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Multiple sclerosis, the most common cause of progressive neurological disability in young adults, is a chronic inflammatory disease. There is solid evidence for a genetic influence in multiple sclerosis, and deciphering the causative genes could reveal key pathways influencing the disease. A genome region on rat chromosome 9 regulates experimental autoimmune encephalomyelitis, a model for multiple sclerosis. Using interval-specific congenic rat lines and association of single-nucleotide polymorphisms with inflammatory phenotypes, we localized the gene of influence to Vav1 , which codes for a signal-transducing protein in leukocytes. Analysis of seven human cohorts (12,735 individuals) demonstrated an association of rs2546133-rs2617822 haplotypes in the first VAV1 intron with multiple sclerosis (CA: odds ratio, 1.18; CG: odds ratio, 0.86; TG: odds ratio, 0.90). The risk CA haplotype also predisposed for higher VAV1 messenger RNA expression. VAV1 expression was increased in individuals with multiple sclerosis and correlated with tumor necrosis factor and interferon-γ expression in peripheral blood and cerebrospinal fluid cells. We conclude that VAV1 plays a central role in controlling central nervous system immune-mediated disease and proinflammatory cytokine production critical for disease pathogenesis.

A single nucleotide polymorphism causing constitutive activation of Vav1 results in increased natural Treg generation and is responsible for the imbalance between Vav1 GEF and adaptor functions., CD4+ regulatory T cells (Treg cells) expressing the transcription factor Foxp3 play a pivotal role in maintaining peripheral tolerance by inhibiting the expansion and function of pathogenic conventional T cells (Tconv cells). In this study, we show that a locus on rat chromosome 9 controls the size of the natural Treg cell compartment. Fine mapping of this locus with interval-specific congenic lines and association experiments using single nucleotide polymorphisms (SNPs) identified a nonsynonymous SNP in the Vav1 gene that leads to the substitution of an arginine by a tryptophan (p.Arg63Trp). This p.Arg63Trp polymorphism is associated with increased proportion and absolute numbers of Treg cells in the thymus and peripheral lymphoid organs, without impacting the size of the Tconv cell compartment. This polymorphism is also responsible for Vav1 constitutive activation, revealed by its tyrosine 174 hyperphosphorylation and increased guanine nucleotide exchange factor activity. Moreover, it induces a marked reduction in Vav1 cellular contents and a reduction of Ca2+ flux after TCR engagement. Together, our data reveal a key role for Vav1-dependent T cell antigen receptor signaling in natural Treg cell development.

Differential cytokine production by T cells plays an important role in the outcome of the immune response. We show that the level of CD45RC expression differentiates rat CD8 T cells in two subpopulations, CD45RChigh and CD45RClow, that have different cytokine profiles and functions. Upon in vitro stimulation, in an Ag-presenting cell-independent system, CD45RChigh CD8 T cells produce IL-2 and IFN-γ while CD45RClow CD8 T cells produce IL-4, IL-10, and IL-13. In vitro, these subsets also exhibit different cytotoxic and suppressive functions. The CD45RChigh/CD45RClow CD8 T cell ratio was determined in Lewis (LEW) and Brown-Norway (BN) rats. These two rat strains differ with respect to the Th1/Th2 polarization of their immune responses and to their susceptibility to develop distinct immune diseases. The CD45RChigh/CD45RClow CD8 T cell ratio is higher in LEW than in BN rats, and this difference is dependent on hemopoietic cells. Linkage analysis in a F2(LEW × BN) intercross identified two quantitative trait loci on chromosomes 9 and 20 controlling the CD45RChigh/CD45RClow CD8 T cell ratio. This genetic control was confirmed in congenic rats. The region on chromosome 9 was narrowed down to a 1.2-cM interval that was found to also control the IgE response in a model of Th2-mediated disorder. Identification of genes that control the CD45RChigh/CD45RClow CD8 T cell subsets in these regions could be of great interest for the understanding of the pathophysiology of immune-mediated diseases.

The development of inflammatory diseases depends on complex interactions between several genes and various environmental factors. Discovering new genetic risk factors and understanding the mechanisms whereby they influence disease development is of paramount importance. We previously reported that deficiency in Themis1, a new actor of TCR signaling, impairs regulatory T cell (Treg) function and predisposes Brown–Norway (BN) rats to spontaneous inflammatory bowel disease (IBD). In this study, we reveal that the epistasis between Themis1 and Vav1 controls the occurrence of these phenotypes. Indeed, by contrast with BN rats, Themis1 deficiency in Lewis rats neither impairs Treg suppressive functions nor induces pathological manifestations. By using congenic lines on the BN genomic background, we show that the impact of Themis1 deficiency on Treg suppressive functions depends on a 117-kb interval coding for a R63W polymorphism that impacts Vav1 expression and functions. Indeed, the introduction of a 117-kb interval containing the Lewis Vav1-R63 variant restores Treg function and protects Themis1-deficient BN rats from spontaneous IBD development. We further show that Themis1 binds more efficiently to the BN Vav1-W63 variant and is required to stabilize its recruitment to the transmembrane adaptor LAT and to fully promote the activation of Erk kinases. Together, these results highlight the importance of the signaling pathway involving epistasis between Themis1 and Vav1 in the control of Treg suppressive function and susceptibility to IBD development.

Summary: The understanding of the mechanisms of immune tolerance and the unravelling of the pathophysiology of autoimmune diseases rely on animal models. In this respect, BN and LEW rats represent models of choice to study immune-mediated diseases from the cellular and genetic points of view. Indeed, BN and LEW rats are extremes with respect to their polarisation of the immune response as well as their susceptibility to autoimmune diseases. LEW rats are susceptible to Th1-mediated autoimmune diseases while BN rats are highly susceptible to Th2-mediated autoimmune disease. Comparison of the T cell compartment between LEW and BN rats revealed several important differences. 1) A MHC-dependent quantitative difference that is due to a defect in the CD8 T cell compartment in BN rats. 2) A qualitative MHC-independent difference that is related to a high frequency of CD45RClow CD4 and CD8 T cell subsets, producing IL-4, IL-13, IL-10 and TGF-β in BN rats as compared to LEW rats. 3) Interestingly, the genetic studies showed that susceptibility to Th1-mediated experimental autoimmune encephalomyelitis, and to Th2-mediated disorders triggered by gold salts as well as the difference in the CD45RChigh/CD45RClow ratio between LEW and BN rats are genetically determined by regions on chromosomes 9, 10 and 20.

Th1 and Th2 cells produce different cytokines and have distinct functions. Th1/Th2 cell differentiation is influenced, among other factors, by the nature of TCR-MHC interactions. However, how the TCR transduces a signal resulting in IFN-gamma or IL-4 production is a matter of debate. For example, some authors reported a loss of calcium signaling pathway in Th2 cells. We used a T cell hybridoma producing IL-4 upon weak TCR stimulation and both IL-4 and IFN-gamma for strong TCR engagement as a model to study how TCR signaling pathways are differentially activated in both conditions of stimulation and how this influences the production of cytokines. We show that: (1) the calcium response is identical following weak and strong TCR stimulation; (2) mitogen-activated protein kinase(MAPK) activation is a gradual phenomenon depending upon the strength of TCR activation; (3) a calcium response, even weak, triggers IL-4 expression; (4) IFN-gamma synthesis requires not only a calcium response but also MAPK activation. The MAPK pathway is dispensable for IL-4 production, although it amplifies IL-4 synthesis upon strong TCR stimulation; (5) TCR-induced IL-4 production also depends on calcium signaling in Th2 cells, while IFN-gamma synthesis is dependent, in addition, on MAPK activation in Th1 cells.

Injection of Brown Norway (BN) rats with gold salts provides a model to analyze the genetic control of the IgE response. A cohort of F2 progeny of susceptible BN and resistant LEW strains has been studied to carry out a genome-wide search for loci controlling the IgE response. Genome scanning identified two previously described loci, Atps1 and Atps2, and a new locus, Atps3. Atps1 linked to the MHC and Atps2 linked to the cytokine gene cluster that included the IL-4 region have been previously associated with serum IgE concentrations and with other Th2-dependent immune manifestations triggered by gold salts. The new interval, Atps3, identified on chromosome 9 (Lod score = 16), appears to play a major role in the control of the IgE response since it accounts for 31% of the genetic variance. Moreover, Atps3 is linked to anti-laminin antibody response and to glomerular immunoglobulin deposits. The identification and functional characterization of genes involved in these regions, particularly in Atps3, may shed light on the pathogenesis of atopic diseases in man.

Resume Deux lipases, la lipoproteine lipase (LPL) et la lipase hepatique (LH) agissent de concert dans l'hydrolyse intravasculaire des particules riches en triglycerides (TG). L'injection intraveineuse d'heparine augmente l'activite plasmatique de la LPL et de la LH. Une dose unique d'heparine (20 UI/100 g) est injectee a des rats qui recoivent ensuite, et sous la forme d'un bolus, 20 mg de TG d'une emulsion lipidique a 20% contenant pour moitie des triglycerides a chaines moyennes (TCM) et des triglycerides a chaines longues (TCL), ou des TCL seuls. Les TG plasmatiques sont significativement moins eleves 4, 6 et 8 min apres injection d'heparine dans le groupe TCM/TCL (p

International audience; Gold or mercury salts trigger a dramatic IgE response and a CD4 T-cell-dependent nephropathy in Brown-Norway (BN), but not in Lewis (LEW) rats. We previously identified the 1.1-Mb Iresp3 (immunoglobin response QTL3) locus on chromosome 9 that controls these gold salt-triggered immune disorders. In the present work, we investigated the genetic control of HgCl2-induced immunological disorders and assessed the relative contribution of the CD45RC(high) and CD45RC(low) CD4 T-cell subpopulations in this control. By using interval-specific congenic lines, we narrowed down Iresp3 locus to 117-kb and showed that BN rats congenic for the LEW 117-kb were protected from HgCl2-triggered IgE response and nephropathy. This 117-kb interval also controls CD45RC expression by CD4 T cells and the ability of CD45RC(high) CD4 T cells to trigger the autoimmune disorders resulting from HgCl2 administration. This 117-kb region contains four genes, including Vav1, a strong candidate gene according to its cellular function and exclusive expression in hematopoietic cells. Thus, this study highlights the role of the CD45RC(high) CD4 T-cell subpopulation in the opposite susceptibility of BN and LEW rats to HgCl2-triggered immune disorders and identifies a 117-kb interval on chromosome 9 that has a key role in their functions.

Male and female rats fed a cystine-rich diet (5% l-cystine) became hypercholesterolemic after 2 months, with 2-fold higher cholesterol levels carried mainly by the HDL1 and HDL2 lipoprotein fractions. Post-heparin lipoprotein lipase activity was increased in male rats only (60%, P < 0.01), while hepatic lipase (HL) activity was increased in both males and females (48%, P < 0.001 and 27%, P < 0.01, respectively). In the liver, HL activity and mRNA levels were increased in males (30%, P < 0.01, and 70%, P < 0.001, respectively), but not in females. A higher correlation between HDL1-cholesterol and liver, HL activity was found in male rats than in female rats. In the latter, although the cystine diet induced a virtually identical increase in HDL1-cholesterol, HL gene expression was not promoted. It is suggested that HL gene expression may be triggered by the uptake of HDL1-cholesterol in male rats, while oestrogens in female rats would counteract this effect.

Resume Les chylomicrons sont synthetises par l'intestin et assurent le transport des triglycerides, du cholesterol et des phospholipides d'origine alimentaire. Les remnants resultent d'une lipolyse extra-hepatique tres rapide et sont principalement captes par le foie. Les mecanismes du captage hepatique de ces particules riches en triglycerides ne sont pas clairement compris. Les facteurs qui modulent le metabolisme intravasculaire des lipoproteines post-prandiales peuvent etre particulierement importants car ces particules sont supposees etre atherogenes.

Congenic and consomic rat strains are inbred strains containing in their genome a given genomic region (congenic) or a whole chromosome (consomic) from another strain. They are nowadays invaluable tools for the identification of genes and mechanisms of multifactorial diseases, one of the main goals in biomedicine. They are produced by repeated backcrosses from a donor inbred strain to a recipient inbred strain, and thereafter maintained by conventional brother-x-sister mating. Although their production is lengthy and costly, it only requires a zootechny unit for breeding and tools for genotyping.

Congenic and consomic rat strains are inbred strains containing in their genome a given genomic region (congenic) or a whole chromosome (consomic) from another strain. They are nowadays invaluable tools for the identification of genes and mechanisms of multifactorial diseases, one of the main goals in biomedicine. They are produced by repeated backcrosses from a donor inbred strain to a recipient inbred strain, and thereafter maintained by conventional brother-x-sister mating. Although their production is lengthy and costly, it only requires a zootechny unit for breeding and tools for genotyping.

Hepatic lipase (HL) and lipoprotein lipase (LPL) were assayed in heparinized plasma from male normocholesterolaemic (SW) and genetically hypercholesterolaemic (RICO) rats. Both strains were fed on either a semi-purified control diet or the same diet enriched with 0.5% or 1% cholesterol. HL activity was similar in both groups of rats fed on the control diet. LPL activity was found to be significantly lower in RICO rats (35% decrease, P less than 0.05). Feeding with a high-cholesterol diet led to a decrease in HL activity (15-23%) in both groups of rats but no change was detected in LPL activity, which remained consistently lower in the RICO rats. Thus, with the control diet, LPL activity is lower in RICO rats but presumably is not rate-limiting for their triacylglycerol clearance, given the normal triacylglycerol levels present. After cholesterol feeding, however, the lower LPL activity may become rate-limiting together with the decrease in HL activity, as in these circumstances hypertriacylglycerolaemia was evident and the hypercholesterolaemia of this strain was further increased.

Toxoplasmosis is a healthcare problem in pregnant women and immunocompromised patients. Like humans, rats usually develop a subclinical chronic infection. LEW rats exhibit total resistance toToxoplasma gondiiinfection, which is expressed in a dominant mode. A genome-wide search carried out in a cohort of F2progeny of susceptible BN and resistant LEW rats led to identify on chromosome 10 a major locus of control, which we calledToxo1. Using reciprocal BN and LEW lines congenic for chromosome 10 genomic regions from the other strain,Toxo1was found to govern the issue ofT. gondiiinfection whatever the remaining genome. Analyzes of rats characterized by genomic recombination withinToxo1, reduced the interval down to a 1.7-cM region syntenic to human 17p13.In vitrostudies showed that theToxo1-mediated refractoriness toT. gondiiinfection is associated with the ability of the macrophage to impede the proliferation of the parasite within the parasitophorous vacuole. In contrast, proliferation was observed in fibroblasts whatever the genomic origin ofToxo1. Furthermore,ex vivostudies indicate that macrophage controls parasitic infection spreading by aToxo1-mediated mechanism. This forward genetics approach should ultimately unravel a major pathway of innate resistance to toxoplasmosis and possibly to other apicomplexan parasitic diseases.

Brown Norway (BN) rats treated with aurothiopropanol-sulfonate (Atps) constitute a model of Th2-mediated immunological disorders associated with elevated IgE responses and renal IgG deposits. Using F2 offspring between Atps-susceptible BN and Atps-resistant Lewis rats, we had previously mapped three quantitative trait loci on chromosomes 9, 10, and 20 for which BN alleles increased susceptibility to Atps-induced immunological disorders (Aiid). In this study we have used congenic lines for the latter two quantitative trait loci, formerly called Atps2 and Atps3 and now named Aiid2 (chromosome 10) and Aiid3 (chromosome 9), for fine mapping and characterization of their impact on Atps-triggered reactions. In Aiid2 congenic lines, the gene(s) controlling part of the IgE response to Atps was mapped to an ∼7-cM region, which includes the IL-4 cytokine gene cluster. Two congenic lines in which the introgressed segments shared only a portion of this 7-cM region, showed an intermediate IgE response, indicating the involvement of several genes within this region. Results from BN rats congenic for the Lewis Aiid3 locus, which we mapped to a 1.2-cM interval, showed a stronger effect of this region. In this congenic line, the Atps-triggered IgE response was 10-fold lower than in the BN parental strain, and glomerular IgG deposits were either absent or dramatically reduced. Further genetic and functional dissections of these loci should provide insights into pathways that lead to Th2-adverse reactions.

In Electricite de France (EDF) probabilistic analyses for Reactor Pressure Vessel (RPV) life management, it’s used to take the temperature of the Reactor Spent fuel Pit cooling and treatment System storage tank as a constant. The aim of our study is to evaluate the stability of this temperature. Since 1999, we collected for several sites, and several nuclear plant units, the temperature of the Reactor Spent fuel Pit cooling and treatment System storage tank. Our results illustrate that this temperature depends on the season and the site. We first proposed to give a modelisation of this temperature dependent of the external temperature; even if this modelisation leads to a good R2 , it’s not optimum. We also proposed to explain the temperature using the temperature of the essential service water or for lack the temperature of the river. In the case of extreme quantile study (meaning low temperature), we proposed to use the normal approximation, which seems to be conservative.Copyright © 2003 by ASME

Autoreactive T cells exist in healthy individuals and represent a potential reservoir of pathogenic effectors which, when stimulated by microbial adjuvants, could trigger an autoimmune disease. Experimental studies have indicated that xenobiotics, well defined from a chemical point of view, could promote the differentiation of autoreactive T cells towards a pathogenic pathway. It is therefore theoretically possible that compounds present in vaccines such as thiomersal or aluminium hydroxyde can trigger autoimmune reactions through bystander effects. Mercury and gold in rodents can induce immunological disorders with autoimmune reactions. In vitro, both activate signal transduction pathways that result in the expression of cytokines, particularly of IL-4 and IFNgamma. In a suitable microenvironment heavy metals could therefore favour the activation of autoreactive T cells. In that respect, genetic background is of major importance. Genome-wide searches in the rat have shown that overlapping chromosomal regions control the immunological disorders induced by gold salt treatment, the development of experimental autoimmune encephalomyelitis and the CD45RC(high)/CD45RC(low)CD4(+)T cells balance. The identification and functional characterization of genes controlling these phenotypes may shed light on key regulatory mechanisms of immune responses. This should help to improve efficacy and safety of vaccines.

The level of CD45RC expression differentiates rat CD4 T cells in two subpopulations, CD45RChigh and CD45RClow, that have different cytokine profiles and functions. Interestingly, Lewis (LEW) and Brown Norway (BN) rats, two strains that differ in their ability to mount type 1 and type 2 immune responses and in their susceptibility to autoimmune diseases, exhibit distinct CD45RChigh/CD45RClow CD4 T cell ratios. The CD45RChigh subpopulation predominates in LEW rats, and the CD45RClow subpopulation in BN rats. In this study, we found that the antiinflammatory cytokines, IL-4, IL-10, and IL-13, are exclusively produced by the CD45RClow CD4 T cells. Using bone marrow chimeras, we showed that the difference in the CD45RChigh/CD45RClow CD4 T cell ratio between naive LEW and BN rats is intrinsic to hemopoietic cells. Furthermore, a genome-wide search for loci controlling the balance between T cell subpopulations was conducted in a (LEW × BN) F2 intercross. Genome scanning identified one quantitative trait locus on chromosome 9 (∼17 centiMorgan (cM); log of the odds ratio (LOD) score 3.9). In addition, two regions on chromosomes 10 (∼28 cM; LOD score 3.1) and 20 (∼40 cM; LOD ratio score 3) that contain, respectively, a cytokine gene cluster and the MHC region were suggestive for linkage. Interestingly, overlapping regions on these chromosomes have been implicated in the susceptibility to various immune-mediated disorders. The identification and functional characterization of genes in these regions controlling the CD45RChigh/CD45RClow Th cell subpopulations may shed light on key regulatory mechanisms of pathogenic immune responses.

[4-14C]Cholesteryl oleyl ether-labeled chylomicron remnants were injected into rats which received a specific goat antibody against rat hepatic lipase or a control serum. Chylomicron remnant cholesterol ether disappeared from circulation with a significantly higher half-life (2-fold) in antibody-treated rats than in controls (P less than 0.001). Recovered radioactivity in the liver was 2-fold lower in antibody-treated rats (22.8% (n = 6) vs. 45% (n = 4) P less than 0.01). These results clearly show that hepatic lipase may strongly promote chylomicron remnant cholesterol ether uptake by the liver.

Natural Foxp3+ regulatory T cells (Treg) are of paramount importance for establishing self-tolerance, controlling inflammatory responses and maintaining immune homeostasis. The identification of new molecules controlling Treg functions is nowadays an important challenge. We previously reported the implication of Themis, a new actor of TCR signaling, in Treg suppressive capacities. Indeed, Treg from Themis deficient BN rats were defective, leading to the development of an inflammatory bowel disease. In the present study, we show that Themis deficiency does not impair Treg suppressive functions and does not induce any pathological manifestations in LEW rats. These findings suggest that an interaction with other(s) gene(s) may contribute to the Treg defect and disease development in Themis deficient BN rats. To test this hypothesis and identify the involved gene(s), we performed a genetic dissection using different congenic lines. We show that the impact of Themis deficiency on Treg suppressive functions requires the expression of a constitutively active Vav1 variant. Together, these studies highlight the importance of Vav1 and Themis signaling hub in regulating the suppressive functions of Treg. Understanding the underlying molecular mechanisms may lead to advances in Treg biology and favor the identification of new pharmacogenetic markers and therapeutic targets in immune mediated diseases.

Female lean Zucker rats were fed for four weeks with either a control diet or the same diet enriched with 2% (w/w) cholesterol and cholic acid (0.5%, w/w). This treatment resulted in a 6-fold increase in plasma total cholesterol. A 30% decrease was observed in plasma post-heparin HL activity, in contrast with lipoprotein lipase, which was unmodified in the cholesterol/cholate-fed rats. HL activity measured in liver homogenate from these rats was also decreased (-30%, p0.05), as was its protein mass, quantified by immunoblot analysis (-57%, (p0.01), whereas HL mRNA levels were 3-fold lower in the cholesterol/cholate-fed rats. We conclude that the cholesterol/cholate-enriched diet decreases the HL gene expression by acting at the transcriptional level and/or by affecting HL mRNA stability, or both.

The effects of a fish oil concentrate on blood lipids and lipoproteins were examined in relation to their effects on liver fatty acid synthase (FAS), 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, adipose tissue lipoprotein lipase (LPL), and hepatic triglyceride lipase (H-TGL). For 15 days, 2-mo-old rats were fed a control diet (10% of calories from fat, 4% fat by weight) or diets with 50% of calories (25% wt/wt) provided by lard, lard and fish oil calories (35%/15%), or lard and corn oil (35%/15%). The high-lard diet increased plasma chylomicron and liver triglycerides. The high-lard diet greatly decreased FAS, HMG-CoA reductase, and LPL activities; it also reduced H-TGL activity. Compared with the lard diet, the lard-fish oil diet decreased plasma TG by drastically lowering chylomicron (4-fold, P < 0.001) and very-low-density lipoprotein levels (P < 0.001). It also reduced high-density lipoprotein levels. The lard-fish oil diet prevented hepatic triglyceride accumulation and decreased FAS activity and mass by 3.5-fold (P < 0.001) but did not further decrease HMG-CoA reductase activity. Adipose tissue LPL activity was 2.5-fold (P < 0.001) higher with the lard-fish oil diet than with the lard diet, and H-TGL activity decreased significantly (-32%, P < 0.01), despite unaltered levels of H-TGL mRNA. These effects were significant with only 10% fish oil concentrate in the lard diet. They were not observed with the lard-corn oil diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Active and heat-inactivated hepatic lipase stimulated to a statistically comparable extent the uptake of chylomicron remnant-like particles by isolated rat hepatocytes by 3-fold and 2.3-fold respectively and, likewise, their binding to hepatic plasma membranes by 5-fold and 4-fold respectively. Hepatic lipase may facilitate uptake of these particles, not only as a lipolytic enzyme, but also as a ligand anchored to extracellular glycosaminoglycans.

The aim of this study was to assess whether diets enriched in cholesterol, sodium cholate and drugs known to modify liver cholesterol biosynthesis can modulate hepatic lipase (H-TGL) expression and activity in vivo. Female lean Zucker rats, known to be good responders to cholesterol, were fed for 7 days with a control C diet or the C diet supplemented (w/w) with either 2% cholesterol, 0.5% sodium cholate, 2% cholestyramine or simvastatin (0.1%) added to the cholestyramine diet or given by gavage (10 mg/ rat) for 3 days. H-TGL activity decreased by 34% with cholesterol, and by 27% when both cholesterol and cholate were administered to the rats. Under these conditions, H-TGL mRNA decreased by 34% and 87%, respectively. The sharp decrease in H-TGL expression was associated with a strong increase in cholesteryl ester in total liver and in the liver microsome fraction. H-TGL activity decreased by 33% with cholestyramine and the mRNA level decreased by 47%. Simvastatin lowered H-TGL activity by 55% when added to the cholestyramine diet, probably because of a reduction in food intake. When administrated by gavage, simvastatin increased both the H-TGL activity (by 28%) and mRNA (by 23%). These variations may be linked to the availability of mevalonate-derived sterol and non-sterol products.

Chylomicron remnants (CM-RM), resulting from intravascular hydrolysis of chylomicrons by the endothelium bound lipoprotein lipases (LPL), are rapidly taken up by the liver. It appears clearly that this uptake is mostly receptor mediated1–3 and that CM-RM are taken up as whole particles. Hepatic lipase (HL), which exerts both a triacylglycerol lipase and phospholipase Al activity, may be involved in with CM-RM uptake. Indeed, its inhibition by a specific anti-HL antibody leads to an accumulation of apo B-48 rich-par ticles in the plasma of rats4,5. We have previously shown that CM-RM prealably hydrolyzed by HL and then added to isolated hepatocytes are taken up at a higher rate than non-treated remnants (6). Moreover, we showed that following hydrolysis by HL, there was no modification in remnant apo E levels (unpublished results). Since apo E is the ligand for remnant binding to their hepatic receptors1–3, we examined herein, whether HL-treatment of CM RM could despite unchanged apo E levels, increase 1) the binding of such particles to liver membranes and 2) their plasma clearance and hepatic uptake in vivo.

Evaluation of the serum lipoprotein profile in non-fasting, adult chimpanzees by analytical ultracentrifugation revealed a lower mean LDL level (269 mg/dl) than typical of man. The major molecular form(s) of low density lipoprotein (LDL) was then isolated in the density interval 1.024-1.050 g/ml by sequential ultracentrifugation. The physicochemical properties of chimpanzee LDL, including net surface charge as judged by electrophoresis, molecular size (220 A) by electron microscopy, and chemical composition closely resembled those of man. The antigenic structures of chimpanzee and human LDL were essentially indistinguishable, since immunodiffusion against antiserum to either the human or ape lipoprotein produced a precipitin reaction of complete identity between the two antigens. By micro-immunoprecipitation, the immunological cross-reactivity of LDL from the two species was in the range 85–97%, depending on the nature of the assay. The protein moieties of chimpanzee VLDL and LDL were dominated by proteins of high M r (180000–380000). The predominant component had M r 330 000–380 000, and migrated similarly to the human B100 protein; a second band ( M r = 235 000–285 000) apparently corresponded to the human B74 protein. Fractionation of apo-LDL by gel filtration chromatography in anionic detergent yielded a void volume fraction which we have termed chimpanzee apo-B H . By SDS-gel electrophoresis, apo-B H contained counterparts (bands I and II) to the human B100 and B74 proteins, with the B100-like component (band 1) dominating. The amino acid composition of apo-B H was markedly similar to that of human B100. The structural characteristics of the major molecular form(s) of circulating LDL in chimpanzee are similar to those of the corresponding particles in man, supporting the contention that P. troglodytes is a highly suitable model for studies of the role of LDL and apo-B H in atherogenesis.

The incorporation of proto-, uro- and hematoporphyrin in low density lipoproteins (LDL) of human blood has been studied by equilibrium dialysis, fluorescence and absorption spectroscopy. The lipoproteins may efficiently compete with albumin in the binding of protoporphyrin to human blood proteins in patients suffering from protoporphyria. It can be concluded that hydrophobic porphyrins bind to blood proteins. The complexation of hydrophobic porphyrins in LDL is responsible not only for efficient photodynamic effect at the lipoprotein level, but also for photoinduced lipid peroxidation and for consumption of β-carotene incorporated into LDL which are one of their natural carriers. The water-soluble uroporphyrin, although an efficient photosensitizer for the LDL apoprotein photoinactivation, is much less efficient for lipid peroxidation and β-carotene bleaching. The 353 nm laser flash photolysis shows that porphyrin triplet states are not affected by the physiological β-carotene content of LDL but are fully accessible to oxygen.

A density gradient ultracentrifugal procedure is described for the rapid and reproducible isolation of the major lipoprotein classes, VLDL, LDL, HDL2, and HDL3, from human serum. A step gradient is constructed from four NaCl/KBr solutions varying in density from 1.006 to 1.24 g/ml and from 3 ml of serum adjusted to d 1.21 g/ml. Separation is achieved after a single ultracentrifugation for some 56 x 10(7) gavg min at 15 degrees C in a swinging bucket rotor, at which time the lipoproteins band isopycnically and albumin and other serum proteins are sedimented. Densitometric scanning of gradients revealed a lipoprotein mass profile distinguished by four absorption maxima which fell within the hydrated density ranges of VLDL (d less than 1.016 g/ml), LDL (1.028-1.050 g/ml), HDL2 (1.066-1.100 g/ml), and HDL3 (1.100-1.153 g/ml). Fractionation of gradients on the basis of band distribution, followed by chemical, physical, and immunological analyses of the four principal fractions (i.e., bands) provided data on their electrophoretic mobility, chemical composition, morphology and size distribution, immunological reactivity and apolipoprotein content, thereby confirming their identities as VLDL, LDL, HDL2, and HDL3. The validity of this separation was supported by the quantitative distribution of apo B and apo A-I as assessed by radial immunodiffusion. Lipoprotein quantitation based on chemical analysis of gradient fractions was compared with that by analytical ultracentrifugation for a group of normolipidemic males; results concorded well, giving a similar HDL2:HDL3 ratio (0.35-0.36). Our procedure thus provides a simple and precise manner in which to assess the lipoprotein and apolipoprotein profile of human serum quantitatively and qualitatively.

The molecular basis of the heterogeneity of plasma low density lipoproteins (LDL, d 1.024-1.050 g/ml) was evaluated in 40 normolipidemic male subjects following fractionation by isopycnic density gradient ultracentrifugation into eight major subspecies. The mass profile of our subjects' LDL uniformly displayed single symmetric or asymmetric peaks as a function of density; the peak occurred most frequently (20 subjects) in subfraction 7 (d 1.0297-1.0327 g/ml). Several physicochemical properties (hydrodynamic behavior, electrophoretic mobility, chemical composition, size and particle heterogeneity, and apolipoprotein heterogeneity) of the LDL subfractions were examined. Hydrodynamic analyses revealed unimodal distributions and distinct peak Sf degree rates in individual subfractions. Such behavior correlated well with particle size and heterogeneity data, in which LDL subspecies were typically resolved as unique narrow bands by gradient gel electrophoresis. Subspecies with average densities of 1.024 to 1.0409 g/ml ranged from 229 to 214 A in particle diameter. LDL protein content increased in parallel with density while the proportion of triglyceride diminished; cholesteryl esters predominated, accounting for approximately 40% or more by weight. Distinct differences in net electric charge were demonstrated by electrophoresis in agarose gel, the subspecies with average density of 1.0314 g/ml displaying the lowest net negative charge. ApoB-100 was the major apoprotein in all subspecies, and constituted the unique protein component over the density interval 1.0271-1.0393 g/ml. ApoE and apo[a] were detected at densities less than 1.0271 and greater than 1.0393 g/ml. While apoE was evenly distributed within these two regions, representing up to 2% of apoLDL, the distribution of apo[a] was skewed towards the denser region, in which it amounted to 3-7% of apoLDL. ApoC-III was detectable as a trace component at densities greater than 1.0358 g/ml. Calculation of the number of molecules of each chemical component per LDL subspecies showed the presence of one copy of apoB-100 per particle, in association with decreasing amounts of cholesteryl ester, free cholesterol, and phospholipid. These data indicate that a similar overall molecular organization and structure is maintained in a unimodal distribution of LDL particle subspecies over the density range approximately 1.02 to 1.05 g/ml. In sum, our data may be interpreted to suggest that microheterogeneity in the physicochemical properties of human LDL subspecies reflects dissimilarities in their origins, intravascular metabolism, tissular fate, and possibly in their atherogenicity.

Limited tryptic digestion of human serum low-density (LD) lipoprotein (rho 1.024-1.045 g/ml) under defined conditions permitted isolation by gel filtration chromatography of a stable, protein-deficient lipoprotein; the liberated protein was separated as a mixture of peptides of low molecular weight (less than 5000). Comparison of the chemical, physical and immunological characteristics of the trypsin-treated LD-lipoprotein with those of the native preparation revealed several differences, including (a) a diminished protein content (loss of some 20-25% of the total protein of LD-lipoprotein) and increased proportions of the various lipid components, except for triglyceride (probably resulting from a loss of bound free fatty acids with the liberated peptides); (b) a greater heterogeneity in particle size and slightly larger mean diameter; (c) a lower hydrated density and greater peak sf rate than the native LD-lipoprotein (d) an increased net negative charge; and (e) a partial immunological identity between LD-lipoprotein and the corresponding trypsin-treated fraction. While the amino acid compositions of the protein moieties of LD-lipoprotein and of trypsin-treated LD-lipoprotein were essentially identical, trypsin-treated apo-LD-lipoprotein was distinct in its complete solubility in urea-containing buffers at high concentrations, and also in its partial solubility in buffers lacking denaturing agents. Comparison of the apoproteins of the native and trypsin-treated LD-lipoproteins by electrophoretic techniques based on molecular weight revealed a transformation of the high-molecular weight material (greater than 250 000) characteristic of apo-LD lipoprotein into several polypeptide species (10 major forms) ranging in size from 161 500 to about 10 000. The largest of these (band b1: 161 500) could be completely dissociated into smaller components (b2: 93 500 and b3: 77 000) upon extensive heat treatment at 90 degrees C. Electrophoresis of the soluble fraction of apo-LD-lipoprotein and of that from its trypsin-treated counterpart in polyacrylamide gels containing urea at basic pH showed the disappearance of the small amounts (less than 5%) of C apoproteins of apo-LD-lipoprotein upon tryptic treatment. These results, which were highly reproducible in LD-lipoprotein preparations from different individuals, suggest that trypsin-treated LD-lipoprotein may provide a model for investigation of the organisation and structural role of the prinicipal apoprotein (apolipoprotein-B) in the LD-lipoprotein molecule.

5-methoxypsoralen (5-MOP) binds to human serum low density lipoproteins (LDL) according to a two-step process. Scatchard analysis of the first step yields K = 1.4 × 10 5 M −1 and 4 binding sites. It involves the LDL apoprotein. The second step corresponds to a solubilization, in the lipidic core, of ⋍ 45 molecules of 5MOP per LDL molecule. It is accompanied by a large blue shift of the 5MOP fluorescence. The ability of LDL to bind 5MOP and to carry it into various cells may explain some biological effects sometimes encountered during PUVA therapy.

Limited tryptic digestion of native low-density lipoprotein (LDL) (ϱ 1.024- 1.045 g/ml) liberates some 20 - 25% of its surface-exposed protein as soluble, lipid-free peptides (T-peptides). The T-peptides were resolved into twelve subfractions (peaks 1 - 12) by gel filtration chromatography on Bio-Gel P4 in 5 % formic acid, and classified into four major groups on the basis of criteria of size, amino acid composition and predominant C-terminal residue. The first group, peaks 1-4, (∼ 30% of total peptide protein; M, 2600-6000), displayed lysine as preponderant C-terminal (Lys : Arg, 3.7-7.3:1), were acidic in nature (basic:acidic residues, 0.37-0.61:1), and more hydrophobic than holo-apo-LDL. Peaks 1 and 2 contained glycopeptides (four or less by fingerprinting), the monosaccharide composition of which resembled that of LDL glycopeptides of type I1 [Swaminathan, N. & Aladjem, F. (1976) Biochemistry, 15, 1516-15211. The second group, peaks 5 and 6, (∼ 30% of the total; Mr, 1500-2000), were also apparently derived predominantly by cleavage at lysyl peptidyl bonds (Lys:Arg, 4.6-4.9:1), but were distinct in their higher proportion of basic residues (acidic:basic, 0.83-1.05:1). The third group (peaks 7 and 8) contained peptides approx. eight residues in length, with equal proportions (∼ 10%) of arginine and lysine. In contrast, the smaller fragments (2-5 residues) of peaks 9-12 exhibited arginine as major C-terminal residue (Lys:Arg, mean 0.45:1), as well as similar amounts of acidic and basic residues. Examination of the quantitative distribution of lysine and arginine showed 80% of arginines to be present in fragments of eight residues or less, with the majority (45 %) in peptides of 2-5 residues. In contrast, ∼ 70% of total lysines were located in fragments of z 10-20 residues; only ∼ 10% of lysines were in small (2-5 residue) peptides. Arginine appears therefore to occur most often at short (2-5 residues) and lysine at longer (six or more residues) distances from the next lysine or arginine in trypsin-accessible LDL protein. Clusters of a minimum of two positive charges, often featuring arginine, can thus be envisaged. The resemblance of the amino acid profiles of the major peptide subfractions (peaks 1-8) to that of apolipoprotein B further supports the contention that this apoprotein possesses a repetitive-type sequence. Finally, despite evaluation of peptide heterogeneity by fingerprinting, the inferred presence of incompletely-cleaved tryptic peptides in peaks 3, 4 and 5 precludes attribution of a minimal unique amino acid sequence to apolipoprotein B.

Chylomicron remnants labelled biologically with [3H]cholesterol were efficiently taken up by freshly isolated hepatocytes during a 3 h incubation in Krebs bicarbonate medium. Their [3H]cholesteryl ester was hydrolysed (74% net hydrolysis), and 0.1 mM-chloroquine could partially inhibit this hydrolysis, provided that hepatocytes were first preincubated for 2 h 30 min at 37 degrees C. This hydrolysis was also measured in preincubated cells with remnants double-labelled (3H and 14C) on their free cholesterol moiety; [3H]cholesterol arising from [3H]cholesteryl ester hydrolysis was recovered in the free [3H]cholesterol pool. A dose-response study showed saturation of remnant uptake at 180 micrograms of remnant protein/10(7) cells. Heparin (10 units/ml) increased remnant uptake by 63% (P less than 0.01), [3H]cholesteryl ester accumulation in the cell pellet by 110% (P less than 0.025) and hepatic lipase activity secreted in the medium by 2.4-fold (P less than 0.01) and by 3.3-fold (P less than 0.01) at the end of the preincubation and incubation periods respectively. Addition of 100 munits of semi-purified hepatic lipase preparation/flask stimulated remnant uptake by 44-69%, and [3H]cholesteryl ester accumulation in the presence of chloroquine by 2.1-fold (P less than 0.025). When hepatic lipase was incubated solely with the remnants, it decreased their triacylglycerol and phospholipid contents by 24% and 26% respectively. Thus freshly isolated hepatocytes may be used to study chylomicron-remnant uptake. Hepatic lipase, which seems to underly the stimulating effect of heparin, facilitates remnant uptake in vitro, and this could be mediated by at least one (or both) of its hydrolytic properties.