Very-high-frequency probes for atomic force microscopy with silicon optomechanics.

Atomic force microscopy (AFM) has been consistently supporting nanosciences and nanotechnologies for over 30 years and is used in many fields from condensed matter physics to biology. It enables the measurement of very weak forces at the nanoscale, thus elucidating the interactions at play in fundamental processes. Here, we leverage the combined benefits of micro/nanoelectromechanical systems and cavity optomechanics to fabricate a sensor for dynamic mode AFM at a frequency above 100 MHz. This frequency is two decades above the fastest commercial AFM probes, suggesting an opportunity for measuring forces at timescales unexplored thus far. The fabrication is achieved using very-large-scale integration technologies derived from photonic silicon circuits. The probe's optomechanical ring cavity is coupled to a 1.55 µm laser light and features a 130 MHz mechanical resonance mode with a quality factor of 900 in air. A limit of detection in the displacement of 3 × 10-16 m/√Hz is obtained, enabling the detection of the Brownian motion of the probe and paving the way for force sensing experiments in the dynamic mode with a working vibration amplitude in the picometer range. When inserted in a custom AFM instrument embodiment, this optomechanical sensor demonstrates the capacity to perform force-distance measurements and to maintain a constant interaction strength between the tip and sample, an essential requirement for AFM applications. Experiments indeed show a stable closed-loop operation with a setpoint of 4 nN/nm for an unprecedented subpicometer vibration amplitude, where the tip-sample interaction is mediated by a stretched water meniscus.

Genetic control of HgCl2-induced IgE and autoimmunity by a 117-kb interval on rat chromosome 9 through CD4 CD45RChigh T cells.

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 HgCl(2)-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 HgCl(2)-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 HgCl(2) 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 HgCl(2)-triggered immune disorders and identifies a 117-kb interval on chromosome 9 that has a key role in their functions.

Weak TCR stimulation induces a calcium signal that triggers IL-4 synthesis, stronger TCR stimulation induces MAP kinases that control IFN-gamma production.

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.

Induction of autoimmunity through bystander effects. Lessons from immunological disorders induced by heavy metals.

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 balance between CD45RChigh and CD45RClow CD4 T cells in rats is intrinsic to bone marrow-derived cells and is genetically controlled.

The level of CD45RC expression differentiates rat CD4 T cells in two subpopulations, CD45RC(high) and CD45RC(low), 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 CD45RC(high)/CD45RC(low) CD4 T cell ratios. The CD45RC(high) subpopulation predominates in LEW rats, and the CD45RC(low) 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 CD45RC(low) CD4 T cells. Using bone marrow chimeras, we showed that the difference in the CD45RC(high)/CD45RC(low) 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 x BN) F(2) intercross. Genome scanning identified one quantitative trait locus on chromosome 9 (approximately 17 centiMorgan (cM); log of the odds ratio (LOD) score 3.9). In addition, two regions on chromosomes 10 (approximately 28 cM; LOD score 3.1) and 20 (approximately 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 CD45RC(high)/CD45RC(low) Th cell subpopulations may shed light on key regulatory mechanisms of pathogenic immune responses.

Cellular and genetic factors involved in the difference between Brown Norway and Lewis rats to develop respectively type-2 and type-1 immune-mediated diseases.

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-beta 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 CD4SRChigh/CD45RClow ratio between LEW and BN rats are genetically determined by regions on chromosomes 9, 10 and 20.

Rat chromosome 9 bears a major susceptibility locus for IgE response.

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.

[Recent data on hepatic lipase gene. Structure and functions of this enzyme]. / Données récentes sur le gène de la lipase hépatique. Structure et fonctions de cette enzyme.

Hepatic lipase (HL) is almost exclusively synthesized in the liver. Its gene is located on chromosome 15 in man, on chromosome 9 in rat, and has 9 exons and 8 introns. Inhibitory and activator sequences have been found at the 5' end of the gene, upstream to the initiation site, as well as several consensus sequences such as TRE, SRE, AP-2, CEBP, ERE, AF1 and OCT-1. Glycosylation is an essential step for full active mature protein. The catalytic site is in the N-terminal part of the lipase gene while the lipid binding site is at its C-terminal end. HL is generally thought to be active as a dimer. This enzyme hydrolyses the acyl ester bonds of glycerides and phospholipids as well as the acyl CoA-thiol ester bonds. After being secreted by the hepatocytes, HL remains on the surface of hepatic endothelial cells and hepatocytes, bound to heparan sulfate proteoglycans, where it acts on the uptake of chylomicron remnants, IDL and HDL cholesterol ester. HL also participates in the VLDL to IDL and LDL cascade and in the conversion of HDL2 to HDL3 and pre-beta 1 HDL. Thus, HL plays an important role in lipoprotein metabolism and in reverse cholesterol transport and may thus be involved in atherogenic processes. Moreover, HL expression is regulated by hormones and nutritional state in the pre- and post-natal periods. Therefore, it appears of interest to gain further insight into the regulation of HL gene expression in order to better understand plasma lipoprotein metabolism.

Hepatic lipase gene expression is upregulated by a cystine-rich diet in male but not in female rats.

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.

Effect of dietary cholesterol on activity and mRNA levels of hepatic lipase in rat.

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%, p < 0.05), as was its protein mass, quantified by immunoblot analysis (-57%, (p < 0.01), 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.

Effects of a fish oil-lard diet on rat plasma lipoproteins, liver FAS, and lipolytic enzymes.

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)

Hepatic lipase may act as a ligand in the uptake of artificial chylomicron remnant-like particles by isolated rat hepatocytes.

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.

In vivo regulation of hepatic lipase activity and mRNA levels by diets which modify cholesterol influx to the liver.

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.

[Role of hepatic lipase in the catabolism of chylomicron remnants in the rat]. / Rôle de la lipase hépatique dans le catabolisme des remnants de chylomicrons chez le rat.

Before being taken up by the liver, chylomicrons are hydrolyzed successively by two lipases. The first one is the lipoprotein lipase which hydrolyzes mainly chylomicron triacylglycerols and gives rise to remnant particles. The latters will be further hydrolyzed by the hepatic lipase which exerts mainly a phospholipase A1-like activity on these particles. Chylomicrons as well as remnants, incubated with hepatic lipase in vitro, lose up to 47% of their phospholipids while triacylglycerols are less or even not hydrolyzed at all. A decrease in phosphatidylcholine (-30% and -34%) and an increase in lysophosphatidylcholine (+260% and +316%) are the main modifications measured in chylomicrons and in their remnants respectively, after hepatic lipase action. From several date (Borensztajn's work and ours), it arises that phospholipolysis of chylomicrons and of chylomicron remnants, is the obligatory metabolic step before these particles are taken up and degraded by the liver.

Lipoprotein lipase and hepatic lipase activities in a hypercholesterolaemic (RICO) strain of rat. Effect of dietary cholesterol.

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.

Inhibition of hepatic lipase activity impairs chylomicron remnant-removal in rats.

[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.

Chylomicron-remnant uptake by freshly isolated hepatocytes. Effect of heparin and of hepatic triacylglycerol lipase.

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.

Further resolution of the low density lipoprotein spectrum in normal human plasma: physicochemical characteristics of discrete subspecies separated by density gradient ultracentrifugation.

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.