Chitosan nanoparticle as protein delivery carrier - Systematic examination of fabrication conditions for efficient loading and release

Chitosan nanoparticles fabricated via different preparation protocols have been in recent years widely studied as carriers for therapeutic proteins and genes with varying degree of effectiveness and drawbacks. This work seeks to further explore the polyionic coacervation fabrication process, and associated processing conditions under which protein encapsulation and subsequent release can be systematically and predictably manipulated so as to obtain desired effectiveness. BSA was used as a model protein which was encapsulated by either incorporation or incubation method, using the polyanion tripolyphosphate (TPP) as the coacervation crosslink agent to form chitosan-BSA-TPP nanoparticles. The BSA-loaded chitosan-TPP nanoparticles were characterized for particle size, morphology, zeta potential, BSA encapsulation efficiency, and subsequent release kinetics, which were found predominantly dependent on the factors of chitosan molecular weight, chitosan concentration, BSA loading concentration, and chitosan/TPP mass ratio. The BSA loaded nanoparticles prepared under varying conditions were in the size range of 200-580 nm, and exhibit a high positive zeta potential. Detailed sequential time frame TEM imaging of morphological change of the BSA loaded particles showed a swelling and particle degradation process. Initial burst released due to surface protein desorption and diffusion from sublayers did not relate directly to change of particle size and shape, which was eminently apparent only after 6 h. It is also notable that later stage particle degradation and disintegration did not yield a substantial follow-on release, as the remaining protein molecules, with adaptable 3-D conformation, could be tightly bound and entangled with the cationic chitosan chains. In general, this study demonstrated that the polyionic coacervation process for fabricating protein loaded chitosan nanoparticles offers simple preparation conditions and a clear processing window for manipulation of physiochemical properties of the nanoparticles (e.g., size and surface charge), which can be conditioned to exert control over protein encapsulation efficiency and subsequent release profile. The weakness of the chitosan nanoparticle system lies typically with difficulties in controlling initial burst effect in releasing large quantities of protein molecules. (C) 2007 Elsevier B.V. All rights reserved.

A role of cellular prion protein in programming T-cell cytokine responses in disease.

The cellular prion protein (PrPC) is widely expressed in neural and non-neural tissues, but its function is unknown. Elucidation of the part played by PrPC in adaptive immunity has been a particular conundrum: increased expression of cell surface PrPC has been documented during T-cell activation, yet the functional significance of this activation remains unclear, with conflicting data on the effects of Prnp gene knockout on various parameters of T-cell immunity. We show here that Prnp mRNA is highly inducible within 8–24 h of T-cell activation, with surface protein levels rising from 24 h. When measured in parallel with CD69 and CD25, PrPC is a late activation antigen. Consistent with its up-regulation being a late activation event, PrP deletion did not alter T-cell-antigen presenting cell conjugate formation. Most important, activated PrP0/0 T cells demonstrated much reduced induction of several T helper (Th) 1, Th2, and Th17 cytokines, whereas others, such as TNF- and IL-9, were unaffected. These changes were investigated in the context of an autoimmune model and a bacterial challenge model. In experimental autoimmune encephalomyelitis, PrP-knockout mice showed enhanced disease in the face of reduced IL-17 responses. In a streptococcal sepsis model, this constrained cytokine program was associated with poorer local control of infection, although with reduced bacteremia. The findings indicate that PrPC is a potentially important molecule influencing T-cell activation and effector function.

A predatory patchwork:membrane and surface structures of Bdellovibrio bacteriovorus

Predatory Bdellovibrio bacteriovorus bacteria are remarkable in that they attach to, penetrate and digest other Gram-negative bacteria, living and replicating within them until all resources are exhausted, when they escape the prey ghost to invade fresh prey. Remarkable remodeling of both predator and prey cell occurs during this process to allow the Bdellovibrio to exploit the intracellular niche they have worked so hard to enter, keeping the prey "bdelloplast" intact until the end of predatory growth. If one views motile non-predatory bacteria in a light microscope, one is immediately struck by how rare it is for bacteria to collide. This highlights how the cell surface of Bdellovibrio must be specialized and adapted to allow productive collisions and further to allow entry into the prey periplasm and subsequent secretion of hydrolytic enzymes to digest it. Bdellovibrio can, however, also be made to grow artificially without prey; thus, they have a large genome containing both predatory genes and genes for saprophytic heterotrophic growth. Thus, the membrane and outer surface layers are a patchwork of proteins encompassing not only those that have a sole purpose in heterotrophic growth but also many more that are specialized or employed to attach to, enter, remodel, kill and ultimately digest prey cells. There is much that is as yet not understood, but molecular genetic and post-genomic approaches to microbial physiology have enhanced the pioneering biochemical work of four decades ago in characterizing some of the key events and surface protein requirements for prey attack.

Identification, characterization and antigenicity of the Plasmodium vivax rhoptry neck protein 1 (PvRON1)

Background: Plasmodium vivax malaria remains a major health problem in tropical and sub-tropical regions worldwide. Several rhoptry proteins which are important for interaction with and/or invasion of red blood cells, such as PfRONs, Pf92, Pf38, Pf12 and Pf34, have been described during the last few years and are being considered as potential anti-malarial vaccine candidates. This study describes the identification and characterization of the P. vivax rhoptry neck protein 1 (PvRON1) and examine its antigenicity in natural P. vivax infections. Methods: The PvRON1 encoding gene, which is homologous to that encoding the P. falciparum apical sushi protein (ASP) according to the plasmoDB database, was selected as our study target. The pvron1 gene transcription was evaluated by RT-PCR using RNA obtained from the P. vivax VCG-1 strain. Two peptides derived from the deduced P. vivax Sal-I PvRON1 sequence were synthesized and inoculated in rabbits for obtaining anti-PvRON1 antibodies which were used to confirm the protein expression in VCG-1 strain schizonts along with its association with detergent-resistant microdomains (DRMs) by Western blot, and its localization by immunofluorescence assays. The antigenicity of the PvRON1 protein was assessed using human sera from individuals previously exposed to P. vivax malaria by ELISA. Results: In the P. vivax VCG-1 strain, RON1 is a 764 amino acid-long protein. In silico analysis has revealed that PvRON1 shares essential characteristics with different antigens involved in invasion, such as the presence of a secretory signal, a GPI-anchor sequence and a putative sushi domain. The PvRON1 protein is expressed in parasite's schizont stage, localized in rhoptry necks and it is associated with DRMs. Recombinant protein recognition by human sera indicates that this antigen can trigger an immune response during a natural infection with P. vivax. Conclusions: This study shows the identification and characterization of the P. vivax rhoptry neck protein 1 in the VCG-1 strain. Taking into account that PvRON1 shares several important characteristics with other Plasmodium antigens that play a functional role during RBC invasion and, as shown here, it is antigenic, it could be considered as a good vaccine candidate. Further studies aimed at assessing its immunogenicity and protection-inducing ability in the Aotus monkey model are thus recommended.

Glycosaminoglycans and protein disulfide isomerase-mediated reduction of HIV Env

Conformational changes within the human immunodeficiency virus-1 (HIV-1) surface glycoprotein gp120 result from binding to the lymphocyte surface receptors and trigger gp41-mediated virus/cell membrane fusion. The triggering of fusion requires cleavage of two of the nine disulfide bonds of gp120 by a cell-surface protein disulfide-isomerase (PDI). Soluble glycosaminoglycans such as heparin and heparan sulfate bind gp120 via V3 and, possibly, a CD4-induced domain. They exert anti-HIV activity by interfering with the HIV envelope glycoprotein ( Env)/cell-surface interaction. Env also binds cell-surface glycosaminoglycans. Here, using surface plasmon resonance, we observed an inverse relationship between heparin binding by gp120 and its thiol content. In vitro, and in conditions in which gp120 could bind CD4, heparin and heparan sulfate reduced PDI-mediated gp120 reduction by approximately 80%. Interaction of Env with the surface of lymphocytes treated using sodium chlorate, an inhibitor of glycosaminoglycan synthesis, led to gp120 reduction. We conclude that besides their capacity to block Env/cell interaction, soluble glycosaminoglycans can effect anti-HIV activity via interference with PDI- mediated gp120 reduction. In contrast, their presence at the cell surface is dispensable for Env reduction during the course of interaction with the lymphocyte surface. This work suggests that the reduction of exofacial proteins in various diseases can be inhibited by compounds targeting the substrates ( not by targeting PDI, as is usually done), and that glycosaminoglycans that primarily protect proteins by preserving them from proteolysis also have a role in preventing reduction.

Binding properties and adhesion-mediating regions of the major sheath protein of Treponema denticola ATCC 35405

There is growing evidence that a number of oral Treponema species, in particular Treponema denticola, are associated with the progression of human periodontal disease. The major sheath (or surface) protein (Msp) of T. denticola is implicated in adhesion of bacteria to host cells and tissue proteins and is likely to be an important virulence factor. However, the binding regions of the Msp are not known. We have purified from Escherichia coli recombinant Msp (rMsp) polypeptides corresponding to the following: full-length Msp (rMsp) minus 13 N-terminal amino acid (aa) residues, an amino-terminal fragment (rN-Msp, 189 aa residues), a 57-aa residue segment from the central region (rV-Msp), and a C-terminal fragment (rC-Msp, 272 aa residues). rMsp (530 aa residues) bound to immobilized fibronectin, keratin, laminin, collagen type 1, fibrinogen, hyaluronic acid, and heparin. The N- and V-region polypeptides, but not rC-Msp, also bound to these substrates. Binding of rMsp to fibronectin was targeted to the N-terminal heparin I/fibrin I domain. Antibodies to the N-region or V-region polypeptides, but not antibodies to the rC-Msp fragment, blocked adhesion of T. denticola ATCC 35405 cells to a range of host protein molecules. These results suggest that the N-terminal half of Msp carries epitopes that are surface exposed and that are involved in mediating adhesion. Binding of rMsp onto the cell surface of low-level fibronectin-binding Treponema isolates conferred a 10-fold increase in fibronectin binding. This confirms that Msp functions autonomously as an adhesin and raises the possibility that phenotypic complementation of virulence functions might occur within mixed populations of Treponema species.

Role of alpha 7 Nicotinic Acetylcholine Receptor in Calcium Signaling Induced by Prion Protein Interaction with Stress-inducible Protein 1

The prion protein (PrP(C)) is a conserved glycosylphosphatidyl-inositol-anchored cell surface protein expressed by neurons and other cells. Stress-inducible protein 1 (STI1) binds PrP(C) extracellularly, and this activated signaling complex promotes neuronal differentiation and neuroprotection via the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP-dependent protein kinase 1 (PKA) pathways. However, the mechanism by which the PrPC-STI1 interaction transduces extracellular signals to the intracellular environment is unknown. We found that in hippocampal neurons, STI1-PrP(C) engagement induces an increase in intracellular Ca(2+) levels. This effect was not detected in PrP(C)-null neurons or wild-type neurons treated with an STI1 mutant unable to bind PrP(C). Using a best candidate approach to test for potential channels involved in Ca(2+) influx evoked by STI1-PrP(C), we found that alpha-bungarotoxin, a specific inhibitor for alpha 7 nicotinic acetylcholine receptor (alpha 7nAChR), was able to block PrP(C)-STI1-mediated signaling, neuroprotection, and neuritogenesis. Importantly, when alpha 7nAChR was transfected into HEK 293 cells, it formed a functional complex with PrP(C) and allowed reconstitution of signaling by PrP(C)-STI1 interaction. These results indicate that STI1 can interact with the PrP(C).alpha 7nAChR complex to promote signaling and provide a novel potential target for modulation of the effects of prion protein in neurodegenerative diseases.

Induction of neutralizing antibodies in mice immunized with an amino-terminal polypeptide of Streptococcus mutans P1 protein produced by a recombinant Bacillus subtilis strain

The oral pathogen Streptococcus mutans expresses a surface protein, P1, which interacts with the salivary pellicle on the tooth surface or with fluid-phase saliva, resulting in bacterial adhesion or aggregation, respectively. P1 is a target of protective immunity. Its N-terminal region has been associated with adhesion and aggregation functions and contains epitopes recognized by efficacious antibodies. In this study, we used Bacillus subtilis, a gram-positive expression host, to produce a recombinant N-terminal polypeptide of P1 (P1(39-512)) derived from the S. mutans strain UA159. Purified P1(39-512) reacted with an anti-full-length P1 antiserum as well as one raised against intact S. mutans cells, indicating preserved antigenicity. Immunization of mice with soluble and heat-denatured P1(39-512) induced antibodies that reacted specifically with native P1 on the surface of S. mutans cells. The anti-P1(39-512) antiserum was as effective at blocking saliva-mediated aggregation of S. mutans cells and better at blocking bacterial adhesion to saliva-coated plastic surfaces compared with the anti-full-length P1 antiserum. In addition, adsorption of the anti-P1 antiserum with P1(39-512) eliminated its ability to block the adhesion of S. mutans cells to abiotic surfaces. The present results indicate that P1(39-512), expressed and purified from a recombinant B. subtilis strain, maintains important immunological features of the native protein and represents an additional tool for the development of anticaries vaccines.

Evolutionary dynamics of the immunodominant repeats of the Plasmodium vivax malaria-vaccine candidate circumsporozoite protein (CSP)

The circumsporozoite protein (CSP) of Plasmodium vivax, a major target for malaria vaccine development, has immunodominant B-cell epitopes mapped to central nonapeptide repeat arrays. To determine whether rearrangements of repeat motifs during mitotic DNA replication of parasites create significant CSP diversity under conditions of low effective meiotic recombination rates, we examined csp alleles from sympatric P. vivax isolates systematically sampled from an area of low malaria endemicity in Brazil over a period of 14 months. Nine unique csp types, comprising six different nona peptide repeats, were observed in 45 isolates analyzed. Identical or nearly identical repeats predominated in most arrays, consistent with their recent expansion. We found strong linkage disequilibrium at sites across the chromosome 8 segment flanking the csp locus, consistent with rare meiotic recombination in this region. We conclude that CSP repeat diversity may not be severely constrained by rare meiotic recombination in areas of low malaria endemicity. New repeat variants may be readily created by nonhomologous recombination even when meiotic recombination is rare, with potential implications for CSP-based vaccine development. (C) 2010 Elsevier B.V. All rights reserved.

Naturally acquired inhibitory antibodies to Plasmodium vivax Duffy binding protein are short-lived and allele-specific following a single malaria infection

The Duffy binding protein of Plasmodium vivax (DBP) is a critical adhesion ligand that participates in merozoite invasion of human Duffy-positive erythrocytes. A small outbreak of P. vivax malaria, in a village located in a non-malarious area of Brazil, offered us an opportunity to investigate the DBP immune responses among individuals who had their first and brief exposure to malaria. Thirty-three individuals participated in the five cross-sectional surveys, 15 with confirmed P. vivax infection while residing in the outbreak area (cases) and 18 who had not experienced malaria (non-cases). In the present study, we found that only 20% (three of 15) of the individuals who experienced their first P. vivax infection developed an antibody response to DBP; a secondary boosting can be achieved with a recurrent P. vivax infection. DNA sequences from primary/recurrent P. vivax samples identified a single dbp allele among the samples from the outbreak area. To investigate inhibitory antibodies to the ligand domain of the DBP (cysteine-rich region II, DBP(II)), we performed in vitro assays with mammalian cells expressing DBP(II) sequences which were homologous or not to those from the outbreak isolate. In non-immune individuals, the results of a 12-month follow-up period provided evidence that naturally acquired inhibitory antibodies to DBP(II) are short-lived and biased towards a specific allele.

MSP119 miniproteins can serve as targets for invasion inhibitory antibodies in plasmodium falciparum provided they contain the correct domains for cell surface trafficking.

Antibodies from malaria-exposed individuals can agglutinate merozoites released from Plasmodium schizonts, thereby preventing them from invading new erythrocytes. Merozoite coat proteins attached to the plasma membrane are major targets for host antibodies and are therefore considered important malaria vaccine candidates. Prominent among these is the abundant glycosylphosphatidylinositol (GPI)-anchored merozoite surface protein 1 (MSP1) and particularly its C-terminal fragment (MSP1(19)) comprised of two epidermal growth factor (EGF)-like modules. In this paper, we revisit the role of agglutination and immunity using transgenic fluorescent marker proteins. We describe expression of heterologous MSP1(19)'miniproteins' on the surface of Plasmodium falciparum merozoites. To correctly express these proteins, we determined that GPI-anchoring and the presence of a signal sequence do not allow default export of proteins from the endoplasmic reticulum to merozoite surface and that extra sequence elements are required. The EGFs are insufficient for correct trafficking unless they are fused to additional residues that normally reside upstream of this fragment. Antibodies specifically targeting the surface-expressed miniprotein can inhibit erythrocyte invasion in vitro despite the presence of endogenous MSP1. Using a line expressing a green fluorescent protein-MSP1 fusion protein, we demonstrate that one mode of inhibition by antibodies targeting the MSP1(19) domain is the rapid agglutinating of merozoites prior to erythrocyte attachment.

Production of recombinant EMA-1 protein and its application for the diagnosis of Theileria equi using an enzyme immuno assay in horses from São Paulo State, Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Lepstospira interrogans shotgun phage display identified LigB as a heparin-binding protein

LigB is an adhesin from pathogenic Leptospira that is able to bind to extracellular matrix and is considered a virulence factor. A shotgun phage display genomic library was constructed and used for panning against Heparan Sulfate Proteoglycan (HSPG). A phage clone encoding part of LigB protein was selected in panning experiments and showed specific binding to heparin. To validate the selected clone, fragments of LigB were produced as recombinant proteins and showed affinity to heparin and to mammalian cells. Heparin was also able to reduce the binding of rLB-Ct to mammalian cells. Our data suggests that the glycosaminoglycan moiety of the HSPG is responsible for its binding and could mediate the attachment of the recombinant protein rLB-Ct. Thus, heparin may act as a receptor for Leptospira to colonize and to invade the host tissue. (C) 2012 Elsevier Inc. All rights reserved.

Die Synthese von Glycopeptiden und Glycopeptid-Protein-Konjugaten mit einer Partialstruktur des tumorassoziierten Mucins MUC1 zur Entwicklung von Tumorvakzinen

„Synthese von Glycopeptiden und Glycopeptid-Protein-Konjugaten mit einer Partialstruktur des tumorassoziierten Mucins MUC1 zur Entwicklung von Tumorvakzinen“ Das Glycoprotein MUC1 ist in Tumorepithelzellen sonderlich stark überexprimiert und wegen der vorzeitig einsetzenden Sialylierung sind die Saccharid-Epitope der O-Glycanketten stark verkürzt (sog. tumorassoziierte Antigene). Dadurch werden auch bisher verborgene Peptidepitope des Glycoprotein-Rückgrates auf der Zelloberfläche der Epithelzellen zugänglich, die als fremd von den Zellen des Immunsystems erkannt werden können. Dies macht das MUC1-Zelloberfächenmolekül zu einem Zielmolekül in der Entwicklung von Tumorvakzinen. Diese beiden strukturellen Besonderheiten wurden in der Synthese von Glycohexadecapeptiden verbunden, indem die veränderten tumorassoziierten Saccharidstrukturen TN-, STN- und T-Antigen als Glycosylaminosäure-Festphasenbausteine synthetisiert wurden und in das Peptidepitop der Wiederholungseinheit des MUC1 durch Glycopeptid-Festphasensynthese eingebaut wurden. Wegen der inhärenten schwachen Immunogenität der kurzen Glycopeptide müssen die synthetisierten Glycopeptidstrukturen an ein Trägerprotein, welches das Immunsystem stimuliert, gebunden werden. Zur Anbindung der Glycopeptide ist ein selektives Kupplungsverfahren nötig, um definierte und strukturell einheitliche Glycopeptid-Protein-Konjugate zu erhalten. Es konnte eine neue Methode entwickelt werden, bei der die Konjugation durch eine radikalische Additionsreaktion von als Allylamide funktionalisierten Glycopeptiden an ein Thiol-modifiziertes Trägerprotein erfolgte. Dazu wurde anhand von synthetisierten, als Allylamide modifizierten Modellaminosäuren untersucht, ob diese Reaktion generell für eine Biokonjugation geeignet ist und etwaige Nebenreaktionen auftreten können. Mit dieser Methode konnten verschiedene MUC1-Glycopeptid-Trägerprotein-Konjugate hergestellt werden, deren immunologische Untersuchung noch bevorsteht. Das tumorassoziierte MUC1 nimmt in der immundominanten Region seiner Wiederholungseinheit eine knaufartige Struktur ein. Für die Entwicklung von selektiven Tumorvakzinen ist es von großer Bedeutung möglichst genau die Struktur der veränderten Zelloberflächenmoleküle nachzubilden. Durch die Synthese von cyclischen (Glyco)Peptiden wurde dieses Strukturelement fixiert. Dazu wurden olefinische Aminosäure Festphasenbausteine hergestellt, die zusammen mit den oben genannten Glycosylaminosäuren mittels einer Glycopeptid-Festphasensynthese in acyclische Glycopeptide eingebaut wurden. Diese wurden dann durch Ringschlussmetathese zyklisiert und im Anschluss reduziert und vollständig deblockiert. In einem dritten Projekt wurde der Syntheseweg zur Herstellung einer C-Glycosylaminosäure mit einer N-Acetylgalactosamin-Einheit entwickelt. Wichtige Schritte bei der von Glucosamin ausgehenden Synthese sind die Keck-Allylierung, eine Epimerisierung, die Herstellung eines Brom-Dehydroalanin-Derivates und eine B-Alkyl-Suzuki-Miyaura-Kreuzkupplung sowie Schutzgruppenoperationen. Der racemische Baustein konnte dann in der Peptid-Festphasensynthese eines komplexen MUC1-Tetanustoxin-Konjugates eingesetzt werden.

Proteomic analysis of ocular surface components by use of HPLC based mass spectrometric strategies

In der vorliegenden Arbeit wurde eine Top Down (TD) und zwei Bottom Up (BU) MALDI/ESI Massenspektrometrie/HPLC-Methoden entwickelt mit dem Ziel Augenoberfächenkomponenten, d.h. Tränenfilm und Konjunktivalzellen zu analysieren. Dabei wurde ein detaillierter Einblick in die Entwicklungsschritte gegeben und die Ansätze auf Eignung und methodische Grenzen untersucht. Während der TD Ansatz vorwiegend Eignung zur Analyse von rohen, weitgehend unbearbeiteten Zellproben fand, konnten mittels des BU Ansatzes bearbeitete konjunktivale Zellen, aber auch Tränenfilm mit hoher Sensitivität und Genauigkeit proteomisch analysiert werden. Dabei konnten mittels LC MALDI BU-Methode mehr als 200 Tränenproteine und mittels der LC ESI Methode mehr als 1000 Tränen- sowie konjunktivale Zellproteine gelistet werden. Dabei unterschieden sich ESI- and MALDI- Methoden deutlich bezüglich der Quantität und Qualität der Ergebnisse, weshalb differente proteomische Anwendungsgebiete der beiden Methoden vorgeschlagen wurden. Weiterhin konnten mittels der entwickelten LC MALDI/ESI BU Plattform, basierend auf den Vorteilen gegenüber dem TD Ansatz, therapeutische Einflüsse auf die Augenoberfläche mit Fokus auf die topische Anwendung von Taurin sowie Taflotan® sine, untersucht werden. Für Taurin konnten entzündungshemmende Effekte, belegt durch dynamische Veränderungen des Tränenfilms, dokumentiert werden. Außerdem konnten vorteilhafte, konzentrationsabhängige Wirkweisen auch in Studien an konjunktival Zellen gezeigt werden. Für die Anwendung von konservierungsmittelfreien Taflotan® sine, konnte mittels LC ESI BU Analyse eine Regenerierung der Augenoberfläche in Patienten mit Primärem Offenwinkel Glaukom (POWG), welche unter einem “Trockenem Auge“ litten nach einem therapeutischen Wechsel von Xalatan® basierend auf dynamischen Tränenproteomveränderungen gezeigt werden. Die Ergebnisse konnten mittels Microarray (MA) Analysen bestätigt werden. Sowohl in den Taurin Studien, als auch in der Taflotan® sine Studie, konnten charakteristische Proteine der Augenoberfläche dokumentiert werden, welche eine objektive Bewertung des Gesundheitszustandes der Augenoberfläche ermöglichen. Eine Kombination von Taflotan® sine und Taurin wurde als mögliche Strategie zur Therapie des Trockenen Auges bei POWG Patienten vorgeschlagen und diskutiert.