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Next generation vaccines have the capability to contribute to and revolutionise the veterinary vaccine industry. African horse sickness (AHS) is caused by an arbovirus infection and is characterised by respiratory distress and/or cardiovascular failure and is lethal to horses. Mandatory annual vaccination in endemic areas curtails disease occurrence and severity. However, development of a next generation AHSV vaccine, which is both safe and efficacious, has been an objective globally for years. In this study, both AHSV serotype 5 chimaeric virus-like particles (VLPs) and soluble viral protein 2 (VP2) were successfully produced in Nicotiana benthamiana DXT/FT plants, partially purified and validated by gel electrophoresis, transmission electron microscopy and liquid chromatography-mass spectrometry (LC-MS/ MS) based peptide sequencing before vaccine formulation. IFNAR-/- mice vaccinated with the adjuvanted VLPs or VP2 antigens in a 10 lg prime-boost regime resulted in high titres of antibodies confirmed by both serum neutralising tests (SNTs) and enzyme-linked immunosorbent assays (ELISA). Although previous studies reported high titres of antibodies in horses when vaccinated with plant-produced AHS homogenous VLPs, this is the first study demonstrating the protective efficacy of both AHSV serotype 5 chimaeric VLPs and soluble AHSV-5 VP2 as vaccine candidates. Complementary to this, coating ELISA plates with the soluble VP2 has the potential to underpin serotype-specific serological assays.

The differentiation of macrophages into lipid-filled foam cells is a hallmark of the lung granuloma that forms in patients with active tuberculosis (TB). Mycolic acids (MAs), the abundant lipid virulence factors in the cell wall of Mycobacterium tuberculosis (Mtb), can induce this foam phenotype possibly as a way to perturb host cell lipid homeostasis to support the infection. It is not exactly clear how MAs allow differentiation of foam cells during Mtb infection. Here we investigated how chemically synthetic MAs, each with a defined stereochemistry similar to natural Mtb-associated mycolates, influence cell foamy phenotype and mycobacterial proliferation in murine host macrophages. Using light and laser-scanning-confocal microscopy, we assessed the influence of MA structure first on the induction of granuloma cell types, second on intracellular cholesterol accumulation, and finally on mycobacterial growth. While methoxy-MAs (mMAs) effected multi-vacuolar giant cell formation, keto-MAs (kMAs) induced abundant intracellular lipid droplets that were packed with esterified cholesterol. Macrophages from mice treated with kMA were permissive to mycobacterial growth, whereas cells from mMA treatment were not. This suggests a separate yet key involvement of oxygenated MAs in manipulating host cell lipid homeostasis to establish the state of TB.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is characterized by the abundance of species specific, antigenic cell wall lipids called mycolic acids. These wax-like molecules all share an identical, amphiphilic mycolic motif, but have different functional groups in a long hydrophobic hydrocarbon mero-chain that divide them into three main classes: alpha-, keto- and methoxy-mycolic acids. Whereas alpha-mycolic acids constitutively maintain an abundance of around 50%, the ratio of methoxy- to keto-mycolic acid types may vary depending on, among other things, the growth stage of M. tuberculosis. In human patients, antibodies to mycolic acids have shown potential as diagnostic serum biomarkers for active TB. Variations in mycolic acid composition affect the antigenic properties and can potentially compromise the precision of detection of anti-mycolic acids antibodies in patient sera to natural mixtures. We demonstrate this here with combinations of synthetic mycolic acid antigens, tested against TB patient and control sera. Combinations of methoxy- and α-mycolic acids are more antigenic than combinations of keto- and α-mycolic acids, showing the former to give a more sensitive test for TB biomarker antibodies. Natural mixtures of mycolic acids isolated from mature cultures of M. tuberculosis H37Rv give the same sensitivity as that with synthetic methoxy- and α-mycolic acids in combination, in a surface plasmon resonance inhibition biosensor test. To ensure that the antigenic activity of isolates of natural mycolic acids is reproducible, we cultured M. tuberculosis H37Rv on Middlebrook 7H10 solid agar plates to stationary growth phase in a standardized, optimal way. The proportions of mycolic acid classes in various batches of the isolates prepared from these cultures were compared to a commercially available natural mycolic acid isolate. LC-MS/MS and NMR data for quantitation of mycolic acids class compositions show that the variation in batches is small, suggesting that the quality of the results for anti-mycolic acid antibody detection in the TB patients should not be affected by different batches of natural mycolic acid antigens if prepared in a standard way.

Nitric oxide reductase (NOR) of the P450 oxidoreductase family accepts electrons directly from its cofactor, NADH, to reduce two nitric oxide (NO) molecules to one nitrous oxide molecule and water. The enzyme plays a key role in the removal of radical NO produced during respiratory metabolism, and applications in bioremediation and biocatalysis have been identified. However, a rapid, accurate, and sensitive enzyme assay has not yet been developed for this enzyme family. In this study, we optimized reaction conditions for the development of a spectrophotometric NOR activity microassay using NOC-5 for the provision of NO in solution. We also demonstrate that the assay is suitable for the quantification and characterization of P450-type NOR. The Km and kcat kinetic constants obtained by this assay were comparable to the values determined by gas chromatography, but with improved convenience and cost efficiency, effectively by miniaturization. To our knowledge, this is the first study to present the quantification of NOR activity in a kinetic microassay format.

Aiming to improve the treatment outcomes of current daily tuberculosis (TB) chemotherapy over several months, we investigated whether nanoencapsulation of existing drugs would allow decreasing the treatment frequency to weekly, thereby ultimately improving patient compliance. Nanoencapsulation of three first-line anti-TB drugs was achieved by a unique, scalable spray-drying technology forming free-flowing powders in the nanometer range with encapsulation efficiencies of 82, 75, and 62% respectively for rifampicin, pyrazinamide, and isoniazid. In a pre-clinical study on TB infected mice, we demonstrate that the encapsulated drugs, administered once weekly for nine weeks, showed comparable efficacy to daily treatment with free drugs over the same experimental period. Both treatment approaches had equivalent outcomes for resolution of inflammation associated with the infection of lungs and spleens. These results demonstrate how scalable technology could be used to manufacture nanoencapsulated drugs. The formulations may be used to reduce the oral dose frequency from daily to once weekly in order to treat uncomplicated TB.

Cell wall mycolic acids (MA) from Mycobacterium tuberculosis (M.tb) are CD1b presented antigens that can be used to detect antibodies as surrogate markers of active TB, even in HIV coinfected patients. The use of the complex mixtures of natural MA is complicated by an apparent antibody cross-reactivity with cholesterol. Here firstly we report three recombinant monoclonal scFv antibody fragments in the chicken germ-line antibody repertoire, which demonstrate the possibilities for cross-reactivity: the first recognized both cholesterol and mycolic acids, the second mycolic acids but not cholesterol, and the third cholesterol but not mycolic acids. Secondly, MA structure is experimentally interrogated to try to understand the cross-reactivity. Unique synthetic mycolic acids representative of the three main functional classes show varying antigenicity against human TB patient sera, depending on the functional groups present and on their stereochemistry. Oxygenated (methoxy- and keto-) mycolic acid was found to be more antigenic than alpha-mycolic acids. Synthetic methoxy-mycolic acids were the most antigenic, one containing a trans-cyclopropane apparently being somewhat more antigenic than the natural mixture. Trans-cyclopropane-containing keto- and hydroxy-mycolic acids were also found to be the most antigenic among each of these classes. However, none of the individual synthetic mycolic acids significantly and reproducibly distinguished the pooled serum of TB positive patients from that of TB negative patients better than the natural mixture of MA. This argues against the potential to improve the specificity of serodiagnosis of TB with a defined single synthetic mycolic acid antigen from this set, although sensitivity may be facilitated by using a synthetic methoxy-mycolic acid.

Mycolic acids (MA) are major, species-specific lipid components of Mycobacteria and related genera. In Mycobacterium tuberculosis, it is made up of alpha-, methoxy- and keto-MA, each with specific biological functions and conformational characteristics. Antibodies in tuberculosis (TB) patient sera respond differently towards the three MA classes and were reported to cross-react with cholesterol. To understand the antigenicity and cholesterol cross-reactivity of MA, we generated three different chicken -derived phage-displayed single-chain variable fragments (scFv) that reacted similarly towards the natural mixture of MA, but the first recognized all three classes of chemically synthetic MAs, the second only the two oxygenated types of MAs and the third only methoxy MA. The cholesterol cross-reactivity was investigated after grafting each of the three scFv types onto two configurations of constant chain domains-CH1-4 and CH2-4. Weak but significant cross-reactivity with cholesterol was found only with CH2-4 versions, notably those two that were also able to recognize the trans-keto MA. The cholesteroid nature of mycobacterial mycolic acids therefore seems to be determined by the trans-keto MA subclass. The significantly weaker binding to cholesterol in comparison to MA confirms the potential TB diagnostic application of these antibodies.

The integrity and properties of mycolic acid (MA) antigens integrated into a self-assembled monolayer (SAM) of N-(2-mercaptoethyl)octadecanamide, (MEODA), on a gold electrode have been interrogated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). EIS data showed that Au-MEODA and Au-MEODA-MA behave as microelectrode arrays, with pinholes acting as the microelectrodes that permit electron transport between a redox-active probe in solution and the underlying gold surface. The average radii of the pinholes (r(a)) and half the distance between the centers of the neighbouring pinholes (r(b)), were estimated from EIS using the pore size model, and discussed. Anti-MA antibodies present in a tuberculosis (TB)-infected patient (co-infected with HIV) strongly interact with Au-MEODA-MA showing a rather compact and stable bio-complex structure that is virtually defect-free. The electrochemical impedimetric properties associated with the ability of the Au-MEODA-MA to discriminate between TB positive and negative human sera are also discussed. We prove that the Au-MEODA and Au-MEODA-MA electrodes, as well as the MA-anti-MA antibody interactions, are characterized with time-constant dispersion, typical of microstructures with grain/grain boundary phases. These crucial physico-electrochemical insights into the behaviour of surface-confined MA should provide a useful basis for the design and development of a potential impedimetric immunosensing platform for active tuberculosis.

The synthesis of three stereoisomers of a major homologue of the methoxymycolic acids present in Mycobacterium tuberculosis is described.

The co-immobilization of enzymes on target surfaces facilitates the development of self-contained, multi-enzyme biocatalytic platforms. This generally entails the co-immobilization of an enzyme with catalytic value in combination with another enzyme that performs a complementary function, such as the recycling of a critical cofactor. In this study, we co-immobilized two enzymes from different biological sources for the continuous reduction of nitric oxide, using epoxide- and carboxyl-functionalized hyper-porous microspheres. Successful co-immobilization of a fungal nitric oxide reductase (a member of the cytochrome P450 enzyme family) and a bacterial glucose dehydrogenase was obtained with the carboxyl-functionalized microspheres, with enzyme activity maintenance of 158% for nitric oxide reductase and 104% for glucose dehydrogenase. The optimal stoichiometric ratio of these two enzymes was subsequently determined to enable the two independent chemical reactions to be catalyzed concomitantly, allowing for near-synchronous cofactor conversion rates. This dual-enzyme system provides a novel research tool with potential for in vitro investigations of nitric oxide, and further demonstrates the successful immobilization of a P450 enzyme with potential application towards the immobilization of other cytochrome P450 enzymes.

The appearance of drug-resistant strains of Mycobacterium tuberculosis (Mtb) poses a great challenge to the development of novel treatment programmes to combat tuberculosis. Since innovative nanotechnologies might alleviate the limitations of current therapies, we have designed a new nanoformulation for use as an anti-TB drug delivery system. It consists of incorporating mycobacterial cell wall mycolic acids (MA) as targeting ligands into a drug-encapsulating Poly dl-lactic-co-glycolic acid polymer (PLGA), via a double emulsion solvent evaporation technique. Bone marrow-derived mouse macrophages, either uninfected or infected with different mycobacterial strains (Mycobacterium avium, Mycobacterium bovis BCG or Mtb), were exposed to encapsulated isoniazid-PLGA nanoparticles (NPs) using MA as a targeting ligand. The fate of the NPs was monitored by electron microscopy. Our study showed that i) the inclusion of MA in the nanoformulations resulted in their expression on the outer surface and a significant increase in phagocytic uptake of the NPs; ii) nanoparticle-containing phagosomes were rapidly processed into phagolysosomes, whether MA had been included or not; and iii) nanoparticle-containing phagolysosomes did not fuse with non-matured mycobacterium-containing phagosomes, but fusion events with mycobacterium-containing phagolysosomes were clearly observed.

Rationale: Mycolic acid (MA) constitutes a major and distinguishing cell wall biolipid from Mycobacterium tuberculosis. MA interferes with the lipid homeostasis of alveolar macrophages, inducing differentiation into foamy macrophages exhibiting increased proinflammatory function.Objectives: We verified the interference of this altered macrophage function with inhaled antigen-triggered allergic airway inflammation and underlying Th2 lymphocyte reactivity.Methods: Using ovalbumin (OVA) as model allergen, C57BL/6 or BALB/C mice were sensitized by OVA-alum immunization. Experimental asthma, triggered subsequently by repetitive nebulized OVA inhalation, was assessed, using as readout parameters eosinophilia, peribronchial inflammation, and Th2 cytokine function. Measurements and Main Results: A single intratracheal treatment of sensitized mice with MA, inserted into liposomes as carriers, prevented the onset of OVA-triggered allergic airway inflammation and promoted unresponsiveness to a secondary set of allergen exposures. The development of this tolerant condition required an 8-d lapse after MA instillation, coinciding with the appearance of foamy alveolar macrophages. MA-conditioned CD11b(+)F4/80(+) macrophages, transferred to the airways, mimicked the tolerogenic function of instilled MA; however, without the 8-d lapse requirement. Indicative of a macrophage-mediated tolerogenic antigen-presenting function, major histocompatibility complex (MHC)-mismatched donor macrophages failed to promote tolerance. Furthermore, Treg markers were strongly increased and established tolerance was lost after in situ depletion of CD25(+) Treg cells. Contrary to the interleukin-10 dependence of tolerogenic dendritic cells, IFN-gamma deficiency but not interleukin-10 deficiency abrogated the tolerogenic capacity of MA-conditioned macrophages.Conclusions: These results document an innate-driven Mycobacterium tuberculosis MA-triggered immune regulatory mechanism in control of pulmonary allergic responses by converting macrophages into IFN-gamma-dependent tolerogenic antigen-presenting cells.

Recognition of conserved pathogen-associated molecular patterns constitutes a crucial step in the initiation of innate immune responses. We studied the contribution to the host-pathogen interaction of mycolic acid (MA), a major lipid component of the cell envelope of the macrophage intracellular pathogen Mycobacterium tuberculosis and other mycobacteria. MA administered to the peritoneal cavity or to the airways induced a unique macrophage morphotype, similar to the foamy macrophage derivatives observed in tuberculous granulomas and characterized by intracellular accumulation of neutral lipids and entry into mitosis. When assayed for production of inflammatory mediators, a conditioning rather than a direct activation of the MA-elicited foamy macrophages was observed. MA enabled production of IFN-gamma and myeloperoxidase, enhanced TNF-alpha production and suppressed IL-10 upon renewed exposure to innate triggers. Intratracheal instillation of MA mimicked additional features of the airway response to M. tuberculosis infection, namely a rapid but transient neutrophil influx and IL-6 production and a chronic IL-12 production. These MA-elicited cellular innate defenses and the accompanying formation of foamy macrophages identify for the first time the foamy macrophage morphotype as part of the host response to a pathogen-associated structure. Furthermore, these results characterize MA as a direct trigger of innate immunity, distinct from Toll-like receptor ligands.

a b s t r a c t Mycolic acids are structural components of the mycobacterial cell wall that have been implicated in the pathogenicity and drug resistance of certain mycobacterial species. They also offer potential in areas such as rapid serodiagnosis of human and animal tuberculosis. It is increasingly recognized that con- formational behavior of mycolic acids is very important in understanding all aspects of their function. Atomistic molecular dynamics simulations, in vacuo, of stereochemically defined Mycobacterium tuber- culosis mycolic acids show that they fold spontaneously into reproducible conformational groupings. One of the three characteristic mycolate types, the keto-mycolic acids, behaves very differently from either -mycolic acids or methoxy-mycolic acids, suggesting a distinct biological role. However, subtle conformational behavioral differences between all the three mycolic acid types indicate that cooperative interplay of individual mycolic acids may be important in the biophysical properties of the mycobacterial cell envelope and therefore in pathogenicity.

Phosphate in wastewater effluent is implicated in eutrophication of water reserves. Enhanced biological phosphate removal by activated sludge is attributed to polyphosphate accumulating bacteria, which release phosphate during anaerobiosis and reincorporate it during aerobiosis. The aim of the study was to investigate whether the process of phosphate removal by activated sludge could be probed immunochemically. Antigen preparations from the aerobic and preceding anoxic zones of a phosphate removing system contained intact and lysed bacterial cells. Neither conventional nor subtractive immunisation strategies, the latter employing cyclophosphamide to immunofocus on unique epitopes in the zones, provided antibodies capable of distinguishing between these zones. However, a putatively protein-directed monoclonal antibody could distinguish between the aerobic zones of two activated sludge systems, differing only in phosphate removal ability: immunoblot showed five discrete bands, with molecular weights appearing to be multiples of 18 kDa, unique to the system successful at phosphate removal.

Sustainable management of economically important squid requires monitoring of changes in their abundance, which are related inter alia, to their success in the food chain. The highest mortality is expected in the paralarval stages, which are prone to starvation. Causes of starvation may be linked to the lack of suitable prey. A multiple detection system was developed for the simultaneous identification of five putative zooplankton prey in the guts of paralarval Chokka squid, Loligo vulgaris reynaudii, by employing polyclonal rabbit antisera in conjunction with solid phase immunoassays. Specificities of antisera were validated by ELISA screening against different zooplankton taxa. Cross-reactions observed with ELISA were minimized through manipulation of antibody and antigen concentrations resulting in more specific detection of target prey antigens when used in an immunodot assay. Application of this optimised immunoassay detected multiple predation in paralarval squid samples collected from diverse areas in the Agulhas Bank ecosystem on the south coast of South Africa.

Patient serum antibodies to mycolic acids have the potential to be surrogate markers of active tuberculosis (TB) when they can be distinguished from the ubiquitously present cross-reactive antibodies to cholesterol. Mycolic acids are known to interact more strongly with antibodies present in the serum of patients with active TB than in patients with latent TB or no TB. Examples of single stereoisomers of mycolic acids with chain lengths corresponding to major homologues of those present in Mycobacterium tuberculosis have now been synthesised with a sulfur substituent on the terminal position of the α-chain; initial studies have established that one of these binds to a gold electrode surface, offering the potential to develop second generation sensors for diagnostic patient antibody detection.

Mycolic acids constitute the waxy layer of the outer cell wall of Mycobacterium spp. and a few other genera. They are diverse in structure, providing a unique chromatographic foot-print for almost each of the more than 70 Mycobacterium species. Although mainly esterified to cell wall arabinogalactan, trehalose or glucose, some free mycolic acid is secreted during in vitro growth of Mycobacterium tuberculosis. In M. tuberculosis, α-, keto- and methoxy-mycolic acids are the main classes, each differing in their ability to attract neutrophils, induce foamy macrophages or adopt an antigenic structure for antibody recognition. Of interest is their particular relationship to cholesterol, discovered by their ability to attract cholesterol, to bind Amphotericin B or to be recognised by monoclonal antibodies that cross-react with cholesterol. The structural elements that determine this diverse functionality include the carboxylic acid in the mycolic motif, as well as the nature and stereochemistry of the two functional groups in the merochain. The functional diversity of mycolic acid classes implies that much information may be contained in the selective expression and secretion of mycolic acids to establish tuberculosis after infection of the host. Their cholesteroid nature may relate to how they utilize host cholesterol for their persistent survival.

Nanoparticulate drug delivery systems offer great promise in addressing challenges of drug toxicity, poor bioavailability and non-specificity for a number of drugs. Much progress has been reported for nano drug delivery systems for intravenous administration, however very little is known about the effects of orally administered nanoparticles. Furthermore, the development of nanoparticulate systems necessitates a thorough understanding of the biological response post exposure. This study aimed to elucidate the in vivo uptake of chitosan and polyethylene glycol (PEG) coated Poly, DL, lactic-co-glycolic Acid (PLGA) nanoparticles and the immunological response within 24 h of oral and peritoneal administration. These PLGA nanoparticles were administered orally and peritoneally to female Balb/C mice, they were taken up by macrophages of the peritoneum. When these particles were fluorescently labelled, intracellular localisation was observed. The expression of pro-inflammatory cytokines IL-2, IL-6, IL-12p70 and TNF-α in plasma and peritoneal lavage was found to remain at low concentration in PLGA nanoparticles treated mice as well as ZnO nanoparticles during the 24 hour period. However, these were significantly increased in lipopolysaccharide (LPS) treated mice. Of these pro-inflammatory cytokines, IL-6 and IL-12p70 were produced at the highest concentration in the positive control group. The anti-inflammatory cytokines IL-10 and chemokines INF-γ, IL-4, IL-5 remained at normal levels in PLGA treated mice. IL-10 and INF-γ were significantly increased in LPS treated mice. MCP-1 was found to be significantly produced in all groups in the first hours, except the saline treated mice. These results provide the first report to detail the induction of cytokine production by PLGA nanoparticles engineered for oral applications. http:dx.doi.org/10.1016/j.taap.2010.09.002 http://www.journals.elsevier.com/toxicology-and-applied-pharmacology/ South African Department of Science and Technology

The remarkable physicochemical properties of particles in the nanometer range have been proven to address many challenges in the field of science. However, the possible toxic effects of these particles have raised some concerns. The aim of this article is to evaluate the effects of poly(lactide-co-glycolide) (PLGA) nanoparticles in vitro and in vivo compared to industrial nanoparticles of a similar size range such as zinc oxide, ferrous oxide, and fumed silica. An in vitro cytotoxicity study was conducted to assess the cell viability following exposure to PLGA nanoparticles. Viability was determined by means of a WST assay, wherein cell viability of greater than 75% was observed for both PLGA and amorphous fumed silica particles and ferrous oxide, but was significantly reduced for zinc oxide particles. In vivo toxicity assays were performed via histopathological evaluation, and no specific anatomical pathological changes or tissue damage was observed in the tissues of Balb/C mice. The extent of tissue distribution and retention following oral administration of PLGA particles was analyzed for 7 days. After 7 days, the particles remained detectable in the brain, heart, kidney, liver, lungs, and spleen. The results show that a mean percentage (40.04%) of the particles were localized in the liver, 25.97% in the kidney, and 12.86% in the brain. The lowest percentage was observed in the spleen. Thus, based on these assays, it can be concluded that the toxic effects observed with various industrial nanoparticles will not be observed with particles made of synthetic polymers such as PLGA when applied in the field of nanomedicine. Furthermore, the biodistribution of the particles warrants surface modification of the particles to avoid higher particle localization in the liver.The aim of this study was to evaluate the effects of poly(lactide-co-glycolide) (PLGA) nanoparticles in vitro and in vivo compared to industrial nanoparticles including zinc oxide, ferrous oxide, and fumed silica. The authors concluded that the toxic effects observed with various industrial nanoparticles is unlikely to be observed with particles made of PLGA. The biodistribution of these particles warrants surface modification to avoid particle accumulation in the liver.

Electrochemical impedimetric recognition by anti-mycolic acid antibodies, present in tuberculosis (TB)-positive human serum co-infected with human immunodeficiency virus (HIV), of mycolic acids (MA) integrated into a self-assembled monolayer of N-(2-mercaptoethyl)octadecanamide on a gold electrode is described, proving that the MA-based electrode can satisfactorily discriminate between a TB-positive and a TB-negative serum, thus offering promise as a potential impedimetric immunosensing platform for active tuberculosis.

Antibodies to mycolic acid (MA) antigens can be detected as surrogate markers of active tuberculosis (TB) with evanescent field biosensors where the lipid antigens are encapsulated in liposomes. Standard immunoassay such as ELISA, where the lipid antigen is not encapsulated, but directly adsorbed to the well-bottoms of microtiter plates, does not yield the required sensitivity and specificity for accurate diagnosis of TB. One reason for this is the cross-reactivity of natural anticholesterol antibodies with MAs. MAs are the major cell wall lipids of mycobacteria. Mycobacterial MA has immunomodulatory properties and elicits specific antibodies in TB patients. Liposomes were optimized for their use as carriers both for the presentation of immobilized purified mycobacterial MA on sensor surfaces, and as a soluble inhibitor of antibody binding in inhibition assays. By using an inhibition assay in the biosensor, the interference by anticholesterol antibodies is reduced. Here, we describe the MA carrying capacity of liposomes with and without cholesterol as a stabilizing agent, optimized concentration and size of liposomes for use in the biosensor assay, comparison of the methods for wave-guide and surface plasmon resonance biosensors and how the cholesteroid nature of MA can be demonstrated by the biosensor when Amphotericin B is allowed to bind to MA in liposomes.

The preparation of monoclonal antibodies raised against antigens of salivary gland extracts of femaleRhipicephalus evertsi evertsi is reported. Nine hybridoma cell lines were established of which eight secreted IgG3 and one IgG1. The immune response was biased towards immunogens unique to the prefed stage of the tick salivary glands by prior cyclophosphamide suppression of the immune response against unfed tick salivary glands. Immunoblots of salivary gland antigens, separated by SDS-PAGE showed reactivity with three of the monoclonal antibodies, all of which had identical specificity for antigens unique to prefed salivary extracts. Each of these monoclonal antibodies identified two prominent bands with relative molecular masses corresponding to 23 and 46 and a third minor band with molecular mass of 70 kDa.

Several methods of coating whole cells of Mycobacterium tuberculosis H37 RV to ELISA microtitre plates were compared with the aim of developing an ELISA screening assay for murine monoclonal antibodies in culture supernatants and human antibodies in patient sera. Undercoats of nylon or poly-L-lysine were compared to polystyrene as adsorptive surfaces for the bacteria, the effect of increased ionic strength and iclusion of SDS in the coating buffer measured, and methanol (70%) and glutaraldehyde (5%) investigated for their efficiency as fixatives of the bacterial monolayers. The results suggest PBS as a satisfactory coating buffer for the bacterial cells on polystyrene, and 70% methanol the preferred fixative for the dried antigen-coated plates.

Spontaneous fusion between lymphoid and carcinoma cells in vivo has been described previously. Splenocytes from mice treated with LPS or mitogen have been reported to fuse better with myeloma cells using PEG as fusion agent than splenocytes from untreated mice. We report a phenomenon where immunization of mice with formalin treated, whole Haemophilus paragallinarum bacteria induced spontaneous fusion of splenocytes with myeloma cells in vitro, without the aid of any fusion agent. Co-immunization of mice with H. paragallinarum and an unrelated antigen (hen's egg white lysozyme), followed by co-culturing of the immune splenocytes with SP2/0 myeloma cells, yielded stable hybridoma cell lines producing anti-lysozyme antibodies. H. paragallinarum may be used in adjuvants to simplify the production of monoclonal antibodies, and the discovery of a promotional activity of a gram negative bacterium on cell fusion and hybridoma formation may shed new light on spontaneous fusion as a natural immune phenomenon in cancer.

Mycolic acids (MAs) are a major component of the cell walls of Mycobacterium tuberculosis and related organisms. These alpha-alkyl beta-hydroxy long fatty acids have been the subject of numerous studies for their immunological properties. We previously reported that an interaction between cholesterol and mycolic acids could be responsible for the low accuracy in the serodiagnosis of TB when using free mycolic acid in an ELISA assay. The aim of this work was to investigate if this interaction could be due to a similarity in the structural properties between mycolic acids and cholesterol. The investigation revealed that patient sera cross-reacted with mycolic acids and cholesterol in an ELISA experiment suggesting that both molecules may present related functionality in a similar structural orientation. This relation was further supported by the interaction of mycolic acids with Amphotericin B (AmB), a known binding agent to ergosterol and cholesterol. Using a resonant mirror biosensor, we observed that AmB recognised both cholesterol and mycolic acids. In addition, a specific attraction was observed between mycolic acid and cholesterol by the accumulation of cholesterol from liposomes in suspension onto immobilized mycolic acids containing liposomes, detected with a biosensor technique. Combined, these results suggest that mycolic acids can assume a three-dimensional conformation similar to a sterol. This requires that mycolic acid exposes its hydroxyl group and assumes rigidity in its chain structure to generate a hydrophobic surface topology matching that of cholesterol. A particular folded conformation would be required for this, of which a few different types have already been proven to exist in monolayers of mycolic acids.

Isolation and purification of mycolic acids from Mycobacterium tuberculosis have allowed them to be applied as antigen in an ELISA-based assay to detect specific antibodies in human sera. Tuberculosis patients have previously been shown to contain anti-mycolic acids antibodies. The aim of this study was to determine whether human immunodeficiency virus (HIV) co-infection increases false-negative testing rate and whether other random diseases for which hospitalisation is normally required will contribute to false-positive results. Sera from 118 human subjects were tested for the presence of antibodies to mycolic acids; 59 were patients with proven pulmonary tuberculosis and 59 were control hospitalised patients without evidence of tuberculosis. Each group consisted of HIV-seropositive and HIV-seronegative subjects. The endpoint was the detection of specific antibodies to mycolic acids in the sera, before and after precipitation of immune complexes. The two groups of subjects were well matched for age, gender, race and HIV status. On average, humans infected with Mycobacterium tuberculosis showed a specific antibody response to mycolic acids that was not affected by low CD4 T-lymphocyte counts in HIV-seropositive patients but was compromised by various other serious diseases.

A new method for the stereoselective synthesis of the (R,R)-β-hydroxy-α-alkyl fatty acid fragment of mycolic acids, via an asymmetric anti-aldol reaction is reported. The ‘mycolic acid motif’ fragment was prepared in three steps and >98% ee.

References Acknowledgements Yolandy Lemmer1,3, Boitumelo Semete1, Laetitia Booysen 1,2, Lonji Kalombo1, Lebogang Katata1, Arwyn T. Jones4,Cameron Alexander5, Makobetsa Khati6, Hulda. S. Swai1, Jan A. Verschoor3 1Council for Scientific and Industrial Research, Polymers and Bioceramics, Pretoria, 0001, South Africa, 2Department of Pharmaceutics, North-West University, Potchefstroom Campus, Potchefstroom, 2520, South Africa, 3Department of Biochemistry, University of Pretoria, Pretoria, 0001, South Africa, 4Welsh School of pharmacy, Cardiff University, United Kingdom, 5School of Pharmacy, University of Nottingham, UK, 6Council for Scientific and Industrial Research, Biosciences, Pretoria, 0001, South Africa

The phenomenon of spontaneous fusion between myeloma cells and splenocytes from mice immunized with formalin-inactivated Haemophilus paragallinarum cells, has been reported on recently (1). The identity and properties of the bacterial inducer of fusogenicity of splenocytes have been further investigated with the aid of a monoclonal antibody VF3 against H. paragallinarum (2), which has a bacterial strain specificity correlating with the ability of the strains to induce spontaneous fusion between splenocytes of immunized mice and myeloma cells. It was shown that the lipopolysaccharide fraction of the bacteria was required for the induction of fusogenicity. LPS involvement was clearly indicated by the parallel effects on VF3 antigenicity and fusogenic inductivity of various treatments such as proteolytic digestion, periodate oxidation and sensitivity towards alkali, acid or freezing.

The sesquiterpene lactones isolated from Geigeria were found to be incapable of inducing rat peritoneal mast cell degranulation at levels of 0.3-1.6 mM. The sulphydryl reagent, N-ethylmaleimide, too was unable to trigger mast cell secretion. Instead, it was observed that these compounds inhibited the release of histamine induced by Compound 48/80. Pretreatment of the lactones and N-ethylmaleimide with the amino acid, L-cysteine, reduced their inhibition ability of histamine release to a considerable extent, but not completely. Geigerin(V), which lacks an alpha-methylene group and the chemically prepared cysteine-adduct of dihydrogriesenin(I), were also capable of inhibiting mast cell secretion by Compound 48/80, but to a lesser extent.

Mycolic acids (MAs) are highly hydrophobic long-chain α-alkyl β-hydroxy fatty acids present in the cell wall of Mycobacterium tuberculosis (Mtb) as a complex mixture of molecules with a common general structure but with variable functional groups in the meromycolate chain. In this study, we addressed the relationship between the MA molecular structure and their contribution to the development of T-cell immune responses. Hereto, we used the model antigen ovalbumin and single synthetic MAs, differing in oxygenation class and cis versus trans proximal cyclopropane configuration, as immune stimulatory agents. Subcutaneous delivery of liposome-formulated MAs with a proximal cis cyclopropane elicited antigen-specific Th1 and cytotoxic T-cell immune responses, whereas intratracheal immunization elicited pulmonary Th17 responses. These immune stimulatory activities depended not only on the cis versus trans proximal cyclopropane configuration but also on the MA oxygenation class. Our study thus shows that both the presence and nature of the functional groups in the meromycolate chain affect the immune stimulatory adjuvant activity of Mtb mycolates and suggests that Mtb bacilli may impact on the host protective immune response by modulating the cis versus trans stereochemistry of its mycolates as well as by altering the oxygenation class of the meromycolate functional group.

An oil-based bacterin, containing strains 083 and 0222 of Haemophilus paragallinarum, is commonly used in South Africa to vaccinate laying flocks against infectious coryza. Two strains of H. paragallinarum, designated M85 and SB86, were isolated from infected but vaccinated commercial laying flocks in two incidental outbreaks of coryza in 1985 and 1986. A panel of five monoclonal antibodies was established which clearly distinguished the vaccine strains from the field isolates. One of these reacted with only vaccine strains A and B, another reacted with only field strains M85 and SB86, and the remaining three cross-reacted to various degrees with all four strains or isolates. Immunoassays were performed by enzyme-linked immunosorbent assay using whole bacteria as solid-phase antigen. These monoclonal antibodies may aid in serotyping new field isolates of H. paragallinarum and in improved standardization of vaccine strains.

The publisher regrets that the author list of the above paper which appeared in Chemistry and Physics of Lipids, vol. 163, pp. 800–808 contained errors. The correct author list is published above. The publisher would like to apologise for any inconvenience this may have caused to the authors of this article and readers of the journal.