Emilia Licarete

Ectodomain Shedding Generates Neoepitopes on Collagen XVII, the Major Autoantigen for Bullous Pemphigoid

As a type II transmembrane protein in basal keratinocytes, collagen XVII provides stable adhesion between epidermis and dermis in the skin. Its ectodomain can be shed from the cell surface, and autoantibodies in certain blistering diseases preferentially recognize the shed form. Major epitopes of collagen XVII are clustered within the juxtamembranous noncollagenous 16th A domain, and ectodomain shedding occurs within this region, suggesting that cleavage generates neoepitopes. However, the candidate cleavage sites have been controversial, and the mechanism of neoepitope generation is unclear. In this study, we investigated cleavage sites in the noncollagenous 16th A domain to understand the generation of neoepitopes and their pathological role. Polyclonal Abs recognizing the stretch Leu524-Gly532 preferentially reacted with the shed ectodomain, but not with the full-length form, indicating that a neoepitope was localized at this site. The neoepitope-specific Ab fixed complement and induced granulocyte-dependent dermal-epidermal separation in cryosections of normal human skin. The physiological cleavage sites were identified using mass spectrometry. N termini were found at Asp514, Leu524, Glu525, and Gly526, among which Asp514 and Glu525 were blocked by acetylation and pyroglutaminate. In silico prediction of B cell epitopes indicated that the antigenicity of the Leu524-Gly532 region increased substantially after shedding, regardless of the cleavage sites. Correspondingly, neoepitopes were found in the skin and blister fluids of patients with bullous pemphigoid, and bullous pemphigoid sera reacted with the peptide Leu524-Gly532. Taken together, these data demonstrate that physiological shedding of collagen XVII generates neoepitopes, which may serve as a target of blister-inducing autoantibodies.

Chitosan-coated triangular silver nanoparticles as a novel class of biocompatible, highly sensitive plasmonic platforms for intracellular SERS sensing and imaging

There is a need for new strategies for noninvasive imaging of pathological conditions within the human body. The approach of combining the unique physical properties of noble-metal nanoparticles with their chemical specificity and an easy way of conjugation open up new routes toward building bio-nano-objects for biomedical tracking and imaging. This work reports the design and assessment of a novel class of biocompatible, highly sensitive SERS nanotags based on chitosan-coated silver nanotriangles (Chit-AgNTs) labeled with para-aminothiophenol (p-ATP). The triangular nanoparticles are used as Raman scattering enhancers and have proved to yield a reproducible and strong SERS signal. When tested inside lung cancer cells (A549) this class of SERS nanotags presents low in vitro toxicity, without interfering with cell proliferation. Easily internalized by the cells, as demonstrated by imaging using both reflected bright-light optical microscopy and SERS spectroscopy, the particles are proved to be detectable inside cells under a wide window of excitation wavelengths, ranging from visible to near infrared (NIR). Their high sensitivity and NIR availability make this class of SERS nanotags a promising candidate for noninvasive imaging of cancer cells.

Prevalence of collagen VII-specific autoantibodies in patients with autoimmune and inflammatory diseases

BackgroundAutoimmunity to collagen VII is typically associated with the skin blistering disease epidermolysis bullosa acquisita (EBA), but also occurs occasionally in patients with systemic lupus erythematosus or inflammatory bowel disease. The aim of our present study was to develop an accurate immunoassay for assessing the presence of autoantibodies against collagen VII in large cohorts of patients and healthy donors.MethodsBased on in silico antigenic analysis and previous wetlab epitope mapping data, we designed a chimeric collagen VII construct containing all collagen VII epitopes with higher antigenicity. ELISA was performed with sera from patients with EBA (n = 50), Crohns disease (CD, n = 50), ulcerative colitis (UC, n = 50), bullous pemphigoid (BP, n = 76), and pemphigus vulgaris (PV, n = 42) and healthy donors (n = 245).ResultsBy ELISA, the receiver operating characteristics analysis yielded an area under the curve of 0.98 (95% CI: 0.9638-1.005), allowing to set the cut-off at 0.32 OD at a calculated specificity of 98% and a sensitivity of 94%. Running the optimized test showed that serum IgG autoantibodies from 47 EBA (94%; 95% CI: 87.41%-100%), 2 CD (4%; 95% CI: 0%-9.43%), 8 UC (16%; 95% CI: 5.8%-26%), 2 BP (2.63%; 95% CI: 0%-6.23%), and 4 PV (9.52%; 95% CI: 0%-18.4%) patients as well as from 4 (1.63%; 95% CI: 0%-3.21%) healthy donors reacted with the chimeric protein. Further analysis revealed that in 34%, 37%, 16% and 100% of sera autoantibodies of IgG1, IgG2, IgG3, and IgG4 isotype, respectively, recognized the recombinant autoantigen.ConclusionsUsing a chimeric protein, we developed a new sensitive and specific ELISA to detect collagen specific antibodies. Our results show a low prevalence of collagen VII-specific autoantibodies in inflammatory bowel disease, pemphigus and bullous pemphigoid. Furthermore, we show that the autoimmune response against collagen VII is dominated by IgG4 autoantibodies. The new immunoassay should prove a useful tool for clinical and translational research and should improve the routine diagnosis and disease monitoring in diseases associated with collagen VII-specific autoimmunity.

Biosynthesized silver nanoparticles performing as biogenic SERS-nanotags for investigation of C26 colon carcinoma cells

In this work, two classes of silver nanoparticles (AgNPs) were biosynthesized with the goal to assess their reliability in vitro as surface-enhanced Raman scattering (SERS) nanotags. Mycosynthesized silver nanoparticles (MAgNPs) and phytosynthesized silver nanoparticles (PAgNPs) were produced through environmentally friendly procedures by reduction of silver nitrate with Fusarium oxysporum cell filtrate and Azadirachta indica extract, respectively. Two cell lines, namely C26 murine colon carcinoma cells as example of cancer cells and human immortalized keratinocyte cells (HaCaT) as representative of healthy cell line, were selected for in vitro investigation. The in vitro toxicity studies show that M(P)AgNPs present lower cytotoxic effect on both cell lines as compared with standard citrate coated AgNPs. The internalization of M(P)AgNPs by colon carcinoma cells and structural alterations induced in the morphology of treated cells were analyzed by dark-field (DF) and differential interference contrast (DIC) microscopy, respectively. The most informative data about the cellular uptake and tracking potential of M(P)AgNPs were provided by scanning Confocal Raman Microscopy (CRM) and multivariate K-means cluster analysis of collected Raman spectra. The analysis reveals the subcellular components and the localization of AgNPs inside the cell via the intrinsic SERS signature of biogenic coating material. The use of unique biological material to perform synthesis, stability, biocompatibility and SERS tagging is relevant both from the point of view of encoding nanoparticles with Raman reporters and further applications in cell investigation via Raman/SERS imaging.

Liposomal simvastatin inhibits tumor growth via targeting tumor- associated macrophages-mediated oxidative stress

Statins possess antitumor actions at doses 100- to 500-fold higher than those needed to lower cholesterol levels. Thus, the antitumor efficacy of statins could be improved greatly by using tumor-targeted delivery systems. Therefore the present work aims to investigate the antitumor activity of long-circulating liposome-encapsulated simvastatin (LCL-SIM) versus free SIM in B16.F10 murine melanoma-bearing mice. Our results showed that LCL-SIM inhibits strongly the B16.F10 melanoma growth (by 85%) whereas free SIM was ineffective. Moreover, the antitumor activity of LCL-SIM depends on the presence of functional tumor-associated macrophages (TAM) in tumor tissue and is mainly based on the reduction of the TAM-mediated oxidative stress as well as of the production of the hypoxia-inducible factor 1 α (HIF-1 α) in tumors. In conclusion, our findings suggest that the antitumor activity of LCL-SIM on B16.F10 melanoma growth is a result of the tumor-targeting property of the liposome formulation and is tightly dependent on the presence of TAM in tumor tissue.

IgG antibodies against immunodominant C-terminal epitopes of BP230 do not induce skin blistering in mice

Bullous pemphigoid, the most common autoimmune blistering disease in Western Europe and the USA is characterized by the presence of circulating and tissue-bound autoantibodies against the hemidesmosomal proteins BP230 and BP180/collagen XVII. After binding to their target antigens at the basement membrane of the dermal-epidermal junction these autoantibodies are thought to trigger an inflammatory cascade comprising complement- and granulocyte-dependent reactions that result in tissue damage. Whereas the role of anti-BP180 antibodies has been extensively characterized, few and conflicting data is available on the contribution of anti-BP230 antibodies to bullous pemphigoid pathogenesis. Therefore, we addressed in the present study the role of autoantibodies to BP230 in experimental bullous pemphigoid. Rabbit polyclonal antibodies generated against epitopes of the C-terminal fragment of murine BP230 bound to the basement membrane and activated the complement system ex vivo. Affinity-purified antibodies were subsequently subcutaneously transferred into neonatal and adult BALB/c mice. In vivo, we observed a dose-dependent binding of transferred antibodies in the murine skin; however, there was no complement activation and these mice showed no clinical or histological signs of inflammatory disease, in contrast to mice receiving anti-BP180 antibodies. We further conducted ex vivo experiments and demonstrated that rabbit IgG anti-BP230-specific antibodies, in contrast to antibodies from bullous pemphigoid patients or rabbit IgG anti-BP180 antibodies used as positive controls, did not activate human granulocytes to induce dermal-epidermal separation in skin cryosections. Our present findings demonstrate that antibodies against BP230 are non-pathogenic in experimental models of bullous pemphigoid and suggest that proper activation of the complement and granulocytes represent prerequisites for conferring bullous pemphigoid autoantibodies their tissue destructive potential.

Optimizing long-circulating liposomes for delivery of simvastatin to C26 colon carcinoma cells

Abstract Simvastatin (SIM) is a lipophilic statin that has potential benefits for prevention and treatment of several types of malignancies. However, its low water solubility and the toxicity associated with administration of high doses recommend it for encapsulation in carriers able to deliver the therapeutic dose in the tumor. In this work, liposomes with long-circulating properties were proposed as delivery systems for SIM. The objective of this study was to optimize the formulation of SIM-loaded long-circulating liposomes (LCL-SIM) by using D-optimal experimental design. The influence of phospholipids concentration, phospholipids to cholesterol molar ratio and SIM concentration was studied on SIM liposomal concentration, encapsulation efficiency and liposomal size. The optimized formulation had liposomal SIM concentration 6238 µg/ml, EE % of 83.4 % and vesicle size of 190.5 nm. Additionally we evaluated the in vitro cytotoxicity of the optimized liposomal SIM (LCL-SIM-OPT) on C26 murine colon carcinoma cells cultivated in monoculture as well as in co-culture with murine peritoneal macrophages at a cell density ratio that provides an approximation of physiological conditions of colon carcinoma development in vivo. Our preliminary studies suggested that LCL-SIM-OPT exerted cytotoxicity on C26 cells probably via enhancement of oxidative stress in co-culture environment.

Optimization of prednisolone-loaded long-circulating liposomes via application of Quality by Design (QbD) approach

Abstract Quality by design principles (QbD) were used to assist the formulation of prednisolone-loaded long-circulating liposomes (LCL-PLP) in order to gain a more comprehensive understanding of the preparation process. This approach enables us to improve the final product quality in terms of liposomal drug concentration, encapsulation efficiency and size, and to minimize preparation variability. A 19-run D-optimal experimental design was used to study the impact of the highest risk factors on PLP liposomal concentration (Y1- μg/ml), encapsulation efficiency (Y2-%) and size (Y3-nm). Out of six investigated factors, four of them were identified as critical parameters affecting the studied responses. PLP molar concentration and the molar ratio of DPPC to MPEG-2000-DSPE had a positive impact on both Y1 and Y2, while the rotation speed at the formation of the lipid film had a negative impact. Y3 was highly influenced by prednisolone molar concentration and extrusion temperature. The accuracy and robustness of the model was further on confirmed. The developed model was used to optimize the formulation of LCL-PLP for efficient accumulation of the drug to tumor tissue. The cytotoxicity of the optimized LCL-PLP on C26 murine colon carcinoma cells was assessed. LCL-PLP exerted significant anti-angiogenic and anti-inflammatory effects on M2 macrophages, affecting indirectly the C26 colon carcinoma cell proliferation and development.

Exploitation of pleiotropic actions of statins by using tumour-targeted delivery systems

Abstract Statins are drugs traditionally used to lower cholesterol levels in blood. At concentrations 100- to 500-fold higher than those needed for reaching cholesterol lowering activity, they have anti-tumour activity. This anti-tumour activity is based on statins pleiotropic effects derived from their ability to inhibit the mevalonate synthesis and include anti-proliferative, pro-apoptotic, anti-angiogenic, anti-inflammatory, anti-metastatic actions and modulatory effects on intra-tumour oxidative stress. Thus, in this review, we summarise the possible pleiotropic actions of statins involved in tumour growth inhibition. Since the administration of these high doses of statins is accompanied by severe side effects, targeted delivery of statins seems to be the appropriate strategy for efficient application of statins in oncology. Therefore, we also present an overview of the current status of targeted delivery systems for statins with possible utilisation in oncology.

Development of antiproliferative long-circulating liposomes co-encapsulating doxorubicin and curcumin, through the use of a quality-by-design approach

The aim of this work was to use the quality-by-design (QbD) approach in the development of long-circulating liposomes co-loaded with curcumin (CUR) and doxorubicin (DOX) and to evaluate the cytotoxic potential of these liposomes in vitro using C26 murine colon carcinoma cell line. Based on a risk assessment, six parameters, namely the phospholipid, CUR and DOX concentrations, the phospholipid:cholesterol molar ratio, the temperature during the evaporation and hydration steps and the pH of the phosphate buffer, were identified as potential risk factors for the quality of the final product. The influence of these variables on the critical quality attributes of the co-loaded liposomal CUR and DOX was investigated: particle size, zeta potential, drug loading and entrapment efficiency. For this, a 26−2 factorial design was employed to establish a proper regression model and to generate the contour plots for the responses. The obtained data served to establish the design space for which different combinations of variables yielded liposomes with characteristics within predefined specifications. The validation of the model was carried out by preparing two liposomal formulations corresponding to the robust set point from within the design space and one outside the design space and calculating the percentage bias between the predicted and actual experimental results. The in vitro antiproliferative test showed that at higher CUR concentrations, the liposomes co-encapsulating CUR and DOX had a greater cytotoxic effect than DOX-loaded liposomes. Overall, this study showed that QbD is a useful instrument for controlling and optimizing the manufacturing process of liposomes co-loaded with CUR and DOX and that this nanoparticulate system possesses a great potential for use in colon cancer therapy.