Surface properties of enterococcus faecalis in relation to infective endocarditis

The effect of growth conditions on both the appearance and the antigenic profile of cells of Enterococcus faecalis was investigated using electron micrographs of ruthenium red stained and sectioned cells and SDS-PAGE and blotting techniques respectively. Three specific antigens of molecular weights 73, 40 and 37 kdaltons were of particular interest being expressed most strongly after growth in serum. This medium was deemed to most closely mimic jn vjvo growth conditions reflecting an environment similar to that which the microorganisms would encounter during bacteraemia, preceding the colonisation of the endocardium and the development of infective endocarditis. The 40 and 37 kdalton antigens were shown by immunoqold labelling to be exposed on the surface of the cells although they did not appear to be connected with the fimbriae shown to exist on some of the E. faecalis cells examined by negative staining. The 73, 40 and 37 kdalton antigens were crudely purified using sarkosyl and ammonium sulphate precipitation, and used as the basis of a serodiagnostic test for E. faecalis endocarditis using an ELISA system. This was tested in a blind trial and the success rates were 94% for positives, 90% for negatives with endocarditis caused by other organisms and 80% for E. faecalis infections other than endocarditis. The binding of E.faecalis cells to the serum proteins fibronectin and albumin was investigated using 125I labelled proteins, followed by Scatchard analysis. This showed that· E.faecalis cells do loosely bind large amounts of both of these proteins, thus surely affecting the way in which the host's immune system perceives the cells. The E.faecalis receptor for fibronectin was partially characterised and appeared to involve protein and/or carbohydrate containing components. but did not involve LTA or the 40 and 37 kdalton species specific antigens.

The effect of haem limitation and iron restriction on outer membrane proteins and on respiratory systems of non typable Haemophilus influenzae

The effects of haem limitation and iron restriction on cells of non typable Haemophilus influenzae were investigated. Haem limitation was achieved by adding concentrations of haem to growth media which resulted in substantial decreases in final cell yields. Iron restriction was achieved by substituting protoporphyrin IX (PPIX) for haem in the growth medium and adding an iron chelator to the system. The effect of these nutrient limitations on a) outer membrane composition, and b) respiratory systems of non typable H.influenzae was investigated. Several of the strains examined produced new PPIX-specific outer membrane proteins when cultured utilising PPIX as a porphyrin source. The immune response of patients with bronchiectasis to outer membrane antigens of H.influenzae cultured under iron-restricted conditions was analysed by ELISA and immunoblotting techniques. ELISA analysis revealed that individuals with severe bronchiectasis had high titres of antibodies directed against H.influenzae OMs in both serum and sputum. Immunoblotting with homologous serum showed that where PPIX-specific OMPs were produced they were antigenic and were recognised by patients' serum. This suggested that these H.influenzae OMPs may be expressed in vivo. Additionally, the development of the immune responses to non typable H.influenzae outer membrane antigens was investigated using a rat lung model. Bacteria encased in agar beads were inoculated intratracheally into rat lungs, infection was established, and the immune response monitored for 6 weeks. The animals developed antibodies to PPIX-specific OMPs during the course of infection, providing further evidence that H.influenzae express these novel OMP antigens when growing in vivo. Studies in vitro on respiratory systems of phenotypically altered H.influenzae showed that bacteria grown utilising PPIX as a porphyrin source, or under conditions of iron-restriction produced ten fold fewer cytochromes than cells grown in nutrient excess, while haem limited H.influenzae produced no detectable cytochromes. Respiration of various substrates was depressed in haem limited and in PPIX-grown cultures as compared with cells grown in nutrient excess.

Static energy analysis of MHC class I and class II peptide-binding affinity

Antigenic peptide is presented to a T-cell receptor (TCR) through the formation of a stable complex with a major histocompatibility complex (MHC) molecule. Various predictive algorithms have been developed to estimate a peptide's capacity to form a stable complex with a given MHC class II allele, a technique integral to the strategy of vaccine design. These have previously incorporated such computational techniques as quantitative matrices and neural networks. A novel predictive technique is described, which uses molecular modeling of predetermined crystal structures to estimate the stability of an MHC class II-peptide complex. The structures are remodeled, energy minimized, and annealed before the energetic interaction is calculated.

The histogenesis of lymphoid organs in Tilapia mossambica and antibody responses in adults and fry

The thymic anlagen appears in Tilapia mossambica at 2 days post hatching and becomes lymphoid at 5 days. Lymphoid cells were first seen in the pronephros at 14 days and in the spleen at approximately five weeks of age. Differentiation into red and white pulp regions was seen by 10 weeks of age. Light and electron microscopic studies of adult lymphoid organ revealed increases in size and lymphoid cell numbers. Adult thymus develops a clearer corticomedullary differentiation of thymic corpuscles in the medulla and in the splenic red and white pulp became more distinct. Melanomacrophage centres were seen in spleen and pronephros. Adult fish gave primary and secondary antibody responses following challenge with sheep red bloods cells (SRBC), Escherichia coli (E. coli) and human gamma globulin (HGG). Plaque forming cell and immunocytoadherence assays revealed that head kidney and spleen were major sites for antibody production and development of antigen reactive cells. Proliferative activity in these organs was revealed using autoradiography and scintillation counting. Increased levels of pyroninophilia were also seen following antigenic challenge. Pilot studies on adults revealed that they were capable of rejecting first and second set allografts and leucocytes from spleen and head kidney proliferated in mixed leucocyte cultures. Antibody responses to SRBC, E. coli and HGG develop at about 10-12 weeks of age. Fry given either a single injection of SRBC at 10 weeks or two injections of the same antigen at 10 weeks and 12 days later, failed to respond to a further challenge with SRBC 56 days after the first injection (A time when animals would normally respond positively to this antigen). Injection of E. coli at the same times resulted in a prolonged antibody response.

Formulation and development of cationic liposomes as adjuvants for subunit protein vaccinese

Liposomes remain at the forefront of vaccine design due to their well documented abilities to act as delivery vehicles and adjuvants. Liposomes have been described to initiate an antigen depot-effect, thereby increasing antigen exposure to circulating antigen-presenting cells. More recently, in-depth reviews have focussed on inherent immunostimulatory abilities of various cationic lipids, the use of which is consequently of interest in the development of subunit protein vaccines which when delivered without an adjuvant are poorly immunogenic. The importance of liposomes for the mediation of an antigen depot-effect was examined by use of a dual-radiolabelling technique thereby allowing simultaneous detection of liposomal and antigenic components and analysis of their pharmacokinetic profile. In addition to investigating the biodistribution of these formulations, their physicochemical properties were analysed and the ability of the various liposome formulations to elicit humoral and cell-mediated immune responses was investigated. Our results show a requirement of cationic charge and medium/strong levels of antigen adsorption to the cationic liposome in order for both a liposome and antigen depot-effect to occur at the injection site. The choice of injection route had little effect on the pharmacokinetics or immunogenicity observed. In vitro, cationic liposomes were more cytotoxic than neutral liposomes due to significantly enhanced levels of cell uptake. With regards to the role of bilayer fluidity, liposomes expressing more rigid bilayers displayed increased retention at the injection site although this did not necessarily result in increased antigen retention. Furthermore, liposome bilayer rigidity did not necessarily correlate with improved immunogenicity. In similar findings, liposome size did not appear to control liposome or antigen retention at the injection site. However, a strong liposome size correlation between splenocyte proliferation and production of IL-10 was noted; specifically immunisation with large liposomes lead to increased levels of splenocyte proliferation coupled with decreased IL-10 production.

Discriminating antigen and non-antigen using proteome dissimilarity:bacterial antigens

It has been postulated that immunogenicity results from the overall dissimilarity of pathogenic proteins versus the host proteome. We have sought to use this concept to discriminate between antigens and non-antigens of bacterial origin. Sets of 100 known antigenic and nonantigenic peptide sequences from bacteria were compared to human and mouse proteomes. Both antigenic and non-antigenic sequences lacked human or mouse homologues. Observed distributions were compared using the non-parametric Mann-Whitney test. The statistical null hypothesis was accepted, indicating that antigen and non-antigens did not differ significantly. Likewise, we were unable to determine a threshold able to separate meaningfully antigen from non-antigen. Thus, antigens cannot be predicted from pathogen genomes based solely on their dissimilarity to the human genome.

Discriminating antigen and non-antigen using proteome dissimilarity III:tumour and parasite antigens

Computational genome analysis enables systematic identification of potential immunogenic proteins within a pathogen. Immunogenicity is a system property that arises through the interaction of host and pathogen as mediated through the medium of a immunogenic protein. The overt dissimilarity of pathogenic proteins when compared to the host proteome is conjectured by some to be the determining principal of immunogenicity. Previously, we explored this idea in the context of Bacterial, Viral, and Fungal antigen. In this paper, we broaden and extend our analysis to include complex antigens of eukaryotic origin, arising from tumours and from parasite pathogens. For both types of antigen, known antigenic and non-antigenic protein sequences were compared to human and mouse proteomes. In contrast to our previous results, both visual inspection and statistical evaluation indicate a much wider range of homologues and a significant level of discrimination; but, as before, we could not determine a viable threshold capable of properly separating non-antigen from antigen. In concert with our previous work, we conclude that global proteome dissimilarity is not a useful metric for immunogenicity for presently available antigens arising from Bacteria, viruses, fungi, parasites, and tumours. While we see some signal for certain antigen types, using dissimilarity is not a useful approach to identifying antigenic molecules within pathogen genomes.

Discriminating antigen and non-antigen using proteome dissimilarity II:viral and fungal antigens

Immunogenicity arises via many synergistic mechanisms, yet the overall dissimilarity of pathogenic proteins versus the host proteome has been proposed as a key arbiter. We have previously explored this concept in relation to Bacterial antigens; here we extend our analysis to antigens of viral and fungal origin. Sets of known viral and fungal antigenic and non-antigenic protein sequences were compared to human and mouse proteomes. Both antigenic and non-antigenic sequences lacked human or mouse homologues. Observed distributions were compared using the non-parametric Mann-Whitney test. The statistical null hypothesis was accepted, indicating that antigen and non-antigens did not differ significantly. Likewise, we could not determine a threshold able meaningfully to separate non-antigen from antigen. We conclude that viral and fungal antigens cannot be predicted from pathogen genomes based solely on their dissimilarity to mammalian genomes.

Toward the atomistic simulation of T cell epitopes automated construction of MHC:peptide structures for free energy calculations

Epitopes mediated by T cells lie at the heart of the adaptive immune response and form the essential nucleus of anti-tumour peptide or epitope-based vaccines. Antigenic T cell epitopes are mediated by major histocompatibility complex (MHC) molecules, which present them to T cell receptors. Calculating the affinity between a given MHC molecule and an antigenic peptide using experimental approaches is both difficult and time consuming, thus various computational methods have been developed for this purpose. A server has been developed to allow a structural approach to the problem by generating specific MHC:peptide complex structures and providing configuration files to run molecular modelling simulations upon them. A system has been produced which allows the automated construction of MHC:peptide structure files and the corresponding configuration files required to execute a molecular dynamics simulation using NAMD. The system has been made available through a web-based front end and stand-alone scripts. Previous attempts at structural prediction of MHC:peptide affinity have been limited due to the paucity of structures and the computational expense in running large scale molecular dynamics simulations. The MHCsim server (http://igrid-ext.cryst.bbk.ac.uk/MHCsim) allows the user to rapidly generate any desired MHC:peptide complex and will facilitate molecular modelling simulation of MHC complexes on an unprecedented scale.

A novel multilocus sequence typing scheme for the opportunistic pathogen Propionibacterium acnes and characterisation of type 1 cell surface-associated antigens

We have developed a novel multilocus sequence typing (MLST) scheme and database (http://pubmlst.org/pacnes/) for Propionibacterium acnes based on the analysis of seven core housekeeping genes. The scheme, which was validated against previously described antibody, single locus and random amplification of polymorphic DNA typing methods, displayed excellent resolution and differentiated 123 isolates into 37 sequence types (STs). An overall clonal population structure was detected with six eBURST groups representing the major clades I, II and III, along with two singletons. Two highly successful and global clonal lineages, ST6 (type IA) and ST10 (type IB1), representing 64?% of this current MLST isolate collection were identified. The ST6 clone and closely related single locus variants, which comprise a large clonal complex CC6, dominated isolates from patients with acne, and were also significantly associated with ophthalmic infections. Our data therefore support an association between acne and P. acnes strains from the type IA cluster and highlight the role of a widely disseminated clonal genotype in this condition. Characterization of type I cell surface-associated antigens that are not detected in ST10 or strains of type II and III identified two dermatan-sulphate-binding proteins with putative phase/antigenic variation signatures. We propose that the expression of these proteins by type IA organisms contributes to their role in the pathophysiology of acne and helps explain the recurrent nature of the disease. The MLST scheme and database described in this study should provide a valuable platform for future epidemiological and evolutionary studies of P. acnes.

Effectiveness of nonpharmacologic treatments for acute seasonal allergic conjunctivitis

Objective - To investigate whether artificial tears and cold compress alone or in combination provide a treatment benefit and whether they were as effective as or could enhance topical antiallergic medication. Design - Randomized, masked clinical trial. Participants - Eighteen subjects (mean age, 29.5±11.0 years) allergic to grass pollen. Intervention - Controlled exposure to grass pollen using an environmental chamber to stimulate an ocular allergic reaction followed by application of artificial tears (ATs), 5 minutes of cold compress (CC), ATs combined with CC, or no treatment applied at each separate visit in random order. A subset of 11 subjects also had epinastine hydrochloride (EH) applied alone and combined with CC in random order or instillation of a volume-matched saline control. Main Outcome Measures - Bulbar conjunctival hyperemia, ocular surface temperature, and ocular symptoms repeated before and every 10 minutes after treatment for 1 hour. Results - Bulbar conjunctival hyperemia and ocular symptoms decreased and temperature recovered to baseline faster with nonpharmaceutical treatments compared with no treatment (P??0.05). At all measurement intervals, symptoms were reduced for both EH and EH combined with CC than CC or ATs alone or in combination (P?antigenic challenge. Signs of allergic conjunctivitis generally were reduced most by a combination of a CC in combination with ATs or EH.

Production, purification and characterization of recombinant, full-length human claudin-1

The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-ß-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1:2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81.

Effectiveness of nonpharmacologic treatments for acute seasonal allergic conjunctivitis

Objective To investigate whether artificial tears and cold compress alone or in combination provide a treatment benefit and whether they were as effective as or could enhance topical antiallergic medication. Design Randomized, masked clinical trial. Participants Eighteen subjects (mean age, 29.5±11.0 years) allergic to grass pollen. Intervention Controlled exposure to grass pollen using an environmental chamber to stimulate an ocular allergic reaction followed by application of artificial tears (ATs), 5 minutes of cold compress (CC), ATs combined with CC, or no treatment applied at each separate visit in random order. A subset of 11 subjects also had epinastine hydrochloride (EH) applied alone and combined with CC in random order or instillation of a volume-matched saline control. Main Outcome Measures Bulbar conjunctival hyperemia, ocular surface temperature, and ocular symptoms repeated before and every 10 minutes after treatment for 1 hour. Results Bulbar conjunctival hyperemia and ocular symptoms decreased and temperature recovered to baseline faster with nonpharmaceutical treatments compared with no treatment (P <0.05). Artificial tears combined with CC reduced hyperemia more than other treatments (P <0.05). The treatment effect of EH was enhanced by combining it with a CC (P <0.001). Cold compress combined with ATs or EH lowered the antigen-raised ocular surface temperature to less than the pre-exposure baseline. Artificial tear instillation alone or CC combined with ATs or EH significantly reduced the temperature (P <0.05). Cold compress combined with ATs or EH had a similar cooling effect (P > 0.05). At all measurement intervals, symptoms were reduced for both EH and EH combined with CC than CC or ATs alone or in combination (P <0.014). Conclusions After controlled exposure to grass pollen, CC and AT treatment showed a therapeutic effect on the signs and symptoms of allergic conjunctivitis. A CC enhanced the use of EH alone and was the only treatment to reduce symptoms to baseline within 1 hour of antigenic challenge. Signs of allergic conjunctivitis generally were reduced most by a combination of a CC in combination with ATs or EH. © 2014 by the American Academy of Ophthalmology.

SVRMHC prediction server for MHC-binding peptides

The binding between antigenic peptides (epitopes) and the MHC molecule is a key step in the cellular immune response. Accurate in silico prediction of epitope-MHC binding affinity can greatly expedite epitope screening by reducing costs and experimental effort. Recently, we demonstrated the appealing performance of SVRMHC, an SVR-based quantitative modeling method for peptide-MHC interactions, when applied to three mouse class I MHC molecules. Subsequently, we have greatly extended the construction of SVRMHC models and have established such models for more than 40 class I and class II MHC molecules. Here we present the SVRMHC web server for predicting peptide-MHC binding affinities using these models. Benchmarked percentile scores are provided for all predictions. The larger number of SVRMHC models available allowed for an updated evaluation of the performance of the SVRMHC method compared to other well- known linear modeling methods. SVRMHC is an accurate and easy-to-use prediction server for epitope-MHC binding with significant coverage of MHC molecules. We believe it will prove to be a valuable resource for T cell epitope researchers.

The development of a novel Bio-MEMS filtration chip for the separation of specific cells in fluid suspension

In the clinical/microbiological laboratory there are currently several ways of separating specific cells from a fluid suspension. Conventionally cells can be separated based on size, density, electrical charge, light-scattering properties, and antigenic surface properties. Separating cells using these parameters can require complex technologies and specialist equipment. This paper proposes new Bio-MEMS (microelectromechanical systems) filtration chips manufactured using deep reactive ion etching (DRIE) technology that, when used in conjunction with an optical microscope and a syringe, can filter and grade cells for size without the requirement for additional expensive equipment. These chips also offer great versatility in terms of design and their low cost allows them to be disposable, eliminating sample contamination. The pumping mechanism, unlike many other current filtration techniques, leaves samples mechanically and chemically undamaged. In this paper the principles behind harnessing passive pumping are explored, modelled, and validated against empirical data, and their integration into a microfluidic device to separate cells from a mixed population suspension is described. The design, means of manufacture, and results from preliminary tests are also presented. © IMechE 2007.