Evaluation of antibiotic and cell-based therapy in preventing S. epidermidis-induced nonunion in rats.

Methicillin-resistant S. epidermidis (MRSE) is responsible for biofilm-related infections (Montanaro,2011; Romanò, 2013) and fracture nonunion, as recently demonstrated by our group (Lovati, 2016).The present study aims to investigate the efficacy of antibiotic or cell-based therapies in preventingbacterial infections and nonunion establishment.Under anesthesia, femoral fractures were performed in 30 rats, then the site of injury was injectedwith a clinical-derived MRSE strain and, finally, synthesized with stainless steel plates. Rats weredifferently treated as follows: MRSE-infected controls (IC); systemically-injected vancomycin (s-VANC);local vancomycin-enriched hydrogel (l-HYD); systemically-injected BMSCs (s-BMSCs); and locallyinjectedBMSCs (l-BMSCs).After 6 weeks, pro-inflammatory cytokines, quantitative micro-CT, histological and microbiologicalanalyses were carried out to investigate the host response to the different treatments.Half of the s-BMSCs rats died closely to the systemic cell injection, thus excluded for further analyses.Our results for the IC group were consistent with previously published data (Lovati, 2016), showingsigns of osteomyelitis and nonunion development. In s-VANC and l-HYD groups, micro-CT detected agood bony bridging and the microbiological counts were significantly lower with respect to the othergroups. Our study suggests that the association of s-VANC and l-HYD is an effective treatment toprevent biofilm-induced nonunions. Differently, we cannot positively support cell therapies for thispurpose due to the high risk related to the systemic cell injection, thus requiring further studies to beeventually proposed in clinics

Phenotypic and genomic identification of Staphylococcus epidermidis GOI1153754-03-14 isolated from an infected orthopedic prosthesis

Introduction: Staphylococcus epidermidis GOI1153754-03-14 is able to colonize orthopedic implants and to cause septic non-unions, as validated in a recent in vivo study (Lovati, 2016). To pore over the mechanisms leading to the biofilm formation on metallic implants, in the present study, we carried out the phenotypic and genotypic characterization of the clinical isolate S. epidermidis GOI1153754-03-14.Materials and Methods: The antimicrobial susceptibility and minimum inhibitory concentration (MIC) of the strain were evaluated through the Vitek2 System (Biomerieux), as well as its ability to form biofilm in vitro through a spectrophotometric assay (Stepanovich, 2000).The genomic DNA was extracted by Bacterial Genomic DNA Isolation Kit (Norgen Biotek Corp.). Libraries were prepared with the ThruPLEX DNA-seq (Rubicon Genomics) and then sequenced on the Illumina MiSeq platform through the MiSeq Reagent Kit v3 (600-cycles) to produce 300 bp paired-end reads (Illumina Inc.). Reads were quality-trimmed and gene annotated thanks to the RAST software (Aziz, 2008).Results: The antimicrobial susceptibility along with the MIC values are reported in Table 1. The outputs resulted in 51 contigs (Average = 50,720.6 Mb) with 396X fold average coverage. The total genome is 2,586,753 bp long with a GC content of 31.84% and an N50 value of 7 bp. The whole genome is composed by 2,467 protein-encoding genes and 64 RNAs (55 tRNAs and 9 rRNAs). The entire genome sequence has been deposited in the European Nucleotide Archive (ENA) under the accession no. FWCG01000000 (Bottagisio, 2017).Discussion: The genotypic and phenotypic characterization of the S. epidermidis GOI1153754-03-14 will enable a better comprehension of the mechanisms involved in the biofilm formation on orthopedic implants paving the way for innovative preventative and therapeutic strategies. Moreover, the sequence of this clinical strain is mandatory to develop dedicated proteomics analysis in order to highlight functional mechanism of biofilm formation

Impact of doxorubicin-loaded ferritin nanocages (FerOX) vs. free doxorubicin on T lymphocytes: a translational clinical study on breast cancer patients undergoing neoadjuvant chemotherapy

Despite the advent of numerous targeted therapies in clinical practice, anthracyclines, including doxorubicin (DOX), continue to play a pivotal role in breast cancer (BC) treatment. DOX directly disrupts DNA replication, demonstrating remarkable efficacy against BC cells. However, its non-specificity toward cancer cells leads to significant side effects, limiting its clinical utility. Interestingly, DOX can also enhance the antitumor immune response by promoting immunogenic cell death in BC cells, thereby facilitating the presentation of tumor antigens to the adaptive immune system. However, the generation of an adaptive immune response involves highly proliferative processes, which may be adversely affected by DOX-induced cytotoxicity. Therefore, understanding the impact of DOX on dividing T cells becomes crucial, to deepen our understanding and potentially devise strategies to shield anti-tumor immunity from DOX-induced toxicity. Our investigation focused on studying DOX uptake and its effects on human lymphocytes. We collected lymphocytes from healthy donors and BC patients undergoing neoadjuvant chemotherapy (NAC). Notably, patient-derived peripheral blood mononuclear cells (PBMC) promptly internalized DOX when incubated in vitro or isolated immediately after NAC. These DOX-treated PBMCs exhibited significant proliferative impairment compared to untreated cells or those isolated before treatment initiation. Intriguingly, among diverse lymphocyte sub-populations, CD8 + T cells exhibited the highest uptake of DOX. To address this concern, we explored a novel DOX formulation encapsulated in ferritin nanocages (FerOX). FerOX specifically targets tumors and effectively eradicates BC both in vitro and in vivo. Remarkably, only T cells treated with FerOX exhibited reduced DOX internalization, potentially minimizing cytotoxic effects on adaptive immunity

Everolimus Nanoformulation in Biological Nanoparticles Increases Drug Responsiveness in Resistant and Low-Responsive Breast Cancer Cell Lines

Everolimus (Eve) is an FDA approved drug that inhibits mammalian target of rapamycin (mTOR). It is employed in breast cancer treatment even if its responsiveness is controversial. In an attempt to increase Eve effectiveness, we have developed a novel Eve nanoformulation exploiting H-ferritin nanocages (HEve) to improve its subcellular delivery. We took advantage of the natural tumor targeting of H-Ferritin, which is mediated by the transferrin receptor-1 (TfR1). Breast cancer cells overexpressing TfR-1 were successfully recognized by H-Ferritin, displaying quick nanocage internalization. HEve has been tested and compared to Eve for in vitro efficacy in sensitive and resistant breast cancer cells. Nanoformulated Eve induced remarkable antiproliferative activity in vitro, making even resistant cell lines sensitive to Eve. Moreover, the antiproliferative activity of HEve is fully in accordance with cytotoxicity observed by cell death assay. Furthermore, the significant increase in anticancer efficacy displayed in HEve-treated samples is due to the improved drug accumulation, as demonstrated by UHPLC-MS/MS quantifications. Our findings suggest that optimizing Eve subcellular delivery, thanks to nanoformulation, determines its improved antitumor activity in a panel of Eve-sensitive or resistant breast cancer cell lines

HDL Dysfunctionality: Clinical Relevance of Quality Rather Than Quantity

High-density lipoproteins (HDLs) represent a class of lipoproteins very heterogeneous in structure, composition, and biological functions, which carry out reverse cholesterol transport, antioxidant, anti-inflammatory, antithrombotic, and vasodilator actions. Despite the evidence suggesting a clear inverse relationship between HDL cholesterol (HDL-c) concentration and the risk for cardiovascular disease, plasma HDL cholesterol levels do not predict the functionality and composition of HDLs. The importance of defining both the amount of cholesterol transported and lipoprotein functionality has recently been highlighted. Indeed, different clinical conditions such as obesity, diabetes mellitus type 2 (T2DM), and cardiovascular disease (CVD) can alter the HDL functionality, converting normal HDLs into dysfunctional ones, undergoing structural changes, and exhibiting proinflammatory, pro-oxidant, prothrombotic, and proapoptotic properties. The aim of the current review is to summarize the actual knowledge concerning the physical–chemical alteration of HDLs related to their functions, which have been found to be relevant in several pathological conditions associated with systemic inflammation and oxidative stress

HER-2-Targeted Nanoparticles for Breast Cancer Diagnosis and Treatment

Human epidermal growth factor receptor-2 (HER-2) overexpressing breast cancer is a breast cancer subtype characterized by high aggressiveness, high frequency of brain metastases and poor prognosis. HER-2, a glycoprotein belonging to the ErbB receptor family, is overexpressed on the outer membrane of cancer cells and has been an important therapeutic target for the development of targeted drugs, such as the monoclonal antibodies trastuzumab and pertuzumab. These therapies have been available in clinics for more than twenty years. However, despite the initial enthusiasm, a major issue emerged limiting HER-2 targeted therapy efficacy, i.e., the evolution of drug resistance, which could be tackled by nanotechnology. The aim of this review is to provide a first critical update on the different types of HER-2-targeted nanoparticles that have been proposed in the literature in the last decade for therapeutic purposes. We focus on the different targeting strategies that have been explored, their relative outcomes and current limitations that still need to be improved. Then, we review the nanotools developed as diagnostic kits, focusing on the most recent techniques, which allow accurate quantification of HER-2 levels in tissues, with the aim of promoting more personalized medicinal approaches in patients

Protein-Based Nanoparticles for the Imaging and Treatment of Solid Tumors: The Case of Ferritin Nanocages, a Narrative Review

Protein nanocages have been studied extensively, due to their unique architecture, exceptional biocompatibility and highly customization capabilities. In particular, ferritin nanocages (FNs) have been employed for the delivery of a vast array of molecules, ranging from chemotherapeutics to imaging agents, among others. One of the main favorable characteristics of FNs is their intrinsic targeting efficiency toward the Transferrin Receptor 1, which is overexpressed in many tumors. Furthermore, genetic manipulation can be employed to introduce novel variants that are able to improve the loading capacity, targeting capabilities and bio-availability of this versatile drug delivery system. In this review, we discuss the main characteristics of FN and the most recent applications of this promising nanotechnology in the field of oncology with a particular emphasis on the imaging and treatment of solid tumors

Metabolic Profiling of Type 2 Diabetes Patients after Bariatric Surgery by Raman Spectroscopy

Background: Bariatric surgery (BS) is an important procedure used for the treatment of morbid obesity and has been proven to improve, or even cure, type 2 diabetes mellitus (T2DM). However, of the patients with T2DM who initially go into remission, a proportion experience a relapse during the follow-up. In this context, Raman spectroscopy (RS) could be a promising technique for monitoring the metabolic profile of patients after surgical treatment with the aim of improving their postsurgical management. Methods: Fourteen obese patients with T2DM were recruited. Clinical parameters, adipokines, ghrelin, Fibroblast growth factor 19 (FGF-19) values, and Raman spectra were collected and analyzed before and after surgery. RS results were compared with profiles obtained from 23 healthy subjects (HC), to observe whether the metabolic fingerprint of bariatric patients normalized during the surgical follow-up. Results: The reduction in anthropometric measures and improved glycemic control and lipid profile after surgical treatment highlighted the benefits of BS. Consequently, adipokines, ghrelin and FGF-19 concentration returned to normal values after surgery. However, RS data highlighted an altered metabolic profile even after BS. Conclusion: RS suggests that BS does not fully restore the metabolic profile of patients in the immediate follow-up after the surgery

Determination of the quality of lipoproteins by Raman spectroscopy in obese and healthy subjects

Lipoproteins (LPs) are multimolecular complexes of lipids and proteins responsible for transporting fatty acids, cholesterol, and micronutrients (carotenoids) through the body. The quantification of triglycerides and cholesterol carried by lipoproteins is a leading clinical parameter to assess the increased risk of cardiovascular events. However, in recent times, the study of the overall "quality" of lipoproteins, defined by their biochemical composition and oxidation state, has emerged as necessary to improve the definition of the cardiovascular risk. In this work, we present Raman spectroscopy (RS) as an effective method to immediately detect the functional groups relative to the principal biochemical components and the level of unsaturated lipids present in LPs. Furthermore, we show how RS can reveal the differences in the biochemical composition and oxidation state of LPs extracted from a cohort of obese patients (Ob) and a control group of healthy subjects (HC). In particular, RS revealed how low-density lipoproteins (LDLs) from obese patients are enriched in triglycerides and more oxidized than those from the control group, while high-density lipoproteins (HDLs) from Ob patients were depleted in cholesterol and phospholipids. RS analysis also allowed the study of the relationship between the levels of carotenoids present in the different classes of LPs highlighting how this parameter depends on the disease severity. Overall, these results demonstrated that RS is a viable approach for quickly and effectively gaining information on LPs' biochemical composition and oxidation state, providing an immediate measure of their quality. Besides, RS further proved the role of LPs in obesity and metabolic dysfunctions