Multivalent human antibody-centyrin fusion protein to prevent and treat Staphylococcus aureus infections.

Treating and preventing infections by antimicrobial-resistant bacterial pathogens is a worldwide problem. Pathogens such as Staphylococcus aureus produce an array of virulence determinants, making it difficult to identify single targets for the development of vaccines or monoclonal therapies. We described a human-derived anti-S. aureus monoclonal antibody (mAb)-centyrin fusion protein ("mAbtyrin") that simultaneously targets multiple bacterial adhesins, resists proteolysis by bacterial protease GluV8, avoids Fc engagement by S. aureus IgG-binding proteins SpA and Sbi, and neutralizes pore-forming leukocidins via fusion with anti-toxin centyrins, while maintaining Fc- and complement-mediated functions. Compared with the parental mAb, mAbtyrin protected human phagocytes and boosted phagocyte-mediated killing. The mAbtyrin also reduced pathology, reduced bacterial burden, and protected from different types of infections in preclinical animal models. Finally, mAbtyrin synergized with vancomycin, enhancing pathogen clearance in an animal model of bacteremia. Altogether, these data establish the potential of multivalent mAbs for treating and preventing S. aureus diseases.

Identification of biologic agents to neutralize the bicomponent leukocidins of Staphylococcus aureus.

A key aspect underlying the severity of infections caused by Staphylococcus aureus is the abundance of virulence factors that the pathogen uses to thwart critical components of the human immune response. One such mechanism involves the destruction of host immune cells by cytolytic toxins secreted by S. aureus, including five bicomponent leukocidins: PVL, HlgAB, HlgCB, LukED, and LukAB. Purified leukocidins can lyse immune cells ex vivo, and systemic injections of purified LukED or HlgAB can acutely kill mice. Here, we describe the generation and characterization of centyrins that bind S. aureus leukocidins with high affinity and protect primary human immune cells from toxin-mediated cytolysis. Centyrins are small protein scaffolds derived from the fibronectin type III-binding domain of the human protein tenascin-C. Although centyrins are potent in tissue culture assays, their short serum half-lives limit their efficacies in vivo. By extending the serum half-lives of centyrins through their fusion to an albumin-binding consensus domain, we demonstrate the in vivo efficacy of these biologics in a murine intoxication model and in models of both prophylactic and therapeutic treatment of live S. aureus systemic infections. These biologics that target S. aureus virulence factors have potential for treating and preventing serious staphylococcal infections.

Liquid Culture Production of Fungal Microsclerotia.

Fungal microsclerotia ("small" sclerotia) are compact hyphal aggregates, typically 50-600 µm in diameter, that are formed under unfavorable nutritional and/or environmental conditions. These structures are often melanized and desiccated to some degree containing endogenous nutritional reserves for use when favorable conditions return. Many fungi, mostly plant pathogens, produce microsclerotia as a survival structure. Liquid culture methods have been developed for producing microsclerotia of the Ascomycota Metarhizium spp, Colletotrichum truncatum, Mycoleptodiscus terrestris, and Trichoderma spp. While these fungi have varying culture conditions that optimize microsclerotia production, all share common nutritional and environmental requirements for microsclerotia formation. Described are the general liquid culture techniques, media components, and harvesting and drying methods necessary to produce stable microsclerotial granules of these fungi.

Enabling occupation at the end of life: A literature review.

OBJECTIVE: Occupation, or meaningful activity, can contribute to the well-being and quality of life of all individuals. It is thus a logical tautology that occupation should be enabled for those at the end of life. Our present review examines current provision of these processes by Occupational Therapist, who can be much-valued members of multidisciplinary palliative care teams. METHOD: Following a literature search and critical selection, 10 global papers were identified examining occupation and occupational therapy at the end of life in the acute, hospice, and community environments. RESULTS: Universally, there appeared to be a dearth of therapists working in end-of-life care. Provision of palliative care in hospitals was found to be compensatory or rehabilitative. Hospice therapy emerged as pleasingly occupational, though the number of hospice places was disappointingly few. Community literature was sparse, so it proved challenging to draw definitive conclusions. Promising research refracted light on occupation at home; however, it also revealed stretched domiciliary services, where clients are not well informed about the potential scope of occupational therapy. SIGNIFICANCE OF RESULTS: A "good death" involving a quality end-of-life experience is the foundational goal overarching all therapy and medicine in the provision of palliative care. Arguably, an occupation-focused approach provided by therapists meets client needs to enable meaningful experiences in the limited time left to them. Current occupational therapy practice environments are not necessarily achieving these goals in commensurate fashion. There is a need to promote the role of occupational therapy and circumscribe what therapists can offer. Further research is necessitated across all environments and future funding for therapist positions in palliative teams. End-of-life care can be complex and challenging; however, therapists can facilitate fulfillment of client-centered occupational goals. In engaging with personally constructed nuances of meaning, quality of life can be improved in those deserving of a significant and emotionally rich daily existence during their final days.

Caspase activation as a versatile assay platform for detection of cytotoxic bacterial toxins.

Pathogenic bacteria produce several virulence factors that help them establish infection in permissive hosts. Bacterial toxins are a major class of virulence factors and hence are attractive therapeutic targets for vaccine development. Here, we describe the development of a rapid, sensitive, and high-throughput assay that can be used as a versatile platform to measure the activities of bacterial toxins. We have exploited the ability of these toxins to cause cell death via apoptosis of sensitive cultured cell lines as a readout for measuring toxin activity. Caspases (cysteine-aspartic proteases) are induced early in the apoptotic pathway, and so we used their induction to measure the activities of Clostridium difficile toxins A (TcdA) and B (TcdB) and binary toxin (CDTa-CDTb), Corynebacterium diphtheriae toxin (DT), and Pseudomonas aeruginosa exotoxin A (PEA). Caspase induction in the cell lines, upon exposure to toxins, was optimized by toxin concentration and intoxication time, and the specificity of caspase activity was established using a genetically mutated toxin and a pan-caspase inhibitor. In addition, we demonstrate the utility of the caspase assay for measuring toxin potency, as well as neutralizing antibody (NAb) activity against C. difficile toxins. Furthermore, the caspase assay showed excellent correlation with the filamentous actin (F-actin) polymerization assay for measuring TcdA and TcdB neutralization titers upon vaccination of hamsters. These results demonstrate that the detection of caspase induction due to toxin exposure using a chemiluminescence readout can support potency and clinical immunogenicity testing for bacterial toxin vaccine candidates in development.

Proteomic analysis of the response of Listeria monocytogenes to bile salts under anaerobic conditions.

Listeria monocytogenes is a food-borne pathogen responsible for the disease listeriosis. The infectious process depends on survival in the high bile-salt conditions encountered throughout the gastrointestinal tract, including the gallbladder. However, it is not clear how bile-salt resistance mechanisms are induced, especially under physiologically relevant conditions. This study sought to determine how the L. monocytogenes strains EGDe (serovar 1/2a), F2365 (serovar 4a) and HCC23 (serovar 4b) respond to bile salts under anaerobic conditions. Changes in the expressed proteome were analysed using multidimensional protein identification technology coupled with electrospray ionization tandem mass spectrometry. In general, the response to bile salts among the strains tested involved significant alterations in the presence of cell-wall-associated proteins, DNA repair proteins, protein folding chaperones and oxidative stress-response proteins. Strain viability correlated with an initial osmotic stress response, yet continued survival for EGDe and F2365 involved different mechanisms. Specifically, proteins associated with biofilm formation in EGDe and transmembrane efflux pumps in F2365 were expressed, suggesting that variations exist in how virulent strains respond and adapt to high bile-salt environments. These results indicate that the bile-salt response varies among these serovars and that further research is needed to elucidate how the response to bile salts correlates with colonization potential in vivo.

Comparison of rhesus and cynomolgus macaques in a Streptococcus pyogenes infection model for vaccine evaluation.

Animal models predictive of human disease are generally difficult to establish and reproduce. In the case of the Group A Streptococcus (GAS) bacterium, which is predominantly a human pathogen, virulence assessment in animal models is problematic. We compared a monkey colonization and pharyngitis model of infection in two macaque species to determine the optimal model for vaccine candidate evaluation. Rhesus and cynomolgus macaques were intranasally infected with a streptomycin resistant (Str(r)) GAS strain. Monkeys were monitored for body weight and temperature changes, throat swabs and sera were collected, and clinical observations were noted throughout the study. Both species exhibited oropharyngeal colonization by GAS, with rhesus macaques demonstrating a more sustained colonization through day 28 post-challenge. Veterinary observations revealed no significant differences between GAS-infected rhesus and cynomolgus macaques. Mock-infected monkeys did not exhibit clinical symptoms or GAS colonization throughout the study. ELISA results demonstrated that both rhesus and cynomolgus macaques developed anti-streptolysin-O antibody titers, with cynomolgus generating higher titers. Sera from infected monkeys produced opsonophagocytic killing and bound to the bacterium in an immunofluorescence assay. Both rhesus and cynomolgus macaques can be used for colonization studies with this GAS M3 strain, yet only mild clinical signs of pharyngitis and tonsillitis were observed.

A novel automated method for enumeration of Chlamydia trachomatis inclusion forming units.

Chlamydia trachomatis is an obligate intracellular pathogen that primarily infects epithelial cells. Traditional methods for quantification of inclusion forming units (IFUs) rely upon infection of epithelial cell monolayers in vitro. Following incubation for approximately 2 days, inclusion bodies that result from infection of cells are detected by immunofluorescent staining with an antibody conjugated to a fluorescent dye. These inclusion bodies are then manually counted by microscopic examination of multiple, randomly selected fields of view. This requires substantial operator time and is subject to investigator bias. We have developed a novel method in which we utilize an automated microplate ImmunoSpot reader to count C. trachomatis IFUs. Following infection of epithelial cells in a 96-well plate and subsequent incubation, IFUs are fixed and detected with an anti-C. trachomatis LPS monoclonal antibody. Immobilized antibody is detected with a biotinylated secondary antibody and visualized enzymatically with streptavidin-alkaline phosphatase and the colorimetric substrate nitro-blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phospate (NBT/BCIP). IFUs are then enumerated with the ImmunoSpot system. This method has been used to quantify IFUs from all cell lines traditionally used for chlamydial propagation, including L929, McCoy, HeLa and HaK cells. IFU numbers obtained are comparable to those determined by traditional microscopic counting. In addition, the method can be applied to rapid determination of serum-neutralizing titers for vaccine studies, and we have also applied this approach to quantify Chlamydia recovered from vaginal swabs collected from infected animals. This method provides for rapid enumeration of IFU counts while minimizing investigator bias and has potential applications for both research and diagnostic use.

Liquid-culture production of blastospores of the bioinsecticidal fungus Paecilomyces fumosoroseus using portable fermentation equipment.

The production of fungal spores using on-site, non-sterile, portable fermentation equipment is technically constrained. Very little information is available on the production requirements, such as medium concentration, inoculum stabilization, required fermentation times, and maintenance of axenic growth. In this study, we developed a two-part, liquid concentrate of the production medium that remains stable and soluble at room temperature. We also examined inoculum stability and showed that freeze- or air-dried blastospore preparations were stable for 7 days after rehydration when stored at 4 degrees C. The use of a low-pH (pH 4), relatively rich complex medium provided a growth environment deleterious to bacterial growth yet conducive to rapid sporulation by Paecilomyces fumosoroseus. High concentrations of blastospores (7.9 x 10(8)/ml) of P. fumosoroseus were produced in a 40-h fermentation with very low levels of bacterial contamination when the fermentor was charged with a blastospore production medium with a starting pH of 4 and inoculated with blastospore concentrations greater than 1 x 10(6) spores/ml. These studies demonstrate that the use of disinfected, portable fermentation equipment has potential for on-site production of high concentrations of blastospores of the bioinsecticidal fungus P. fumosoroseus.