Summary Human trophoblast cells express an unusual repertoire of human leucocyte antigen (HLA) molecules which has been difficult to define. Close homology between and extreme polymorphism at the classical HLA class‐I (HLA‐I) loci has made it difficult to generate locus‐specific monoclonal antibodies (mAbs). The problem of defining an antibody’s reactivity against the thousands of existing HLA‐I allotypes has often made it impossible to determine the HLA bound by a mAb in biological samples from a normal outbred population. Here we have used commercially available beads coated with individual HLA‐I to characterize experimentally the reactivity of nine mAb against 96 common HLA‐I allotypes. In conjunction with donor HLA‐I genotyping, we could then define the specific HLA molecules bound by these antibodies in normal individuals. We used this approach to analyse the HLA expression of primary trophoblast cells from normal pregnancies; the choriocarcinoma cells JEG‐3 and JAR; and the placental cell lines HTR‐8/SVneo, Swan‐71 and TEV‐1. We confirm that primary villous trophoblast cells are HLA null whereas extravillous trophoblast cells express HLA‐C, HLA‐G and HLA‐E, but not HLA‐A, HLA‐B or HLA‐DR molecules in normal pregnancy. Tumour‐derived JEG‐3 and JAR cells reflect extravillous and villous trophoblast HLA phenotypes, respectively, but the HLA repertoire of the in vitro derived placental cell lines is not representative of either in vivo trophoblast phenotype. This study raises questions regarding the validity of using the placental cell lines that are currently available as model systems for immunological interactions between fetal trophoblast and maternal leucocytes bearing receptors for HLA molecules.

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. Effects of sirolimus on mesangial cell cholesterol homeostasis: a novel mechanism for its action against lipid-mediated injury in renal allografts.

Inflammation and dyslipidaemia both play important roles in the development of glomerular atherosclerosis in renal diseases. We have demonstrated that inflammatory mediators induced Scr (scavenger receptor) expression and the formation of foam cells, and that AP-1 (activator protein 1)/ets were necessary transcriptional factors for Scr induction in HMCs (human kidney mesangial cells). Most cells are protected from excessive native LDL (low-density lipoprotein) accumulation by tight feedback regulation of the LDLr (LDL receptor). However, we observed that HMCs formed foam cells via the LDLr pathway when incubated with IL-1beta (interleukin-1beta; 5 ng/ml) and unmodified LDL (200 microg/ml), suggesting that inflammatory mediators may disrupt the cholesterol-mediated feedback regulation. This feedback involves cholesterol-mediated down-regulation of LDLr controlled by SCAP [SREBP (sterol responsive element-binding protein) cleavage-activating protein]. We have also demonstrated that both tumour necrosis factor alpha and IL-1beta increased nuclear SREBP-1 levels by increasing SCAP mRNA expression, even in the presence of a high concentration of LDL. Since intracellular lipid content is governed by both influx and efflux mechanisms, we set out to examine the impact of inflammatory cytokines on cholesterol efflux, a process mediated by the protein ABCA1 (ATP binding cassette A1). IL-1beta inhibited [(3)H]cholesterol efflux from HMCs by inhibition of the peroxisome-proliferator-activated receptor/LXR (liver X receptor)/ABCA1 pathway. Taken together, our results suggest that inflammatory mediators increase lipid accumulation in HMCs not only by promoting increased lipoprotein uptake by Scr and LDLr, but also by inhibiting ABCA1-mediated cholesterol efflux to high-density lipoprotein.

Mesangial cell lipid accumulation is a recognised feature of glomerular disease and has been implicated as a factor in the pathogenesis of renal injury. To investigate possible mechanisms of such accumulation, binding of 125I-labelled human low-density lipoprotein (LDL) to rat mesangial cells was studied in vitro. Experiments were performed at 4 degrees C to prevent ligand internalisation. LDL remained associated with the cells after repeated washing. Binding was time-dependent, was inhibited by addition of an excess of unlabelled LDL, but to a much lesser extent by apoprotein-A-rich high-density lipoprotein particles devoid of apoprotein E (HDL-A). Specific binding reached saturation at an LDL concentration of 21 micrograms/ml, required the presence of calcium, and was inhibited by heparin and dextran sulphate. Scatchard analysis suggested a single class of binding site (Kd 22.7 micrograms protein/ml). Higher binding affinities were obtained when rat LDL was substituted for human LDL (Kd 1.3 micrograms/ml) and when human fibroblasts were exposed to human LDL under identical experimental conditions (Kd 3.0 micrograms/ml). Further experiments at 37 degrees C demonstrated degradation of LDL by cells. These results suggest that mesangial cells possess apoprotein B, E receptors. Mesangial cell lipid accumulation may therefore result from receptor-mediated endocytosis of LDL particles.

Calcineurin inhibitors enhance low-density lipoprotein oxida-complexes with their respective immunophilins and tion in transplant patients.these complexes then inhibit a key enzyme serine/threo-Background. Our objective was to assess the pro-oxidant nine phosphatase, calcineurin, which is involved in the status of neoral and tacrolimus in renal transplant patients and dephosphorylation of the transcription factor NFA-T. monitor the protection provided by vitamin C and vitamin E in normalizing low density lipoprotein (LDL) oxidation lag Calcineurin-mediated dephosphorylation of NFA-T is a time of tacrolimus-treated patients. pivotal step in the transcription and translation of the Methods. Plasma LDL was isolated by density gradient ul-IL-2 gene. The susceptibility of low density lipoprotein tracentrifugation from renal transplant patients receiving neo-(LDL) to oxidation is increased by cyclosporine (CsA) ral, tacrolimus and tacrolimus with vitamin C and vitamin E.[2, 3]. Oxidized LDL (Ox-LDL) may be of critical impor-Oxidation was initiated by the addition of CuCl 2 at 37ЊC and monitored at 234 nm over 480 minutes and oxidation lag time tance in triggering a cascade of cellular processes that was computed. Total antioxidant capacity of serum was mealeads to the formation of fatty streak and eventually sured using the enhanced chemiluminescent method. atherosclerotic lesions in the artery wall [4, 5]. The sus-Results. LDL from tacrolimus-treated patients had signifi-

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