Detecting intratumoral heterogeneity of EGFR activity by liposome-based in vivo transfection of a fluorescent biosensor.

Despite decades of research in the epidermal growth factor receptor (EGFR) signalling field, and many targeted anti-cancer drugs that have been tested clinically, the success rate for these agents in the clinic is low, particularly in terms of the improvement of overall survival. Intratumoral heterogeneity is proposed as a major mechanism underlying treatment failure of these molecule-targeted agents. Here we highlight the application of fluorescence lifetime microscopy (FLIM)-based biosensing to demonstrate intratumoral heterogeneity of EGFR activity. For sensing EGFR activity in cells, we used a genetically encoded CrkII-based biosensor which undergoes conformational changes upon tyrosine-221 phosphorylation by EGFR. We transfected this biosensor into EGFR-positive tumour cells using targeted lipopolyplexes bearing EGFR-binding peptides at their surfaces. In a murine model of basal-like breast cancer, we demonstrated a significant degree of intratumoral heterogeneity in EGFR activity, as well as the pharmacodynamic effect of a radionuclide-labeled EGFR inhibitor in situ. Furthermore, a significant correlation between high EGFR activity in tumour cells and macrophage-tumour cell proximity was found to in part account for the intratumoral heterogeneity in EGFR activity observed. The same effect of macrophage infiltrate on EGFR activation was also seen in a colorectal cancer xenograft. In contrast, a non-small cell lung cancer xenograft expressing a constitutively active EGFR conformational mutant exhibited macrophage proximity-independent EGFR activity. Our study validates the use of this methodology to monitor therapeutic response in terms of EGFR activity. In addition, we found iNOS gene induction in macrophages that are cultured in tumour cell-conditioned media as well as an iNOS activity-dependent increase in EGFR activity in tumour cells. These findings point towards an immune microenvironment-mediated regulation that gives rise to the observed intratumoral heterogeneity of EGFR signalling activity in tumour cells in vivo.

Imaging the paediatric lung: what does nanotechnology have to offer?

This review will provide an overview of current research into lung imaging with nanoparticles, with a focus on the use of nanoparticles as molecular imaging agents to observe pathological processes and to monitor the effectiveness of nanoparticulate drug delivery systems. Various imaging modalities together with their advantages and limitations for lung imaging will be discussed. We will also explore the range of nanoparticles used, as well as active or passive targeting of nanoparticles.

Flowering catch crops used as forage plants for dairy cows: influence on fatty acids and tocopherols in milk.

The effect of several flowering dicotyledonous catch crop plants (dicots) on milk fat quality in cows was investigated to test the hypothesis that their phenolic compounds may inhibit ruminal biohydrogenation and thus enhance the transfer to milk of intact, plant-derived polyunsaturated fatty acids. Berseem clover (Trifolium alexandrinum), buckwheat (Fagopyrum esculentum), and phacelia (Phacelia tanacetifolia) were sown in mixture with ryegrass (Lolium multiflorum; intended biomass proportion of 0.2) on 1ha. For comparison, nonflowering chicory (Cichorium intybus, also sown in mixture with ryegrass) and pure ryegrass were cultivated. Realized biomass proportions (wet weight) were 91% for berseem clover, 69% for buckwheat, 54% for phacelia, and 51% for chicory. At the start of flowering (or from d 47 after sowing onward), cultures were harvested daily and fed for 20 d ad libitum to groups of 6 midlactation cows each. Additionally, 1 kg each of energy and protein concentrate and pure ryegrass hay were fed. Individual intake and milk yield of the cows were measured daily. Milk samples were obtained twice daily 5 d before and from 11 to 20 d after the start of treatment feeding. Feed samples were drawn twice a week from the fresh feeds. Apart from standard traits, feeds and milk were analyzed for fatty acids, tocopherols, and phenolic fractions. Only a few substantial treatment effects on intake and performance were observed. All diets based on dicots increased α-linolenic acid (ALA) concentrations in milk fat compared with the ryegrass diet even though the corresponding swards were not generally richer in ALA. The highest ALA concentration in milk fat (1.3 g/100g of fatty acids) occurred with the berseem clover diet. Transfer rate of ALA from feed to milk was highest with the buckwheat diet (0.09) and lowest with ryegrass (0.05). This was congruent with the differences in total extractable phenols, being high in the buckwheat sward (2.6% of dry matter) and low in the ryegrass sward (1.2% of dry matter). Intermediates of ALA biohydrogenation were lowest in the milk fat of the buckwheat group, indicating an inhibitory effect of this treatment, which provided the highest dietary levels of phenols. The α-tocopherol concentration in milk was higher with the buckwheat diet than with berseem clover and phacelia diets. The study provides evidence that the ALA concentration in milk fat could be enhanced by feeding flowering dicots; however, this was due to different modes of action.

Expansion of CD4+ T cells with a cytotoxic phenotype in patients with B-chronic lymphocytic leukaemia (B-CLL).

Abnormal CD4/CD8 ratios and T-cell function have previously been shown in patients with B-chronic lymphocytic leukaemia (B-CLL). We have demonstrated that CD4+ T cells containing both serine esterase and perforin (PF) are increased in the blood of these patients. Using flow cytometry, we have shown that the CD4+ PF+ cells were CD57+ but lacked expression of CD28, suggesting a mature population. The same phenotype in CD8+ T cells is characteristic of mature cytotoxic T cells. However, in contrast to the CD8+ T cells, the CD4+ T cells were more frequently CD45RO positive than CD45RA positive, indicating prior antigen experience. In contrast, this population lacked expression of either CD69 or HLA-DR, arguing that they were not activated or that they are an abnormal population of T cells. Their constitutive cytokine levels showed them mainly to contain IL4 and not IFNgamma, suggesting a Th2 phenotype. The role of the CD4+ PF+ T-cell population is at present uncertain. However, this potentially cytotoxic T-cell population could contribute both to enhancing survival of the B-CLL tumour cells through production of IL4, and to the immunodeficient state frequently seen in patients with this tumour, independent of drug treatment.