Lipid nanoparticles (SLN & NLC) for delivery of vitamin E: a comprehensive review.

The antioxidative and photoprotective properties of vitamin E have caused it to be included as an active agent in various pharmaceutical and cosmetic products. However, its lipophilicity, chemical instability and poor skin penetration have limited the effectiveness of these formulations. For that reason, many attempts to include it in different drug delivery systems have been made. In recent decades, lipid nanoparticles have received special attention due to their advantages of compatibility with the skin, ability to enhance penetration of drugs in the stratum corneum, protection of the encapsulated substance against degradation induced by the external medium and control of drug release. This work reviews the current status of the encapsulation of vitamin E in lipid nanoparticles. We describe the most important methods for obtaining and characterizing lipid nanoparticles containing vitamin E (LNP-VE), various techniques for the evaluation of vitamin E's properties after encapsulation, the main in vitro and in vivo studies of the potential effectiveness or toxicity of LNP-VE, the formulations and stability studies of this delivery system, the commercial products based on LNP-VE and the regulatory aspects related to lipid nanoparticles. Finally, we discuss the most relevant advantages of encapsulating vitamin E in such particles and critical aspects that still demand attention to enhance the potential of solid lipid nanoparticles to deliver vitamin E.

Comparing in vivo biodistribution with radiolabeling and Franz cell permeation assay to validate the efficacy of both methodologies in the evaluation of nanoemulsions: a safety approach.

The Franz cells permeation assay has been performed for over 25 years. However, the advent of nanotechnology created a whole new world, especially with regard to topical products. In this new global scenario an increasing number of nanostructure-based delivery systems (NDSs) have emerged and a global warning relating to the safety of these NDSs is arising. This work studied the efficacy of the Franz cells assay, comparing it with the radiolabeling biodistribution test. For this purpose a formulation of sunscreen based on an NDS was developed and characterized. The results demonstrated both that the NDS did not present in vitro cytotoxicity and that the radiolabeling biodistribution test is more precise for the evaluation of NDS cosmetics than the Franz cells assay, since it detected the permeation of the NDS at a picogram order. Due to this fact, and considering all the concerns related to NDSs and nanoparticles in general, more precise methods must be used in order to guarantee the safe use of these new classes of products.

A sham stimulation-controlled trial of rTMS of the unaffected hemisphere in stroke patients.

The authors investigated the use of slow-frequency repetitive transcranial magnetic stimulation (rTMS) to the unaffected hemisphere to decrease interhemispheric inhibition of the lesioned hemisphere and improve motor function in patients within 12 months of a stroke. Patients showed a significant decrease in simple and choice reaction time and improved performance of the Purdue Pegboard test with their affected hand after rTMS of the motor cortex in the intact hemisphere as compared with sham rTMS.

Relationship between spore germination kinetics and lag time during growth of Mucor racemosus.

AIMS: Spore germination requires microscopic observation whereas fungal growth results in a macroscopic examination. This paper aims at establishing a relationship between the percentage of germinated spores and parameters easily available from visible development. METHODS AND RESULTS: About 225 spores of Mucor racemosus were inoculated on PDA medium and incubated at 15 degrees and 25 degrees C. Germination kinetics were modelled by a logistic function. Fungal development provided two parameters, a growth rate, micro, and a lag period, lambda, defined as the slope of the straight line of the graph radius (mm) vs time (h) and the intercept of this line with the X-axis, respectively. CONCLUSIONS: It was found that the lag period coincided with the completion of the germination process, although the number of spores inoculated should be controlled carefully. SIGNIFICANCE AND IMPACT OF THE STUDY: Providing that this result can be generalized, this procedure would constitute a significant breakthrough for predicting food spoilage by moulds.

Comparison of the effects of temperature and water activity on growth rate of food spoilage moulds.

The influence of temperature (T) and water activity (aw) on the growth rate (mu) of seven moulds (Alternaria alternata, Aspergillus flavus, Cladosporium cladosporioides, Mucor racemosus, Penicillium chrysogenum, Rhizopus oryzae and Trichoderma harzianum) was assessed in suboptimal conditions. Firstly, the dependence of fungal growth on temperature, at aw 0.99, was modelled through an approach described previously for bacteria. A dimensionless growth rate variable: mu(dimalpha)=mu/mu (optalpha) depended on the following normalised temperature: T(dim)=(T-T(min))/(T(opt)- T(min)) according to a power function: mu(dimalpha)=[T(dim)]alpha, where alpha was an exponent to be estimated. Secondly, the same approach was used to describe the influence of aw on fungal growth, at the respective optimum temperatures for each mould. Similarly, mu(dimbeta)=mu/mu(optbeta) depended on the following normalised water activity: a(wdim)=(aw-a(wmin))/(a(wopt)-a(wmin)) according to a power function: mu(dimbeta)=[a(wdim)](beta). Results show: (i) for each mould, the alpha-value is significantly less than the beta-value, confirming that water activity has a greater influence than temperature on fungal development; (ii) the alpha-values and the beta-values depend on the mould; (iii) the alpha-value is less than 1 for the mesophilic mould A. flavus, whereas the other moulds are characterised by higher alpha-values ranging from 1.10 to 1.54; (iv) the mesophilic A. flavus exhibits a low beta-value, 1.50, compared to the hydrophilic T. harzianum, beta=2.44, while beta-values are within the range (1.71-2.37) for the other moulds.

AMF1 (GPS2) modulates p53 transactivation.

We have reported that the papillomavirus E2 protein binds the nuclear factor AMF1 (also called G-protein pathway suppressor 2 or GPS2) and that their interaction is necessary for transcriptional activation by E2. It has also been shown that AMF1 can influence the activity of cellular transcription factors. These observations led us to test whether AMF1 regulates the functions of p53, a critical transcriptional activator that integrates stress signals and regulates cell cycle and programmed cell death. We report that AMF1 associates with p53 in vivo and in vitro and facilitates the p53 response by augmenting p53-dependent transcription. Overexpression of AMF1 in U2OS cells increases basal level p21(WAF1/CIP1) expression and causes a G(1) arrest. U2OS cells stably overexpressing AMF1 show increased apoptosis upon exposure to UV irradiation. These data demonstrate that AMF1 modulates p53 activities.

Multiple functions of human papillomavirus type 16 E6 contribute to the immortalization of mammary epithelial cells.

The E6 proteins from cervical cancer-associated human papillomavirus (HPV) types such as HPV type 16 (HPV-16) induce proteolysis of the p53 tumor suppressor protein through interaction with E6-AP. We have previously shown that human mammary epithelial cells (MECs) immortalized by HPV-16 E6 display low levels of p53. HPV-16 E6 as well as other cancer-related papillomavirus E6 proteins also binds the cellular protein E6BP (ERC-55). To explore the potential functional significance of these interactions, we created and analyzed a series of E6 mutants for their ability to interact with E6-AP, p53, and E6BP in vitro. While there was a similar pattern of binding among these E6 targets, a subset of mutants differentiated E6-AP binding, p53 binding, and p53 degradation activities. These results demonstrated that E6 binding to E6-AP is not sufficient for binding to p53 and that E6 binding to p53 is not sufficient for inducing p53 degradation. The in vivo activity of these HPV-16 E6 mutants was tested in MECs. In agreement with the in vitro results, most of these p53 degradation-defective E6 mutants were unable to reduce the p53 level in early-passage MECs. Interestingly, several mutants that showed severely reduced ability for interacting with E6-AP, p53, and E6BP in vitro efficiently immortalized MECs. These immortalized cells exhibited low p53 levels at late passage. Furthermore, mutants defective for p53 degradation but able to immortalize MECs were also identified, and the immortal cells retained normal levels of p53 protein. These results imply that multiple functions of HPV-16 E6 contribute to MEC immortalization.

p300/MDM2 complexes participate in MDM2-mediated p53 degradation.

Control of p53 turnover is critical to p53 function. E1A binding to p300/CBP translates into enhanced p53 stability, implying that these coactivator proteins normally operate in p53 turnover control. In this regard, the p300 C/H1 region serves as a specific in vivo binding site for both p53 and MDM2, a naturally occurring p53 destabilizer. Moreover, most of the endogenous MDM2 is bound to p300, and genetic analysis implies that specific interactions of p53 and MDM2 with p300 C/H1 are important steps in the MDM2-directed turnover of p53. A specific role for p300 in endogenous p53 degradation is underscored by the p53-stabilizing effect of overproducing the p300 C/H1 domain. Taken together, the data indicate that specific interactions between p300/CBP C/H1, p53, and MDM2 are intimately involved in the MDM2-mediated control of p53 abundance.

The domain of p53 required for binding HPV 16 E6 is separable from the degradation domain.

The E6 proteins of specific cancer-associated human papillomaviruses (HPVs) complex with and mediate degradation of the cellular anti-oncogene p53 in vitro. A critical property of p53 is its ability to stimulate transcription from promoters containing its recognition sequence. HPV E6, mutant p53 proteins, and several DNA tumor virus oncogenes inhibit the transcriptional activity of wild-type p53. In this report, the structural requirements for the interaction between HPV 16 E6 and p53 were examined both in vivo and in vitro. p53-stimulated transcription was efficiently inhibited by wild-type HPV 16 E6 and E6 mutants competent for p53 binding and degradation. A series of p53 deletions and hybrid proteins with heterologous DNA binding, dimerization and transactivation domains were analysed for transcriptional interaction with HPV 16 E6 to determine the domains of p53 required for transcriptional inhibition. These chimeric proteins were also analysed for E6 binding and E6-mediated degradation in vitro. In both assays, complex formation with E6 was mediated through the amino-terminal 345 amino acids of p53 without a specific requirement for its C-terminus. Hybrid proteins containing residues 161-345 of p53 also bound E6, but this segment of p53 was not susceptible to E6 induced proteolysis. A second region of p53, within its N-terminal 160 aa, is required for E6 induced degradation of complexed p53. Taken together, these results suggest that the complex formation between E6 and p53 is not mediated through the C-terminus of p53 and that binding and degradation are separable.

Placental and fetal Doppler velocimetry in pregnancies complicated by maternal diabetes mellitus.

OBJECTIVE: Our purpose was to investigate placental and fetal circulation in pregnancies complicated by maternal diabetes mellitus and to relate any changes to fetal blood pH, Po2, and hematocrit. STUDY DESIGN: Doppler measurements of both uterine arteries, one umbilical artery, the fetal descending thoracic aorta, and one fetal middle cerebral artery were performed in 65 well-controlled diabetic pregnancies in a cross-sectional study at the Harris Birthright Research Centre for Fetal Medicine, London. In 41 cases cordocentesis was also performed for the measurement of umbilical venous blood pH, Po2, and hematocrit. RESULTS: The mean umbilical venous blood pH was significantly lower and the hematocrit significantly higher than the appropriate normal mean for gestation. However, the Doppler indices of the placental and fetal circulations were essentially normal, except in some of the cases complicated by preeclampsia or intrauterine growth retardation. CONCLUSIONS: Maternal diabetes mellitus is not associated with abnormalities in Doppler indexes of the placental or fetal circulations.

Fetal B lymphocyte subpopulations in normal pregnancies.

In 190 pregnancies undergoing cordocentesis for prenatal diagnosis (n = 174) or elective caesarean section (n = 16), fetal peripheral blood B lymphocyte subpopulations were measured using a fluorescence-activated cell sorter (FACScan). The total number of B lymphocytes and polyreactive CD5+ B cells increased exponentially with gestation from respective means of 0.33 x 10(9)/l and 0.25 x 10(9)/l at 17 weeks to a plateau of 0.66 x 10(9)/l and 0.54 x 10(9) at 36 weeks, remaining at that level thereafter. The number of mature CD10- and active CD23+ B lymphocytes increased linearly from a mean of 0.07 x 10(9)/l and 0.11 x 10(9)/l at 17 weeks to 0.24 x 10(9)/l and 0.37 x 10(9)/l, respectively, at 40 weeks. As expected, all B lymphocytes expressed the HLA-DR antigen from as early as 16 weeks gestation. These alterations in specific B lymphocyte subpopulations reflect the pattern of maturation and development of the fetal humoral immune system.

Fetal nuchal translucency: ultrasound screening for chromosomal defects in first trimester of pregnancy.

OBJECTIVE: To examine the significance of fetal nuchal translucency at 10-14 weeks' gestation in the prediction of abnormal fetal karyotype. DESIGN: Prospective screening study. SETTING: The Harris Birthright Research Centre for Fetal Medicine, King's College Hospital, London. SUBJECTS: 827 fetuses undergoing first trimester karyotyping by amniocentesis or chorionic villus sampling. MAIN OUTCOME MEASURE: Incidence of chromosomal defects. RESULTS: The incidence of chromosomal defects was 3% (28 of 827 cases). In the 51 (6%) fetuses with nuchal translucency 3-8 mm thick the incidence of chromosomal defects was 35% (18 cases). In contrast, only 10 of the remaining 776 (1%) fetuses were chromosomally abnormal. CONCLUSION: Fetal nuchal translucency > or = 3 mm is a useful first trimester marker for fetal chromosomal abnormalities.

Fetal T-lymphocyte subpopulations in normal pregnancies.

Peripheral blood T lymphocyte subpopulations were measured, using a fluorescence-activated cell sorter, in fetal blood samples obtained either by cordocentesis (n = 118) or at elective caesarean section (n = 14). Both the numbers and percentages of the total T lymphocytes (CD3+) and T-helper lymphocytes (CD4+) increased exponentially with gestation from respective means of 46% (1.15 x 10(9)/l) and 29% (0.70 x 10(9)/l) at 16 weeks to a plateau of 75% (3.11 x 10(9)/l) and 54% (2.10 x 10(9)/l) at 34 weeks. Similarly, the number of suppressor/cytotoxic T lymphocytes (CD8+) increased linearly with gestation from a mean of 22% (0.55 x 10(9)/l) at 16 weeks to 24% (0.96 x 10(9)/l) at 40 weeks; there were no natural cytotoxic T lymphocytes (CD3+CD56+) in any of the fetal blood samples. The helper-to-suppressor T lymphocyte ratio (CD4/CD8) increased exponentially with gestation from a mean of 1.22 at 16 weeks to 2.57 at 28 weeks. The alterations in T lymphocyte subpopulations were accompanied by changes in the expression of CD45RA, L-selectin, CD25 and HLA-DR. These alterations in T lymphocyte subpopulations with gestation reflect the pattern of maturation and development of the fetal cell-mediated immune system.

Regulation of human melanocyte growth, dendricity, and melanization by keratinocyte derived factors.

Epidermal pigmentation involves the synthesis of melanin in melanocytes and its transfer to surrounding keratinocytes, where it functions in photoprotection. To investigate the possible role of the keratinocyte in regulating pigmentation, human keratinocytes were incubated for 24 h in a defined culture medium, which was then transferred to pure human melanocyte cultures. After 1 week, the conditioned medium produced a fourfold increase in melanocyte yield and a seven-fold increase in total melanin. Increased melanocyte dendricity was clearly visible within 24 h as well. Ultrafiltration of the keratinocyte-conditioned medium suggested approximately one-half of the growth promoting activity as well as most of the dendricity and melanization stimulating activities were of low molecular weight (less than 500 Da). High molecular weight fractions stimulated only melanocyte growth. Of the several known keratinocyte-derived factors tested, none could be implicated as a mediator of the observed effects. Basic fibroblast growth factor, known to stimulate melanocyte growth in some culture systems, failed to stimulate growth, dendricity, or melanin content when added to the complete non-conditioned medium. Interleukin-1 alpha, interleukin-1 beta, 12-hydroxyeicosatetraenoic acid, prostaglandin E2, leukotriene B4, and adenosine 3',5'-cyclic monophosphate analogues also had no effect. These studies demonstrate that keratinocytes in vitro release factors that modulate melanocyte behavior and expand our understanding of controls for human epidermal pigmentation.

Induction of nerve growth factor receptors on cultured human melanocytes.

Normal differentiation and malignant transformation of human melanocytes involve a complex series of interactions during which both genetic and environmental factors play roles. At present, the regulation of these processes is poorly understood. We have induced the expression of nerve growth factor (NGF) receptors on cultured human melanocytes with phorbol 12-tetradecanoate 13-acetate and have correlated this event with the appearance of a more differentiated, dendritic morphology. Criteria for NGF receptor expression included protein accumulation and cell-surface immunofluorescent staining with a monoclonal antibody directed against the human receptor and induction of the messenger RNA species as determined by blot-hybridization studies. The presence of the receptor could also be induced by UV irradiation or growth factor deprivation. The NGF receptor is inducible in cultured human melanocytes, and we suggest that NGF may modulate the behavior of this neural crest-derived cell in the skin.

Vitamin D, its precursors, and metabolites do not affect melanization of cultured human melanocytes.

Exposure of the skin to sunlight results in both tanning and vitamin D3 production. It has therefore been suggested that vitamin D3 or its active metabolite 1,25-dihydroxyvitamin D3 may be the mediator of UV-induced melanogenesis. To test this hypothesis, newborn foreskin-derived melanocytes were cultured in paired dishes in hormone-supplemented medium with 2% serum containing no detectable vitamin D3 or in the same medium containing 10(-8) or 10(-10) M of either provitamin D3, lumisterol, previtamin D3, vitamin D3, 25-hydroxyvitamin D3, or 1,25-dihydroxyvitamin D3. After 10 days, cell number in cultures containing vitamin D compounds was 93%-140% of unsupplemented controls and melanin content was 60%-120% of control, with no significant difference in either parameter for any compound tested. In separate experiments, human melanocytes and Cloudman S91 melanoma cells were repeatedly irradiated with physiologic doses of simulated sunlight and incubated between irradiations with provitamin D3, previtamin D3, vitamin D3, or 1,25-dihydroxyvitamin D3. Irradiated cultures had a 90%-95% inhibition of cell growth associated with a 200%-800% increase in melanin content per cell relative to controls, but there was no effect of any vitamin D compound on either cell type. Neither cultured human melanocytes nor S91 cells showed evidence of the cytosolic 1,25-dihydroxy-vitamin D3 receptor binding by sucrose density gradient analysis using radiolabeled 1,25-dihydroxyvitamin D3. The combined data strongly suggest that neither vitamin D3 nor its precursors or metabolites directly mediate melanogenesis in these cells.