Role of Nano-miRNAs in Diagnostics and Therapeutics.

MicroRNAs (miRNA) are key regulators of gene expression, controlling different biological processes such as cellular development, differentiation, proliferation, metabolism, and apoptosis. The relationships between miRNA expression and the onset and progression of different diseases, such as tumours, cardiovascular and rheumatic diseases, and neurological disorders, are well known. A nanotechnology-based approach could match miRNA delivery and detection to move beyond the proof-of-concept stage. Different kinds of nanotechnologies can have a major impact on the diagnosis and treatment of miRNA-related diseases such as cancer. Developing novel methodologies aimed at clinical practice represents a big challenge for the early diagnosis of specific diseases. Within this context, nanotechnology represents a wide emerging area at the forefront of research over the last two decades, whose potential has yet to be fully attained. Nanomedicine, derived from nanotechnology, can exploit the unique properties of nanometer-sized particles for diagnostic and therapeutic purposes. Through nanomedicine, specific treatment to counteract only cancer-cell proliferation will be improved, while leaving healthy cells intact. In this review, we dissect the properties of different nanocarriers and their roles in the early detection and treatment of cancer.

Low Concentrated Fractionalized Nanofibers as Suitable Fillers for Optimization of Structural-Functional Parameters of Dead Space Gel Implants after Rectal Extirpation.

Dead space after rectal resection in colorectal surgery is an area with a high risk of complications. In this study, our goal was to develop a novel 3D implant based on composite hydrogels enriched with fractionalized nanofibers. We employed, as a novel approach in abdominal surgery, the application of agarose gels functionalized with fractionalized nanofibers on pieces dozens of microns large with a well-preserved nano-substructure. This retained excellent cell accommodation and proliferation, while nanofiber structures in separated islets allowed cells a free migration throughout the gel. We found these low-concentrated fractionalized nanofibers to be a good tool for structural and biomechanical optimization of the 3D hydrogel implants. In addition, this nano-structuralized system can serve as a convenient drug delivery system for a controlled release of encapsulated bioactive substances from the nanofiber core. Thus, we present novel 3D nanofiber-based gels for controlled release, with a possibility to modify both their biomechanical properties and drug release intended for 3D lesions healing after a rectal extirpation, hysterectomy, or pelvic exenteration.

Lanreotide Induces Cytokine Modulation in Intestinal Neuroendocrine Tumors and Overcomes Resistance to Everolimus.

Somatostatin analogs mantain their major role in the treatment of patients with advanced neuroendocrine tumors (NETs) and have multiple modulatory effects on the immune system. Here, we evaluated the effects of lanreotide treatment on expression of Th1, Th2 cytokine patterns in serum of patients with NETs and in bronchial and pancreatic NET cell lines. Our results showed that lanreotide treatment promoted a Th1 cytotoxic immune-phenotype in patients with NETs originated by intestinal sites. Similar results were obtained also in vitro where lanreotide induced expression of Th1 cytokines only in pancreatic and not in bronchial-derived NET cell lines. It seems, therefore, that cytokinomics can represent a useful tool for the identification of tumor biomarkers for the early diagnosis and evaluation of the response to therapy in NET patients. To avoid the drug-resistance induced by everolimus (mTOR inhibitor), we made the pancreatic NET cell line resistant to this drug. After treatment with lanreotide we found that the drug reduced its viability compared to that of sensitive cells. These data may have direct implications in design of future translation combination trial on NET patients.

Distinctive germline expression of class I human leukocyte antigen (HLA) alleles and DRB1 heterozygosis predict the outcome of patients with non-small cell lung cancer receiving PD-1/PD-L1 immune checkpoint blockade.

BACKGROUND: Nivolumab is a human monoclonal antibody against programmed cell death receptor-1 (PD-1) able to rescue quiescent tumor infiltrating cytotoxic T lymphocytes (CTLs) restoring their ability to kill target cells expressing specific tumor antigen-derived epitope peptides bound to homologue human leukocyte antigen (HLA) molecules. Nivolumab is currently an active but expensive therapeutic agent for metastatic non-small cell lung cancer (mNSCLC), producing, in some cases, immune-related adverse events (irAEs). At the present, no reliable biomarkers have been validated to predict either treatment response or adverse events in treated patients. METHODS: We performed a retrospective multi-institutional analysis including 119 patients with mNSCLC who received PD-1 blockade since November 2015 to investigate the predictive role of germinal class I HLA and DRB1 genotype. We investigated the correlation among patients' outcome and irAEs frequency with specific HLA A, B, C and DRB1 alleles by reverse sequence-specific oligonucleotide (SSO) DNA typing. RESULTS: A poor outcome in patients negative for the expression of two most frequent HLA-A alleles was detected (HLA: HLA-A*01 and or A*02; progression-free survival (PFS): 7.5 (2.8 to 12.2) vs 15.9 (0 to 39.2) months, p=0.01). In particular, HLA-A*01-positive patients showed a prolonged PFS of 22.6 (10.2 to 35.0) and overall survival (OS) of 30.8 (7.7 to 53.9) months, respectively. We also reported that HLA-A and DRB1 locus heterozygosis (het) were correlated to a worse OS if we considered het in the locus A; in reverse, long survival was correlated to het in DRB1. CONCLUSIONS: This study demonstrate that class I and II HLA allele characterization to define tumor immunogenicity has relevant implications in predicting nivolumab efficacy in mNSCLC and provide the rationale for further prospective trials of cancer immunotherapy.

Comparative Study of NGS Platform Ion Torrent Personal Genome Machine and Therascreen Rotor-Gene Q for the Detection of Somatic Variants in Cancer.

Molecular profiling of a tumor allows the opportunity to design specific therapies which are able to interact only with cancer cells characterized by the accumulation of several genomic aberrations. This study investigates the usefulness of next-generation sequencing (NGS) and mutation-specific analysis methods for the detection of target genes for current therapies in non-small-cell lung cancer (NSCLC), metastatic colorectal cancer (mCRC), and melanoma patients. We focused our attention on EGFR, BRAF, KRAS, and BRAF genes for NSCLC, melanoma, and mCRC samples, respectively. Our study demonstrated that in about 2% of analyzed cases, the two techniques did not show the same or overlapping results. Two patients affected by mCRC resulted in wild-type (WT) for BRAF and two cases with NSCLC were WT for EGFR according to PGM analysis. In contrast, these samples were mutated for the evaluated genes using the therascreen test on Rotor-Gene Q. In conclusion, our experience suggests that it would be appropriate to confirm the WT status of the genes of interest with a more sensitive analysis method to avoid the presence of a small neoplastic clone and drive the clinician to correct patient monitoring.

miR-125b Upregulates miR-34a and Sequentially Activates Stress Adaption and Cell Death Mechanisms in Multiple Myeloma.

miR-125b, ubiquitously expressed and frequently dysregulated in several tumors, has gained special interest in the field of cancer research, displaying either oncogenic or oncosuppressor potential based on tumor type. We have previously demonstrated its tumor-suppressive role in multiple myeloma (MM), but the analysis of molecular mechanisms needs additional investigation. The purpose of this study was to explore the effects of miR-125b and its chemically modified analogs in modulating cell viability and cancer-associated molecular pathways, also focusing on the functional aspects of stress adaptation (autophagy and senescence), as well as programmed cell death (apoptosis). Based on the well-known low microRNA (miRNA) stability in therapeutic application, we designed chemically modified miR-125b mimics, laying the bases for their subsequent investigation in in vivo models. Our study clearly confirmed an oncosuppressive function depending on the repression of multiple targets, and it allowed the identification, for the first time, of miR-125b-dependent miR-34a stimulation as a possible consequence of the inhibitory role on the interleukin-6 receptor (IL-6R)/signal transducer and activator of transcription 3 (STAT3)/miR-34a feedback loop. Moreover, we identified a pattern of miR-125b-co-regulated miRNAs, shedding light on possible new players of anti-MM activity. Finally, functional studies also revealed a sequential activation of senescence, autophagy, and apoptosis, thus indicating, for the first two processes, an early cytoprotective and inhibitory role from apoptosis activation.

Identification of Sarcosine as a Target Molecule for the Canine Olfactory Detection of Prostate Carcinoma.

The hypothesis that dogs can detect malignant tumours through the identification of specific molecules is nearly 30 years old. To date, several reports have described the successful detection of distinct types of cancer. However, is still a lack of data regarding the specific molecules that can be recognized by a dog's olfactory apparatus. Hence, we performed a study with artificially prepared, well-characterized urinary specimens that were enriched with sarcosine, a widely reported urinary biomarker for prostate cancer (PCa). For the purposes of the study, a German shepherd dog was utilized for analyses of 60 positive and 120 negative samples. Our study provides the first evidence that a sniffer dog specially trained for the olfactory detection of PCa can recognize sarcosine in artificial urine with a performance [sensitivity of 90%, specificity of 95%, and precision of 90% for the highest amount of sarcosine (10 µmol/L)] that is comparable to the identification of PCa-diagnosed subjects (sensitivity of 93.5% and specificity of 91.6%). This study casts light on the unrevealed phenomenon of PCa olfactory detection and opens the door for further studies with canine olfactory detection and cancer diagnostics.

Needleless emulsion electrospinning for the regulated delivery of susceptible proteins.

In the present work, we developed a novel needleless emulsion electrospinning technique that improves the production rate of the core/shell production process. The nanofibres are based on poly-ε-caprolactone (PCL) as a continuous phase combined with a droplet phase based on Pluronic F-68 (PF-68). The PCL-PF-68 nanofibres show a time-regulated release of active molecules. Needleless emulsion electrospinning was used to encapsulate a diverse set of compounds to the core phase [i.e. 5-(4,6-dichlorotriazinyl) aminofluorescein -PF-68, horseradish peroxidase, Tetramethylrhodamine-dextran, insulin growth factor-I, transforming growth factor-ß and basic fibroblast growth factor]. In addition, the PF-68 facilitates the preservation of the bioactivity of delivered proteins. The system's potential was highlighted by an improvement in the metabolic activity and proliferation of mesenchymal stem cells. The developed system has the potential to deliver susceptible molecules in tissue-engineering applications.

Designing of PLA scaffolds for bone tissue replacement fabricated by ordinary commercial 3D printer.

BACKGROUND: The primary objective of Tissue engineering is a regeneration or replacement of tissues or organs damaged by disease, injury, or congenital anomalies. At present, Tissue engineering repairs damaged tissues and organs with artificial supporting structures called scaffolds. These are used for attachment and subsequent growth of appropriate cells. During the cell growth gradual biodegradation of the scaffold occurs and the final product is a new tissue with the desired shape and properties. In recent years, research workplaces are focused on developing scaffold by bio-fabrication techniques to achieve fast, precise and cheap automatic manufacturing of these structures. Most promising techniques seem to be Rapid prototyping due to its high level of precision and controlling. However, this technique is still to solve various issues before it is easily used for scaffold fabrication. In this article we tested printing of clinically applicable scaffolds with use of commercially available devices and materials. Research presented in this article is in general focused on "scaffolding" on a field of bone tissue replacement. RESULTS: Commercially available 3D printer and Polylactic acid were used to create originally designed and possibly suitable scaffold structures for bone tissue engineering. We tested printing of scaffolds with different geometrical structures. Based on the osteosarcoma cells proliferation experiment and mechanical testing of designed scaffold samples, it will be stated that it is likely not necessary to keep the recommended porosity of the scaffold for bone tissue replacement at about 90%, and it will also be clarified why this fact eliminates mechanical properties issue. Moreover, it is demonstrated that the size of an individual pore could be double the size of the recommended range between 0.2-0.35 mm without affecting the cell proliferation. CONCLUSION: Rapid prototyping technique based on Fused deposition modelling was used for the fabrication of designed scaffold structures. All the experiments were performed in order to show how to possibly solve certain limitations and issues that are currently reported by research workplaces on the field of scaffold bio-fabrication. These results should provide new valuable knowledge for further research.

Highly efficient mesenchymal stem cell proliferation on poly-ε-caprolactone nanofibers with embedded magnetic nanoparticles.

In this study, we have developed a combined approach to accelerate the proliferation of mesenchymal stem cells (MSCs) in vitro, using a new nanofibrous scaffold made by needleless electrospinning from a mixture of poly-ε-caprolactone and magnetic particles. The biological characteristics of porcine MSCs were investigated while cultured in vitro on composite scaffold enriched with magnetic nanoparticles. Our data indicate that due to the synergic effect of the poly-ε-caprolactone nanofibers and magnetic particles, cellular adhesion and proliferation of MSCs is enhanced and osteogenic differentiation is supported. The cellular and physical attributes make this new scaffold very promising for the acceleration of efficient MSC proliferation and regeneration of hard tissues.

Abdominal closure reinforcement by using polypropylene mesh functionalized with poly-ε-caprolactone nanofibers and growth factors for prevention of incisional hernia formation.

Incisional hernia affects up to 20% of patients after abdominal surgery. Unlike other types of hernia, its prognosis is poor, and patients suffer from recurrence within 10 years of the operation. Currently used hernia-repair meshes do not guarantee success, but only extend the recurrence-free period by about 5 years. Most of them are nonresorbable, and these implants can lead to many complications that are in some cases life-threatening. Electrospun nanofibers of various polymers have been used as tissue scaffolds and have been explored extensively in the last decade, due to their low cost and good biocompatibility. Their architecture mimics the natural extracellular matrix. We tested a biodegradable polyester poly-ε-caprolactone in the form of nanofibers as a scaffold for fascia healing in an abdominal closure-reinforcement model for prevention of incisional hernia formation. Both in vitro tests and an experiment on a rabbit model showed promising results.

Nanotechnology and mesenchymal stem cells with chondrocytes in prevention of partial growth plate arrest in pigs.

INTRODUCTION: This study describes the results achieved using a combination of allogeneic mesenchymal stem cells (MSCs) with chondrocytes (CHC) and a new scaffold consisting of type-I collagen and chitosan nanofibers in the prevention of partial growth plate arrest after iatrogenic injury in pigs. MATERIAL AND METHODS: The miniature pig was selected as an experimental model to compare the results in the left femoral bones (MSCs and CHC in scaffold transplantation into the iatrogenic partial distal growth plate defect) and right femoral bones (scaffold alone transplantation). The experimental group consisted of 10 animals. Bone marrow from os ilium as the source of MSCs was used. A porous cylinder consisting of 0.5% by weight type-I collagen and 30% by weight chitosan, was the optimal choice. The length of the bone and angular deformity of distal femur after the healing period was measured and the quality and structure of the newly formed cartilage was histologically examined. RESULTS: Transplantation of the composite scaffold in combination with MSCs and chondrocytes led to the prevention of growth disorder and angular deformity in the distal epiphysis of the left femur. Compared to the right (control) femur, tissue similar to hyaline cartilage with signs of columnar organization typical of the growth plate occurred in most cases. CONCLUSIONS: The promising results of this study reveal the new and effective means for the prevention of bone bridge formation after growth plate injury.

Novel biodegradable polydioxanone stents in a rabbit airway model.

OBJECTIVE: This study was undertaken to evaluate safety and biocompatibility of a novel biodegradable polydioxanone stent in a rabbit tracheal model. Metallic and silicone stents represent standard therapeutic approaches for hollow organ stenosis, although complications have been reported repeatedly. Biodegradable stents could reduce the risks associated with this procedure while still achieving the purpose of maintaining lumen patency. METHODS: A commercially available polydioxanone suture strand with a long safety record was used to manufacture the self-expanding stents. The polydioxanone stents were then implanted bronchoscopically and under fluoroscopic guidance into the tracheas of white rabbits (N = 25). Periodic clinical examination was performed. Histopathologic examination concluded the study for the 5 experimental groups at 3, 4, 5, 10, and 15 weeks after implantation. RESULTS: There were no unexpected deaths and no stent displacements during the study. The animals remained in good condition, without stent debris expectoration. Macroscopic examination revealed that the tracheal lumen stayed open. Histologic examination showed that tracheal damage score was highest 5 weeks after stenting, including in-stent necrosis of the epithelium. Stent degradation was complete with no remnants after 10 weeks, leaving the trachea completely healed at 15 weeks after implantation. CONCLUSIONS: This animal airway model has demonstrated acceptable safety and biocompatibility of this novel biodegradable polydioxanone stent. We suggest that polydioxanone stenting be used for further clinical studies for cases in which complete stent degradation after temporary airway treatment is desirable.

Quantification of humeroulnar incongruity in labrador retrievers with and without medial coronoid disease.

OBJECTIVE: To quantify humeroulnar incongruity on elbow radiographs in Labrador Retrievers with or without medial coronoid disease (MCD). STUDY DESIGN: Retrospective study of 92 elbows. SAMPLE POPULATION: Radiographic projections of elbow joints from Labrador Retrievers with MCD (n = 42 elbows; 26 dogs) and without MCD (n = 50 elbows; 25 dogs). PROCEDURE: The congruity of the humeroulnar joint was measured using an index of subluxation (SI) for each elbow. SI was defined as the distance between the centers of 2 circles drawn along the margins of the incisura trochlearis and the trochlea of humerus on mediolateral digital radiographic projections, normalized by the radius of the circle circumscribing the humeral trochlea. SI was compared between right and left elbows with and without pathology using a Wilcoxon test for paired data, and between normal and abnormal groups with a Wilcoxon test for unpaired data. Mismatch between ulnar curvature and curvature of humeral trochlea and radioulnar incongruency were also noted (Wilcoxon test). The intraobserver repeatability, correlation between SI and radioulnar incongruency, and between SI and mismatch elbow curvature were estimated with a Pearson's correlation coefficient. RESULTS: Intraobserver repeatability of SI measurement was high (r = 0.97). Mean ± SD humeroulnar incongruity (SI) was greater in elbows with MCD (18.5 ± 6.6) than in the normal elbows (1.7 ± 2.0, P < 0.001). The difference between the diameters of the curvatures of the ulnar and humeral trochlea was greater in elbows with MCD (12.5 ± 4.4) than in the normal group (10.7 ± 4.1, P < 0.05). A moderate correlation was found between the degree of humeroulnar incongruity and a radioulnar step (r = 0.63); however, no correlation was identified between SI and the difference between the diameters of the curvatures of the ulnar and humeral trochleae (r = 0.14). CONCLUSION: We propose a radiographic index to measure humeroulnar incongruity on mediolateral digital radiographic projections. This index (SI) supports the presence of humeroulnar incongruity in Labrador Retrievers with MCD. Further evaluation of its reproducibility and clinical importance are warranted. Although there is a moderate correlation between humeroulnar incongruity and radioulnar incongruency, causation has not been established.

Allogeneic and autogenous transplantations of MSCs in treatment of the physeal bone bridge in rabbits.

BACKGROUND: The aim of this experimental study on New Zealand's white rabbits was to find differences in the results of treating the distal physeal femoral defect by the transplantation of autologous or allogeneic mesenchymal stem cells (MSCs). After the excision of a created bone bridge in the distal physis of the right femur, modified composite scaffold with MSCs was transplanted into the defect. In animal Group A (n = 11) autogenous MSCs were implanted; in animal Group B (n = 15) allogeneic MSCs were implanted. An iatrogenic physeal defect of the left femur of each animal not treated by MSCs transplantation served as control. The rabbits were euthanized four months after the transplantation. The treatment results were evaluated morphometrically (femoral length and valgus deformity measurement) and histologically (character and quality of the new cartilage). RESULTS: Four months after the transplantation, the right femurs of the animals in Group A were on average longer by 0.50 +/- 0.04 cm (p = 0.018) than their left femurs, the right femurs of rabbits in Group B were on average longer by 0.43 +/- 0.01 cm (p = 0.028) than their left femurs.4 months after the therapeutic transplantation of MSCs valgus deformity of the distal part of the right femur of animals in Group A was significantly lower (by 4.45 +/- 1.86 degrees ) than that of their left femur (p = 0.028), in Group B as well (by 3.66 +/- 0.95 degrees than that of their left femur p = 0.001). However, no significant difference was found between rabbits with transplanted autogenous MSCs (Group A) and rabbits with transplanted allogeneic MSCs (Group B) either in the femur length (p = 0.495), or in its valgus deformity (p = 0.1597). After the MSCs transplantation the presence of a newly formed hyaline cartilage was demonstrated histologically in all the animals (both groups). The ability of transplanted MSCs to survive in the damaged physis was demonstrated in vivo by magnetic resonance, in vitro by Perls reaction and immunofluorescence. CONCLUSION: The transplantation of both autogenous and allogeneic MSCs into a defect of the growth plate appears as an effective method of surgical treatment of physeal cartilage injury. However, the Findings point to the conclusion that there is no clear difference in the final effect of the transplantation procedure used.

Radiographic, computed tomographic, and arthroscopic evaluation of experimental radio-ulnar incongruence in the dog.

OBJECTIVE: To compare the diagnostic value of arthroscopy, computed tomography (CT), and radiography for evaluation of radio-ulnar incongruence (RUI). STUDY DESIGN: Experimental evaluation of induced progressive RUI. SAMPLE POPULATION: Cadaveric Labrador forelimbs (n=11). METHODS: The radius was shortened by 1, 2, and 3 mm with a surgical model of RUI. RUI was scored on radiographs, CT (2 radiologists), and arthroscopy (2 surgeons) before and after each modification. The sensitivity and specificity of each modality were compared. The effects of arthroscope and elbow position on arthroscopy observations were evaluated. Agreement between surgeons, radiologists, and each imaging technique and the known status of the elbow was calculated. RESULTS: Complete arthroscopic sessions had an averaged sensitivity of 94% and specificity of 81.9%. The ability to detect mild incongruity (1 mm step) was greater at the incisure than other locations (P<.001). The average sensitivity and specificity of radiography were 99.3% and 42.4%, and for CT were 85.05% and 45.8%, respectively. The average agreement between imaging techniques and the known status of the elbows was greater with complete arthroscopic sessions (89.75%) than radiography (70.1%) and CT (76.85%). Inter-investigator agreement was greater between surgeons scoring arthroscopic examinations (88.6%) than radiologists scoring CT studies (43.9%). CONCLUSIONS: Evaluation of arthroscopic images allows sensitive and reproducible detection of experimental RUI, especially at the incisure. Arthroscopic evaluation of experimental RUI reached a higher diagnostic value than radiographs and CT images, because of its specificity and reproducibility. CLINICAL RELEVANCE: The diagnostic value and reproducibility of arthroscopy may compare favorably with those of CT when evaluating RUI in dogs with elbow disease.