Cholesteryl Ester Transfer Protein (CETP) Genotype and Reduced CETP Levels Associated With Decreased Prevalence of Hypertension

OBJECTIVES: To clarify whether reduced cholesteryl ester transfer protein (CETP) activity carries inherent blood pressure risks and to infer whether the increased blood pressure and elevated mortality associated with torcetrapib are idiosyncratic or characteristic of this class of drugs.PATIENTS AND METHODS: We examined the associations among CETP genotype, phenotype, and blood pressure in a cohort of 521 older adults (who have complete data for the variables required in our primary analysis) enrolled between November 1, 1998, and June 30, 2003, in our ongoing studies of genes associated with longevity, including a cohort with a high prevalence of a genotype coding for a reduced activity variant of CETP and low levels of CETP.RESULTS: The prevalence of hypertension was actually lower among homozygotes for the variant CETP (48% vs 60% among those with wild-type and 65% among heterozygotes; P=.03). Low levels of CETP were associated with reduced prevalence of hypertension (65% in highest tertile, 59% in middle tertile, and 55% in lowest tertile; P=.04) and lower systolic blood pressure (140.8, 138.1, 136.2 mm Hg, respectively; P=.03).CONCLUSION: Reduced levels of CETP are associated with lower, not higher, blood pressure. The adverse results with torcetrapib, if mediated through blood pressure, are likely to represent effects of this specific drug, rather than a result of lower CETP levels.CETP = cholesteryl ester transfer protein; CI = confidence interval; HDL = high-density lipoprotein; LDL = low-density lipoprotein; SNP = single nucleotide polymorphismA decrease in cholesteryl ester transfer protein (CETP) leads to increased high-density lipoprotein (HDL) cholesterol levels and lipoprotein particle size, which may provide significant cardiovascular protection.¹ Earlier work by our group has shown that homozygosity for a single nucleotide polymorphism (SNP), resulting in substitution of valine (V) for isoleucine (I) at codon 405 in the CETP gene, is overrepresented in centenarians.² Therefore, this finding, validated in an independent study,³ suggests that this mutation may contribute to healthy aging by protection from several age-related diseases.⁴ Individuals homozygous for this SNP have decreased CETP levels, increased HDL cholesterol levels, and increased lipoprotein particle sizes. Homozygosity for this SNP is seen significantly more often in the offspring of parents with exceptional longevity than in the offspring of parents with shorter life spans.² Furthermore, a recent review found a consistent association between lower CETP levels, caused by several different genotypes, and decreased risk of coronary artery disease.⁵ These observations suggest that interventions that reduce CETP levels might promote cardiovascular health and contribute to longevity.Despite the potential cardiovascular benefits of lower CETP levels, a phase 3 trial of torcetrapib, a CETP inhibitor, was terminated early because of increased risk of death due to cardiovascular disease in the active treatment group.⁶ Torcetrapib has been associated with increased blood pressure,⁷ which is suspected of contributing to the observed increase in mortality. Other investigators have called for investigation of the connection between low CETP levels and blood pressure.⁸To examine whether naturally occurring CETP polymorphism, decreased CETP levels, increased HDL cholesterol levels, and increased lipoprotein particle sizes are associated with increased blood pressure, we analyzed data from our cohorts of older adults. We reasoned that if blood pressure is independent of spontaneous CETP levels, the problems found in the torcetrapib trial may be specific collateral effects of that drug and not generalizable to its pharmacological class. However, if reduction in CETP levels, due to genetic or other causes, is associated with increased blood pressure, beneficial effects of low CETP levels may apply only to those who have lived to old age or it may be that resulting higher blood pressure levels are tolerated because of other beneficial effects.     

Abnormal regulation of LDL receptor activity and abnormal cellular metabolism of hypertriglyceridaemic low density lipoprotein: normalization with bezafibrate therapy

The regulation of LDL (B,E) receptor activity and of cellular LDL protein metabolism by hypertriglyceridaemic (HTG) low density lipoprotein before and during hypolipidaemic therapy (with bezafibrate (BZ] were determined in cultured human skin fibroblasts. Defective binding and subnormal capacity to regulate LDL receptor activity was found for HTG-LDL. Binding affinity (Kd) of HTG-LDL to the receptor was 4.97 x 10(-8) M and of N-LDL, 1.74 x 10(-8) M. When assayed with normal 125I-LDL, the capacity of HTG-LDL to down-regulate receptor activity was 46-68% less than N-LDL. Both abnormalities reverted towards normal during treatment. The cellular metabolism of HTG-, BZ- and N-LDL in cells grown for 48 h with the respective lipoproteins was determined. In spite of their defective binding to the receptor, the metabolism of HTG-LDL in the regulated cells was accelerated in comparison to N-LDL, and equal to that of BZ-LDL. That observation is explained by the inefficient ability of HTG-LDL to depress LDL receptor activities in the cells.     

噬菌体表面呈现人红细胞血型McAb 50A单链抗体的研究

目的 构建表达抗人红细胞血型A抗原50A杂交瘤细胞的单链抗体(ScFv)。方法 应用重组噬菌体抗体技术,从50A杂交瘤细胞中分离、构建单链抗体基因,并将其克隆入噬粒pCANTAB5E中,转化E.coli XL-Blue,辅助噬菌体援救构建50A噬菌体单链抗体库;采用完整红细胞亲和富集法淘选阳性重组噬菌体,鉴定重组噬菌体并进行序列测定分析;免疫印迹试验检测重组单链抗体的特异性抗原活动。结果 用M13KO7援救XL-Blue转化菌中的pCANTAB5E重组噬菌体,得到滴度为4×10~9 pfu/ml噬菌体单链抗体库;免疫印迹试验证实表达产物保留了亲本单抗对人红细胞血型A抗原的特异性亲和力。结论 成功构建50A McAb单链噬菌体抗体库,为进一步研制高特异性、高亲和力的基因工程原核血型检定抗体试剂奠定了基础。     

Anti-atherosclerosis effect of different doses of CETP vaccine in rabbit model of atherosclerosis

AimTo evaluate atheroprotective effects of different doses of cholesteryl ester transfer protein (CETP) vaccine, three doses of Tetanus toxoid-CETP (TT-CETP) peptide including 10, 50 and 100/rabbit, termed FA10, FA50, FA100, respectively, were administered in rabbit model of atherosclerosis.MethodsAnimals were vaccinated subcutaneously (S.C.) with 100 μl of vaccine in presence of complete Freund’s adjuvant (CFA) for the first administration. Rabbits were boosted 4 times at 3 weeks intervals with the same peptide dose formulated in incomplete Freund’s adjuvant (IFA). Animals were fed with diet supplemented with 2% cholesterol from week 11 to week 19. Anti-TT-CETP specific antibody and CETP activity in sera were determined. Therapeutic response was examined by tracking plasma lipoprotein levels (HDL-C, LDL-C and total cholesterol), and pathologic observation of intima/media thickness at the site of aortic lesions.ResultsAll TT-CETP vaccine doses generated strong anti TT-CETP antibody response. CETP activity reduced in rabbits vaccinated with FA100 (P = 0.031). FA100 showed significant increase in level of HDL-C rather than control group (P = 0.006). However, no significant reduction were found in atherosclerotic lesion when compared to control.ConclusionInhibition of CETP activity and increased HDL-C were found with FA100, but the vaccine failed to prevent aortic lesion development in immunized rabbits when compared to control. Our result supports the hypothesis stated that CETP may not be an attractive therapeutic target for the prevention of cardiovascular disease.     

Multiple B-cell clones producing antibodies directed to the spacer and disintegrin/thrombospondin type-1 repeat 1 (TSP1) of ADAMTS13 in a patient with acquired thrombotic thrombocytopenic purpura

BACKGROUND: The cysteine-rich/spacer domains of ADAMTS13 contain a major binding site for antibodies in patients with acquired thrombotic thrombocytopenic purpura (TTP). OBJECTIVE: To study the heterogeneity of the antibody response towards these domains an immunoglobulin V-gene phage-display library was constructed to isolate monoclonal anti-ADAMTS13 antibodies from the immunoglobulin repertoire of a patient with acquired TTP. METHODS: Combined variable heavy chain (VH) and variable light chain (VL) segments, expressed as single-chain Fv fragments (scFv), were selected for binding to an ADAMTS13 fragment consisting of the disintegrin/thrombospondin type-1 repeat 1 (TSP1)/cysteine-rich/spacer domains. RESULTS: Seven different scFv antibody clones were identified that were assigned to four groups based on their homology to VH germline gene segments. Epitope-mapping revealed that scFv I-9 (VH1-69), I-26 (VH1-02), and I-41 (VH3-09) bind to an overlapping binding site in the ADAMTS13 spacer domain, whereas scFv I-16 (VH3-07) binds to the disintegrin/TSP1 domains. The affinity of scFv for the disintegrin/TSP1/cysteine-rich/spacer domain was determined by surface plasmon resonance analysis and the dissociation constants ranged from 3 to 254 nM. The scFv partially inhibited ADAMTS13 activity. However, full-length IgG prepared from the variable domains of scFv I-9 inhibited ADAMTS13 activity more profoundly. Plasma of six patients with acquired TTP competed for binding of scFv I-9 to ADAMTS13. CONCLUSION: Our data indicate that multiple B-cell clones producing antibodies directed against the spacer domain are present in the patient analyzed in this study. Our findings also suggest that antibodies with a similar epitope specificity as scFv I-9 are present in plasma of other patients with acquired TTP.     

Characterization of binding of the ATP-sensitive potassium channel ligand, [3H]glyburide, to neuronal and muscle preparations

Binding of the hypoglycemic sulfonylurea, [3H]glyburide, to crude membrane fractions from brain, heart and smooth (intestinal) muscle was saturable, linear with protein concentration and reversible. Saturation analysis revealed high affinity sites (KH values, 7 x 10(-11) M, 5 x 10(-11) M and 6 x 10(-11) M), with Bmax-H values 209, 36 and 23 fmol/mg protein in the brain, heart and smooth muscle, respectively. High affinity [3H]glyburide binding was pharmacologically specific, insensitive to a variety of receptor-active ligands, but sensitive to a series of sulfonylureas, and good, essentially 1:1, correlations were obtained between binding affinities and literature-derived pharmacologic activities. The K+ channel activators, cromakalim, nicorandil, pinacidil and minoxidil were not effective as inhibitors of [3H]glyburide binding. However, diazoxide was a modestly effective inhibitor. Putative low affinity sites (KL values, 3 x 10(-7) M, 1 x 10(-7) M and 2 x 10(-9) M) with Bmax-L values 4956, 336 and 53 fmol/mg protein in brain, heart and smooth muscle, respectively, were identified. Their significance remains to be established. Except for ATP gamma S, the ability of nucleotide triphosphates to inhibit high affinity [3H] glyburide binding was dependent on the presence of Mg++. ADP, in the presence of Mg++, inhibited binding with an IC50 value of 6.3 x 10(-4) M. Nucleotide monophosphates did not inhibit [3H] glyburide binding in the presence or absence of Mg++, whereas in the presence of Mg++, nucleotide triphosphates were equally potent inhibitors of binding. The rank order potency for nucleotide diphosphate inhibition of binding, in the presence of Mg++, is ADP greater than GDP greater than IDP = UDP. In the absence of Mg++, [3H]glyburide binding shows a biphasic response to ADP, and the inhibition of binding by ADP was prevented by ATP. It is suggested that this biphasic response is the result of a second nucleotide binding site.     

CETP inhibition in cardiovascular risk management: a critical appraisal

In view of the cardioprotective effect of high-density lipoproteins (HDL) and the limited effects of statin and fibrate therapy on HDL cholesterol, it is clinically relevant to test whether pharmacological treatment aimed at raising HDL lowers cardiovascular risk. Cholesteryl ester transfer protein (CETP) is a new therapeutic target, because the cholesteryl ester transfer process lowers HDL cholesterol and contributes to an atherogenic lipoprotein profile, particularly when plasma triglycerides are high. Clinical evidence suggests that coronary artery calcification as well as intima media thickness is positively related to plasma cholesteryl ester transfer, and that high plasma CETP concentration is associated with increased cardiovascular risk in hypertriglyceridaemia. However, CETP could also have anti-atherogenic potential, since it provides a potentially beneficial route for delivery of HDL-derived cholesteryl esters to the liver. In addition, CETP could also favourably stimulate peripheral cell cholesterol removal and enhance hepatic cholesterol uptake. Recent evidence suggests that a high CETP level may confer lower cardiovascular risk in the context of low triglycerides. At maximal doses, the CETP inhibitors JTT-705 and torcetrapib elicit a marked rise in HDL cholesterol of up to 34% and 91-106%, respectively. The effectiveness of these drugs on (intermediate) clinical outcome measures is currently being tested in large-scale phase III clinical trials, with torcetrapib being only evaluated in combination therapy with atorvastatin. When and how to use CETP inhibitors, e.g. in combination with a statin or a fibrate, is a major challenge. We propose that low HDL cholesterol in the context of high triglycerides, such as found in type 2 diabetes mellitus, could become an important indication area for this new class of drugs.     

Ouabain-specific antibodies: immunochemical properties and reversal of Na+, K+-activated adenosine triphosphatase inhibition

Antibodies with high affinity and specificity for the cardiac glycoside ouabain were raised in rabbits. The antigen used was a conjugate of ouabain linked through its rhamnose moiety to terminal alpha-amino groups of poly D,L alanyl-human serum albumin. Ouabain-specific antibodies were present as early as 3 wk, and rose steadily in titer over the initial 20-33 wk of immunization. Levels as high as 6.5 mg specific immunoglobulin per ml antiserum were reached in one rabbit at the end of 45 wk. The average intrinsic association constants for ouabain were 1.3 x 10(9) M(-1) and 1.6 x 10(9) M(-1) in antisera studied in detail, and there was evidence of restricted heterogeneity of binding site affinities. A high degree of specificity was demonstrated. Significant cross-reactivity occurred only with other cardioactive steroid compounds such as acetyl strophanthidin, digoxin, and digitoxin, while endogenous steroids did not cross-react even when present in 1000-fold excess. A rapid and convenient radioimmunoassay procedure for plasma or urine ouabain concentrations was developed using these antibodies. Competition between ouabain-(3)H tracer and unlabeled ouabain for specific antibody binding sites allowed the measurement of ouabain concentrations as low as 0.1 ng/ml or less without need for extraction procedures. The high association constants observed in these studies permit antibody reversal of established myocardial effects of ouabain. Both blockade and reversal of ouabain inhibition of canine myocardial microsomal Na(+), K(+)-activated ATPase by antibody were documented, suggesting a possible mechanism for reversal of cellular effects.     

Supersensitive time-resolved immunofluorometric assay of free prostate-specific antigen with nanoparticle label technology.

BACKGROUND: The extreme specific activity of the long-lifetime fluorescent europium(III) chelate nanoparticles and the enhanced monovalent binding affinity of multivalent nanoparticle-antibody bioconjugates are attractive for noncompetitive immunoassay. METHODS: We used a noncompetitive, two-step immunoassay design to measure free prostate-specific antigen (PSA). Europium(III) chelate nanoparticles (107 nm in diameter) were coated with a monoclonal anti-PSA antibody (intrinsic affinity, 6 x 10(9) L/mol). The nanoparticle-antibody bioconjugates had an average of 214 active binding sites per particle and a monovalent binding affinity of 7 x 10(10) L/mol. The assay was performed in a low-fluorescence microtitration well passively coated with an another monoclonal anti-PSA antibody (affinity, 2 x 10(10) L/mol), and the europium(III) fluorescence was measured directly from the bottom of the well by a standard time-resolved microtitration plate fluorometer. RESULTS: The detection limit (mean + 2 SD) was 0.040 ng/L (7.3 x 10(5) molecules/mL), and the dynamic detection range covered four orders of magnitude in a 3-h total assay time. The imprecision (CV) over the whole assay range was 2-10%. The detection limit of the assay was limited by the fractional nonspecific binding of the bioconjugate to the solid phase (0.05%), which was higher than the nonspecific binding of the original antibody (<0.01%). CONCLUSIONS: The sensitivity of the new assay is equal to that of the ambient-analyte, microspot immunoassay and will be improved by use of optimized, high binding-site density nanoparticle-antibody bioconjugates with reduced nonspecific binding and improved monovalent binding affinity.     

Human cholesteryl ester transfer protein expression enhances the mouse survival rate in an experimental systemic inflammation model: a novel role for CETP

Mice expressing human cholesteryl ester transfer protein (huCETP) are more resistant to Escherichia coli bacterial wall LPS because death rates 5 days after intraperitoneal inoculation of LPS were higher in wild-type than in huCETP+/+ mice, whereas all huCETP+/+ mice remained alive. After LPS inoculation, plasma concentrations of TNF-alpha and IL-6 increased less in huCETP+/+ than in wild-type mice. LPS in vitro elicited lower TNF-alpha production by CETP expressing than by wild-type macrophages. In addition, TNF-alpha production by RAW 264.7 murine macrophages increased on incubation with LPS but decreased in a dose-dependent manner when human CETP was added to the medium. Human CETP in vitro enhanced the LPS binding to plasma high-density lipoprotein/low-density lipoprotein. The liver uptake of intravenous infused 14C-LPS from Salmonella typhimurium was greater in huCETP+/+ than in wild-type mice. Present data indicate for the first time that CETP is an endogenous component involved in the first line of defense against an exacerbated production of proinflammatory mediators.     

Bradykinin-stimulated inositol phosphate production in NG108-15 cells is mediated by a small population of binding sites which rapidly desensitize.

[3H]Bradykinin (BDK) binds to two distinct binding sites (P less than .01, N = 12) in NG108-15 cell membranes; (site 1: Kd1 = 3.09 x 10(-10) M, Bmax1 = 242 +/- 24 fmol/mg protein) and (site 2: Kd2 = 1.94 x 10(-8) M, Bmax2 = 491 +/- 75 fmol/mg protein). Although site 1 comprises only 33 +/- 4% (N = 12) of the total binding site population, comparison of the binding affinity and functional potency for BDK agonist analogs exhibiting differential selectivity for the two sites reveals that this high affinity site is the receptor mediating inositol monophosphate (IP) production in this cell line. BDK-stimulated IP production undergoes a very rapid (5 min) desensitization that is characterized by both a loss in agonist potency (EC50 = 3.57 x 10(-9) M vs. 1.94 x 10(-10) M in controls; P less than .001, N = 12) and a decrease in amplitude of response (fold stimulation = 1.45 +/- 0.06 vs. 1.80 +/- 0.09 in controls; P less than .01, N = 12). Only the decrease in response amplitude is attenuated by down-regulation of protein kinase C by prior long term treatment of the cells with 12-O-tetradecanoylphorbol 13-acetate (TPA), indicating an involvement of protein kinase C activation in the desensitization process. Desensitization is accompanied by down-regulation of site 1 only (Bmax1 = 71 +/- 8 fmol/mg (N = 10; P less than .001 vs. controls)); Bmax2 and the Kd for BDK at both sites remain unchanged, further supporting the contention that site 1 is the functionally relevant receptor. In contrast to the functional data, long term TPA treatment does not attenuate the receptor down-regulation, indicating that the rapid desensitization involves both receptor-related and postreceptor mechanisms. The implications of this property of the BDK receptor for analog design and receptor classification are discussed.     

CETP and oxidized LDL levels increase in dyslipidemic subjects

OBJECTIVES: To explore the possible associations among cholesteryl ester transfer protein (CETP), contents of lipids in low-density lipoprotein (LDL), and in vivo oxidized LDL (Ox-LDL). DESIGN AND METHODS: CETP and Ox-LDL were both detected by ELISA. Their levels and the lipid contents of LDL were investigated in 200 subjects with various dyslipidaemias. RESULTS: Compared to the control, CETP levels were significantly increased in subjects with mixed hyperlipidaemia and hypercholesterolaemic. Ox-LDL levels were only significantly increased in mixed hyperlipidaemia subjects. Triacyglycerols to cholesterol ratio in LDL was significantly increased in various dyslipidaemias subjects, of which, hypertriglyceridaemic subjects exhibited the most significant change, while hypercholesterolaemic subjects the least. Multiple linear regression analysis showed that total cholesterol and triacyglycerols levels in very low-density lipoprotein were significantly related with CETP (R(2)=0.066), and triacyglycerols and total cholesterol levels were significantly related with Ox-LDL (R(2)=0.094), respectively. CONCLUSIONS: High CETP promotes the transport of lipids among lipoproteins, which changed the lipid composition of LDL, resulting in the increase of in vivo Ox-LDL level, and subsequently contributing to the atherogenic process in dyslipidaemias subjects.     

Prolonged caloric restriction in obese patients with type 2 diabetes mellitus decreases plasma CETP and increases apolipoprotein AI levels without improving the cholesterol efflux properties of HDL

OBJECTIVE

Using a mouse model for human-like lipoprotein metabolism, we observed previously that reduction of the hepatic triglyceride (TG) content resulted in a decrease in plasma cholesteryl ester transfer protein (CETP) and an increase in HDL levels. The aim of the current study was to investigate the effects of prolonged caloric restriction in obese patients with type 2 diabetes mellitus, resulting in a major reduction in hepatic TG content, on plasma CETP and HDL levels.

RESEARCH DESIGN AND METHODS

We studied 27 obese (BMI: 37.2 ± 0.9 kg/m²) insulin-dependent patients with type 2 diabetes mellitus (14 men and 13 women, aged 55 ± 2 years) who received a 16-week very low calorie diet (VLCD). At baseline and after a 16-week VLCD, plasma lipids, lipoproteins, and CETP were measured. Furthermore, functionality of HDL with respect to inducing cholesterol efflux from human monocyte cells (THP-1) was determined.

RESULTS

A 16-week VLCD markedly decreased plasma CETP concentration (−18%; P < 0.01) and increased plasma apolipoprotein (apo)AI levels (+16%; P < 0.05), without significantly affecting plasma HDL-cholesterol and HDL-phospholipids. Although a VLCD results in HDL that is less lipidated, the functionality of HDL with respect to inducing cholesterol efflux in vitro was unchanged.

CONCLUSIONS

The marked decrease in hepatic TG content induced by a 16-week VLCD is accompanied by a decrease in plasma CETP concentration and an increase in apoAI levels, without improving the cholesterol efflux properties of HDL in vitro.Patients with type 2 diabetes mellitus display a typical atherogenic dyslipidemia marked by increased plasma triglycerides (TG) and VLDL-cholesterol concentrations and decreased HDL-cholesterol levels. Furthermore, hepatic steatosis, which is also strongly associated with cardiovascular disease risk (1,2), is frequently observed in patients with type 2 diabetes mellitus (3–5).We demonstrated previously that the HDL-raising effect of various classical lipid-lowering drugs was caused by a reduction in plasma cholesteryl ester transfer protein (CETP) that mediates the net transfer of cholesteryl esters from HDL to (V)LDL. In APOE*3-Leiden.CETP mice, a well-established animal model for human-like lipoprotein metabolism, statins (6), fibrates (7), and niacin (8) decrease the hepatic lipid content (i.e., both TG and cholesterol), resulting in a decreased hepatic CETP expression accompanied by decreased plasma CETP levels and a consequently increased plasma HDL. Recently, we showed that a similar mechanism may account for the HDL-raising effect of pioglitazone in humans. In patients with type 2 diabetes mellitus, pioglitazone decreased hepatic TG content (9), accompanied by a decrease in plasma CETP concentration and increase in HDL level (10). In contrast, metformin did not affect either hepatic TG, plasma CETP, or HDL levels (10).Lifestyle interventions such as diet-induced weight reduction and exercise are very important in the treatment of obese patients with type 2 diabetes mellitus. Recently, we reported that a 16-week very low calorie diet (VLCD) in obese patients with type 2 diabetes mellitus significantly decreased plasma total cholesterol and TG levels and markedly reduced hepatic TG content (11), but the potential beneficial effect of a VLCD on plasma CETP and HDL levels has not been studied. Therefore, using plasma samples from that study (11), we investigated whether prolonged caloric restriction reduces CETP concentration and thereby increases HDL levels in obese patients with type 2 diabetes mellitus.     

Identification and characterization of specific receptors for monocyte- derived neutrophil chemotactic factor (MDNCF) on human neutrophils

Specific receptors for a recently purified and cloned monocyte-derived neutrophil chemotactic factor (MDNCF) have been identified on the surface of normal human peripheral blood neutrophils using 125I-labeled recombinant human MDNCF (125I-MDNCF). Competitive binding of 125I-MDNCF to human neutrophils reached a maximal level at 1-3 h at 4 degrees C. The Scatchard analysis showed that there are approximately 20,000 receptors per cell with a single type of high affinity binding (Kd, 8 x 10(-10) M). The receptors for MDNCF are clearly distinct from the receptors for other cytokines and chemotactic agents, e.g., IL-1 alpha, TNF-alpha, and FMLP, C5a, leukotriene B4, and platelet activating factor. Based on the SDS-PAGE analysis of chemically crosslinked 125I-MDNCF receptor complex, there are two polypeptides that bind MDNCF; the molecular weight of these two MDNCF receptors were estimated to be 67,000 and 59,000. Treatment of a promyelocytic cell line, HL60, with 1.25% DMSO for 5 d in vitro increased the number of receptors up to 7,000 receptors/cell with a Kd of 1.2 x 10(-9) M.     

Cholesteryl ester transfer protein inhibitor (JTT-705) and the development of atherosclerosis in rabbits with severe hypercholesterolaemia

Cholesteryl ester transfer protein (CETP) is a major determinant of plasma levels of high-density lipoprotein-cholesterol (HDL-C) in humans. The anti-atherogenic effect of lowering CETP levels is dependent not only on HDL-C levels but also on a metabolic background of increased low-density lipoprotein or very-low-density lipoprotein. Here we investigated the effects of JTT-705, a chemical inhibitor of CETP, on the development of atherosclerosis in Japanese white rabbits fed on a high cholesterol diet. After 4 weeks on a diet of 0.25% cholesterol-containing chow, 100 mg/kg (low dose) or 300 mg/kg (high dose) JTT-705 was given, and the animals were monitored at weeks 0, 4, 8 and 12. Aortic atherosclerotic lesions were determined at the end of this period. JTT-705 induced a significant increase in HDL-C in the high-dose group [from 21+/-3 to 50+/-7 mg/dl (mean+/-S.E.M.); P <0.0001] compared with the control group (from 21+/-2 to 27+/-2 mg/dl). The atheromatous area was 60+/-9% in the high-dose group and 58+/-9% in the control group. Moreover, correlation analysis showed that triacylglycerol and non-HDL-C levels had a direct relationship with the development of atherosclerosis, but CETP activity and HDL-C levels did not. Thus the CETP inhibitor JTT-705 alone did not have an anti-atherogenic effect in our rabbit model, of severe hypercholesterolaemia suggesting a relatively minor effect of HDL-elevating therapy as compared with decreases in non-HDL-C (or triacylglycerol) levels in patients with severe hypercholesterolaemia, such as familial hypercholesterolaemia.     

Lectins from Triticum vulgaris and Limax flavus are universal antagonists of botulinum neurotoxin and tetanus toxin.

Lectins from Anguilla anguilla, Artocarpus integrifolia, Canavalia ensiformis, Datora stramonium, Glycine max, Limax flavus, Ricinus communis and Triticum vulgaris were tested for their abilities to antagonize the binding of botulinum neurotoxin and tetanus toxin to rat brain membranes and to antagonize the ability of these toxins to block neuromuscular transmission in mouse phrenic nerve-hemidiaphragm preparations. Lectins from Limax flavus and Triticum vulgaris, both of which have affinity for sialic acid, were antagonists of the various serotypes of botulinum neurotoxin and tetanus toxin. When tested against the high affinity binding site for botulinum neurotoxin type B, the lectin from Limax flavus had a Ki of 3.1 x 10(-7) M and the lectin from Triticum vulgaris had a Ki of 3.75 x 10(-7) M. When tested against the high affinity binding site for tetanus toxin, the lectins from Limax flavus and Triticum vulgaris had Ki values of 1.5 x 10(-7) and 1 x 10(-6) M, respectively. In all cases the lectins behaved as competitive antagonists. In reverse experiments, neither botulinum toxin nor tetanus toxin was a very effective antagonist of lectin binding to brain membranes. Studies on isolated neuromuscular preparations showed that the lectin from Triticum vulgaris did not affect transmission at concentrations of 10(-6) to 10(-3) M, but at a concentration of 3 x 10(-5) M the lectin produced highly statistically significant antagonism of the neuromuscular blocking properties of botulinum neurotoxin types A, B, C, D, E and F as well as tetanus toxin. The lectin did not antagonize beta-bungarotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced phagocytic cell respiratory burst induced by spinal manipulation: potential role of substance P.

The effect of spinal manipulation on the respiratory burst of polymorphonuclear neutrophils (PMN) and monocytes from treated adults was measured by zymosan-stimulated chemiluminescence (CL). Peripheral blood was collected 15 min before and 15 min after treatment (sham manipulation, thoracic spine manipulation, or soft tissue manipulation), the cells were isolated, challenged with a standardized, opsonized luminol-containing suspension of zymosan, and monitored for CL. Plasma from two subsets of subjects was radioimmunoassayed for Substance P (SP). PMN were also preincubated with SP in vitro over the dose range 5 x 10(-12) M to 5 x 10(-8) M and the CL response monitored. The CL responses of both PMN and monocytes from subjects who received spinal manipulation were significantly higher after than before treatment, and significantly higher than the response in sham or soft-tissue treated subjects. Measurement of the force applied by sham and spinal manipulation suggested a force threshold for the enhancement of the CL response. Plasma levels of SP before and after treatment in sham treated subjects did not differ significantly; however, elevated plasma SP was observed in subjects after spinal manipulation. Preincubation of PMN with 1 x 10(-11) M, 5 x 10(-11) M or 1 x 10(-10) M SP in vitro primed PMN for an enhanced respiratory burst when the cells were subsequently challenged.     

CETP gene mutation (D442G) increases low-density lipoprotein particle size in patients with coronary heart disease

BACKGROUND: Small, dense low-density lipoprotein (LDL) in subjects with the atherogenic pattern B has been established as a risk factor of atherosclerosis. Cholesteryl ester transfer protein (CETP) plays an important role in the transfer and exchange of cholesteryl esters and triglycerides between the lipoprotein classes of human plasma. It has been shown that CETP can also change the particle size of high-density lipoprotein (HDL) and LDL subfractions in vitro. Previous clinical studies about CETP gene mutations mainly focused on abnormalities in HDL, few involved those in LDL. OBJECTIVES: To investigate the effect of the D442G mutation in the CETP gene on major peak size of LDL particles in patients with coronary heart diseases (CHD). METHODS: D442G mutation in the CETP gene was detected using the PCR-RFLP. LDL particles sizes were analyzed by 2-16% nondenaturing polyacrylamide gradient gels in CHD patients with D442G mutation in the CETP gene. RESULTS: Six heterozygotes and one homozygote were found to have the D442G mutation among 200 CHD patients. The frequency of this mutation was 3.5%. The major peak size of LDL in patients with gene mutation (n=7) was significantly larger than that in patients without the mutation (n=40) (26.92 +/- 0.79 nm vs. 25.71 +/- 0.66 nm, respectively; P<0.01). All the patients with the gene mutation expressed pattern A, whereas only about half of the patients without the mutation expressed this pattern. The patients with gene mutation had decreased plasma CETP concentration, while increased concentration of HDL-C and apolipoprotein A-I compared with controls. CONCLUSIONS: CETP gene mutation (D442G) increases LDL particle size. This suggests that CETP play an antiatherogenic role.     

Anacetrapib, a novel CETP inhibitor: pursuing a new approach to cardiovascular risk reduction

Cholesteryl ester transfer protein (CETP) inhibition is a promising experimental strategy to raise high-density lipoprotein cholesterol (HDL-C) and reduce cardiovascular risk. This review focuses on the highly selective and potent CE TP inhibitor anacetrapib and discusses the available preclinical and clinical information pertaining to it. We also describe strategies to target HDL-C, discuss the mechanism underlying CETP inhibition and its effects on lipid biology, and give an overview of other CETP inhibitors that are currently in development.     

Distinct abnormalities in the interaction of purified types IIA and IIB von Willebrand factor with the two platelet binding sites, glycoprotein complexes Ib-IX and IIb-IIIa.

We have studied the interaction of the congenitally abnormal type IIA and IIB von Willebrand factor (vWF) molecules, both lacking the larger multimeric forms, with the two vWF binding sites on platelets, the glycoprotein (GP) Ib-IX and GP IIb-IIIa complexes. Variant as well as normal (N) vWF were purified from plasma. Estimates for binding of subunit molecules per platelet at saturation (Bmax) and dissociation constant in moles/liter (Kd), respectively, were obtained from binding isotherms of 125I-labeled vWF, with the following results. In the presence of ristocetin (binding to GP Ib-IX): N, 25,693 and 0.5 x 10(-8); IIA, both parameters not measurable; IIB, 17,708 and 0.87 x 10(-8). After thrombin stimulation (binding to GP IIb-IIIa): N, 17,059 and 1.12 x 10(-8); IIA, 23,751 and 4.87 x 10(-8); IIB, 19,890 and 2.52 x 10(-8). Distinct experiments based on measuring the ability of the variant species (from the same patients and one additional IIB patient) to inhibit the binding of normal 125I-vWF to platelets gave results in agreement with those reported above. Other studies showed that only IIB vWF bound to platelets in the absence of any mediating substance (Kd = 5.21 x 10(-8) mol/liter and Bmax = 9,599 subunits per platelet) and induced aggregation at a concentration of 10 micrograms/ml (3.6 x 10(-8) M). Thus, IIB vWF binds to GP Ib-IX with high affinity and induces platelet aggregation, whether with or without ristocetin, in spite of the absence of larger multimers. In contrast, the binding of IIA vWF to GP Ib-IX occurs with very decreased affinity, and this defective function may result from specific structural abnormalities rather than just being a reflection of the absence of larger multimeric forms. Both IIA and IIB vWF exhibit decreased affinity for GP IIb-IIIa. In this case, the extent of the defect correlates with the absence of larger multimers.