to HepG2 cell cultures yielded CAT expression intransfected cells. The transfection efficiency was significantly reducedin the absence of galactose ligand or removal of endosomolytic peptide.Intravenous administration of DNA/GM245.3 peptide complexes (1:0.5(–/+)) into the tail vein of normal rats yielded DNA uptake in theliver. Substitution of GM245.3 by galactosylated lipopeptide GM246.3resulted in more stable DNA particles, and a 10-fold enhancement inliver plasmid uptake. CAT expression was detectable in liver followingintravenous administration of DNA/GM246.3 complexes. Addition ofendosomolytic lipopeptide GM227.3 into the complexes(DNA/GM246.3/GM227.3 (1:0.5:1 (–/+/–))) yielded a 5-fold increase inCAT expression. Liver expression was 8-fold and 40-fold higher thanlung and spleen, respectively, and localized in the hepatocytes only.The transfection efficiency in liver was enhanced by increasing DNAdose and injection volume. The plasmid uptake and expression in liverusing DNA/GM246.3/GM227.3 complexes was 100-200-fold higherthan DNA formulated in glucose. Tissue examination and serumbiochemistry did not show any adverse effect of the DNA/GM246.3/GM227.3 (1:0.5:1 (–/+/–)) complexes after intravenous delivery.Conclusions. Gene targeting to hepatocytes was achieved by systemicadministration of a well-tolerated synthetic glycopeptide-baseddelivery system. The transfection efficiency of this glycopeptide deliverysystem was dependent on peptide structure, endosomolytic activity,colloidal particle stability, and injection volume.     

Advantages of Day 4 Embryo Transfer in Patients Undergoing Preimplantation Genetic Diagnosis of Aneuploidy

Purpose: Following preimplantation genetic diagnosis of aneuploidy, embryo transfer was executed on day 4, with the aim of providing more time for expanding from six to nine the number of diagnosed chromosomes per single cell (Group 2; 45 cycles). The results obtained were compared to those derived from conventional day 3 transfer (Group 1; 71 cycles). Methods: For multicolor fluorescence in situ hybridization analysis, two panels of probes were used: the first, specific for chromosomes XY, 13, 16, 18, and 21, was tested in all patients (Groups 1 and 2); the second was implemented only in Group 2 patients for the detection of chromosomes 14, 15, and 22. Results: A total of 406 embryos underwent fluorescence in situ hybridization analysis in Group 1, and 236 in Group 2. Comparable percentages of both chromosomal abnormalities (61% and 62%) and pregnancy and implantation rates (36% and 24.5% in Group 1, 41% and 23.6% in Group 2) resulted, regardless of the higher mean age in Group 2. Conclusions: The diagnosis of the nine chromosomes which are most frequently associated with aneuploidy in humans could have an immediate impact on the rate of spontaneous abortions. Additional advantages are represented by the more accurate morphological evaluation of euploid embryos; the advanced compaction, which means that embryos are less exposed to damage during the transfer procedure; and the possibility of performing a reanalysis in cases where a fluorescence in situ hybridization diagnosis is not obtained.     

Annexin V inhibits the 12-O-tetradecanoylphorbol-13-acetate-induced activation of Ras/extracellular signal-regulated kinase (ERK) signaling pathway upstream of Shc in MCF-7 cells

Annexin V is a Ca2+-dependent phospholipid binding protein. Although it has been shown to inhibit protein kinase C (PKC) in cell-free systems, its role in the intact cell is unclear. A stable MCF-7 human breast cancer cell overexpression system was established to investigate the function of annexin V. In these cells, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation and kinase activity of ERK1/2 were suppressed. Morphological changes induced by TPA were reduced by annexin V overexpression as well as by the pan-PKC inhibitor, bisindolylmaleimide I, and by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor, PD98059. TPA-induced MEK1/2 and Raf-1 phosphorylation were reduced in these cells. The TPA-enhanced active Ras, and its association with Raf-1, were reduced. TPA treatment of MCF-7 cells caused an increased association of Shc with Grb2. However, this increased association was prevented in the annexin V-overexpressors. p21WAF/CIP1 is responsible for inhibition of cell cycle progression in MCF-7 cells. TPA induced the expression of p21WAF/CIP1 to a greater extent in MCF-7 parent and control plasmid cells than in annexin V overexpressors. PD98059 inhibited this increase, suggesting that TPA upregulation of p21WAF/CIP1 occurs via the MEK pathway, and that annexin V overexpression blunts it. This work shows that annexin V overexpression suppresses the TPA-induced Ras/ERK signaling by inhibiting at/or upstream of Shc, possibly through the inhibition of PKCs. Oncogene (2000).     

Prevalence of Infections After In-Office Hysteroscopy in Premenopausal and Postmenopausal Women

Study Objective

To estimate the incidence of infection after diagnostic and operative hysteroscopic procedures performed in an in-office setting with different distension media (saline solution or CO₂).

Transitions in auditory rehabilitation with bone conduction implants (BCI)

Background: The bone conductive implants (BCI) are nowadays a reliable alternative for rehabilitation of specific forms of hearing loss, i.e. conductive, mixed or single sided deafness (SSD).

Aims/Objective: To analyse the various factors in play when considering an auditory rehabilitation with a bone-conductive device (BCI).

Materials and Methods: The clinical charts of subjects who underwent BCI application at the same Implanting Center from 2005 to 2018 were retrieved analysing also the reason for eventual explantation and the alternative option (transition) for hearing rehabilitation.

Results: Nine BAHA Compact, 4 BAHA Intenso, 21 BAHA Divino, 3 BAHA BP100, 4 Ponto, 2 Sophono, 5 Bonebridge, 5 BAHA5 Attract; 11 BAHA5 Connect were used in 12 unilateral COM; 16 bilateral COM; 3 unilateral cholesteatoma; 6 bilateral cholesteatoma; 2 unilateral otosclerosis; 5 bilateral otosclerosis; 9 congenital malformations; 6 major otoneurosurgical procedures; 5 sudden deafness. Explantation was necessary for five subjects.

Conclusions: Middle ear pathology and sequels from surgery represent the most common reason for BCI implantation, both in unilateral and in bilateral cases. Transition from one implantable device to another one can be predictable, mostly when explantation is necessary.

Significance: The role of BCI for rehabilitation in middle ear pathology may be extremely important.     

Smoking habits, exposure to passive smoking and attitudes to a non-smoking policy among hospital staff

A survey was carried out into the smoking habits and exposure to passive smoking among health staff in the hospitals of Faenza, Forli and Rimini (Emilia-Romagna, Northern Italy). 2453 subjects answered anonymously a 41 question questionnaire. 53% of the subjects were professional nurses, 16% doctors, 15% maintenance staff, 10% ancillary staff, 1% non-medical graduates, 2% were administrators and 3% were assigned to the category ‘others’.Of the subjects answering the questionnaire 39% were smokers, 19% ex-smokers and 42% non smokers. The highest number of smokers was found among women (41%) compared to men (37%) and among ancillary staff (48%) compared to nurses (41%) and doctors (31%). The males were mostly heavy smokers (≥20 cigarettes/d) and smoked strong cigarettes (≥12 mg/cig condensate content). The females were mostly light smokers (<10 cigarettes/d) and smoked light cigarettes (1–6 mg/cig condensate content). A high percentage of subjects (87%) smoked at work especially in areas reserved for staff. 43% and 26% of shift workers and non-shift workers tended not to modify their habit when on morning or afternoon shifts. During night shifts the majority of them increased their tobacco consumption.Around 87% of hospital employees stated they were exposed to passive smoking inside the hospital especially in cooking areas, at information desks and corridors. Nurses, ancillaries and maintenance staff were those most exposed and for a greater number of hours per day compared to doctors. Almost all subjects were aware of the harm caused by passive smoking. 56% of smokers, 65% of ex-smokers and 72% of non smokers said they were willing to participate in future campaigns to limit smoking in their hospitals.     

Pure red cell aplasia of pregnancy: a distinct clinical entity

Summary. We describe a 31-year-old patient with pure red cell aplasia of pregnancy, successfully managed with regular blood transfusions. In vitro studies showed specific inhibition of day 14 erythroid colonies (BFU-E) using serum and purified immunoglobulin G (IgG) obtained from the patient at diagnosis (before blood transfusion). The inhibition of BFU-E disappeared when haematological remission occurred 3 weeks after delivery and she remains clinically well with a normal haemoglobin 4 years later.     

Different neurologic outcomes in two patients with neonatal hyperinsulinemic hypoglycemia

The authors report the cases of two patients with neonatal onset of hypoketotic hypoglycemia with hyperinsulinism and a poor response to diazoxide. Pancreatic venous sampling showed a diffuse pancreatic hyperplasia in one patient and a focal lesion in the other. The second patient, diagnosed after a significant delay, suffered severe long-term neurological sequelae, despite having more limited hyperplasia; the brain MRI also showed severe pathological changes in cortex and white matter, predominantly in the parietooccipital region. Early and accurate diagnosis is critical in these patients in whom hypoglycemia is compounded by a lack of the ketone bodies which represent a vital alternative source of energy for the central nervous system.