Erythrocytes as Carriers for Drugs and Contrast Agents

Erythrocytes, also known as Red Blood Cells (RBC), are typically used in transfusion medicine to replace lost blood in patients who underwent different kinds of medical treatments as well as those involved in accidents resulting in blood loss. In addition to these common uses, RBC are being used for a variety of new applications either as therapeutics or as diagnostics. Most of these novel approaches are made possible due to the peculiar properties of these cells. We have invented a technology that allows cells to be opened and resealed without affecting their main physiological characteristics with a minimal amount of patient blood.  Uses of processed RBCs in biomedical engineering include work with drugs, biomedical compounds and/or nanomaterials. These constructs are a new armamentarium available to the physicians for the release of drugs in circulation, for targeting drugs to selected sites in the body, or for in vivo diagnostic procedures based on magnetic and/or optical methods. Autologous human RBC loaded with dexamethasone (EryDex), a common corticosteroid,  have been used in the treatment of Cystic Fibrosis, Crohn’s Disease, and other severe inflammatory conditions. Benefits and safety of this technology have been documented in over 2,500 treatments. EryDel SpA is a company focused on developing and commercializing innovative therapies and diagnostics based on the use of autologous RBCs as agent carriers. More recently, EryDel SpA completed a Phase II Proof of Concept study in patients with Ataxia Telangiectasia (AT), a rare progressive neurological autosomal recessive disorder that leads to mortality in most patients at an early age, with significant benefit seen on primary and secondary end-points. EryDex treatment has received Orphan Drug Designation by EMA for the treatment of Cystic Fibrosis and both by EMA and FDA for the treatment of AT.The encapsulation of superparamagnetic nanoparticles within RBC has lead to the generation of new biomimetic constructs that now permits the use of these nanomaterials in vivo avoiding their rapid sequestration and their accumulation in unwanted districts (PCT WO 2008/003524 A3). Similarly, the encapsulation of infrared fluorescent agents into RBC gives opportunity to the measurement of vasomotion in the human retinal vasculature suggesting a possible correlation with retinal edema.In summary, the newly developed RBC-based drug delivery system is an innovative technology platform that could be used in a wide range of applications opening to unlimited new therapeutic approaches. Furthermore, the same system has been adapted to deliver contrasting agents within the body enabling the improvement of current fluorangiographic procedures and the imaging by Magnetic Resonance (MRI) and Magnetic Particle (MPI). EryDel S.p.A. has recently completed trials to bring EryDex treatment to the market and to implement the clinical applications of the RBC technology

Solving a Real-Life Distributor's Pallet Loading Problem

We consider the distributor's pallet loading problem where a set of different boxes are packed on the smallest number of pallets by satisfying a given set of constraints. In particular, we refer to a real-life environment where each pallet is loaded with a set of layers made of boxes, and both a stability constraint and a compression constraint must be respected. The stability requirement imposes the following: (a) to load at level k+1 a layer with total area (i.e., the sum of the bottom faces' area of the boxes present in the layer) not exceeding α times the area of the layer of level k (where α≥1), and (b) to limit with a given threshold the difference between the highest and the lowest box of a layer. The compression constraint defines the maximum weight that each layer k can sustain; hence, the total weight of the layers loaded over k must not exceed that value. Some stability and compression constraints are considered in other works, but to our knowledge, none are defined as faced in a real-life problem. We present a matheuristic approach which works in two phases. In the first, a number of layers are defined using classical 2D bin packing algorithms, applied to a smart selection of boxes. In the second phase, the layers are packed on the minimum number of pallets by means of a specialized MILP model solved with Gurobi. Computational experiments on real-life instances are used to assess the effectiveness of the algorithm

The expression and modulation of CEACAM1 and tumor cell transformation

BACKGROUND: In this review, we focus our discussion on one class of carcinoembryonic antigen-related cell adhesion molecules, Carcinoembryonic antigen-related cell adhesion molecules 1 (CEACAM1). This has been observed in several malignant transformations to be subjected to complex mechanisms of modulation and dysregulation. AIMS: Restoration of CEACAM1 expression in tumor cell lines often abolishes their oncogenicity in vivo, and therefore, this adhesion molecule has been regarded as a tumor suppressor. In contrast, de novo expression of CEACAM1 is found with the progression of malignancy and metastatic spread in a large array of cancer tissues which include melanoma, Non Small cell Lung carcinoma (NSCLC) as well as bladder, prostate, thyroid, breast, colon and gastric carcinomas. DISCUSSION: We report and discuss the most significant findings confirming at immunohistochemical and clinical level the correlation between poor prognosis and expression of CEACAM1 on the cell surface of tumors

Gastrointestinal Tumors: Phytochemical and Drug Combinations Targeting the Hallmarks of Cancer

Cancer is a worldwide burden resulting in millions of deaths each year. In particular, gastrointestinal tumors are life-threatening malignancies and one of the leading reasons for death in developed countries. Phytochemicals can be found in grains, vegetables, fruits and several foods. Many phytochemicals, such as curcumin, genistein, luteolin, vitexin-2-O-xyloside, avenanthramides, quercetin, epigallocatechin-3-gallate (EGCG), resveratrol, sulforaphane, piperine and thymoquinone have been used in combination with different chemotherapeutic agents for their synergistic anticancer effects against various forms of cancer. In this review, we describe the antitumor properties and biological effects of combinations of phytochemicals and anticancer drugs against gastrointestinal tumors: colon cancer, gastric cancer, liver cancer, pancreatic cancer. We focus on the molecular pathways, oncoproteins and tumor suppressors modulated by the combination of phytochemicals with antitumor drugs and on the biomarkers of the hallmarks of cancer influenced by these therapeutic strategies in cancer cell lines, xenograft models and clinical trials. The increased knowledge of biomarkers and molecular pathways regulated by the combination of phytochemicals and conventional anticancer drugs in both in vitro and in vivo models will remarkably improve the efficacy of these therapeutic strategies against gastrointestinal tumors in future innovative clinical applications

Human Red Blood Cells Modulate Cytokine Expression in Monocytes/Macrophages Under Anoxic Conditions

In the bone marrow (BM) hematopoietic niche, the oxygen tension is usually very low. Such condition affects stem and progenitor cell proliferation and differentiation and, at cellular level regulates hematopoietic growth factors, chemokines and adhesion molecules expression. In turn, these molecules affect the proliferation and maturation of other cellular components of the niche. Due to the complexity of the system we started the in vitro investigations of the IL-6, IL-8, TNFα cytokines expression and the vascular endothelial growth factor (VEGF), considered key mediators of the hematopoietic niche, in human macrophages and macrophage cell line. Since in the niche the oxygen availability is mediated by red blood cells (RBCs), we have influenced the anoxic cell cultures by the administration of oxygenated or deoxygenated RBCs (deoxy RBCs). The results reported in this brief paper show that the presence of RBCs up-regulates IL-8 mRNA while IL-6 and VEGF mRNA expression appears down-regulated. This does not occur when deoxy RBCs are used. Moreover, it appears that the administration of RBCs leads to an increase of TNFα expression levels in MonoMac 6 (MM6). Interestingly, the modulation of these factors likely occurs in a hypoxia-inducible factor-1α (HIF-1α) independent manner. Considering the role of oxygen in the hematopoietic niche further studies should explore these preliminary observations in more details

ddit4 gene expression is switched on by a new hdac4 function in ataxia telangiectasia

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Preclinical developments of enzyme-loaded red blood cells

Therapeutic enzymes are currently used in the treatment of several diseases. In most cases, the benefits are limited due to poor in vivo stability, immunogenicity, and drug-induced inactivating antibodies. A partial solution to the problem is obtained by masking the therapeutic protein by chemical modifications. Unfortunately, this is not a satisfactory solution because frequent adverse events, including anaphylaxis, can arise

Identification of the main ubiquitination site in human erythroid α-spectrin

AbstractErythroid spectrin is the main component of the red cell membrane skeleton, which is very important in determining the shape, resistance to mechanical stresses and deformability of red cells. Previously we demonstrated that human erythroid α-spectrin is ubiquitinated in vitro and in vivo, and using recombinant peptides we identified on repeat 17 the main ubiquitination site of α-spectrin. In order to identify the lysine(s) involved in the ubiquitination process, in the present study we mutated the lysines by site-directed mutagenesis. We found that ubiquitination was dramatically inhibited in peptides carrying the mutation of lysine 27 on repeat 17 (mutants K25,27R and K27R). We also demonstrated that the correct folding of this protein is fundamental for its recognition by the ubiquitin conjugating system. Furthermore, the region flanking lysine 27 showed a 75% similarity with the leucine zipper pattern present in many regulatory proteins. Thus, a new potential ubiquitin recognition motif was identified in α-spectrin and may be present in several other proteins

Identification of ä-Spectrin Domains Susceptible to Ubiquitination

Previously, we demonstrated that alpha-spectrin is a substrate for the ubiquitin system and that this conjugation is a dynamic process (Corsi, D., Galluzzi, L., Crinelli, R., and Magnani, M. (1995) J. Biol. Chem. 270, 8928-8935). In this study, we mapped the sites of ubiquitination on erythrocyte alpha-spectrin. A peptide map of digested alpha-spectrin, previously submitted to in vitro 125I-ubiquitin conjugation, revealed the presence of four distinct labeled bands with Mr 40,000, 36,000, 29,000, and 25,500. Western blotting experiments using antibodies against each alpha-spectrin domain revealed that only IgG anti-alphaIII domain recognized the 125I-labeled ubiquitin peptide of 29 kDa, whereas the IgG anti-alphaV domain recognized the Mr 40,000 125I-ubiquitin-labeled peptide. The other two labeled bands of Mr 36,000 and Mr 25,500 were identified as tetra and tri multiubiquitin chains. Ubiquitination of the alphaIII and alphaV domains was further confirmed by anti-alpha-spectrin domain immunoaffinity chromatography. Endoprotease Lys C-digested spectrin conjugated previously to 125I-ubiquitin was incubated with antibodies against each trypsin-resistant domain of alpha-spectrin. Gamma counting of the radiolabeled antigen-antibody complexes purified by protein A chromatography showed labeling in the IgG anti-alphaIII and anti-alphaV complexes alone. Domain alphaIII is not associated with any known function, whereas domain alphaV contains the nucleation site for the association of the alpha and beta chains. Ubiquitination of the latter domain suggests a role for ubiquitin in the modulation of the stability, deformability, and viscoelastic properties of the erythrocyte membrane

Multitalented Synthetic Antimicrobial Peptides and Their Antibacterial, Antifungal and Antiviral Mechanisms

: Despite the great strides in healthcare during the last century, some challenges still remained unanswered. The development of multi-drug resistant bacteria, the alarming growth of fungal infections, the emerging/re-emerging of viral diseases are yet a worldwide threat. Since the discovery of natural antimicrobial peptides able to broadly hit several pathogens, peptide-based therapeutics have been under the lenses of the researchers. This review aims to focus on synthetic peptides and elucidate their multifaceted mechanisms of action as antiviral, antibacterial and antifungal agents. Antimicrobial peptides generally affect highly preserved structures, e.g., the phospholipid membrane via pore formation or other constitutive targets like peptidoglycans in Gram-negative and Gram-positive bacteria, and glucan in the fungal cell wall. Additionally, some peptides are particularly active on biofilm destabilizing the microbial communities. They can also act intracellularly, e.g., on protein biosynthesis or DNA replication. Their intracellular properties are extended upon viral infection since peptides can influence several steps along the virus life cycle starting from viral receptor-cell interaction to the budding. Besides their mode of action, improvements in manufacturing to increase their half-life and performances are also taken into consideration together with advantages and impairments in the clinical usage. Thus far, the progress of new synthetic peptide-based approaches is making them a promising tool to counteract emerging infections