Sialic acid blockade in dendritic cells enhances CD8+ T cell responses by facilitating high-avidity interactions.

Sialic acids are negatively charged carbohydrates that cap the glycans of glycoproteins and glycolipids. Sialic acids are involved in various biological processes including cell-cell adhesion and immune recognition. In dendritic cells (DCs), the major antigen-presenting cells of the immune system, sialic acids emerge as important regulators of maturation and interaction with other lymphocytes including T cells. Many aspects of how sialic acids regulate DC functions are not well understood and tools and model systems to address these are limited. Here, we have established cultures of murine bone marrow-derived DCs (BMDCs) that lack sialic acid expression using a sialic acid-blocking mimetic Ac53FaxNeu5Ac. Ac53FaxNeu5Ac treatment potentiated BMDC activation via toll-like receptor (TLR) stimulation without affecting differentiation and viability. Sialic acid blockade further increased the capacity of BMDCs to induce antigen-specific CD8+ T cell proliferation. Transcriptome-wide gene expression analysis revealed that sialic acid mimetic treatment of BMDCs induces differential expression of genes involved in T cell activation, cell-adhesion, and cell-cell interactions. Subsequent cell clustering assays and single cell avidity measurements demonstrated that BMDCs with reduced sialylation form higher avidity interactions with CD8+ T cells. This increased avidity was detectable in the absence of antigens, but was especially pronounced in antigen-dependent interactions. Together, our data show that sialic acid blockade in BMDCs ameliorates maturation and enhances both cognate T cell receptor-MHC-dependent and independent T cell interactions that allow for more robust CD8+ T cell responses.

Generation of αCD11b-CpG antibody conjugates for the targeted stimulation of myeloid cells.

CpG oligonucleotides are short single-stranded synthetic DNA molecules. Upon binding to Toll-like receptor 9 (TLR9), CpG activates immune cells in humans and mice. This results in robust Th1 type immunity potentially resulting in clearance of pathogens, reduction of allergy and anti-tumor immunity. However, the effectiveness of CpG as an adjuvant depends on its administration route, with only strong effects seen when CpG is administered locally. As local administration is not always feasible, we generated conjugates to specifically deliver CpG to myeloid cells often abundantly present in tumors. For this we coupled CpG (3'-Thiol-modified phosphorothioate (PTO) CpG-ODN1826 type B (5'-tccatgacgttcctgacgtt-3')) to monoclonal antibodies (mAbs) directed against the myeloid cell marker CD11b using maleimide-thiol coupling. The CD11b-CpG mAb (αCD11b-CpG) conjugates contained about four CpG molecules/conjugate and displayed binding and internalization characteristics similar to unconjugated CD11b mAbs (αCD11b). The αCD11b-CpG conjugates readily induced maturation of murine dendritic cells (DCs) in a TLR9-dependent manner in vitro. Following intravenous injection, αCD11b-CpG conjugates efficiently targeted CD11b+ immune cells in the blood, lymph nodes and spleen. Finally, injection of αCD11b-CpG conjugates, but not untargeted conjugates, induced maturation of CD11b+ cell subsets in vivo. In conclusion, conjugating CpG to αCD11b enabled specific targeting and activation of myeloid cells in vivo.

Metabolism of norethisterone and norethisterone derivatives in rat uterus, vagina, and aorta.

The 19-nor-progestogen norethisterone is used as a progestogen component in contraceptives and in continuous- and sequential combined hormone replacement therapy (HRT) in postmenopausal women. Metabolism of norethisterone in HRT target tissues may play a role in its biological response. The aim of this study was to investigate which steroid-metabolizing enzymes are present in rat uterus, vagina, and aorta, three HRT target tissues. Next, the ability of the tissues to metabolize norethisterone was assessed. Furthermore, to investigate the effect of substituents at the 7- and 11-position, the metabolism of Org OM38 (7alpha-methyl-norethisterone), Org 4060 (11beta-ethyl-norethisterone), and Org 34694 (7alpha-methyl,11-ethylidene-norethisterone) was studied. Using radiolabeled progesterone, the presence of 20alpha-hydroxysteroid dehydrogenase, 5alpha-reductase, and 3alpha-hydroxysteroid dehydrogenase activity could be demonstrated in uterus, vagina, and to a lesser extent in aorta. The combined action of the latter two enzyme activities resulted in 3alpha-OH,5alpha-H-norethisterone as the major metabolite of radiolabeled norethisterone in uterus (26.9%), vagina (37.1%), and aorta (1.4%). The norethisterone derivatives, however, were metabolized to a much lesser extent (1.0-7.6%). No formation of 5alpha-reduced forms of Org 4060, Org OM38, or Org 34694 was found, while formation of minor amounts of 3alpha-OH-Org 4060 and 3alpha-OH-Org OM38 could be demonstrated in both uterus, vagina, and aorta. These findings confirm the role of 5alpha-reductase as a rate-limiting step in the metabolism of norethisterone derivatives and show important inhibitory effects of substituents at the 7alpha- and 11-position of the steroid skeleton on 5alpha-reduction.

Metabolism of estradiol, ethynylestradiol, and moxestrol in rat uterus, vagina, and aorta: influence of sex steroid treatment.

Estrogen replacement therapy for postmenopausal women consists of an estrogenic and a progestagenic compound. The treatment has a positive estrogenic effect on bone, the cardiovascular system, and vagina but is dependent of the estrogen-progestagen balance in uterus to prevent unwanted proliferation. We were interested in the influence of estrogens and progestagens on estrogen metabolism in target tissues of estrogen replacement therapy. Therefore, we studied the metabolism of estradiol, 17alpha-ethynylestradiol, and moxestrol (11beta-methoxy-17alpha-ethynylestradiol) in rat uterus, vagina, and aorta. In uterus and vagina, estradiol was converted to estrone, estradiol-3-glucuronide, and estrone-3-glucuronide. These metabolites demonstrate the presence of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and UDP-glucuronosyl transferase (UDP-GT) in uterus and vagina. We found that the conversion of estradiol by 17beta-HSD in uterus was increased in animals treated with estradiol or with a combination of estradiol and progesterone. The conversion of estradiol in uterus by UDP-GT was estradiol-induced and in contrast, progesterone-suppressed. In the vagina, steroid hormone treatment had no effect on estradiol conversion by 17beta-HSD or UDP-GT. Ethynylestradiol was glucuronidated only, and this was not affected by steroid treatment. Moxestrol was not converted in any of the three organs that were studied, indicating that the 11beta-methoxy substituent renders it a poor substrate for glucuronidation. Overall, the estrogen metabolism, and its regulation by sex steroids, in rat uterus is different compared with human uterus. Therefore, the rat may not be the best-suited model to investigate uterine effects of estradiol-progestagen combined treatment.

The influence of a fish oil-enriched diet on the phospholipid fatty acid turnover in the rabbit red cell membrane in vivo.

The influence of the polyunsaturated fatty acid (PUFA) composition of the diet on the rate of fatty acid turnover of individual phospholipids in the erythrocyte membrane in vivo was studied. Following modification of the fatty acid composition of the membrane phospholipids by the use of a fish oil or a linoleic acid enriched diet, phospholipids--labelled in the unsaturated fatty acid at the 2-position of the glycerol moiety--were introduced into the membrane of freshly isolated rabbit erythrocytes. Thereafter, the labelled erythrocytes were reinjected into the bloodstream of the animal. It appears that, with the exception of 1-palmitoyl,2-linoleoyl phosphatidylcholine, all other phosphatidylcholines disappear faster from the erythrocytes of fish oil-fed rabbits than from the red cells of linoleic acid-fed rabbits. Another parameter, which possibly influences the turnover rates of PUFA containing phospholipids, can be peroxidation. An attempt was made to measure peroxidative damage of lipids in vivo by the introduction of 1-palmitoyl,2-cis-parinaroyl phosphatidylcholine (PnPC)--a probe to measure oxidative stress--into the membrane of freshly isolated erythrocytes, in the same way as is described for the radioactive phospholipids. The data demonstrate that the fluorescent signal from the PnPC decreases at a fast rate which is independent of the dietary conditions.