Detection of clustered circulating tumour cells in early breast cancer.

Circulating tumour cell (CTC) clusters have been proposed to be major players in the metastatic spread of breast cancer, particularly during advanced disease stages. Yet, it is unclear whether or not they manifest in early breast cancer, as their occurrence in patients with metastasis-free primary disease has not been thoroughly evaluated. In this study, exploiting nanostructured titanium oxide-coated slides for shear-free CTC identification, we detect clustered CTCs in the curative setting of multiple patients with early breast cancer prior to surgical treatment, highlighting their presence already at early disease stages. These results spotlight an important aspect of metastasis biology and the possibility to intervene with anti-cluster therapeutics already during the early manifestation of breast cancer.

Efficacy of Teeth Whitening with a Mouthwash: In Vitro and In Vivo Approaches.

OBJECTIVES: Two different studies were conducted to evaluate the whitening efficacy of a mouthwash versus a placebo using in vitro and in vivomodels. The tested mouthwash was formulated with no oxidizing or abrasive agents containing chlorhexidine (CHX) and polyvinylpyrrolidone (PVP). METHODS: The purpose of the in vitro study was to determine whether the mouthwash formulation OC15AB could reduce the accumulation of staining in an accepted stain model. Bovine central incisors were cut to obtain enamel specimens of ~8 × 8 mm2. The specimens were then immersed in human saliva (room temperature, slight stirring) for one hour to allow a pellicle film to form. They were then placed in contact with a staining solution containing coffee and tea. The amount of stain (tooth color) was quantified photometrically (Minolta C221 colorimeter) using the L* value of the L*a*b* scale. The purpose of the in vivo study was to evaluate the whitening power and tolerability of OC15AB versus a placebo mouthwash in a double-blind, randomized clinical study. In total, 40 subjects were divided randomly into two homogeneous groups. Each group used a different mouthwash (OC15AB or placebo) for 56 consecutive days. During this period, clinical and instrumental parameters, namely variations in tooth color and mucosal and gum alterations, were evaluated. The in vivo study analyses used a two-sided Student's t-test. Evaluations within groups used t-tests for paired data. RESULTS: From the in vitro test, OC15AB had a significant effect in reducing stain accumulation over the entire treatment period. The in vivo test showed that OC15AB was well tolerated and had whitening power in the subjects. OC15AB demonstrated a statistically significant reduction in extrinsic tooth staining from baseline and versus the placebo. CONCLUSIONS: The in vitro and in vivo methods used to investigate the whitening efficacy of the mouthwash formulation produced similar and consistent results. The experimental model used is an important tool in the search for new technologies for teeth whitening. Our preliminary experimental data confirm the possibility of achieving a whitening effect using a mouthwash formulation with no oxidizing or abrasive agents containing CHX and PVP. The formulation tested demonstrated a significant reduction, in vitro and in vivo, in extrinsic tooth staining from baseline and versus the placebo.

Acne RA-1,2, a novel UV-selective face cream for patients with acne: Efficacy and tolerability results of a randomized, placebo-controlled clinical study.

BACKGROUND: General skincare measures such as the use of moisturisers and products containing adequate photoprotection are important components of acne patients' management to complement the pharmacological regimen. Acne RA-1,2 is a novel dermato-cosmetic product which contains selective photofilters and active ingredients against the multifactorial pathophysiology of acne. OBJECTIVES: To evaluate the tolerability of Acne RA-1,2 and its effect on the clinical signs of acne. METHODS: This double-blind, placebo-controlled study randomized 40 adult patients with 10-25 comedones per half face to once-daily application of Acne RA-1,2 or placebo for 8 weeks. Evaluations after 4 and 8 weeks included the number of comedones, transepidermal water loss (TEWL), sebum production, and tolerability. RESULTS: In the Acne RA-1,2 group, there was a significant 35% decrease in the mean number of comedones from 26 at baseline to 17 at Week 8 (P<.001), a 7% significant reduction in TEWL (9.32 to 8.66 g/h/m2 ; P<.001), and a 24% significant reduction in sebum production (154.8 to 117.6 µg/cm2 ; P<.001). The reductions in TEWL and sebum production were significantly greater than those in the placebo group at Weeks 4 and 8 (P<0.05). There were no adverse events. CONCLUSIONS: Acne RA-1,2 was well tolerated and effective at reducing comedones and sebum production and improving epidermal barrier function. These results suggest that Acne RA-1,2 is useful against acne-prone facial skin, particularly as it targets sebum production, which topical pharmacological acne therapies do not address.

In vitro Percutaneous Absorption of Niacinamide and Phytosterols and in vivo Evaluation of their Effect on Skin Barrier Recovery.

In this study, we evaluated different strategies to optimize the percutaneous absorption of niacinamide (NA) and soy phytosterols (FITO) by making use of solid lipid nanoparticles (SLN) and penetration enhancers, such as the hydrogenated lecithin. The evaluation of the skin permeation of NA and FITO has been effected in vitro using excised human skin (i.e., stratum corneum-epidermis or SCE). Furthermore, we evaluated the in vivo effect that NA and FITO has on skin barrier recovery after the topical application; using the extent of methyl nicotinate (MN)-induced erythema in damaged skin as a parameter to determine the rate of stratum corneum recovery. Results pointed out the importance of these strategies as valid tools for NA and FITO topical delivery. In fact, soy lecithin based formulations were able to increase the percutaneous absorption of the two active ingredients, while SLN guaranteed an interesting delayed and sustained release of FITO. In vivo evaluation showed clearly that the formulation containing both the actives (NA and FITO) is able to recover about 95% of skin barrier integrity eight days after tape stripping. This effect is probably due to the "synergistic effect" of NA and FITO.

Wound healing properties of hyaluronan derivatives bearing ferulate residues.

HAFA macromolecules were designed as graft copolymers combining ferulic acid (FA) structure and the hyaluronic acid (HA) backbone linked through an ester bond. These materials were prepared by feruloylation of HA with bisimidazolide 3 [i.e. (E)-4-(3-(1H-imidazol-1-yl)-3-oxoprop-1-enyl)-2-methoxyphenyl 1H-imidazole-1-carboxylate] and obtained with different grafting degree (GD) values, which could be tuned by applying suitable reaction conditions. Among the numerous applications envisioned for HAFA graft copolymers on the basis of the physico-chemical, biological, and pharmacological properties of the starting natural products and the grafting-derived features such as physical cross-linking, potential wound healing properties have been evaluated in vitro and in vivo in preclinical models. In human keratinocyte (HaCaT) cells, our data showed the ability of HAFA-17 (GD = 7%) to ameliorate the in vitro scratch wound significantly with respect to the control HA and FA alone, and this effect was associated with the ability of HAFA-17 to also induce keratinocyte proliferation as determined by BrdU assay. In addition, experiments on wound healing in SKH1 mice confirmed the ability of HAFA-17 to improve the wound closure rate also in vivo. Overall, the data presented herein suggest HAFA-17 as a possible future drug for the therapeutic treatment of acute and chronic wounds.

Correction: Wound healing properties of hyaluronan derivatives bearing ferulate residues.

Correction for 'Wound healing properties of hyaluronan derivatives bearing ferulate residues' by Giuseppe Valacchi et al., J. Mater. Chem. B, 2015, DOI: .

microFIND(®) approach to fluorescent in situ hybridization (FISH).

FISH technology has gained increasing attention in the management of cancer disease, either for predictive or prognostic indications. Molecular cytogenetics has greatly improved diagnostic capability of classical cytogenetics analysis of metaphase-based chromosome for the identification of genetic aberrations. The availability of a large number of fluorescent probes, each specific for different genetic lesions, together with a robust protocol for interphase FISH, provide the pathologist with the essential tools for an accurate evaluation of patient's disease. Hemato-oncological and many of the solid tumors have been comprehensively characterized by peculiar genetic defects and are now routinely evaluated by interphase FISH. Despite the reliability of the method, which has undergone only minor changes since the 1970s, FISH assay is still hampered by reagents cost, preventing its adoption in large-scale oncological screening. In this chapter we describe a major improvement of interphase FISH assay for cytological samples through the description of the miniaturized device microFIND(®) that offers, besides reduction of cost per assay, a completely novel vision to the FISH technology, thanks to the perspective of full automation of FISH assay using a dedicated robotic platform for microFIND(®) handling, (not presently described in the chapter).

Miniaturized FISH for screening of onco-hematological malignancies.

Fluorescence in situ hybridization (FISH) represents a major step in the analysis of chromosomal aberrations in cancer. It allows the precise detection of specific rearrangements, both for diagnostic and prognostic purposes. Here we present a miniaturized FISH method performed on fresh and fixed hematological samples. This procedure has been developed together with a microfluidic device that integrates cluster-assembled nanostructured TiO2 (ns-TiO2) as a nanomaterial promoting hematopoietic cell immobilization in conditions of shear stress. As a result of miniaturization, FISH can be performed with at least a 10-fold reduction in probe usage and minimal cell requirements, creating the possibility of using FISH in genetic screening applications. We developed the protocol on tumor cells and bone marrow (BM) from a normal donor using commercially sex-specific and onco-hematology probes. The procedure was then validated using either BM or peripheral blood (PB) from six patients with hematological diseases, each associated with different genetic lesions. Miniaturized FISH demonstrated comparable performance to standard FISH, indicating that it is suitable for genetic screenings, in research, and in clinical settings for the diagnosis of samples from onco-hematological malignancies.

Pirin delocalization in melanoma progression identified by high content immuno-detection based approaches.

BACKGROUND: Pirin (PIR) is a highly conserved nuclear protein originally isolated as an interactor of NFI/CTF1 transcription/replication factor. It is a member of the functionally diverse cupin superfamily and its activity has been linked to different biological and molecular processes, such as regulation of transcription, apoptosis, stress response and enzymatic processes. Although its precise role in these functions has not yet been defined, PIR expression is known to be deregulated in several human malignancies. RESULTS: We performed immunohistochemical analysis of PIR expression in primary samples from normal human tissues and tumors and identified a dislocation of PIR to the cytoplasm in a subset of melanomas, and a positive correlation between cytoplasmic PIR levels and melanoma progression. PIR localization was subsequently analyzed in vitro in melanoma cell lines through a high content immunofluorescence based approach (ImmunoCell-Array). CONCLUSIONS: The high consistency between in vivo and in vitro results obtained by immunohistochemistry and ImmunoCell-Array provides a validation of the potential of ImmunoCell-Array technology for the rapid screening of putative biological markers, and suggests that cytoplasmic localization of PIR may represent a characteristic of melanoma progression.

Characterization of cluster-assembled nanostructured titanium oxide coatings as substrates for protein arrays.

Protein microarray technologies are rapidly expanding to fulfill current needs of proteome discovery for disease management. Nanostructured materials have been shown to present interesting features when used in biological settings: nanostructured titanium oxide film (ns-TiOx), synthesized by supersonic cluster beam deposition (SCBD), has recently emerged as a biocompatible substrate in different biological assays. The ns-TiOx surface is characterized by a morphology at the nanoscale that can be tuned to modulate specific biomolecule-material interactions. Here we present a systematic characterization of ns-TiOx coatings as protein binding surfaces, comparing their performances with those of most common commercial substrates in protein and antibody microarray assays. Through a robust statistical evaluation of repeatability in terms of coefficient of variation (CV) analysis, we demonstrate that ns-TiOx can be used as reliable substrate for biochips in analytical protein microarray application.

ImmunoCell-Array: a novel technology for pathway discovery and cell profiling.

A major challenge in drug discovery is the definition of the activity of therapeutic compounds before they enter clinical trials. Currently, many tools are available to profile drug response at a multiparametric level. Furthermore, the novel discipline of systems biology is offering interpretative cues to the amount of data generated by the so-called omics technologies. Nonetheless, novel approaches are needed to comprehensively evaluate drug response. This review will describe a recent technology for protein profiling, the ImmunoCell-Array, which, in to the authors' opinion, can enter the scenario of profiling technologies with the goal of offering a comprehensive description of proteome dynamics in a cellular context.

Immunocell-array for molecular dissection of multiple signaling pathways in mammalian cells.

The knowledge of signaling pathways that are triggered by physiological and pathological conditions or drug treatment is essential for the comprehension of the biological events that regulate cellular responses. Recently novel platforms based on "reverse-phase protein arrays" have proven to be useful in the study of different pathways, but they still lack the possibility to detect events in the complexity of a cellular context. We developed an "immunocell-array" of cells on chip where, upon cell plating, growing, drug treatment, and fixation, by spotting specific antibodies we can detect the localization and state of hundreds of proteins involved in specific signaling pathways. By applying this technology to mammalian cells we analyzed signaling proteins involved in the response to DNA damage and identified a chromatin remodeling pathway following bleomycin treatment. We propose our technology as a new tool for the array-based multiplexed analysis of signaling pathways in drug response screening, for the proteomics of profiling patient cells, and ultimately for the high throughput screening of antibodies for immunofluorescence applications.

Retroviral microarray-based platform on nanostructured TiO2 for functional genomics and drug discovery.

Living-cell microarrays are powerful tools for functional genomics and drug discovery. However, despite several attempts to improve this technology, it is still a challenge to obtain microarrays of cells efficiently overexpressing or downregulating specific genes to address complex phenotypes. Here, we present a cell-based microarray for phenotype screening on primary and cancer cells based on the localized reverse infection by retroviruses. Viral vectors are immobilized on a nanostructured titanium dioxide (ns-TiO2) film obtained by depositing a supersonic beam of titania clusters on a glass substrate. We validated the retroviral cell array by overexpression of GFP reporter genes in primary and cancer cells, and by RNA interference of p53 in primary cells by analyzing effects in cell growth. We demonstrate that ns-TiO2 retroviral arrays are an enabling tool for the study of gene function of families of genes for complex phenotypes and for the identification of novel drug targets.

Biocompatibility of cluster-assembled nanostructured TiO2 with primary and cancer cells.

We have characterized the biocompatibility of nanostructured TiO2 films produced by the deposition of a supersonic beam of TiOx clusters. Physical analysis shows that these films possess, at the nanoscale, a granularity and porosity mimicking those of typical extracellular matrix structures and adsorption properties that could allow surface functionalization with different macromolecules such as DNA, proteins, and peptides. To explore the biocompatibility of this novel nanostructured surface, different cancer and primary cells were analyzed in terms of morphological appearance (by bright field microscopy and immunofluorescence) and growth properties, with the aim to evaluate cluster-assembled TiO2 films as substrates for cell-based and tissue-based applications. Our results strongly suggest that this new biomaterial supports normal growth and adhesion of primary and cancer cells with no need for coating with ECM proteins; we thus propose this new material as an optimal substrate for different applications in cell-based assays, biosensors or microfabricated medical devices.