Immunocolloidal Targeting of the Endocytotic Siglec-7 Receptor Using Peripheral Attachment of Siglec-7 Antibodies to Poly(Lactide-co-Glycolide) Nanoparticles

Purpose: To prepare a nanoparticulate formulation expressing variable peripheral carboxyl density using non-endcapped and endcapped poly(lactide-co-glycolide), conjugated to antibodies recognising the siglec-7 receptor, which is expressed on most acute myeloid leukaemias. The aim is to exploit this receptor as a therapeutic target by constructing an internalising drug-loaded nanoparticle able to
translocate into cytoplasm by siglec receptor-mediated internalisation.

Materials and Methods: Antibodies to the siglec-7 (CD33-like) receptor were conjugated to dye-loaded nanoparticles using carbodiimide chemistry, giving 32.6 mg protein per mg of nanoparticles using 100% of the non-endcapped PLGA. Binding studies using cognate antigen were used to verify preservation of antibody function following conjugation.

Results: Mouse embryonic fibroblasts expressing recombinant siglec-7 receptor and exposed to NileRed-loaded nanoparticles conjugated to antibody accumulated intracellular fluorescence, which was not observed if either antibody or siglec-7 receptor was absent. Confocal microscopy revealed internalised perinuclear cytoplasmic staining, with an Acridine Orange-based analysis showing red staining in localised foci, indicating localisation within acidic endocytic compartments.

Conclusions: Results show antibody-NP constructs are internalised via siglec-7 receptor-mediated internalisation. If loaded with a therapeutic agent, antibody-NP constructs can cross into cytoplasmic
space and delivery drugs intracellularly to cells expressing CD33-like receptors, such as natural killer cells and monocytes.

Range-edge effects promote clonal growth in peripheral populations of the one-sided wintergreen Orthilia secunda

Existing in suboptimal conditions is a frequent occurrence for species inhabiting the cusp of their ecological range. In range-edge populations of plants, the scarcity of suitable habitat may be reflected in small population sizes which may result in increased self-pollination and/or inbreeding and an increase in the incidence of clonal reproduction. These factors may result in a decrease in levels of genetic diversity and a loss of potential adaptive variation that may compromise species' ability to cope with changes in their environment, an issue that is particularly relevant today with the current concern surrounding global climate change and its effect on species' distributional ranges. In the present study, we have compared the levels of clonal reproduction in the one-sided wintergreen Orthilia secunda (L.) House in (1) populations from its main continuous distribution range, (2) populations occurring on the limits of the continuous range, and (3) peripheral populations outwith the species' continuous distribution range. Range-edge populations in Scotland and Sweden displayed significantly lower genotypic richness and diversity than those from the main area of the species' distribution in these countries. Populations from Ireland, which occur in the temperate zone rather than the boreal conditions that are the preferred habitat for the species, and which represent relict populations left over from cooler periods in the Earth's history, displayed no within-population genetic diversity, suggesting a complete lack of sexual reproduction. Furthermore, the genetic distinctiveness of the Irish populations, which contained alleles not found in either the Scottish or the Swedish populations, highlights the value of 'trailing edge' populations and supports the concept of 'parochial conservation', namely the conservation of species that are locally rare but globally common.

Role of peripheral afference during acquisition of a complex coordination task

It has long been supposed that the interference observed in certain patterns of coordination is mediated, at least in part, by peripheral afference from the moving limbs. We manipulated the level of afferent input, arising from movement of the opposite limb, during the acquisition of a complex coordination task. Participants learned to generate flexion and extension movements of the right wrist, of 75degrees amplitude, that were a quarter cycle out of phase with a 1-Hz sinusoidal visual reference signal. On separate trials, the left wrist either was at rest, or was moved passively by a torque motor through 50degrees, 75degrees or 100degrees, in synchrony with the reference signal. Five acquisition sessions were conducted on successive days. A retention session was conducted I week later. Performance was initially superior when the opposite limb was moved passively than when it was static. The amplitude and frequency of active movement were lower in the static condition than in the driven conditions and the variation in the relative phase relation across trials was greater than in the driven conditions. In addition, the variability of amplitude, frequency and the relative phase relation during each trial was greater when the opposite limb was static than when driven. Similar effects were expressed in electromyograms. The most marked and consistent differences in the accuracy and consistency of performance (defined in terms of relative phase) were between the static condition and the condition in which the left wrist was moved through 50degrees. These outcomes were exhibited most prominently during initial exposure to the task. Increases in task performance during the acquisition period, as assessed by a number of kinematic variables, were generally well described by power functions. In addition, the recruitment of extensor carpi radialis (ECR), and the degree of co-contraction of flexor carpi radialis and ECR, decreased during acquisition. Our results indicate that, in an appropriate task context, afferent feedback from the opposite limb, even when out of phase with the focal movement, may have a positive influence upon the stability of coordination.

Comparative analysis of dna methylation profiles in peripheral blood leukocytes versus lymphoblastoid cell lines

Previous reports have shown that DNA methylation profiles within primary human malignant tissues are altered when these cells are transformed into cancer cell lines. However, it is unclear if similar differences in DNA methylation profiles exist between DNA derived from peripheral blood leukocytes (PBLs) and corresponding Epstein-Barr Virus transformed lymphoblastoid cell lines (LCLs). To assess the utility of LCLs as a resource for methylation studies we have compared DNA methylation profiles in promoter and 5' regions of 318 genes in PBL and LCL sample pairs from patients with type 1 diabetes with or without nephropathy. We identified a total of 27 (similar to 8%) genes that revealed different DNA methylation profiles in PBL compared with LCL-derived DNA samples. In conclusion, although the profiles for most promoter regions were similar between PBL-LCL pairs, our results indicate that LCL-derived DNA may not be suitable for DNA methylation studies at least in diabetic nephropathy.

Distinct patterns of peripheral HIV-1-specific interferon- gamma responses in exposed HIV-1-seronegative individuals.

It is unclear how human immunodeficiency virus (HIV) type 1–specific immune responses in exposed seronegative (ESN) individuals differ from those in HIV-1–infected subjects. By use of overlapping peptides spanning Gag, Tat, Nef, Vif, Vpr, and Vpu, peripheral blood mononuclear cells from ESN individuals, their seropositive (SP) partners, and unexposed seronegative control subjects were screened for interferon-? production. Responses were more frequent (95.7% vs. 20%), of a higher magnitude (9-fold), and of wider breadth (median number of peptides recognized, 18 vs. 2.5) in SP than in ESN individuals. Peptides recognized by ESN individuals were less frequently recognized by their SP partners. SP subjects infrequently recognized peptides from Vif, and such responses were subdominant; among ESN individuals, this HIV-1 protein was most frequently recognized. Immunodominant peptides recognized by SP subjects tended to be from relatively conserved regions, whereas peptides recognized by ESN individuals were associated with slow disease progression.

The Effects of Cilostazol on Exercise-induced Ischaemia-reperfusion Injury in Patients with Peripheral Arterial Disease

Objectives: Cilostazol improves walking distance in peripheral arterial disease (PAD) patients. The study objectives were to assess the effects of cilostazol on walking distance, followed by the additional assessment of cilostazol on exercise-induced ischaemiaereperfusion injury in such patients.

Methods: PAD patients were prospectively recruited to a double-blinded, placebo-controlled trial. Patients were randomised to receive either cilostazol 100 mg or placebo twice a day. The primary end-point was an improvement in walking distance. Secondary end-points included the assessment of oxygen-derived free-radical generation, antioxidant consumption and other markers of the in?ammatory cascade. Initial and absolute claudication distances (ICDs and ACDs, respectively) were measured on a treadmill. In?ammatory response was assessed before and 30 min post-exercise by measuring lipid hydroperoxide, ascorbate, atocopherol, b-carotene, P-selectin, intracellular and vascular cell-adhesion molecules (I-CAM and V-CAM), thromboxane B2 (TXB2), interleukin-6, interleukin-10, high-sensitive C-reactive protein (hsCRP), albuminecreatinine ratio (ACR) and urinary levels of p75TNF receptor. All tests were performed at baseline and 6 and 24 weeks.

Results: One hundred and six PAD patients (of whom 73 were males) were recruited and successfully randomised from December 2004 to January 2006. Patients who received cilostazol demonstrated a more signi?cant improvement in the mean percentage change from baseline in ACD (77.2% vs. 26.6% at 6 weeks, pZ0.026 and 161.7% vs. 79.0% at 24 weeks, pZ0.048) as compared to the placebo. Cilostazol reduced lipid hydroperoxide levels compared to a placebo-related increase before and after exercise (6 weeks: pre-exercise: 11.8% vs.

Competing O-H insertion and  -elimination in rhodium carbenoid reactions; synthesis of 2-alkoxy-3-arylpropanoates

Rhodium(II) carboxylate catalyzed decompn. of diazo esters 3 (shown as I) and PhCH2C(CO2Et)N2 4 in the presence of alcs. or water results in formation of 2-alkoxy- or 2-hydroxy-3-arylpropanoates, resp., by O-H insertion in competition with cinnamates by elimination; the ratio of insertion to elimination is dramatically affected by the carboxylate ligand on rhodium. Use of methanol-d as the alc. confirms that the alkene does not arise by elimination from the initial alkoxyester product.