Changes in the blood level, efficacy, and safety of tacrolimus in pregnancy and the lactation period in patients with systemic lupus erythematosus.

OBJECTIVES: We investigated the efficacy and safety of tacrolimus (TAC) by monitoring its serum concentration for mothers and infants in pregnant patients with systemic lupus erythematosus (SLE). METHODS: We measured trough concentrations of TAC in 25 pregnant patients with SLE to assess influence of TAC on the disease activity. Additionally, we measured the concentrations of TAC in umbilical arterial blood, breast milk, and breastfed infants to investigate the safety of TAC for the mothers and infants. RESULTS: The trough concentrations of TAC in the mothers significantly decreased in the second trimester as compared with those before pregnancy. However, the decrease in the trough concentrations of TAC did not lead to the deterioration of SLE. When examined, the doses of TAC were significantly lower in the second trimester and postpartum in the deteriorating group than those in the non-deteriorating group. There were no adverse events by TAC in mothers and fetuses. The concentrations of TAC in the umbilical cord blood were lower than those in the maternal blood. The relative infant dose in breastfed infants of TAC was < 1%. The level of TAC in infant bloods was below detectable limits. CONCLUSION: These findings suggest that TAC is one of the most effective and safest immunosuppressive drugs for use in pregnant patients with SLE.

Cholesterol-sphingomyelin interaction in membrane and apolipoprotein-mediated cellular cholesterol efflux.

Helical apolipoproteins interact with cellular surface and generate high density lipoprotein (HDL) by removing phospholipid and cholesterol from cells. We have reported that the HDL is generated by this reaction with the fetal rat astrocytes and meningeal fibroblasts but cholesterol is poorly available to this reaction with the astrocytes (Ito et al. 1999. J. Neurochem. 72: 2362;-2369). Partial digestion of the membrane by extracellular sphingomyelinase increased the incorporation of cholesterol into thus-generated HDL from both types of cell. This increase was diminished by supplement of endogenous or exogenous sphingomyelin to the cells. The sphingomyelinase treatment decreased cholesterol in the membrane mainly in the detergent-resisting domain. The intracellular cholesterol used by acylCoA:cholesterol acyltransferase increased by the sphingomyelinase treatment in the absence of apoA-I, more remarkably in the fibroblast than in the astrocytes. ApoA-I suppressed this increase completely in the astrocytes, but only partially in the fibroblast. The effect of the sphingomyelin digestion was more prominent for the apolipoprotein-mediated reaction than the diffusion-mediated cellular cholesterol efflux. Thus, cholesterol molecules restricted by sphingomyelin in the domain of the plasma membrane appear to be primarily used for the HDL assembly upon the apolipoprotein;-cell interaction.

Participation of syndecan 2 in the induction of stress fiber formation in cooperation with integrin alpha5beta1: structural characteristics of heparan sulfate chains with avidity to COOH-terminal heparin-binding domain of fibronectin.

The present study provides direct evidence that syndecan 2 participates selectively in the induction of stress fiber formation in cooperation with integrin alpha5beta1 through specific binding of its heparan sulfate side chains to the fibronectin substrate. Our previous study with Lewis lung carcinoma-derived P29 cells demonstrated that the cell surface heparan sulfate proteoglycan, which binds to fibronectin, is syndecan 2 (N. Itano et al., 1996, Biochem. J. 315, 925-930). We here report that in vitro treatment of the cells by antisense oligonucleotide for syndecan 2 resulted in a failure to form stress fibers on fibronectin substrate in association with specific suppression of its cell surface expression. Instead, localization of actin filaments in the cytoplasmic cortex occurred. A similar response of the cells was observed when the cells were treated to eliminate functions of cell surface heparan sulfates, including exogenous addition of heparin and pretreatment with anti-heparan sulfate antibody, F58-10E4, and with proteinase-free heparitinase I. Size- and structure-defined oligosaccharides prepared from heparin and chemically modified heparins were utilized as competitive inhibitors to examine the structural characteristics of the cell surface heparan sulfates involved in organization of the actin cytoskeleton. Their affinity chromatography on a column linked with a recombinant H-271 peptide containing a C-terminal heparin-binding domain of fibronectin demonstrated that 2-O-sulfated iduronates were essential for the binding. Inhibition studies revealed that a heparin-derived dodecasaccharide sample enriched with an IdoA(2OS)-GlcNS(6OS) disaccharide completely blocked binding of the syndecan 2 ectodomain to immobilized H-271 peptide. Finally, the dodecasaccharide sample was shown to inhibit stress fiber formation, triggered by adhesion of P29 cells to a CH-271 polypeptide consisting of both the RGD cell-binding and the C-terminal heparin-binding domains of fibronectin in a fused form. All these results consistently suggest that syndecan 2 proteoglycan interacts with the C-terminal heparin-binding domain of fibronectin at the highly sulfated cluster(s), such as [IdoA(2OS)-GlcNS(6OS)](6) present in its heparan sulfate chains, to result in the induction of stress fiber formation in cooperation with integrin alpha5beta1.

Phosphorylation of a membrane-intercalated proteoglycan, syndecan-2, expressed in a stroma-inducing clone from a mouse Lewis lung carcinoma.

We previously reported that a mouse Lewis lung carcinoma-derived stroma-inducing clone, P29, highly expresses a syndecan-like proteoglycan exhibiting specific binding to fibronectin, a major constituent of the interstitial matrix formed by the induced stromal cells, via its heparan sulphate chains [Itano, Oguri, Nakanishi and Okayama (1993) J. Biochem. (Tokyo) 114, 862-873]. On metabolic labelling of the proteoglycan with [32P]Pi, followed by identification of the radiolabelled material using glycanases, almost all the isotope was found to have been incorporated into a core portion of molecular mass 48 kDa, which was generated by digestion with heparan sulphate lyase I plus chondroitin ABC lyase. Immunoblotting of the core protein with a monoclonal antibody, F58-6G12, demonstrated that the proteoglycan was mouse syndecan-2. CsCl-density-gradient centrifugation after mild treatment of liposome-intercalated 32P-labelled syndecan-2 with trypsin resulted in clear separation of the radioactivity into a bottom fraction containing all the glycosaminoglycans (accounting for 40% of the total radioactivity) and a top fraction containing liposome-associated peptides (60%). The former isotope was shown to be linked covalently to both heparan sulphate and chondroitin sulphate chains, probably at their bridge regions. The latter was mostly attributed to phosphoserine, the one and only phosphorylated amino acid released on acid hydrolysis of this proteoglycan, strongly suggesting that the phosphorylation occurs at a specific serine residue(s) in the cytoplasmic domain of the core protein.