Vibrio proteases for biomedical applications: Modulating the proteolytic secretome of v. alginolyticus and v. parahaemolyticus for improved enzymes production

Proteolytic enzymes are of great interest for biotechnological purposes, and their large-scale production, as well as the discovery of strains producing new molecules, is a relevant issue. Collagenases are employed for biomedical and pharmaceutical purposes. The high specificity of collagenase-based preparations toward the substrate strongly relies on the enzyme purity. However, the overall activity may depend on the cooperation with other proteases, the presence of which may be essential for the overall enzymatic activity, but potentially harmful for cells and tissues. Vibrios produce some of the most promising bacterial proteases (including collagenases), and their exo-proteome includes several enzymes with different substrate specificities, the production and relative abundances of which strongly depend on growth conditions. We evaluated the effects of different media compositions on the proteolytic exo-proteome of Vibrio alginolyticus and its closely relative Vibrio parahaemolyticus, in order to improve the overall proteases production, as well as the yield of the desired enzymes subset. Substantial biological responses were achieved with all media, which allowed defining culture conditions for targeted improvement of selected enzyme classes, besides giving insights in possible regulatory mechanisms. In particular, we focused our efforts on collagenases production, because of the growing biotechnological interest due to their pharmaceutical/biomedical applications

Native rodent species are unlikely sources of infection for Leishmania (Viannia) braziliensis along the Transoceanic Highway in Madre de Dios, Peru.

An estimated 2.3 million disability-adjusted life years are lost globally from leishmaniasis. In Peru's Amazon region, the department of Madre de Dios (MDD) rises above the rest of the country in terms of the annual incidence rates of human leishmaniasis. Leishmania (Viannia) braziliensis is the species most frequently responsible for the form of disease that results in tissue destruction of the nose and mouth. However, essentially nothing is known regarding the reservoirs of this vector-borne, zoonotic parasite in MDD. Wild rodents have been suspected, or proven, to be reservoirs of several Leishmania spp. in various ecosystems and countries. Additionally, people who live or work in forested terrain, especially those who are not regionally local and whose immune systems are thus naïve to the parasite, are at most risk for contracting L. (V.) braziliensis. Hence, the objective of this study was to collect tissues from wild rodents captured at several study sites along the Amazonian segment of the newly constructed Transoceanic Highway and to use molecular laboratory techniques to analyze samples for the presence of Leishmania parasites. Liver tissues were tested via polymerase chain reaction from a total of 217 rodents; bone marrow and skin biopsies (ear and tail) were also tested from a subset of these same animals. The most numerous rodent species captured and tested were Oligoryzomys microtis (40.7%), Hylaeamys perenensis (15.7%), and Proechimys spp. (12%). All samples were negative for Leishmania, implying that although incidental infections may occur, these abundant rodent species are unlikely to serve as primary reservoirs of L. (V.) braziliensis along the Transoceanic Highway in MDD. Therefore, although these rodent species may persist and even thrive in moderately altered landscapes, we did not find any evidence to suggest they pose a risk for L. (V.) braziliensis transmission to human inhabitants in this highly prevalent region

3d collagen hydrogel promotes in vitro langerhans islets vascularization through ad-mvfs angiogenic activity

Adipose derived microvascular fragments (ad-MVFs) consist of effective vascularization units able to reassemble into efficient microvascular networks. Because of their content in stem cells and related angiogenic activity, ad-MVFs represent an interesting tool for applications in regenerative medicine. Here we show that gentle dissociation of rat adipose tissue provides a mixture of ad-MVFs with a length distribution ranging from 33–955 µm that are able to maintain their original morphology. The isolated units of ad-MVFs that resulted were able to activate transcriptional switching toward angiogenesis, forming tubes, branches, and entire capillary networks when cultured in 3D collagen type-I hydrogel. The proper involvement of metalloproteases (MMP2/MMP9) and serine proteases in basal lamina and extracellular matrix ECM degradation during the angiogenesis were concurrently assessed by the evaluation of alpha-smooth muscle actin (αSMA) expression. These results suggest that collagen type-I hydrogel provides an adequate 3D environment supporting the activation of the vascularization process. As a proof of concept, we exploited 3D collagen hydrogel for the setting of ad-MVF–islet of Langerhans coculture to improve the islets vascularization. Our results suggest potential employment of the proposed in vitro system for regenerative medicine applications, such as the improving of the islet of Langerhans engraftment before transplantation

Neural Crest-Derived Chondrocytes Isolation for Tissue Engineering in Regenerative Medicine

Chondrocyte transplantation has been successfully tested and proposed as a clinical procedure aiming to repair articular cartilage defects. However, the isolation of chondrocytes and the optimization of the enzymatic digestion process, as well as their successful in vitro expansion, remain the main challenges in cartilage tissue engineering. In order to address these issues, we investigated the performance of recombinant collagenases in tissue dissociation assays with the aim of isolating chondrocytes from bovine nasal cartilage in order to establish the optimal enzyme blend to ensure the best outcomes of the overall procedure. We show, for the first time, that collagenase H activity alone is required for effective cartilage digestion, resulting in an improvement in the yield of viable cells. The extracted chondrocytes proved able to grow and activate differentiation/dedifferentiation programs, as assessed by morphological and gene expression analyses

Physical and biological properties of electrospun poly(d,l-lactide)/nanoclay and poly(d,l-lactide)/nanosilica nanofibrous scaffold for bone tissue engineering

Electrospun scaffolds exhibiting high physical performances with the ability to support cell attachment and proliferation are attracting more and more scientific interest for tissue engineering applications. The inclusion of inorganic nanoparticles such as nanosilica and nanoclay into electrospun biopolymeric matrices can meet these challenging requirements. The silica and clay incorporation into polymeric nanofibers has been reported to enhance and improve the mechanical properties as well as the osteogenic properties of the scaffolds. In this work, for the first time, the physical and biological properties of polylactic acid (PLA) electrospun mats filled with different concentrations of nanosilica and nanoclay were evaluated and compared. The inclusion of the particles was evaluated through morphological investigations and Fourier transform infrared spectroscopy. The morphology of nanofibers was differently affected by the amount and kind of fillers and it was correlated to the viscosity of the polymeric suspensions. The wettability of the scaffolds, evaluated through wet contact angle measurements, slightly increased for both the nanocomposites. The crystallinity of the systems was investigated by differential scanning calorimetry highlighting the nucleating action of both nanosilica and nanoclay on PLA. Scaffolds were mechanically characterized with tensile tests to evaluate the reinforcing action of the fillers. Finally, cell culture assays with pre-osteoblastic cells were conducted on a selected composite scaffold in order to compare the cell proliferation and morphology with that of neat PLA scaffolds. Based on the results, we can convince that nanosilica and nanoclay can be both considered great potential fillers for electrospun systems engineered for bone tissue regeneration

A Fibrillar Biodegradable Scaffold for Blood Vessels Tissue Engineering

In recent years there has been a growing interest for the development of tubular scaffolds employed to assist the replacement of small blood vessels. Materials designed for this purpose need to be biodegradable, have good mechanical properties and improve cell adhesion, proliferation and differentiation. To obtain biomaterials with these properties, electrospinning seems to be one of the most useful technique. Several biodegradable synthetic polymers or constituents of the extracellular matrix (ECM) have been electrospun showing optimal mechanical properties and biodegradability. However, such polymers are lacking in versatile chemical structure affordable to immobilize growth factors or chemokines. The glycosaminoglycan heparin is able to bind several growth factors like vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) and, when grafted onto the scaffold surface it is able to attract cells thus improving their proliferation and differentiation. Aim of this research was the production and the preliminary in vitro biological characterization of a new biodegradable material, obtained by electrospun a polyaminoacid-graft-polyester copolymer. The electrospun biomaterial has been successfully grafted with heparin exploiting the better chemical reactivity of the polyaminoacid portions of the graft copolymer. Then its morphology has been investigated by scanning electron microscopy (SEM) and the potential biodegradability of the material has been studied until 60 days. Preliminary biological data in vitro, on human endothelial cells, show a good compatibility of the scaffold obtained by electrospinning, with regard to cell adhesion and proliferation. Experiments are in progress to evaluate the effects of heparin on cell differentiation

Epstein-Barr Virus Epitope-Major Histocompatibility Complex Interaction Combined with Convergent Recombination Drives Selection of Diverse T Cell Receptor alpha and beta Repertoires

Recognition modes of individual T cell receptors (TCRs) are well studied, but factors driving the selection of TCR repertoires from primary through persistent human virus infections are less well understood. Using deep sequencing, we demonstrate a high degree of diversity of Epstein-Barr virus (EBV)-specific clonotypes in acute infectious mononucleosis (AIM). Only 9% of unique clonotypes detected in AIM persisted into convalescence; the majority (91%) of unique clonotypes detected in AIM were not detected in convalescence and were seeming replaced by equally diverse de novo clonotypes. The persistent clonotypes had a greater probability of being generated than nonpersistent clonotypes due to convergence recombination of multiple nucleotide sequences to encode the same amino acid sequence, as well as the use of shorter complementarity-determining regions 3 (CDR3s) with fewer nucleotide additions (i.e., sequences closer to germ line). Moreover, the two most immunodominant HLA-A2-restricted EBV epitopes, BRLF1109 and BMLF1280, show highly distinct antigen-specific public (i.e., shared between individuals) features. In fact, TCRalpha CDR3 motifs played a dominant role, while TCRbeta played a minimal role, in the selection of TCR repertoire to an immunodominant EBV epitope, BRLF1. This contrasts with the majority of previously reported repertoires, which appear to be selected either on TCRbeta CDR3 interactions with peptide/major histocompatibility complex (MHC) or in combination with TCRalpha CDR3. Understanding of how TCR-peptide-MHC complex interactions drive repertoire selection can be used to develop optimal strategies for vaccine design or generation of appropriate adoptive immunotherapies for viral infections in transplant settings or for cancer. IMPORTANCE Several lines of evidence suggest that TCRalpha and TCRbeta repertoires play a role in disease outcomes and treatment strategies during viral infections in transplant patients and in cancer and autoimmune disease therapy. Our data suggest that it is essential that we understand the basic principles of how to drive optimum repertoires for both TCR chains, alpha and beta. We address this important issue by characterizing the CD8 TCR repertoire to a common persistent human viral infection (EBV), which is controlled by appropriate CD8 T cell responses. The ultimate goal would be to determine if the individuals who are infected asymptomatically develop a different TCR repertoire than those that develop the immunopathology of AIM. Here, we begin by doing an in-depth characterization of both CD8 T cell TCRalpha and TCRbeta repertoires to two immunodominant EBV epitopes over the course of AIM, identifying potential factors that may be driving their selection