George Lagoumintzis

Muscle and neuronal nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are integral membrane proteins and prototypic members of the ligand‐gated ion‐channel superfamily, which has precursors in the prokaryotic world. They are formed by the assembly of five transmembrane subunits, selected from a pool of 17 homologous polypeptides (α1–10, β1–4, γ, δ, and ε). There are many nAChR subtypes, each consisting of a specific combination of subunits, which mediate diverse physiological functions. They are widely expressed in the central nervous system, while, in the periphery, they mediate synaptic transmission at the neuromuscular junction and ganglia. nAChRs are also found in non‐neuronal/nonmuscle cells (keratinocytes, epithelia, macrophages, etc.). Extensive research has determined the specific function of several nAChR subtypes. nAChRs are now important therapeutic targets for various diseases, including myasthenia gravis, Alzheimers and Parkinsons diseases, and schizophrenia, as well as for the cessation of smoking. However, knowledge is still incomplete, largely because of a lack of high‐resolution X‐ray structures for these molecules. Nevertheless, electron microscopy studies on 2D crystals of nAChR from fish electric organs and the determination of the high‐resolution X‐ray structure of the acetylcholine binding protein (AChBP) from snails, a homolog of the extracellular domain of the nAChR, have been major steps forward and the data obtained have important implications for the design of subtype‐specific drugs. Here, we review some of the latest advances in our understanding of nAChRs and their involvement in physiology and pathology.

Transcutaneous delivery of a nanoencapsulated antigen: Induction of immune responses

We investigated the influence of antigen entrapment in PLA nanoparticles on the immune responses obtained after transcutaneous immunization. OVA-loaded PLA nanoparticles were prepared using a double emulsion process. Following application onto bare skin of mice in vivo, fluorescence-labeled nanoparticles were detected in the duct of the hair follicles indicating that the nanoparticles can penetrate the skin barrier through the hair follicles. Although the OVA-loaded nanoparticles elicited lower antibody responses than those induced by OVA in aqueous solution they were more efficient in inducing cytokine responses. In vitro re-stimulation of cultured splenocytes with OVA elicited a little higher levels of IFN-gamma (difference statistically insignificant, p>0.05) and significantly higher levels of IL-2 (p<0.001) in mice immunized with OVA-loaded nanoparticles compared to those immunized with OVA in solution. In the presence of CT, the OVA-loaded nanoparticles induced significantly higher IFN-gamma and IL-2 than all other formulations. Transcutaneous administration of OVA encapsulated in the PLA nanoparticles exhibited priming efficacy to a challenging dose of OVA given via different route. These findings indicate the potential of nanoparticles to deliver antigens via the transcutaneous route and prime for antibody and strong cellular responses. The co-administration of an adjuvant such as CT had the added advantage of modulating the immune response, a desirable characteristic within the context of vaccination against intracellular versus extracellular pathogens.

A critical view of the general public’s awareness and physicians’ opinion of the trends and potential pitfalls of genetic testing in Greece

AIM Progress in deciphering the functionality of the human genome sequence in the wake of technological advances in the field of genomic medicine have dramatically reduced the overall costs of genetic analysis, thereby facilitating the incorporation of genetic testing services into mainstream clinical practice. Although Greek genetic testing laboratories offer a variety of different genetic tests, relatively little is known about how either the general public or medical practitioners perceive genetic testing services. MATERIALS & METHODS We have therefore performed a nationwide survey of the views of 1717 members of the general public, divided into three age groups, from all over Greece, and residing in both large and small cities and villages, in order to acquire a better understanding of how they perceive genetic testing. We also canvassed the opinions of 496 medical practitioners with regard to genetic testing services in a separate survey that addressed similar issues. RESULTS Our subsequent analysis indicated that a large proportion of the general public is aware of the nature of DNA, genetic disorders and the potential benefits of genetic testing, although this proportion declines steadily with age. Furthermore, a large proportion of the interviewed individuals would be willing to undergo genetic testing even if the cost of analysis was not covered by healthcare insurance. However, a relatively small proportion of the general public has actually been advized to undergo genetic testing, either by relatives or physicians. Most physicians believe that the regulatory and legal framework that governs genetic testing services in Greece is rather weak. Interestingly, the vast majority of the general public strongly opposes direct-access genetic testing, and most would prefer referral from a physician than from a pharmacist. CONCLUSION Overall, our results provide a critical evaluation of the views of the general public with regard to genetics and genetic testing services in Greece and should serve as a model for replication in other populations.

Synergistic regulation of Pseudomonas aeruginosa- induced cytokine production in human monocytes by mannose receptor and TLR2

The immune response to pathogen is regulated by a combination of specific PRR, which are involved in pathogen recognition. Pseudomonas aeruginosa, a bacterium that causes life‐threatening disease in immuno‐compromised host, is recognized by distinct members of the TLR family. We have previously shown that viable P. aeruginosa bacteria are recognized by human monocytes mainly through TLR2. Using ligand‐specific blocking antibodies, we herein show that the mannose receptor (MR), a phagocytic receptor for unopsonized P. aeruginosa bacteria, contributes equally to TLR2 in proinflammatory cytokine production by human monocytes in response to P. aeruginosa infection. Synergy of both receptors totally controls the immune response. Viable P. aeruginosa bacteria activate NF‐κB and MAPK pathways and enhance TLR2‐mediated signaling in MR‐transfected human embryonic kidney 293 cells. Moreover, MR follows the same kinetics and colocalizes with TLR2 in the endosome during in vivo infection of human macrophages with P. aeruginosa. The studies provide the first demonstration of a significant role for MR, synergistic with TLR2, in activating a proinflammatory response to P. aeruginosa infection.

Direct Proof of the In Vivo Pathogenic Role of the AChR Autoantibodies from Myasthenia Gravis Patients

Several studies have suggested that the autoantibodies (autoAbs) against muscle acetylcholine receptor (AChR) of myasthenia gravis (MG) patients are the main pathogenic factor in MG; however, this belief has not yet been confirmed with direct observations. Although animals immunized with AChR or injected with anti-AChR monoclonal Abs, or with crude human MG Ig fractions exhibit MG symptoms, the pathogenic role of isolated anti-AChR autoAbs, and, more importantly, the absence of pathogenic factor(s) in the autoAb-depleted MG sera has not yet been shown by in vivo studies. Using recombinant extracellular domains of the human AChR α and β subunits, we have isolated autoAbs from the sera of four MG patients. The ability of these isolated anti-subunit Abs and of the Ab-depleted sera to passively transfer experimental autoimmune MG in Lewis rats was investigated. We found that the isolated anti-subunit Abs were at least as efficient as the corresponding whole sera or whole Ig in causing experimental MG. Abs to both α- and β-subunit were pathogenic although the anti-α-subunit were much more efficient than the anti-β-subunit ones. Interestingly, the autoAb-depleted sera were free of pathogenic activity. The later suggests that the myasthenogenic potency of the studied anti-AChR MG sera is totally due to their anti-AChR autoAbs, and therefore selective elimination of the anti-AChR autoAbs from MG patients may be an efficient therapy for MG.

ETHNOS: A versatile electronic tool for the development and curation of national genetic databases

National and ethnic mutation databases (NEMDBs) are emerging online repositories, recording extensive information about the described genetic heterogeneity of an ethnic group or population. These resources facilitate the provision of genetic services and provide a comprehensive list of genomic variations among different populations. As such, they enhance awareness of the various genetic disorders. Here, we describe the features of the ETHNOS software, a simple but versatile tool based on a flat-file database that is specifically designed for the development and curation of NEMDBs. ETHNOS is a freely available software which runs more than half of the NEMDBs currently available. Given the emerging need for NEMDB in genetic testing services and the fact that ETHNOS is the only off-the-shelf software available for NEMDB development and curation, its adoption in subsequent NEMDB development would contribute towards data content uniformity, unlike the diverse contents and quality of the available gene (locus)-specific databases. Finally, we allude to the potential applications of NEMDBs, not only as worldwide central allele frequency repositories, but also, and most importantly, as data warehouses of individual-level genomic data, hence allowing for a comprehensive ethnicity-specific documentation of genomic variation.

Pseudomonas aeruginosa Slime Glycolipoprotein Is a Potent Stimulant of Tumor Necrosis Factor Alpha Gene Expression and Activation of Transcription Activators Nuclear Factor κB and Activator Protein 1 in Human Monocytes

ABSTRACT Pseudomonas aeruginosa, an opportunistic pathogen, causes infections associated with a high incidence of morbidity and mortality in immunocompromised hosts. Production of tumor necrosis factor alpha (TNF-α), primarily by cells of monocytic lineage, is a crucial event in the course of these infections. During in vivo infections with P. aeruginosa, both lipopolysaccharide (LPS) and extracellular slime glycolipoprotein (GLP) produced by mucoid and nonmucoid strains are released. In the present study, we sought to explore the relative contributions of these two bacterial products to TNF-α production by human monocytes. To this end, fresh human monocytes and THP-1 human monocytic cells were stimulated with P. aeruginosa LPS or GLP. GLP was found to be a more potent stimulus for TNF-α production (threefold higher) by human monocytes than LPS. Moreover, its effect was comparable to that of viable bacteria. Quantitative mRNA analysis revealed predominantly transcriptional regulation. Electrophoretic mobility shift assays and transfection assays demonstrated activation of NF-κB and activator protein 1 (AP-1). NF-κB activation by GLP was rapid and followed the same time course as that by viable bacteria, suggesting that bacteria could directly activate NF-κB through GLP. Moreover P. aeruginosa GLP induced the formation of AP-1 complex with delayed kinetics compared with NF-κB but much more efficiently than the homologous LPS. These results identify GLP as the most important stimulant for TNF-α production by human monocytes. Activation of NF-κB and AP-1 by P. aeruginosa GLP may be involved not only in TNF-α induction but also in many of the inflammatory responses triggered in the course of infection with P. aeruginosa.

Isolation and functional characterization of anti-acetylcholine receptor subunit-specific autoantibodies from myasthenic patients: Receptor loss in cell culture

The muscle nicotinic acetylcholine receptor (nAChR) is the major autoantigen in the autoimmune disease myasthenia gravis (MG), in which autoantibodies bind to, and cause loss of, nAChRs. Antibody-mediated nAChR loss is caused by the action of complement and by the acceleration of nAChR internalization caused by antibody-induced cross-linking of nAChR molecules (antigenic modulation). To obtain an insight into the role of the various anti-nAChR antibody specificities in MG, we have studied nAChR antigenic modulation caused by isolated anti-subunit autoantibodies. Autoantibodies against the nAChR alpha or beta subunits were isolated from four MG sera by affinity chromatography on columns carrying immobilized recombinant extracellular domains of human nAChR expressed in the yeast Pichia pastoris. The isolated anti-alpha and anti-beta autoantibodies, as well as untreated MG sera, induced nAChR antigenic modulation in TE671 cells. Partially antibody-depleted sera exhibited reduced modulating activity, whereas a serum completely depleted of anti-nAChR antibodies exhibited no nAChR modulation. Interestingly, the anti-alpha autoantibodies were, on average, approximately 4.3 times more effective than the anti-beta autoantibodies. The present work supports the notion that anti-nAChR autoantibodies may be the sole nAChR-reducing factor in anti-nAChR antibody-seropositive MG, and that anti-alpha-subunit autoantibodies are the dominant pathogenic autoantibody specificity.

Scale up and safety parameters of antigen specific immunoadsorption of human anti-acetylcholine receptor antibodies.

Myasthenia gravis is an autoimmune disease usually caused by autoantibodies against the muscle nicotinic acetylcholine receptor (nAChR). Current treatments are not specific, and thus often cause side effects. Here, we elaborate on our previous findings on antigen specific immunoadsorption towards scaling up the method as well as testing whole blood apheresis. The average percent of plasma or whole blood immunoadsorption was up to 79.5%±2.9. Moreover, neither pyrogens were co-administered nor did complement activation occur after immunoadsorption. Thus, antigen-specific apheresis of anti-AChR autoantibodies seems a safe and effective treatment for myasthenia gravis that can be scaled up for clinical testing.

TNF-alpha induction by Pseudomonas aeruginosa lipopolysaccharide or slime-glycolipoprotein in human monocytes is regulated at the level of Mitogen-activated Protein Kinase activity: a distinct role of Toll-like receptor 2 and 4.

TNF‐α production has a central role in the development and progression of Pseudomonas aeruginosa septic shock. We have previously shown that P. aeruginosa slime‐glycolipoprotein (slime‐GLP) is the most potent stimulant compared to P. aeruginosa lipopolysaccharide (LPS), for TNF‐α production and NF‐kB activation in human monocytes. Herein, we show that secretion of TNF‐α by fresh human monocytes, induced by P. aeruginosa slime‐GLP, LPS or viable bacteria, was paralleled by phosphorylation and/or activation of Mitogen‐activated Protein Kinases (MAPKs) ERK1/2, p38 as well as c‐Jun NH2‐terminal kinase. TNF‐α levels were significantly reduced by ERK1/2 inhibitor (PD98059), or p38 inhibitor (SB203580). Combination of both inhibitors almost abolished TNF‐α induction. Pseudomonas aeruginosa slime‐GLP differed from the P. aeruginosa‐LPS only regarding the strength of p38 and ERK1/2 activation, with slime‐GLP leading to a stronger activation of p38 and ERK1/2. Involvement of TLR2 and TLR4 for phosphorylation of p38 and ERK1/2 was shown using specific blocking anti‐TLR2 and anti‐TLR4 antibodies. Activation of both p38 and ERK1/2 induced by P. aeruginosa slime‐GLP was dramatically reduced in the presence of anti‐TLR2 and to a lesser degree in the presence of anti‐TLR4, whereas the P. aeruginosa‐LPS‐induced stimulation was inhibited only in the presence of anti‐TLR4. Our data show that P. aeruginosa viable bacteria, through slime‐GLP, stimulate specific members of the MAPKs more efficiently than the P. aeruginosa‐LPS, involving mainly TLR2.