Proteomic profile of extracellular vesicles from plasma and CSF of multiple sclerosis patients reveals disease activity-associated EAAT2.

BACKGROUND AND OBJECTIVES: There is an urgent need to discover blood-based biomarkers of multiple sclerosis (MS) to better define the underlying biology of relapses and monitor disease progression. The main goal of this study is to search for candidate biomarkers of MS relapses associated with circulating extracellular vesicles (EVs), an emerging tool for biomarker discovery. METHODS: EVs, purified from unpaired plasma and CSF samples of RRMS patients by size-exclusion chromatography (SEC), underwent proteomic analysis to discover novel biomarkers associated with MS relapses. The candidate biomarkers of disease activity were detected by comparison approach between plasma- and CSF-EV proteomes associated with relapses. Among them, a selected potential biomarker was evaluated in a cohort of MS patients, using a novel and highly reproducible flow cytometry-based approach in order to detect low abundant EV subsets in a complex body fluid such as plasma. RESULTS: The proteomic profiles of both SEC-purified plasma EVs (from 6 patients in relapse and 5 patients in remission) and SEC-purified CSF EVs (from 4 patients in relapse and 3 patients in remission) revealed a set of proteins associated with MS relapses significant enriched in the synaptic transmission pathway. Among common proteins, excitatory amino-acid transporter 2, EAAT2, responsible for the majority of the glutamate uptake in CNS, was worthy of further investigation. By screening plasma samples from 110 MS patients, we found a significant association of plasma EV-carried EAAT2 protein (EV-EAAT2) with MS relapses, regardless of disease-modifying therapies. This finding was confirmed by investigating the presence of EV-EAAT2 in plasma samples collected longitudinally from 10 RRMS patients, during relapse and remission. Moreover, plasma EV-EAAT2 levels correlated positively with Expanded Disability Status Scale (EDSS) score in remitting MS patients but showed a negative correlation with age in patients with secondary progressive (SPMS). CONCLUSION: Our results emphaticize the usefulness of plasma EVs as a source of accessible biomarkers to remotely analyse the CNS status. Plasma EV-EAAT2 showed to be a promising biomarker for MS relapses. Further studies are required to assess the clinical relevance of this biomarker also for disability progression independent of relapse activity and transition from RRMS towards SPMS.

Microbiota-Derived Extracellular Vesicle as Emerging Actors in Host Interactions.

The human microbiota is an intricate micro-ecosystem comprising a diverse range of dynamic microbial populations mainly consisting of bacteria, whose interactions with hosts strongly affect several physiological and pathological processes. The gut microbiota is being increasingly recognized as a critical player in maintaining homeostasis, contributing to the main functions of the intestine and distal organs such as the brain. However, gut dysbiosis, characterized by composition and function alterations of microbiota with intestinal barrier dysfunction has been linked to the development and progression of several pathologies, including intestinal inflammatory diseases, systemic autoimmune diseases, such as rheumatic arthritis, and neurodegenerative diseases, such as Alzheimer's disease. Moreover, oral microbiota research has gained significant interest in recent years due to its potential impact on overall health. Emerging evidence on the role of microbiota-host interactions in health and disease has triggered a marked interest on the functional role of bacterial extracellular vesicles (BEVs) as mediators of inter-kingdom communication. Accumulating evidence reveals that BEVs mediate host interactions by transporting and delivering into host cells effector molecules that modulate host signaling pathways and cell processes, influencing health and disease. This review discusses the critical role of BEVs from the gut, lung, skin and oral cavity in the epithelium, immune system, and CNS interactions.

Extracellular vesicles as contributors in the pathogenesis of multiple sclerosis.

Extracellular vesicles (EVs) are a heterogeneous family of extracellular structures bounded by a phospholipid bilayer, released by all cell types in various biological fluids, such as blood and cerebrospinal fluid (CSF), playing important roles in intercellular communication, both locally and systemically. EVs carry and deliver a variety of bioactive molecules (proteins, nucleic acids, lipids and metabolites), conferring epigenetic and phenotypic changes to the recipient cells and thus resulting as important mediators of both homeostasis and pathogenesis. In neurological diseases, such as multiple sclerosis (MS), the EV ability to cross Blood-Brain Barrier (BBB), moving from central nervous system (CNS) to the peripheral circulation and vice versa, has increased the interest in EV study in the neurological field. In the present review, we will provide an overview of the recent advances made in understanding the pathogenic role of EVs regarding the immune response, the BBB dysfunction and the CNS inflammatory processes.

Construction of Human Naïve Antibody Gene Libraries.

Size and variability often represent an obstacle in generating an effective antibody gene library for the detection of an abundant repertoire of antigens. Therefore, optimizing the construction of a large library is essential for the selection of high-affinity reactive fragments. Here, we report a highly efficient method for the construction of a human naïve antibody gene library for the selection of antibodies as single-chain variable fragments. This protocol is based on many different sets of oligonucleotide primers and multistep electroporation and ligation reactions.This advanced method can be adopted by any molecular biology laboratory to generate a naïve library for use in isolating single-chain fragment variables against specific targets.

Design and construction of a new human naïve single-chain fragment variable antibody library, IORISS1.

Human monoclonal antibodies are a powerful tool with increasingly successful exploitations and the single chain fragment variable format can be considered the building block for the implementation of more complex and effective antibody-based constructs. Phage display is one of the best and most efficient methods to isolate human antibodies selected from an efficient and variable phage display library. We report a method for the construction of a human naïve single-chain variable fragment library, termed IORISS1. Many different sets of oligonucleotide primers as well as optimized electroporation and ligation reactions were used to generate this library of 1.2×10(9) individual clones. The key difference is the diversity of variable gene templates, which was derived from only 15 non-immunized human donors. The method described here, was used to make a new human naïve single-chain fragment variable phage display library that represents a valuable source of diverse antibodies that can be used as research reagents or as a starting point for the development of therapeutics. Using biopanning, we determined the ability of IORISS1 to yield antibodies. The results we obtained suggest that, by using an optimized protocol, an efficient phage antibody library can be generated.

Peripheral blood biomarkers in multiple sclerosis.

Multiple sclerosis is the most common autoimmune disorder affecting the central nervous system. The heterogeneity of pathophysiological processes in MS contributes to the highly variable course of the disease and unpredictable response to therapies. The major focus of the research on MS is the identification of biomarkers in biological fluids, such as cerebrospinal fluid or blood, to guide patient management reliably. Because of the difficulties in obtaining spinal fluid samples and the necessity for lumbar puncture to make a diagnosis has reduced, the research of blood-based biomarkers may provide increasingly important tools for clinical practice. However, currently there are no clearly established MS blood-based biomarkers. The availability of reliable biomarkers could radically alter the management of MS at critical phases of the disease spectrum, allowing for intervention strategies that may prevent evolution to long-term neurological disability. This article provides an overview of this research field and focuses on recent advances in blood-based biomarker research.

Generation of human single-chain antibody to the CD99 cell surface determinant specifically recognizing Ewing's sarcoma tumor cells.

The survival of pediatric patients with cancer entities including osteosarcoma and Ewing's sarcoma (ES), remains extremely low hence novel treatment approaches are urgently needed. Therefore, based on the concept of targeted therapy, numerous potential targets for the treatment of these cancers have been evaluated pre-clinically or in some cases even clinically during the last decade. In ES the CD99 protein is an attractive target antigen. In this respect, a new entry site for therapeutic intervention may derive from specific human antibodies against CD99. Human scFvC7 was isolated from a semi-synthetic ETH-2 antibody phage library panned on the extracellular portion of recombinant human CD99 protein. The scFvC7 was genetically sequenced, tested for CD99 recognition on an array of recombinant CD99 fragments and measured for binding affinity by ELISA. Finally, it was tested for staining CD99 antigen on a large panel of tumor and normal cells and tissues by cytofluorimetric and immunohistochemical assays. The new antibody scFvC7 recognizes the CD99 extracellular domain included between residues 50 and 74 with a binding affinity of 2.4 x 10(-8) M. In contrast with all other antibodies to CD99 so far isolated, scFvC7 shows a unique specificity in cancer cell recognition: It stained prevalently ES cells while no or weak reactivity was observed on the majority of the other tumor and normal cells and tissues. Thanks to its properties the new anti-CD99 antibody here described represents the first step towards the construction of new selective ES therapeutics.

P-glycoprotein binds to ezrin at amino acid residues 149-242 in the FERM domain and plays a key role in the multidrug resistance of human osteosarcoma.

Overexpression of the mdr1 gene encoding P-glycoprotein (Pgp) exerts a major role in reducing the effectiveness of cytotoxic therapy in osteosarcoma. The interaction between actin and Pgp has been shown to be instrumental in the establishment of multidrug resistance (MDR) in human tumor cells. The cytoskeleton linker ezrin exerts a pivotal role in maintaining the functional connection between actin and Pgp. We investigated the role of ezrin in a human multidrug-resistant osteosarcoma cell line overexpressing Pgp and compared it to its counterpart that overexpresses an ezrin deletion mutant. The results showed that Pgp binds at amino acid residues 149-242 of the N-terminal domain of ezrin. The interaction between ezrin and Pgp occurs in the plasma membrane of MDR cells, where they also co-localize with the ganglioside G(M1) located in lipid rafts. The overexpression of the ezrin deletion mutant entirely restored drug susceptibility of osteosarcoma cells, consistent with Pgp dislocation to cytoplasmic compartments and abrogation of G(M1) /Pgp co-localization at the plasma membrane. Our study provides evidence that ezrin exerts a key role in MDR of human osteosarcoma cells through a Pgp-ezrin-actin connection that is instrumental for the permanence of Pgp into plasma membrane lipid rafts. We also show for the first time that Pgp-binding site is localized to amino acid residues 149-242 of the ezrin Band 4.1, Ezrin/Radixin/Moesin (FERM) domain, thus proposing a specific target for future molecular therapy aimed at counteracting MDR in osteosarcoma patients.

Human monoclonal antibodies in single chain fragment variable format with potent neutralization activity against influenza virus H5N1.

Effective diagnostic and therapeutic strategies are needed to control and combat the highly pathogenic avian influenza virus (AIV) subtype H5N1. To this end, we developed human monoclonal antibodies (mAbs) in single chain fragment variable (scFv) format towards the H5N1 avian influenza virus to gain new insights for the development of immunotherapy against human cases of H5N1. Using a biopanning based approach a large array of scFvs against H5N1 virus were isolated from the human semi-synthetic ETH-2 phage antibody library. H5N1 ELISA-positive scFvs with unique variable heavy (VH) and light (VL) chain gene sequences showed different biochemical properties and neutralization activity across H5N1 viral strains. In particular, the scFv clones AV.D1 and AV.C4 exerted a significant inhibition of the H5N1 A/Vietnam/1194/2004 virus infection in a pseudotype-based neutralization assay. Interestingly, these two scFvs displayed a cross-clade neutralizing activity versus A/whooping swan/Mongolia/244/2005 and A/Indonesia/5/2005 strains. These studies provide proof of the concept that human mAbs in scFv format with well-defined H5N1 recognition patterns and in vitro neutralizing activity can be easily and rapidly isolated by biopanning selection of an entirely artificial antibody repertoire using inactivated H5N1 virus as a bait.

Protection by anti-beta-glucan antibodies is associated with restricted beta-1,3 glucan binding specificity and inhibition of fungal growth and adherence.

Anti-beta-glucan antibodies elicited by a laminarin-conjugate vaccine confer cross-protection to mice challenged with major fungal pathogens such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. To gain insights into protective beta-glucan epitope(s) and protection mechanisms, we studied two anti-beta-glucan monoclonal antibodies (mAb) with identical complementarity-determining regions but different isotypes (mAb 2G8, IgG2b and mAb 1E12, IgM). C. albicans, the most relevant fungal pathogen for humans, was used as a model.Both mAbs bound to fungal cell surface and to the beta1,3-beta1,6 glucan of the fungal cell wall skeleton, as shown by immunofluorescence, electron-microscopy and ELISA. They were also equally unable to opsonize fungal cells in a J774 macrophage phagocytosis and killing assay. However, only the IgG2b conferred substantial protection against mucosal and systemic candidiasis in passive vaccination experiments in rodents. Competition ELISA and microarray analyses using sequence-defined glucan oligosaccharides showed that the protective IgG2b selectively bound to beta1,3-linked (laminarin-like) glucose sequences whereas the non-protective IgM bound to beta1,6- and beta1,4-linked glucose sequences in addition to beta1,3-linked ones. Only the protective IgG2b recognized heterogeneous, polydisperse high molecular weight cell wall and secretory components of the fungus, two of which were identified as the GPI-anchored cell wall proteins Als3 and Hyr1. In addition, only the IgG2b inhibited in vitro two critical virulence attributes of the fungus, hyphal growth and adherence to human epithelial cells.Our study demonstrates that the isotype of anti-beta-glucan antibodies may affect details of the beta-glucan epitopes recognized, and this may be associated with a differing ability to inhibit virulence attributes of the fungus and confer protection in vivo. Our data also suggest that the anti-virulence properties of the IgG2b mAb may be linked to its capacity to recognize beta-glucan epitope(s) on some cell wall components that exert critical functions in fungal cell wall structure and adherence to host cells.

Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from yeast.

BACKGROUND: The ability of cytosine deaminase (CD) to convert the antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme to model gene or antibody - directed enzyme-prodrug therapy (GDEPT/ADEPT) aiming to improve the therapeutic ratio (benefit versus toxic side-effects) of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv) format represents a powerful reagent to allow in in vitro and in vivo detection of CD expression in GDEPT/ADEPT studies. RESULTS: An enzymatic active recombinant CD from yeast (yCD) was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU. CONCLUSION: The construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function.

Genetic construction, expression, and characterization of a single chain anti-CEA antibody fused to cytosine deaminase from yeast.

We report the genetic construction and expression of a fusion protein between a single chain fragment variable (scFv) human antibody (E8) specific for CEA cell surface antigen and yeast cytosine deaminase (yCD). Sequences encoding for the scFvE8 human monoclonal antibody recognizing an epitope shared by CEACAM1, CEACAM3 and CEACAM5 isoforms were assembled with a monomer of yCD. The construct was placed under the transcriptional regulation of the lac promoter, and in frame with 6xHis tag for protein purification. After transformation and induction of E. coli, the protein was recovered from cell lysates and processed for purification. The scFvE8:yCD fusion protein possessed the binding specificity for melanoma (Mel P5) and colon carcinoma (LoVo) cell lines similar to its cognate human scFv antibody. The scFv8:yCD system showed the ability to render tumor cells susceptible to the far less toxic substrate 5-fluorocytosine (5-FC) by its enzymatic conversion into 5-fluorouracil (5-FU). In vitro pre-treatment of Mel P5 and LoVo cell lines with scFvE8:yCD followed by cell washing and incubation with 5-FC, resulted in significant cell killing supporting the utility of this fusion protein as an agent for tumor-selective prodrug activation. This study shows the feasibility of constructing fusion proteins in a prokaryotic cell based system consisting of a human scFv antibody and yCD to convert the antifungal agent 5-FC to 5-FU, one of the widely used anticancer agent.

Generation of human scFvs antibodies recognizing a prion protein epitope expressed on the surface of human lymphoblastoid cells.

BACKGROUND: A hallmark of prion disease is the transformation of normal cellular prion protein (PrPc) into an infectious disease-associated isoform, (PrPsc). Anti-prion protein monoclonal antibodies are invaluable for structure-function studies of PrP molecules. Furthermore recent in vitro and in vivo studies indicate that anti-PrP monoclonal antibodies can prevent the incorporation of PrPc into propagating prions. In the present article, we show two new human phage antibodies, isolated on recombinant hamster prion protein (rHaPrP). RESULTS: We adopted an antibody phage display strategy to isolate specific human antibodies directed towards rHaPrP which has been used as a bait for panning the synthetic ETH-2 antibody phage library. Two phage antibodies clones named MA3.B4 and MA3.G3 were isolated and characterized under genetic biochemical and immunocytochemical aspects. The clones were found to recognize the prion protein in ELISA studies. In flow-cytometry studies, these human single chain Fragment variable (scFv) phage-antibodies show a well defined pattern of reactivity on human lymphoblastoid and myeloid cells. CONCLUSION: Sequence analysis of the gene encoding for the antibody fragments and antigen recognition patterns determined by flow-cytometry analysis indicate that the isolated scFvs recognize novel epitopes in the PrPc molecule. These new anti PrPc human antibodies are unique reagents for prion protein detection and may represent a biologic platform to develop new reagents to treat PrPsc associated disease.

Generation of human antibody fragments recognizing distinct epitopes of the nucleocapsid (N) SARS-CoV protein using a phage display approach.

BACKGROUND: Severe acute respiratory syndrome (SARS)-CoV is a newly emerging virus that causes SARS with high mortality rate in infected people. Successful control of the global SARS epidemic will require rapid and sensitive diagnostic tests to monitor its spread, as well as, the development of vaccines and new antiviral compounds including neutralizing antibodies that effectively prevent or treat this disease. METHODS: The human synthetic single-chain fragment variable (scFv) ETH-2 phage antibody library was used for the isolation of scFvs against the nucleocapsid (N) protein of SARS-CoV using a bio panning-based strategy. The selected scFvs were characterized under genetics-molecular aspects and for SARS-CoV N protein detection in ELISA, western blotting and immunocytochemistry. RESULTS: Human scFv antibodies to N protein of SARS-CoV can be easily isolated by selecting the ETH-2 phage library on immunotubes coated with antigen. These in vitro selected human scFvs specifically recognize in ELISA and western blotting studies distinct epitopes in N protein domains and detect in immunohistochemistry investigations SARS-CoV particles in infected Vero cells. CONCLUSION: The human scFv antibodies isolated and described in this study represent useful reagents for rapid detection of N SARS-CoV protein and SARS virus particles in infected target cells.

Application of a synthetic phage antibody library (ETH-2) for the isolation of single chain fragment variable (scFv) human antibodies to the pathogenic isoform of the hamster prion protein (HaPrPsc).

To overcome the limitation represented by the poor immunogenicity of prion protein (PrP) for conventional monoclonal antibodies preparation, we adopted an antibody phage display strategy to isolate specific human single chain fragment variable (scFv) directed towards the pathogenic isoform of the hamster prion protein (HaPrPsc). Phage-displaying HaPrPsc reactive scFvs were obtained after three rounds of selection of the ETH- 2 synthetic antibody library on HaPrPsc-coated immunotubes and subsequent amplification in TG1 E. coli cells. These phage-antibodies bind in ELISA to HaPrPsc and do not cross-react with the recombinant hamster prion protein (rHaPrP). Sequence analyses of the gene encoding for the antibody fragments and antigen recognition patterns determined by flow-cytometry on lymphoid cells indicate that the selected scFv recognize distinct epitopes in the PrPsc molecule. The results of this study demonstrate that display of scFvs on filamentous phage offers the possibility of producing phage antibodies showing immunoglobulin-like functions using only in vitro procedures, thus overcoming limitations of conventional hybridoma technology.

Isolation and characterization of the human monoclonal antibodies C10 in single-chain fragment variable (scFv) format to glucose oxidase from Aspergillus niger.

Despite biotechnological and clinical applications very few monoclonal antibodies (MAbs) directed to the enzyme glucose oxidase, have been produced so far because of the heavy side effects of the immunization schedule for conventional MAb preparation. In contrast, the phage display method allows for the selection of monoclonal human antibody fragments against any antigens, including toxic proteins. Furthermore, cDNA sequences encoding selected antibody fragments are readily identified, facilitating various molecular targeting approaches. In order to obtain such human fragments recognizing glucose oxidase, we used the large synthetic ETH-2 library based on the principle of protein design. Phage displaying glucose oxidase reactive scFvs were obtained after three rounds of selection on glucose oxidase-coated immunotubes and subsequent amplification in TG1 E. coli cells. Eventually, one high reactive scFv clone was selected and further examined. The anti-glucose oxidase scFv C10 was found suitable for Western blot; Biacore analysis showed that the binding affinity of the glucose oxidase-reactive scFv is almost equal that of MAbs prepared with conventional hybridoma technology. Finally, the cDNA sequence of this human scFv may be exploited to generate bispecific antibodies to target in the tumor environment-specific toxic enzymatic reaction.