Hybrid supramolecular gels of Fmoc-F/halloysite nanotubes: systems for sustained release of camptothecin.

Supramolecular gel hybrids obtained by self-assembly of Fmoc-l-phenylalanine (Fmoc-F) in the presence of functionalized halloysite nanotubes (f-HNT) were obtained in biocompatible solvents and employed as carriers for the delivery of camptothecin (CPT) molecules. The synthesis of the new f-HNT material as well as its characterization are described. The properties of the hybrid hydrogels and organogels were analyzed by several techniques. The presence of small amounts of f-HNT allows good dispersion of the tubes and the subsequent formation of homogeneous gels. The experimental results show that f-HNT functions only as an additive in the hybrid gels and does not demonstrate gelator behavior. The in vitro kinetic release from both f-HNT/CPT and Fmoc-F/f-HNT/CPT was studied in media that imitates physiological conditions, and the factors controlling the release process were determined and discussed. Furthermore, the antiproliferative in vitro activities of the gels were evaluated towards human cervical cancer HeLa cells. A comparison of data collected in both systems shows the synergistic action of f-HNT and the gel matrix in controlling the release of CPT in the media and maintaining the drug in its active form. Finally, a comparison with pristine HNT is also reported. This study suggests a suitable strategy to obtain two-component gel hybrids based on nanocarriers with controlled drug carrier capacity for biomedical applications.

Halloysite nanotubes loaded with peppermint essential oil as filler for functional biopolymer film.

The purpose of this paper is to show how a functional bionanocomposite film with both antioxidant and antimicrobial activities was successfully prepared by the filling of a pectin matrix with modified Halloysite nanotubes (HNT) containing the essential peppermint oil (PO). Firstly, HNT surfaces were functionalized with cucurbit[6]uril (CB[6]) molecules with the aim to enhance the affinity of the nanofiller towards PO, which was estimated by means of HPLC experiments. The HNT/CB[6] hybrid was characterized by several methods (thermogravimetry, FT-IR spectroscopy and scanning electron microscopy) highlighting the influence of the supramolecular interactions on the composition, thermal behavior and morphology of the filler. Then, a pectin+HNT/CB[6] biofilm was prepared by the use of the casting method under specific experimental conditions in order to favor the entrapment of the volatile PO into the nanocomposite structure. Water contact angle measurements, thermogravimetry and tensile tests evidenced the effects of the modified filler on the thermo-mechanical and wettability properties of pectin, which were correlated to the microscopic structure of the biocomposite film. In addition, PO release in food simulant solvent was investigated at different temperatures (4 and 25°C), whereas the antioxidant activity of the nanocomposite film was estimated using the DPPH method. Finally, we studied the in vitro antibacterial activity of the biofilm against Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive), which were isolated by beef and cow milk, respectively. These experiments were carried out at specific temperatures (4, 37 and 65°C) that can be useful for a multi-step food conservation. This paper puts forwards an easy strategy to prepare a functional sustainable edible film with thermo-sensitive antioxidant/antimicrobial activity.

Differential regulation of Fas-mediated apoptosis in both thyrocyte and lymphocyte cellular compartments correlates with opposite phenotypic manifestations of autoimmune thyroid disease.

Several mechanisms are probably involved in determining the evolution of autoimmune thyroid disease (AITD) towards either hypothyroidism and the clinical syndrome known as Hashimoto's thyroiditis (HT) or toward hyperthyroidism and the symptoms of Graves' disease (GD). To gain further insight into such mechanisms we performed an exhaustive comparative analysis of the expression of key molecules regulating cell death (Fas, Fas ligand [FasL], Bcl-2) and apoptosis in both thyrocytes and thyroid infiltrating lymphocytes (TILs) from patients with either GD or HT. GD thyrocytes expressed less Fas/FasL than HT thyrocytes, whereas GD TILs had higher levels of Fas/FasL than HT TILs. GD thyrocytes expressed increased levels of the antiapoptotic molecule Bcl-2 compared to the low levels detected in HT thyrocytes. The opposite pattern was observed in GD (low Bcl-2) and HT (high Bcl-2) TILs. The patterns of apoptosis observed were consistent with the regulation of Fas, FasL, and Bcl-2 described above. Our findings suggest that in GD thyroid the regulation of Fas/FasL/Bcl2 favors apoptosis of infiltrating lymphocytes, possibly limiting their autoreactive potential and impairing their ability to mediate tissue damage. Moreover, the reduced levels of Fas/FasL and increased levels of Bcl-2 should favor thyrocyte survival and favor the thyrocyte hypertrophy associated with immunoglobulins stimulating the thyrotropin (TSH) receptor. In contrast, the regulation of Fas/FasL/Bcl2 expression in HT promotes thyrocyte apoptosis, tissue damage, and a gradual reduction in thyrocyte numbers leading to hypothyroidism. These findings help define key molecular mechanisms contributing to the clinical outcome of thyroid autoimmunity.

Inhibitory effect of neuraminidase on SP-induced histamine release and TNF-alpha mRNA in rat mast cells: evidence of a receptor-independent mechanism.

The neuropeptide substance P (SP) is a mediator of neuro-inflammation and can play a role by induction of histamine release (HR) and TNF-alpha. However, its effect on the heterogeneous response of mast cells (MC) has not been completely studied. We have established that the SR can induce 25% of HR in highly purified rat uterine MC at diestrous but not at proestrous phases of the reproductive cycle and 88% of HR in peritoneal mast cells (PMC). We also found 2.2 fold increase in TNF-alpha mRNA at diestrous, in SP stimulated uterine MC versus control and 2.7 fold increase in PMC; RT and competitive PCR were used to amplify the TNF-alpha mRNA. We have thereafter investigated the mechanism whereby the binding of SP to sialic acid on the MC membrane, could trigger secretion of histamine and induction of TNF-alpha mRNA. The neuraminidase pretreatment (0.1 U/ml) inhibited SP-stimulated HR from either uterine MC and PMC (98% and 50%, respectively) and totally inhibited SP-stimulated TNF-alpha mRNA levels. The neuraminidase effect was not toxic, since it was not observed in IgE mediated HR and TNF-alpha mRNA levels. In conclusion, the inhibitory effect of the neuraminidase on the SP-mediated increase of histamine and TNF-alpha mRNA, suggests that the SP-sialic acid interaction could have a role in the MC heterogeneous response.

A factor secreted by human embryo stimulates cytokine release by uterine mast cell.

The existence of a biochemical network of embryo-maternal communication implies that various secreted molecules constitute a signal-response mechanism, important for the process of embryo implantation in mammals. Here we report the purification of a protein with an apparent molecular weight of 136 kDa, responsible for a 2000-fold increase in embryo-derived histamine-releasing factor (EHRF) activity. This protein, purified from medium from the in-vitro culture of 2-8-cell human embryos, by means of affinity chromatography, was capable of binding immunoglobulin (Ig)E as demonstrated by immunoblotting and enzyme-linked immunosorbent assays. We found EHRF was capable of inducing release of histamine and cytokines in vitro from rat uterine tissue, collected on day 4 of pregnancy (preimplantation stage of embryo development). When EHRF was used as a secretagogue, granulocyte macrophage-colony stimulating factor (GM-CSF) release increased from 3 to 55 pg/g (P < 0.01) and tumour necrosis factor-alpha (TNF-alpha) release increased from 0 to 2.1 ng/g (P < 0.01), as detected by enzyme-linked immunosorbent assay. A simple method was used to purify uterine mast cells using an IgE-Sepharose affinity chromatography column and the purity (90%) was checked with Dynabeads coupled to specific rat IgE antibody. When purified mast cells were stimulated with EHRF in the same way as the uterine explants, a similar pattern of GM-CSF and TNF-alpha release was obtained. We also describe the reverse transcription-polymerase chain reaction (RT-PCR) of GM-CSF and TNF-alpha mRNA from purified uterine mast cells. On day 4 of pregnancy only the mRNA of TNF-alpha was found and this increased after stimulation with the EHRF. In conclusion, the data presented suggest that uterine mast cells isolated during the preimplantation stage release cytokines in vitro following interaction with an embryo factor.

Effect of substance P on uterine mast cell cytokine release during the reproductive cycle.

There is increasing evidence that neuropeptides, steroid hormones and inflammatory cytokines influence the immune response during the reproductive cycle. In the present study, we focus on the effects of neuropeptide Substance P (SP) during the pre-implantation stage of embryo development (day 4 of pregnancy), at pro-estrus and di-estrus (two phases with different hormonal states). We found heterogeneous responses to SP and anti-IgE by the rat uterine mast cells (MCs), as detected by ELISA. In fact, MCs purified from uteri on day 4 of pregnancy released histamine, granulocyte macrophage-colony stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) in response to anti-IgE, but not to SP. When pre-incubated with SP, the release to anti-IgE was significantly enhanced compared to anti-IgE alone. Exposure of SP to antibodies to SP, prior to pre-incubation with MCs, negated the SP effect on IgE-mediated release. At the pro-estrus phase SP showed similar behavior as on day 4 of pregnancy, whereas at the di-estrus phase SP alone was capable of inducing release of histamine and cytokines from purified uterine MCs. Moreover, non-quantitative RT-PCR analysis of the TNF-alpha mRNA level suggested an SP stimulation at the di-estrus phase, but neither on day 4 of pregnancy nor at the pro-estrus phase. Taken together, these data strongly suggest that SP can modulate IgE-mediated uterine MC release of histamine and inflammatory cytokines in different ways, depending on the phase of the reproductive cycle.

Mutagenic alteration of the distal switch II region of RAS blocks CDC25-dependent signaling functions.

We have explored the role of the distal switch II region of the yeast RAS2 protein in determining the response to the nucleotide exchange factor CDC25. We first constructed yeast tester strains in which the deletion of the chromosomal CDC25, RAS1, and RAS2 genes, in combination with the chromosomal suppressor CRI4, resulted in detectable phenotypes in vivo and in vitro. Phenotypes included impaired growth at 37 degrees C, defective glucose-induced cyclic AMP signaling, and low adenylyl cyclase activity of membrane preparations. Tester strains were subsequently used for the reintroduction of various combinations of wild-type and mutated RAS2 and CDC25 genes by genetic techniques, as well as for in vitro reconstitution assays with the corresponding proteins. CDC25 restored both growth and glucose-induced cyclic AMP signaling in the presence, but not in the absence of wild-type RAS2. A gene encoding a RAS2 protein with a mutationally altered switch II region was expressed but was ineffective in reintegrating exchange factor-dependent responses in vivo. Wild-type, but not mutagenically altered, RAS2 proteins were stimulated by exchange factors in vitro. We conclude that the conserved distal switch II region is required for CDC25-dependent activation of RAS.

Smouldering hepatitis B virus replication in patients with chronic liver disease and hepatitis delta virus superinfection.

Hepatitis B virus deoxyribonucleic acid (HBV-DNA) was studied by Southern blot analysis in liver biopsy specimens from 75 HBsAg-positive patients with chronic liver disease living in southern Italy. Twenty-seven of the patients were hepatitis delta virus (HDV) superinfected. Intrahepatic HBV-DNA was detected in 54 (72%) patients, 32 (59%) of them with replicative forms. The presence of replicative forms was directly related to liver HBcAg and inversely related to liver HDAg, as shown by multivariate analysis. However, 14 patients with intrahepatic HBV-DNA non-replicative pattern and about half of HDV-infected patients were liver HBcAg and/or serum HBV-DNA positive, mostly in low amounts. Histological inflammatory activity was strongly related to liver HBcAg expression regardless of HDV superinfection, as confirmed by multivariate analysis. Our results confirm previous studies about the concordance between intrahepatic HBV-DNA replicative pattern and liver HBcAg expression and about inhibition by HDV of high-level HBV replication. However, they suggest that low-level HBV replication may have an important role in causing liver damage also among HDV-infected patients, in a population where the spreading of HBV and HDV is a naturally occurring event.