Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2/neu-positive tumour cells

Trastuzumab (Herceptin), a humanized IgG1 antibody raised against the human epidermal growth factor receptor 2 (HER2/neu), is the main antibody in clinical use against breast cancer. Pre-clinical evidence and clinical studies indicate that trastuzumab employs several anti-tumour mechanisms that most likely contribute to enhanced survival of patients with HER2/neu-positive breast carcinomas. New strategies are aimed at improving antibody-based therapeutics like trastuzumab, e.g. by enhancing antibody-mediated effector function mechanisms. Based on our previous findings that a chimaeric ovarian tumour antigen-specific IgE antibody showed greater efficacy in tumour cell killing, compared to the corresponding IgG1 antibody, we have produced an IgE homologue of trastuzumab. Trastuzumab IgE was engineered with the same light- and heavy-chain variable-regions as trastuzumab, but with an epsilon in place of the gamma-1 heavy-chain constant region. We describe the physical characterisation and ligand binding properties of the trastuzumab IgE and elucidate its potential anti-tumour activities in functional assays. Both trastuzumab and trastuzumab IgE can activate monocytic cells to kill tumour cells, but they operate by different mechanisms: trastuzumab functions in antibody-dependent cell-mediated phagocytosis (ADCP), whereas trastuzumab IgE functions in antibody-dependent cell-mediated cytotoxicity (ADCC). Trastuzumab IgE, incubated with mast cells and HER2/neu-expressing tumour cells, triggers mast cell degranulation, recruiting against cancer cells a potent immune response, characteristic of allergic reactions. Finally, in viability assays both antibodies mediate comparable levels of tumour cell growth arrest. These functional characteristics of trastuzumab IgE, some distinct from those of trastuzumab, indicate its potential to complement or improve upon the existing clinical benefits of trastuzumab.     

Embryonic rotational behaviour in the pond snail Lymnaea stagnalis: influences of environmental oxygen and development stage

Responses of freshwater organisms to environmental oxygen tensions (PO₂) have focused on adult (i.e. late developmental) stages, yet responses of embryonic stages to changes in environmental PO₂ must also have implications for organismal biology. Here we assess how the rotational behaviour of the freshwater snail Lymnaea stagnalis changes during development in response to conditions of hypoxia and hyperoxia. As rotation rate is linked to gas mixing in the fluid surrounding the embryo, we predicted that it would increase under hypoxic conditions but decrease under hyperoxia. Contrary to predictions, early, veliger stage embryos showed no change in their rotation rate under hyperoxia, and later, hippo stage embryos showed only a marginally significant increase in rotation under these conditions. Predictions for hypoxia were broadly supported, however, with both veliger and hippo stages showing a marked hypoxia-related increase in their rotation rates. There were also subtle differences between developmental stages, with hippos responding at PO₂s (50% air saturation) greater than those required to elicit a similar response in veligers (20% air saturation). Differences between developmental stages also occurred on return to normoxic conditions following hypoxia: rotation in veligers returned to pre-exposure levels, whereas there was a virtual cessation in embryos at the hippo stage, likely the result of overstimulation of oxygen sensors driving ciliary movement in later, more developed embryos. Together, these findings suggest that the spinning activity of L. stagnalis embryos varies depending on environmental PO₂s and developmental stage, increasing during hypoxia to mix capsular contents and maintain a diffusive gradient for oxygen entry into the capsule from the external environment (“stir-bar” theory of embryonic rotational behaviour).     

Comparative phylogeography of woodland reptiles in California: repeated patterns of cladogenesis and population expansion

The ultimate goal of comparative phylogeographical analyses is to infer processes of diversification from contemporary geographical patterns of genetic diversity. When such studies are employed across diverse groups in an array of communities, it may be difficult to discover common evolutionary and ecological processes associated with diversification. In order to identify taxa that have responded in a similar fashion to historical events, we conducted comparative phylogeographical analyses on a phylogenetically and ecologically limited set of taxa. Here, we focus on a group of squamate reptiles (snakes and lizards) that share similar ecological requirements and generally occupy the same communities in the western USA. At a gross level, deep genetic division in Contia tenuis, Diadophis punctatus, Elgaria multicarinata, the Charina bottae complex, and Lampropeltis zonata are often concordant in the Transverse Ranges, the Monterey Bay and Sacramento-San Joaquin Delta region, and the southern Sierra Nevada in California. Molecular clock estimates suggest that major phyletic breaks within many of these taxa roughly coincide temporally, and may correspond to important geological events. Furthermore, significant congruence between the phylogeographies of E. multicarinata and L. zonata suggests that the succession of vicariance and dispersal events in these species progressed in concert. Such congruence suggests that E. multicarinata and L. zonata have occupied the same communities through time. However, across our entire multi-taxon data set, the sequence of branching events rarely match between sympatric taxa, indicating the importance of subtle differences in life history features as well as random processes in creating unique genetic patterns. Lastly, coalescent and noncoalescent estimates of population expansion suggest that populations in the more southerly distributed clades of C. tenuis, D. punctatus, E. multicarinata, and L. zonata have been stable, while populations in more northerly clades appear to have recently expanded. This concerted demographic response is consistent with palaeontological data and previous phylogeographical work that suggests that woodland habitat has become more restricted in southern California, but more widespread in the North during Holocene warming. Future phylogeographical work focusing on allied and ecologically associated taxa may add insight into the ecological and evolutionary processes that yield current patterns of genetic diversity.     

Transgenic ipt tobacco overproducing cytokinins overaccumulates phenolic compounds during in vitro growth.

We present evidence that overproduction of endogenous cytokinins (CK) caused stress response in non-rooting Pssu-ipt transgenic tobacco (Nicotiana tabacum L.) grown in vitro. It was demonstrated by overaccumulation of phenolic compounds, synthesis of pathogenesis related proteins (PR proteins), and increase in peroxidase (POD) activities. Immunolocalization of zeatin and also PR-1b protein on leaf cryo-sections proved their accumulation in all mesophyll cells of transgenic tobacco contrary to control non-transgenic plants. Intensive blue autofluorescence of phenolic compounds induced by UV in cross-sections of leaf midrib showed enhanced contents of phenolics in transgenic tobacco compared with controls, nevertheless, no significant difference between both plant types was found in leaf total lignin content. Transgenic plantlets exhibited higher peroxidase activities of both soluble and ionically bound fractions compared with controls. HPLC analysis of phenolic acids confirmed the increase in all phenolic acids in transgenic tobacco except for salicylic acid (SA). The effect of high phenolic content on rooting of transgenic tobacco is discussed.     

Structural basis for the differential regulation of DNA by the methionine repressor MetJ

The Met regulon in Escherichia coli encodes several proteins responsible for the biosynthesis of methionine. Regulation of the expression of most of these proteins is governed by the methionine repressor protein MetJ and its co-repressor, the methionine derivative S-adenosylmethionine. Genes controlled by MetJ contain from two to five sequential copies of a homologous 8-bp sequence called the metbox. A crystal structure for one of the complexes, the repressor tetramer bound to two metboxes, has been reported (Somers, W. S., and S. E. Phillips (1992) Nature 359, 387-393), but little structural work on the larger assemblies has been done presumably because of the difficulties in crystallization and the variability in the number and sequences of metboxes for the various genes. Small angle neutron scattering was used to study complexes of MetJ and S-adenosylmethionine with double-stranded DNA containing two, three, and five metboxes. Our results demonstrate that the crystal structure of the two-metbox complex is not the native solution conformation of the complex. Instead, the system adopts a less compact conformation in which there is decreased interaction between the adjacent MetJ dimers. Models built of the higher order complexes from the scattering data show that the three-metbox complex is organized much like the two-metbox complex. However, the five-metbox complex differs significantly from the smaller complexes, providing much closer packing of the adjacent MetJ dimers and allowing additional contacts not available in the crystal structure. The results suggest that there is a structural basis for the differences observed in the regulatory effectiveness of MetJ for the various genes of the Met regulon.     

Synthesis, molecular modeling and biological evaluation of aza-proline and aza-pipecolic derivatives as FKBP12 ligands and their in vivo neuroprotective effects

Nonimmunosuppressant ligands, exemplified by GPI 1046 (1), for the peptidyl-prolyl isomerase FKBP12 have been found to unexpectedly possess powerful neuroprotective and neuroregenerative effects in vitro and in vivo. We have extensively explored the therapeutic utility of FKBP12 ligands based on analogues of proline and pipecolic acid. As part of our ongoing program to explore novel structural classes of FKBP12 ligands, we herein wish to report a new class of FKBP12 ligands containing aza-proline and aza-pipecolic acid analogues. Details of the synthetic studies, together with biological activity will be presented.     

Application of Balanced Scorecard in the Evaluation of a Complex Health System Intervention: 12 Months Post Intervention Findings from the BHOMA Intervention: A Cluster Randomised Trial in Zambia

Introduction

In many low income countries, the delivery of quality health services is hampered by health system-wide barriers which are often interlinked, however empirical evidence on how to assess the level and scope of these barriers is scarce. A balanced scorecard is a tool that allows for wider analysis of domains that are deemed important in achieving the overall vision of the health system. We present the quantitative results of the 12 months follow-up study applying the balanced scorecard approach in the BHOMA intervention with the aim of demonstrating the utility of the balanced scorecard in evaluating multiple building blocks in a trial setting.

Methods

The BHOMA is a cluster randomised trial that aims to strengthen the health system in three rural districts in Zambia. The intervention aims to improve clinical care quality by implementing practical tools that establish clear clinical care standards through intensive clinic implementations. This paper reports the findings of the follow-up health facility survey that was conducted after 12 months of intervention implementation. Comparisons were made between those facilities in the intervention and control sites. STATA version 12 was used for analysis.

Results

The study found significant mean differences between intervention(I) and control (C) sites in the following domains: Training domain (Mean I:C; 87.5.vs 61.1, mean difference 23.3, p = 0.031), adult clinical observation domain (mean I:C; 73.3 vs.58.0, mean difference 10.9, p = 0.02 ) and health information domain (mean I:C; 63.6 vs.56.1, mean difference 6.8, p = 0.01. There was no gender differences in adult service satisfaction. Governance and motivation scores did not differ between control and intervention sites.

Conclusion

This study demonstrates the utility of the balanced scorecard in assessing multiple elements of the health system. Using system wide approaches and triangulating data collection methods seems to be key to successful evaluation of such complex health intervention.

Trial number

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01942278","term_id":"NCT01942278"}}NCT01942278     

Late paleocene flora of the northern Alaska Peninsula: the role of transberingian plant migrations and climatic change

For the first time, the Late Sagwon Flora is described from the upper beds of the Prince Creek Formation (Upper Paleocene) at the Sagavanirktok River (northern Alaska Peninsula). The flora is dominated by the angiosperm Tiliaephyllum brooksense Moiseeva et Herman sp. nov. and conifer Metasequoia occidentalis (Newb.) Chaney. The Late Sagwon Flora is most similar to the Danian or Danian-Selandian flora from the middle part of the Upper Tsagayan Subformation (Amur Region) and lower part of the Wuyun Formation (Heilongjiang Province, China). This similarity allows us to hypothesize that the genus Tiliaephyllum, which dominated in the Late Tsagayan Flora, migrated via the Bering Land Bridge from southern paleolatitudes of the Far East to high latitudes of the Arctic Pacific, due to the progressively warming climate of the Paleocene. Additional new angiosperm species are described from the Late Sagwon Flora: Archeampelos mullii Moiseeva et Herman sp. nov., Tiliaephyllum brooksense Moiseeva et Herman sp. nov., and Dicotylophyllum sagwonicum Moiseeva et Herman sp. nov.