Five birds,one stone: Neutralization of α-hemolysin and 4 bi-component leukocidins of Staphylococcus aureus with a single human monoclonal antibody

Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component leukocidins that share only ∼26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by α-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by α-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to α-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis.     

Flavonoid chemistry of Chenopodium fremontii. Infraspecific variation and systematic implications at the interspecific level

Four flavonoid geographical races based on twenty flavonol 3-O-glycosides were found to exist in Chenopodium fremontii with those populations from the northern part of the range (northern Colorado, Wyoming. and western Nebraska) producing 7-methyl ethers and 3-O-galactosides and glucosides. Plants from Arizona. southern Colorado and New Mexico lack 7-methyl ethers and contain 3-O-rhamnogalactosides and rhamnoglucosides (rutinosides). California populations are chemically similar to those from Arizona, southern Colorado and New Mexico but contain arabinosides while lacking rutinosides. No morphological features could be correlated with the chemical races. Chenopodium fremontii can be distinguished chemically from other closely related diploid species of the western U.S., all of which exhibit simpler flavonoid patterns. It is suggested that the simpler chemical patterns for the latter species (which include C. atrovirens, C. desiccatum, C. hians. C. incanum, C. leptophyllum, and C. pratericola) are a derived condition relative to C. fremontii.     

Systematic implications of flavonoids in Hazardia

The flavonoid patterns in Hazardia species support species delimitations and relationships based on morphology and geography. The compounds thus far elucidated are glycosides of quercetin, kaempferol, isorhamnetin, luteolin, and apigenin, glycoflavones of apigenin, and methoxylated flavonol aglycones.     

Polarized migration of lymphatic endothelial cells is critically dependent on podoplanin regulation of Cdc42

We have shown previously that T1α/podoplanin is required for capillary tube formation by human lung microvascular lymphatic endothelial cells (HMVEC-LLy) and that cells with decreased podoplanin expression fail to properly activate the small GTPase RhoA shortly after the beginning of the lymphangiogenic process. The objective of this study was to determine whether podoplanin regulates HMVEC-LLy migration and whether this regulation is via modulation of small GTPase activation. In analysis of scratch wound assays, we found that small interfering RNA (siRNA) depletion of podoplanin expression in HMVEC-LLy inhibits VEGF-induced microtubule-organizing center (MTOC) and Golgi polarization and causes a dramatic reduction in directional migration compared with control siRNA-transfected cells. In addition, a striking redistribution of cortical actin to fiber networks across the cell body is observed in these cells, and, remarkably, it returns to control levels if the cells are cotransfected with a dominant-negative mutant of Cdc42. Moreover, cotransfection of a dominant-negative construct of Cdc42 into podoplanin knockdown HMVEC-LLy completely abrogated the effect of podoplanin deficiency, rescuing MTOC and Golgi polarization and cell migration to control level. Importantly, expression of constitutively active Cdc42 construct, like podoplanin knockdown, decreased RhoA-GTP level in HMVEC-LLy, demonstrating cross talk between both GTPases. Taken together, the results indicate that polarized migration of lymphatic endothelial cells in response to VEGF is mediated via a pathway of podoplanin regulation of small GTPase activities, in particular Cdc42.     

Isolation of engineered, full-length antibodies from libraries expressed in Escherichia coli

We describe facile isolation of full-length IgG antibodies from combinatorial libraries expressed in E. coli. Full-length heavy and light chains are secreted into the periplasm, where they assemble into aglycosylated IgGs that are captured by an Fc-binding protein that is tethered to the inner membrane. After permeabilizing the outer membrane, spheroplast clones expressing so-called E-clonal antibodies, which specifically recognize fluorescently labeled antigen, are selected using flow cytometry. Screening of a library constructed from an immunized animal yielded several antibodies with nanomolar affinities toward the protective antigen of Bacillus anthracis.     

TRIP-1 via AKT modulation drives lung fibroblast/myofibroblast trans-differentiation

Background

Myofibroblasts are the critical effector cells in the pathogenesis of pulmonary fibrosis which carries a high degree of morbidity and mortality. We have previously identified Type II TGFβ receptor interacting protein 1 (TRIP-1), through proteomic analysis, as a key regulator of collagen contraction in primary human lung fibroblasts—a functional characteristic of myofibroblasts, and the last, but critical step in the process of fibrosis. However, whether or not TRIP-1 modulates fibroblast trans-differentiation to myofibroblasts is not known.

Methods

TRIP-1 expression was altered in primary human lung fibroblasts by siRNA and plasmid transfection. Transfected fibroblasts were then analyzed for myofibroblast features and function such as α-SMA expression, collagen contraction ability, and resistance to apoptosis.

Results

The down-regulation of TRIP-1 expression in primary human lung fibroblasts induces α-SMA expression and enhances resistance to apoptosis and collagen contraction ability. In contrast, TRIP-1 over-expression inhibits α-SMA expression. Remarkably, the effects of the loss of TRIP-1 are not abrogated by blockage of TGFβ ligand activation of the Smad3 pathway or by Smad3 knockdown. Rather, a TRIP-1 mediated enhancement of AKT phosphorylation is the implicated pathway. In TRIP-1 knockdown fibroblasts, AKT inhibition prevents α-SMA induction, and transfection with a constitutively active AKT construct drives collagen contraction and decreases apoptosis.

Conclusions

TRIP-1 regulates fibroblast acquisition of phenotype and function associated with myofibroblasts. The importance of this finding is it suggests TRIP-1 expression could be a potential target in therapeutic strategy aimed against pathological fibrosis.     

Total cost estimation for implementing genome-enabled selection in a multi-level swine production system

Background

Determining an animal’s genetic merit using genomic information can improve estimated breeding value (EBV) accuracy; however, the magnitude of the accuracy improvement must be large enough to recover the costs associated with implementing genome-enabled selection. One way to reduce costs is to genotype nucleus herd selection candidates using a low-density chip and to use high-density chip genotyping for animals that are used as parents in the nucleus breeding herd. The objective of this study was to develop a tool to estimate the cost structure associated with incorporating genome-enabled selection into multi-level commercial breeding programs.

Results

For the purpose of this deterministic study, it was assumed that a commercial pig is created from a terminal line sire and a dam that is a cross between two maternal lines. It was also assumed that all male and female selection candidates from the 1000 sow maternal line nucleus herds were genotyped at low density and all animals used for breeding at high density. With the assumptions used in this analysis, it was estimated that genome-enabled selection costs for a maternal line would be approximately US$0.082 per weaned pig in the commercial production system. A total of US$0.164 per weaned pig is needed to incorporate genome-enabled selection into the two maternal lines. Similarly, for a 600 sow terminal line nucleus herd and genotyping only male selection candidates with the low-density panel, the cost per weaned pig in the commercial herd was estimated to be US$0.044. This means that US$0.21 per weaned pig produced at the commercial level and sired by boars obtained from the nucleus herd breeding program needs to be added to the genetic merit value in order to break even on the additional cost required when genome-enabled selection is used in both maternal lines and the terminal line.

Conclusions

By modifying the input values, such as herd size and genotyping strategy, a flexible spreadsheet tool developed from this work can be used to estimate the additional costs associated with genome-enabled selection. This tool will aid breeders in estimating the economic viability of incorporating genome-enabled selection into their specific breeding program.     

Antibody Engineering and Therapeutics: December 8-12, 2013, Huntington Beach,CA

The 24^th Antibody Engineering & Therapeutics meeting brought together a broad range of participants who were updated on the latest advances in antibody research and development. Organized by IBC Life Sciences, the gathering is the annual meeting of The Antibody Society, which serves as the scientific sponsor. Preconference workshops on 3D modeling and delineation of clonal lineages were featured, and the conference included sessions on a wide variety of topics relevant to researchers, including systems biology; antibody deep sequencing and repertoires; the effects of antibody gene variation and usage on antibody response; directed evolution; knowledge-based design; antibodies in a complex environment; polyreactive antibodies and polyspecificity; the interface between antibody therapy and cellular immunity in cancer; antibodies in cardiometabolic medicine; antibody pharmacokinetics, distribution and off-target toxicity; optimizing antibody formats for immunotherapy; polyclonals, oligoclonals and bispecifics; antibody discovery platforms; and antibody-drug conjugates.     

River macrophyte indices: not the Holy Grail!

1. Recent studies have demonstrated that there is generally no unambiguous relationship between plant species composition and specific environmental conditions in rivers. Nevertheless, indices of environmental pressures based on macrophytes are flourishing, because of the requirements of the Water Framework Directive (WFD). 2. We first reviewed nine such indices against 13 criteria for bioindicators. Then, using data from France and England, we tested whether the IBMR (Macrophyte Biological Index for Rivers) and LEAFPACS (predictions and classification system for macrophytes) methods could reliably indicate nutrient and hydromorphological pressures. Finally, we used an improved bootstrapping method to estimate accuracy. 3. Currently, most indices lack ecological meaning for a variety of reasons, including partial sampling (backwaters are excluded); reliance on list of taxa (there are identification difficulties) rather than structure and functions; correlation rather than causation; application within a limited biogeographical area; reliance on ‘expert’ judgement; high precision but poor accuracy; poorly defined reference conditions; lack of independent tests; and an inability to discriminate reliably between the target pressures of interest from confounding background variables. 4. IBMR was a far better indicator of pH (or HCO₃‐pCO₂) than it was of soluble reactive phosphorus, SRP (or SRP‐NH₄). While there was a highly significant correlation between IBMR and SRP after removing the effect of pH, the relationship was weak (r² = 0.08, n = 215, P < 0.001). 5. LEAFPACS is a multi‐metric method summing up five individual indices, each compliant with the WFD. Its individual metrics were not better correlated with nutrient and hydromorphological pressures (with r² < 0.1, n = 62, P < 0.05) than was the IBMR. The meaning of the overall metric is questionable. 6. There are problems in determining the precision of the indices, owing to uncertainties in recording, but they are less than the uncertainties in determining accuracy (because species optima and tolerances are sometimes poorly known). 7. Reliable information is needed to improve the state of our rivers. Macrophyte indices are able to detect statistically significant pressures from a large population of sites but cannot be applied at specific sites, as required by the WFD, owing to large uncertainties and low explanatory power. Typically, more than 90% of the variability in macrophyte indices is attributed to factors other than human pressure. The WFD would be better served by a simpler, holistic approach based on our current mechanistic understanding of river processes. These findings are likely to apply also to other taxonomic groups (macroinvertebrates, diatoms, fish) used in the assessment of purported ecological quality and to palaeolimnological measures of reference status.