A general process for the development of peptide-based immunoassays for monoclonal antibodies

Monoclonal antibodies (mAb) are an important and growing class of cancer therapeutics, but pharmacokinetic analyses have in many cases been constrained by the lack of standard and robust pharmacologic assays. The goal of this project was to develop a general method for the production of immunoassays that can measure the levels of therapeutic monoclonal antibodies in biologic samples at relevant concentrations. Alemtuzumab and rituximab are monoclonal approved for the treatment of B-cell malignancies and were used as a model system. Phage-displayed peptide libraries were screened for peptide sequences recognized by alemtuzumab (anti-CD52) or rituximab (anti-CD20). Synthetic biotinylated peptides were used in enzyme-linked immunosorbent assays (ELISA). Peptides directly synthesized on polymer resin beads were used in an immunofluorescent-based assay. Peptide mimetope sequences were recovered for both mAb and confirmed by competitive staining and kinetic measurements. A peptide-based ELISA method was developed for each. The assay for rituximab had a limit of detection of 4 μg/ml, and the assay for alemtuzumab had a limit of detection of 1 μg/ml. Antibody-specific staining of peptide conjugated beads could be seen in a dose-dependent manner. Phage-displayed peptide libraries can be a source of highly specific mimetopes for therapeutic mAb. The biotinylated forms of those peptides are compatible with conventional ELISA methods with sensitivities comparable to other assay methods and sufficient for pharmacological studies of those mAb given at high dose. The process outlined here can be applied to any mAb to enable improved pharmacokinetic analysis during the development and clinical use of this class of therapies

Assessment of the anthelmintic activity of medicinal plant extracts and purified condensed tannins against free-living and parasitic stages of Oesophagostomum dentatum

Background: Plant-derived condensed tannins (CT) show promise as a complementary option to treat gastrointestinal helminth infections, thus reducing reliance on synthetic anthelmintic drugs. Most studies on the anthelmintic effects of CT have been conducted on parasites of ruminant livestock. Oesophagostomum dentatum is an economically important parasite of pigs, as well as serving as a useful laboratory model of helminth parasites due to the ability to culture it in vitro for long periods through several life-cycle stages. Here, we investigated the anthelmintic effects of CT on multiple life-cycles stages of O. dentatum. Methods: Extracts and purified fractions were prepared from five plants containing CT and analysed by HPLC-MS. Anthelmintic activity was assessed at five different stages of the O. dentatum life cycle; the development of eggs to infective third-stage larvae (L3), the parasitic L3 stage, the moult from L3 to fourth-stage larvae (L4), the L4 stage and the adult stage. Results: Free-living larvae of O. dentatum were highly susceptible to all five plant extracts. In contrast, only two of the five extracts had activity against L3, as evidenced by migration inhibition assays, whilst three of the five extracts inhibited the moulting of L3 to L4. All five extracts reduced the motility of L4, and the motility of adult worms exposed to a CT-rich extract derived from hazelnut skins was strongly inhibited, with electron microscopy demonstrating direct damage to the worm cuticle and hypodermis. Purified CT fractions retained anthelmintic activity, and depletion of CT from extracts by pre-incubation in polyvinylpolypyrrolidone removed anthelmintic effects, strongly suggesting CT as the active molecules. Conclusions: These results suggest that CT may have promise as an alternative parasite control option for O. dentatum in pigs, particularly against adult stages. Moreover, our results demonstrate a varied susceptibility of different life-cycle stages of the same parasite to CT, which may offer an insight into the anthelmintic mechanisms of these commonly found plant compounds

An isoperimetric inequality in the plane with a log-convex density

Given a positive lower semi-continuous density $f$ on $\mathbb{R}^2$ the weighted volume $V_f:=f\mathscr{L}^2$ is defined on the $\mathscr{L}^2$-measurable sets in $\mathbb{R}^2$. The $f$-weighted perimeter of a set of finite perimeter $E$ in $\mathbb{R}^2$ is written $P_f(E)$. We study minimisers for the weighted isoperimetric problem $$I_f(v):=\inf\Big\{ P_f(E):E\text{ is a set of finite perimeter in }\mathbb{R}^2\text{ and }V_f(E)=v\Big\}$$ for $v>0$. Suppose $f$ takes the form $f:\mathbb{R}^2\rightarrow(0,+\infty);x\mapsto e^{h(|x|)}$ where $h:[0,+\infty)\rightarrow\mathbb{R}$ is a non-decreasing convex function. Let $v>0$ and $B$ a centred ball in $\mathbb{R}^2$ with $V_f(B)=v$. We show that $B$ is a minimiser for the above variational problem and obtain a uniqueness result

Influenza A Virus Infection of Human Primary Dendritic Cells Impairs Their Ability to Cross-Present Antigen to CD8 T Cells

Influenza A virus (IAV) infection is normally controlled by adaptive immune responses initiated by dendritic cells (DCs). We investigated the consequences of IAV infection of human primary DCs on their ability to function as antigen-presenting cells. IAV was internalized by both myeloid DCs (mDCs) and plasmacytoid DCs but only mDCs supported viral replication. Although infected mDCs efficiently presented endogenous IAV antigens on MHC class II, this was not the case for presentation on MHC class I. Indeed, cross-presentation by uninfected cells of minute amounts of endocytosed, exogenous IAV was ∼300-fold more efficient than presentation of IAV antigens synthesized by infected cells and resulted in a statistically significant increase in expansion of IAV-specific CD8 T cells. Furthermore, IAV infection also impaired cross-presentation of other exogenous antigens, indicating that IAV infection broadly attenuates presentation on MHC class I molecules. Our results suggest that cross-presentation by uninfected mDCs is a preferred mechanism of antigen-presentation for the activation and expansion of CD8 T cells during IAV infection

Epidemiologic heterogeneity of common mood and anxiety disorders over the lifecourse in the general population: a systematic review

Background Clinical evidence has long suggested there may be heterogeneity in the patterns and predictors of common mood and anxiety disorders; however, epidemiologic studies have generally treated these outcomes as homogenous entities. The objective of this study was to systematically review the epidemiologic evidence for potential patterns of heterogeneity of common mood and anxiety disorders over the lifecourse in the general population. Methods We reviewed epidemiologic studies examining heterogeneity in either the nature of symptoms experienced ( symptom syndromes ) or in patterns of symptoms over time ( symptom trajectories ). To be included, studies of syndromes were required to identify distinct symptom subtypes, and studies of trajectories were required to identify distinct longitudinal patterns of symptoms in at least three waves of follow-up. Studies based on clinical or patient populations were excluded. Results While research in this field is in its infancy, we found growing evidence that, not only can mood and anxiety disorders be differentiated by symptom syndromes and trajectories, but that the factors associated with these disorders may vary between these subtypes. Whether this reflects a causal pathway, where genetic or environmental factors influence the nature of the symptom or trajectory subtype experienced by an individual, or whether individuals with different subtypes differed in their susceptibility to different environmental factors, could not be determined. Few studies addressed issues of comorbidity or transitions in symptoms between common disorders. Conclusion Understanding the diversity of these conditions may help us identify preventable factors that are only associated with some subtypes of these common disorders

Gene Discovery in the Threatened Elkhorn Coral: 454 Sequencing of the Acropora palmata Transcriptome

BACKGROUND: Cnidarians, including corals and anemones, offer unique insights into metazoan evolution because they harbor genetic similarities with vertebrates beyond that found in model invertebrates and retain genes known only from non-metazoans. Cataloging genes expressed in Acropora palmata, a foundation-species of reefs in the Caribbean and western Atlantic, will advance our understanding of the genetic basis of ecologically important traits in corals and comes at a time when sequencing efforts in other cnidarians allow for multi-species comparisons. RESULTS: A cDNA library from a sample enriched for symbiont free larval tissue was sequenced on the 454 GS-FLX platform. Over 960,000 reads were obtained and assembled into 42,630 contigs. Annotation data was acquired for 57% of the assembled sequences. Analysis of the assembled sequences indicated that 83-100% of all A. palmata transcripts were tagged, and provided a rough estimate of the total number genes expressed in our samples (~18,000-20,000). The coral annotation data contained many of the same molecular components as in the Bilateria, particularly in pathways associated with oxidative stress and DNA damage repair, and provided evidence that homologs of p53, a key player in DNA repair pathways, has experienced selection along the branch separating Cnidaria and Bilateria. Transcriptome wide screens of paralog groups and transition/transversion ratios highlighted genes including: green fluorescent proteins, carbonic anhydrase, and oxidative stress proteins; and functional groups involved in protein and nucleic acid metabolism, and the formation of structural molecules. These results provide a starting point for study of adaptive evolution in corals. CONCLUSIONS: Currently available transcriptome data now make comparative studies of the mechanisms underlying coral's evolutionary success possible. Here we identified candidate genes that enable corals to maintain genomic integrity despite considerable exposure to genotoxic stress over long life spans, and showed conservation of important physiological pathways between corals and bilaterians

Immunological Change in a Parasite-Impoverished Environment: Divergent Signals from Four Island Taxa

Dramatic declines of native Hawaiian avifauna due to the human-mediated emergence of avian malaria and pox prompted an examination of whether island taxa share a common altered immunological signature, potentially driven by reduced genetic diversity and reduced exposure to parasites. We tested this hypothesis by characterizing parasite prevalence, genetic diversity and three measures of immune response in two recently-introduced species (Neochmia temporalis and Zosterops lateralis) and two island endemics (Acrocephalus aequinoctialis and A. rimitarae) and then comparing the results to those observed in closely-related mainland counterparts. The prevalence of blood parasites was significantly lower in 3 of 4 island taxa, due in part to the absence of certain parasite lineages represented in mainland populations. Indices of genetic diversity were unchanged in the island population of N. temporalis; however, allelic richness was significantly lower in the island population of Z. lateralis while both allelic richness and heterozygosity were significantly reduced in the two island-endemic species examined. Although parasite prevalence and genetic diversity generally conformed to expectations for an island system, we did not find evidence for a pattern of uniformly altered immune responses in island taxa, even amongst endemic taxa with the longest residence times. The island population of Z. lateralis exhibited a significantly reduced inflammatory cell-mediated response while levels of natural antibodies remained unchanged for this and the other recently introduced island taxon. In contrast, the island endemic A. rimitarae exhibited a significantly increased inflammatory response as well as higher levels of natural antibodies and complement. These measures were unchanged or lower in A. aequinoctialis. We suggest that small differences in the pathogenic landscape and the stochastic history of mutation and genetic drift are likely to be important in shaping the unique immunological profiles of small isolated populations. Consequently, predicting the impact of introduced disease on the many other endemic faunas of the remote Pacific will remain a challenge

Beyond trastuzumab: novel therapeutic strategies in HER2-positive metastatic breast cancer

The use of trastuzumab, a monoclonal antibody that targets the human epidermal growth factor receptor 2 (HER2) alteration present in 25 to 30% of breast cancers, has been associated with improved survival outcomes in both the adjuvant and metastatic settings. However, despite the robust clinical efficacy of trastuzumab in HER2-positive metastatic breast cancer (MBC), primary and secondary resistance remains a clinical challenge. Although lapatinib has demonstrated modest activity in this setting, trials reported to date have yet to demonstrate improvements in overall survival with its use. Novel therapeutic strategies to circumvent trastuzumab resistance are warranted, and agents targeting the HER, vascular endothelial growth factor, heat shock protein 90, phosphoinositide 3 kinase/Akt/mammalian target of rapamycin, and insulin-like growth factor-1 receptor pathways represent rational approaches in the management of HER2-positive disease. In this review, early-phase and emerging trial data surrounding the use of these promising agents in HER2-positive MBC will be discussed

Increased peri-ductal collagen micro-organization may contribute to raised mammographic density

BACKGROUND: High mammographic density is a therapeutically modifiable risk factor for breast cancer. Although mammographic density is correlated with the relative abundance of collagen-rich fibroglandular tissue, the causative mechanisms, associated structural remodelling and mechanical consequences remain poorly defined. In this study we have developed a new collaborative bedside-to-bench workflow to determine the relationship between mammographic density, collagen abundance and alignment, tissue stiffness and the expression of extracellular matrix organising proteins. METHODS: Mammographic density was assessed in 22 post-menopausal women (aged 54–66 y). A radiologist and a pathologist identified and excised regions of elevated non-cancerous X-ray density prior to laboratory characterization. Collagen abundance was determined by both Masson’s trichrome and Picrosirius red staining (which enhances collagen birefringence when viewed under polarised light). The structural specificity of these collagen visualisation methods was determined by comparing the relative birefringence and ultrastructure (visualised by atomic force microscopy) of unaligned collagen I fibrils in reconstituted gels with the highly aligned collagen fibrils in rat tail tendon. Localised collagen fibril organisation and stiffness was also evaluated in tissue sections by atomic force microscopy/spectroscopy and the abundance of key extracellular proteins was assessed using mass spectrometry. RESULTS: Mammographic density was positively correlated with the abundance of aligned periductal fibrils rather than with the abundance of amorphous collagen. Compared with matched tissue resected from the breasts of low mammographic density patients, the highly birefringent tissue in mammographically dense breasts was both significantly stiffer and characterised by large (>80 μm long) fibrillar collagen bundles. Subsequent proteomic analyses not only confirmed the absence of collagen fibrosis in high mammographic density tissue, but additionally identified the up-regulation of periostin and collagen XVI (regulators of collagen fibril structure and architecture) as potential mediators of localised mechanical stiffness. CONCLUSIONS: These preliminary data suggest that remodelling, and hence stiffening, of the existing stromal collagen microarchitecture promotes high mammographic density within the breast. In turn, this aberrant mechanical environment may trigger neoplasia-associated mechanotransduction pathways within the epithelial cell population. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13058-015-0664-2) contains supplementary material, which is available to authorized users