Efficient, chemoselective synthesis of immunomicelles using single-domain antibodies with a C-terminal thioester

Background  

Classical bioconjugation strategies for generating antibody-functionalized nanoparticles are non-specific and typically result in heterogeneous compounds that can be compromised in activity. Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester, providing a unique handle for site-specific conjugation using native chemical ligation (NCL). However, current methods to generate antibody fragments with C-terminal thioesters require cumbersome refolding procedures, effectively preventing application of NCL for antibody-mediated targeting and molecular imaging.     

Ultrastructural localisation of intramuscular expression of BDNF mRNA by silver-gold intensified non-radioactive in situ hybridisation

A non-radioactive in situ hybridisation method is described for the detection of low intramuscular levels of brain-derived neurotrophic factor (BDNF) mRNA at the electron microscope level. Application of high-grade silver-gold intensification of the diaminobenzidine end product of in situ hybridisation revealed that in adult rat muscle the constitutive expression of muscular BDNF is confined to the myofibres; satellite cells, Schwann cells, endothelial cells, fibroblasts or axons do not appear to contribute to BDNF production in normal muscle. Although muscular BDNF is a neurotrophic factor for innervating motoneurons and supposedly released only at the motor endplates, the production of BDNF mRNA appears to occur along the entire length of the myofibres and is not confined to nuclei in the postsynaptic regions.     

Changes in peripheral immune cell numbers and functions in octogenarian walkers – an acute exercise study

Background

Age-related changes of the immune system, termed immunosenescence, may underlie the increased risk of infections and morbidity in the elderly. Little is known about the effects of acute exercise on peripheral immune parameters in octogenarians. Therefore, we investigated acute exercise-induced changes in phenotype and function of the immune system in octogenarians participating in the 2013 edition of the Nijmegen Four Days Marches. Blood sampling was performed at baseline and immediately after 4 days of the walking exercise (30 km/day). A comprehensive set of adaptive and innate immune traits were enumerated and analyzed by flow-cytometry. Peripheral blood mononuclear cells, isolated before and after walking were stimulated with LPS and supernatants were analysed for IL-1β, IL-6, IL-8 and TNF-α concentrations by ELISA. CMV serostatus was determined by ELISA.

Results

The walking exercise induced a clear leucocytosis with numerical increases of granulocytes, monocytes and lymphocytes. These exercise-induced changes were most profound in CMV seropositive subjects. Within lymphocytes, numerical increases of particularly CD4+ T cells were noted. Further T cell differentiation analysis revealed profound increases of naïve CD4+ T cells, including naïve Treg. Significant increases were also noted for CD4+ memory T cell subsets. In contrast, only slight increases in naïve and memory CD8+ T cell subsets were detected. Exercise did not affect markers of immune exhaustion in memory T cell subsets. NK cells demonstrated a numerical decline and a change in cellular composition with a selective decrease of the mature CD56^dim NK cells. The latter was seen in CMV seronegative subjects only. Also, a higher IL-6 and IL-8 production capacity of LPS-stimulated PBMC was seen after walking.

Conclusion

In this exceptional cohort of octogenarian walkers, acute exercise induced changes in immune cell numbers and functions. A clear response of CD4+ T cells, rather than CD8+ T cells or NK cells was noted. Remarkably, the response to exercise within the CD4+ T cell compartment was dominated by naïve CD4+ subsets.

     

Electron microscopic studies on the interaction of rat Kupffer cells and Plasmodium berghei sporozoites

The interactions between Plasmodium berghei sporozoites and Kupffer cells in rat liver were studied by transmission electron microscopy. Between 10 and 45 min after inoculation, sporozoites were found in the process of entering Kupffer cells and inside phagolysosomes. The sporozoites entered the Kupffer cells by phagocytosis as determined by the presence of pseudopods and local accumulations of aggregated microfilaments and the resulting exclusion of other organelles in the phagocyte cytoplasm beneath the attached parasite. Sporozoites were taken up either with their anterior end first, or backwards. Scanning electron microscopy of in vitro sporozoite Kupffer cell interaction confirmed these observations. It was concluded that sporozoites are taken up in a normal phagocytic way by the Kupffer cells, regardless of their initial place of contact or position. Thirty min after inoculation sporozoites found in phagolysosomes were still morphologically intact but after 45 min we could encounter completely digested sporozoites.     

Susceptibility of Cut Rose Flower Cultivars to Infections by Different Isolates of Botrytis cinerea

A method was developed for the inoculation of flowers of cut roses with conidia of Botrytis cinerea. Flower buds were inoculated by spraying of conidial supensions that were ultrasonicated for 10 s. The differences in susceptibility between 8 rose cvs to infections of isolate Bc-33 and the differences in pathogenicity between 14 5. cinerea isolates to cv. ‘Sonia’ were evaluated. Isolates obtained from rose flowers caused higher infection rates than those obtamed from various other hosts. The CVS ‘Madelon’, ‘Melody’ and ‘Sonia’ were found to be highly susceptible, whereas the cvs ‘Caramboie’, ‘Gabriella’, ‘Pasadena’ and ‘Rubinette’ were only slightly susceptible. The lower disease severity in the less susceptible cvs was based on a retardation of the growth of mfection hyphae in the petals, which may have been due to partial resistance. The formation of symptoms is effectuated already by the initial stages in the infection process. Thus, the apparent existing partial resistance to infections of B. cinerea cannot prevent the loss of ornamental value.     

The effect of three years of TNF alpha blocking therapy on markers of bone turnover and their predictive value for treatment discontinuation in patients with ankylosing spondylitis: a prospective longitudinal observational cohort study

Introduction

Statins (hydroxymethylglutaryl coenzyme A reductase inhibitors) are effective in reducing the risk of cardiovascular morbidity and mortality in patients with hyperlipidemia, hypertension, or type II diabetes. Next to their cholesterol-lowering activity, statins have immunomodulatory properties. Based on these properties, we hypothesized that statin use may eventually lead to dysregulation of immune responses, possibly resulting in autoimmunity. We have recently shown in an observational study that statin use was associated with an increased risk of developing rheumatoid arthritis. Our objective was to investigate whether a causal relationship could be established for this finding.

Methods

The mouse collagen type II (CII)-induced arthritis (CIA) model was used, with immunization, challenge, and euthanasia at days 0, 21, and 42, respectively. Statins were given orally before (day -28 until day 21) or after (day 21 until day 42) CIA induction. Atorvastatin (0.2 mg/day) or pravastatin (0.8 mg/day) was administered. Arthritis was recorded three times a week. Serum anti-CII autoantibodies and cytokines in supernatants from Concanavalin-A-stimulated lymph node cells and CII-stimulated spleen cells were measured.

Results

Statin administration accelerated arthritis onset and resulted in 100% arthritic animals, whereas only seven out of 12 nonstatin control animals developed arthritis. Atorvastatin administration after CIA induction resulted in earlier onset than atorvastatin administration before induction, or than pravastatin administration before or after induction. The arthritic score of animals given pravastatin before CIA induction was similar to that of the nonstatin controls, whereas the other groups that received statins showed higher arthritic scores. Atorvastatin administration, especially before CIA induction, increased anti-CII autoantibody production. IL-2 and IL-17 production by lymph node and spleen cells was higher in CIA animals than in PBS controls, but was not affected by statin administration. While IFN?? production was not affected by CIA induction, atorvastatin administration before CIA induction increased the production of this cytokine.

Conclusion

These data support previous results from our observational studies, indicating a role for statins in the induction of autoimmunity.     

The PDZ Domain of the Guanine Nucleotide Exchange Factor PDZGEF Directs Binding to Phosphatidic Acid during Brush Border Formation

PDZGEF is a guanine nucleotide exchange factor for the small G protein Rap. It was recently found that PDZGEF contributes to establishment of intestinal epithelial polarity downstream of the kinase Lkb1. By binding to phosphatidic acid enriched at the apical membrane, PDZGEF locally activates Rap2a resulting in induction of brush border formation via a pathway that includes the polarity players TNIK, Mst4 and Ezrin. Here we show that the PDZ domain of PDZGEF is essential and sufficient for targeting PDZGEF to the apical membrane of polarized intestinal epithelial cells. Inhibition of PLD and consequently production of phosphatidic acid inhibitis targeting of PDZGEF to the plasma membrane. Furthermore, localization requires specific positively charged residues within the PDZ domain. We conclude that local accumulation of PDZGEF at the apical membrane during establishment of epithelial polarity is mediated by electrostatic interactions between positively charged side chains in the PDZ domain and negatively charged phosphatidic acid.     

Relevance of the leaving group for antitumor activity of new platinum(II) compounds containing anthracene derivatives as a carrier ligand

A new anticancer-active platinum(II) compound [Pt(A9pyp)(dmso)(cbdca)], containing the E-1-(9-anthryl)-3-(2-pyridyl)-2-propenone ligand (abbreviated as A9pyp) has been synthesized by the replacement of the anionic chloride ligands in cis-[Pt(A9pyp)(dmso)Cl₂] by the dianionic chelating cyclobutanedicarboxylate ligand (abbreviated as cbdca). The in vitro relevance of the leaving group of these new platinum(II) compounds has been investigated. Measurements of the time-dependent intracellular accumulation of both compounds in human ovarian carcinoma cell lines show that the leaving group affects their cellular uptake. In addition, the leaving group also influences DNA platination, and, therefore, has an effect on the biological activity against a pair of human ovarian carcinoma cell lines, i.e. sensitive and resistant to cisplatin.