Biomedical subjects

T W De Bruin

Publications and source records attributed to T W De Bruin.

14 recordsLinked to original sources

PubMed2001

Two polymorphisms in the apo A-IV gene and familial combined hyperlipidemia.

Linkage and association of the apo AI-CIII-IV gene region to familial combined hyperlipidemia (FCHL) was reported previously, based on the presence of genetic variants in the apo CIII and apo AI gene. No data were available yet on the contribution of the apo A-IV locus. Two DNA variants in exon 3 of the apo A-IV gene, A (Thr)(347)T (Ser) and [CTGT](3-4) were characterized by sequencing the coding region of the apo A-IV gene and were analyzed in our Dutch FCHL cohort (30 probands, 159 affected relative, 317 unaffected relatives and 218 spouses). The genotype frequency of the A(347)T variant was different in probands and spouses. In probands no 2/2 carriers were found, resulting in a significant decreased frequency of the 2-allele (P<0.05). This was suggestive for a protective role of the presence of the serine (T) allele on the prevalence of FCHL. No difference in frequency distribution was found for the [CTGT](3-4) variant between the groups. Homozygous 4/4 carriers in spouses had a more favorable lipid profile (LDL-cholesterol and apo B, P<0.05). The absence of linkage disequilibrium of the A(347)T with other markers in the gene cluster, and the absence of linkage disequilibrium with [CTGT](3-4) marker and the MspI-AI marker in the apo A-I promoter showed that these two apo A-IV variants reside on different haplotypes from the apo A-I and apo C-III markers. This was illustrated by extensive haplotype analysis. The present data on the contribution of DNA variants in the apo A-IV gene support our previous observations that the apo AI-CIII-AIV gene cluster has a complex genetic contribution to FCHL both by conferring susceptibility and protection.

Adult↗

PubMed2001

New genetic variants in the apoA-I and apoC-III genes and familial combined hyperlipidemia.

Linkage and association between the apolipoprotein (apo) A-I/C-III/A-IV gene region on chromosome 11 and familial combined hyperlipidemia (FCHL) has been observed previously. Using sequence analysis two new allelic variants were identified, C(317) -T in intron 2 of the apoA-I gene and C(1100)-T in exon 3 of the apoC-III gene. These variants were studied in 30 FCHL probands, 159 hyperlipidemic relatives, 327 normolipidemic relatives, and 218 spouses. The allele frequencies of both variants were significantly different in probands and spouses (P < 0.002 and P < 0.001, respectively), with increased frequency of the minor alleles in the probands. The minor genotypes (TT) were associated with elevated plasma triglyceride and apoC-III. Both variants were in strong, although not complete, linkage disequilibrium with each other and with the MspI site in the promoter region of the apoA-I gene and the SstI site in the 3' untranslated region of exon 4 of the apoC-III gene. Haplotypes based on these four variants were constructed and the distributions of haplotype combinations were significantly different (P < 0.0001). Two distinct haplotypes predisposing to FCHL were found: 2-2-1-2 and 1-2-2-2 (MspI, C(317) -T; SstI, C(1100)-T). The haplotype combinations carrying one of these high risk alleles are associated with elevated lipid levels in probands and in spouses. While these effects can be attributed to the presence of the M2 and S2 minor alleles, these results suggest that the importance of specific allelic haplotypes may be greater than single genotypic effects.

Adult↗

PubMed2001

Genetic heterogeneity in the apolipoprotein C-III promoter and effects of insulin.

In the present study, we have investigated the in vivo and in vitro role of two newly identified variants (G(-)944A and A(-)1180C) located in the upstream promoter region of the apolipoprotein C-III (apoC-III) gene. These variants were studied in 30 probands diagnosed with FCHL, their relatives, and spouses. The allele frequencies of both variants were not different between the groups. No significant associations between plasma lipid traits and DNA variants were observed. We further analyzed the effect of the presence of these variants in the upstream enhancing region of the apoC-III gene, as five different in vivo occurring haplotypes, on the transcriptional activity of apoC-III in both HepG2 and Caco-2 cells. All five promoter constructs, including the wild type, showed similar enhancing activity of the apoC-III gene. The average transcription efficiency was enhanced 19-fold in HepG2 cells and 11-fold in Caco-2 cells. Previous studies have shown in vitro insulin-dependent down-regulation of the apoC-III gene transcription in HepG2 cells by DNA variation in an insulin response element (IRE) in the apoC-III promoter. We observed a 30% insulin-dependent down-regulation of apoC-III expression that was, however, independent of the presence of the two IRE variants. In contrast, in Caco-2 cells, a more variable insulin-dependent up-regulation was found that was also independent of the presence of the IRE variants.In conclusion, our data suggested that the apoC-III gene transcription in vitro is regulated by insulin but independent of the presence of the two IRE variants at -455 and -482. We were unable to detect associations between these apoC-III variants and plasma lipids and insulin in our FCHL population. This means that in vivo apoC-III transcription not only depends upon insulin but appears to be mediated by other mechanisms.

Apolipoprotein C-III↗

PubMed1999

Combined hyperlipidemia is associated with increased exercise-induced muscle protein release which is improved by triglyceride-lowering intervention.

Although myopathy is considered an adverse effect of treatment with 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors and fibrates in combined hyperlipidemia, the present study was performed to investigate whether combined hyperlipidemia itself is associated with skeletal muscle pathology and whether lipid-lowering intervention has beneficial effects. To investigate whether combined hyperlipidemia is associated with skeletal muscle pathology, 10 male patients and 15 normolipidemic controls underwent a 45-minute standardized bicycle ergometer test at a load of 2 W/kg lean body mass (parallel study). One- and 8-hour postexercise increments in the plasma level of the muscle proteins creatine kinase (CK), myoglobin (Mb), and fatty acid-binding protein (FABP) were assessed as parameters for (subclinical) skeletal muscle pathology. The 8-hour postexercise increments in CK and Mb and 1-hour postexercise increment in Mb were significantly higher in patients than in controls, thus indicating increased exercise-induced muscle membrane permeability in combined hyperlipidemia. To investigate the effects of lipid-lowering intervention on skeletal muscle in combined hyperlipidemia, 21 subjects with combined hyperlipidemia were randomized double-blindly to receive 6 weeks of treatment with fluvastatin 40 mg/d, gemfibrozil 600 mg twice daily, or combination therapy. All subjects underwent an ergometer test before and after treatment. Gemfibrozil treatment alone reduced the CK increments 8 hours postexercise by 47% and the FABP increments 1 and 8 hours postexercise by 83% and 101%, respectively (all P < .05). Combined treatment reduced Mb increments 1 hour postexercise by 54% and FABP increments 8 hours postexercise by 44% (all P < .05). A highly significant correlation existed between therapy-induced changes in plasma triglycerides and changes in postexercise increments of FABP and Mb. In conclusion, combined hyperlipidemia is associated with an increased exercise-induced release of muscle proteins, which is ameliorated by triglyceride-lowering intervention. As FABP is an indicator for ischemia-induced skeletal muscle pathology, a possible explanation is the impaired muscle blood flow during hypertriglyceridemia, which may be reversed by triglyceride-lowering intervention. The mechanism and clinical relevance of these findings remain to be investigated.

Double-Blind Method↗

PubMed1999

Limitations of the semisynthetic library approach for obtaining human monoclonal autoantibodies to the thyrotropin receptor of Graves' disease.

Graves' disease (GD) is characterized by the presence of autoantibodies against the TSH-receptor (TSH-R) which are pathogenic and, upon binding to the receptor, trigger intracellular signal transduction. The autoantibodies are oligoclonal and as they are responsible for disease activity, their characterization would lead to a better understanding of the development of GD. Attempts to isolate anti-TSH-R antibodies from patients have proved to be difficult due to the exceedingly low serum levels due to rarity of these B cells, together with difficulties in obtaining purified TSH-R capable of interacting with patients autoantibodies. We employed phage antibody display technology and performed selection with a previously characterized semisynthetic antibody library on the purified extracellular ectodomain of the TSH-R. We report the isolation of six different anti-TSH-R monoclonal phage antibodies (moPhabs) from this library. All the moPhabs recognized TSH-R and its recombinant fragments by Western blotting, but failed to recognize the native TSH-R by flow cytometry. Consequently, the moPhabs did not lead to TSH-R activation. As these were the first moPhabs to TSH-R, they were analysed in terms of nucleotide and amino acid sequence and epitope specificity on the receptor. The moPhabs used immunoglobulin VH1 and VH3 germ line genes, all associated with Vlambda3 genes. Interestingly, the CDR3 regions of all moPhabs were remarkably similar, though not identical. In light of the common CDR3 usage, the epitopes recognized on TSH-R appeared to be restricted to amino acids residues 405-411 and 357-364. In summary, our results show that semisynthetic libraries may be limited in isolating human monoclonal antibodies that resemble pathogenic antithyrotropin receptor autoantibodies present in patients with GD. It is likely that until preparations of purified TSH-R that can be recognized by patients autoantibodies become available, similar to the recently described glycosylphosphatidylinositol (GPI) anchored TSH-R ectodomain, monoclonal antibodies from phage antibody display to TSH-R will be limited for isolating the rare, pathogenic antibodies of GD.

Animals↗

PubMed1995

Effects of alcohol and fluvastatin on lipid metabolism and hepatic function.

OBJECTIVE: To determine the effects of fluvastatin, a synthetic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, combined with moderate alcohol consumption on lipid profiles and hepatic function in patients with primary hypercholesterolemia. DESIGN: Randomized, placebo-controlled, crossover study. SETTING: Lipid clinic of a university hospital. PATIENTS: 31 patients with primary hypercholesterolemia (low-density lipoprotein cholesterol levels > or = 4.2 mmol/L) who had previously received a lipid-lowering diet. INTERVENTIONS: After a dietary baseline period, 26 patients were randomly assigned to receive 6 weeks of treatment with either 1) fluvastatin, 40 mg/d, added to 20 g of ethanol and diluted to 20% with orange juice or 2) fluvastatin added to orange juice alone. After a 6-week washout period, the two groups crossed over. MAIN OUTCOME MEASURES: Plasma fluvastatin levels, lipid levels, and clinical variables were determined at the end of each treatment period. RESULTS: Six patients left the study prematurely. The remaining patients (15 men, 5 women; mean age +/- SD 49.1 +/- 14.5 years; mean body mass index +/- SD 24.5 +/- 2.2 kg/m2) completed the study. Fluvastatin, alone and combined with alcohol, resulted in similar decreases in levels of total cholesterol (22% and 23%, respectively; P < 0.001 when compared with baseline), low-density lipoprotein cholesterol (28% and 29%, respectively; P < 0.001 compared with baseline), and apolipoprotein B (17% and 20%, respectively; P < 0.001 compared with baseline). High-density lipoprotein cholesterol and triglyceride levels were not changed. Fluvastatin with alcohol resulted in a significantly greater area under the plasma concentration curve (23.4 +/- 4.7 compared with 18.2 +/- 3.2 x 10(3) ng.min/mL) and in a greater time to maximum concentration (187.5 +/- 16.6 min compared with 130.9 +/- 7.0 min) than fluvastatin alone. Terminal half-life tended to increase. No important adverse clinical effects were observed. CONCLUSION: Six weeks of daily, moderate alcohol consumption influenced the metabolism of fluvastatin but did not interfere with its lipid-lowering efficacy and had no adverse effects.

Adult↗

PubMed1995

Apolipoprotein E2/E3/E4 genotyping with agarose gels.

Here we report an improved, simple method to assign the human apolipoprotein (apo) E genotype and its isoforms, apo E2, apo E3, and apo E4. Genomic DNA was amplified with specific primers that included the polymorphic region of amino acids 112 and 158. Digestion of the product with Hhal resulted in unique fragments that were separated on Meta-Phor agarose instead of polyacrylamide. The pattern of unique DNA fragments obtained unequivocally characterizes the different apo E alleles.

Apolipoprotein E2↗

PubMed1994

Postprandial apolipoprotein B100 and B48 metabolism in familial combined hyperlipidaemia before and after reduction of fasting plasma triglycerides.

Hepatic VLDL overproduction in familial combined hyperlipidaemia (FCH) may delay the clearance of atherogenic apolipoprotein (apo) B containing particles. We investigated if normalization of fasting plasma triglycerides (TG) by hypolipidaemic treatment results in improved metabolism of apo B48 and apo B100 in six male subjects with FCH and compared them to six normolipidaemic controls. The FCH patients were studied before (TG, 5.2 +/- 1.2 mmol l-1; mean +/- SEM) and after therapy (TG, 2.1 +/- 0.3 mmol l-1) with either simvastatin (n = 4) or combined therapy with gemfibrozil (n = 2). The postprandial changes of apo B100 and apo B48 were studied after a single oral fat meal (24 h; 50 gram fat m-2). Changes in triglyceride rich particles (TRP; d < 1.006 g ml-1) and remnant fractions (REM; d:1.006-1.019 g ml-1) of apo B were quantitated by scanning silverstained SDS-PAGE (4-15%). Apo B48 in fasting TRP in untreated and treated FCH was 15% and 14% of total apo B, and 6% in controls (P < 0.05). In controls, postprandial B48 increased maximally at 4 h by 81% in TRP and by 137% in REM compared to baseline. In treated FCH, the postprandial apo B48 pattern normalized in TRP compared to the untreated state. Postprandial apo B100 in controls decreased in TRP and REM by 33% and 18% (P < 0.05). In untreated and treated FCH, postprandial apo B100 remained unchanged vs. baseline in TRP and in REM suggesting hypersecretion of VLDL. The elimination of B100--assessed as area under the curve--in TRP (32.5 +/- 3.6 au.h; mean +/- SEM) and REM fractions (33.2 +/- 3.1 au.h), improved significantly after treatment (21.0 +/- 2.8 and 20.4 +/- 3.3 au.h, respectively). The apo B48 clearance in TRP fractions was improved after treatment (4.3 +/- 1.4 au.h vs. 2.9 +/- 1.2 au.h; P = 0.06), but not in REM fractions (2.8 +/- 1.0 au.h vs. 1.8 +/- 0.5 au.h; NS). In conclusion, in FCH subjects with apo B100 hypersecretion and increased fasting plasma apo B48 levels, reduction of fasting plasma TG improved, but did not normalize, TRP apo B48 and B100 metabolism. However, therapy normalized postprandial apo B100 remnant metabolism. Impaired postprandial apo B metabolism may be instrumental in the development of premature atherosclerosis in FCH subjects.

Adult↗

PubMed1991

Postprandial decrease in HDL cholesterol and HDL apo A-I in normal subjects in relation to triglyceride metabolism.

The postprandial lipoprotein metabolism is important since it determines the circulation of potentially atherogenic particles and influences the metabolism of high-density lipoproteins (HDL) in a complex manner that is at present not completely understood. Therefore, the short-term (24-h) changes in postprandial lipoprotein metabolism, including retinyl palmitate (RP), apolipoprotein A-I (apo A-I), and apolipoprotein B, were studied in relation to postheparin lipolytic activities in six healthy normolipidemic men after an oral RP fat tolerance test. The fat load (98 g) was cleared in 7 h, because the triglyceride (TG) concentrations had returned to initial values (0.72 +/- 0.31 mmol/l) at that time. RP showed a peak in plasma at 4 and 5 h but remained present in chylomicron (remnants) in low concentrations after 8 and 24 h. After the fat load, HDL cholesterol and HDL-associated apo A-I showed a significant decrease in concentration of 35 and 29%, respectively. The decrease coincided with the increase in chylomicron remnants and the transient appearance of TG-enriched HDL. Hepatic lipase was correlated to both the initial HDL cholesterol concentration as well as the peak concentration of TG in chylomicron remnants, suggesting that it could be one of the regulating common physiological pathways in postprandial HDL and TG metabolism. In the subjects studied, the atherogenic potential of plasma increased in response to an oral fat load, characterized by a decrease in HDL cholesterol and HDL-associated apo A-I.

Adult↗

PubMed1990

Proteolysis of human apolipoprotein B: effect on quantitative immunoturbidimetry.

Apolipoprotein (Apo) B100 is the obligatory structural protein of low and very low density lipoproteins. We studied the appearance of a kallikrein-induced proteolytic fragment of Apo B100, identified by sodium dodecyl sulphate (SDS)-polyacrylamide electrophoresis as Apo B74, in plasma samples obtained from 7 men and 7 women. We also studied the effect of Apo B74 on total Apo B immunoreactivity by immunoturbidimetry. Apo B74 was found in plasma from 4 of the 7 women when the samples had been stored at 4 degrees C in glass or plastic tubes, but in some of the samples from the men. These 4 women used oral estrogens. The plasma samples which contained Apo B74 showed evidence of kallikrein activation. Presence of Apo B74 resulted in an underestimation of total Apo B immunoreactivity by 13% (P less than 0.05).

Adult↗

PubMed1990

Heterogeneity of TSH receptor-binding antibodies in Hashimoto's thyroiditis and Graves' disease.

The possible heterogeneity of TSH receptor antibodies in Graves' disease (GD) and Hashimoto's thyroiditis (HT) with respect to the binding site on the receptor and corresponding biological effect was studied. Employing an immunoprecipitation assay (IPA), the sera of 80% of the patients with GD (24 out of 30) and 76% of the patients with HT (16 out of 21) contained TSH receptor-binding antibodies, compared to none of the sera from 17 normal volunteers and 8 patients with nontoxic multinodular goiter. TSH inhibited immunoprecipitation by GD and HT sera. In HT sera (n = 9), but not in GD sera (n = 5), heterogeneity of the TSH-induced inhibition was observed. Four HT sera showed complete inhibition of immunoprecipitation at a saturating concentration (19.8 nM) of TSH. Five HT sera, like the 5 GD sera, showed partial inhibition of immunoprecipitation by 19.8 nM TSH. Thyroid stimulating immunoglobulins (TSI) were found in four of the five GD sera and in only one of the nine HT sera. The results suggest that different subpopulations of TSH receptor antibodies, characterized by other receptor binding sites or different affinities, are associated with autoimmune thyroid disease.

Adult↗

PubMed1990

Thyrotropin dependent and independent thyroid cell lines selected from FRTL-5 derived tumors grown in nude mice.

FRTL-5 cells were used to set up a thyroid tumor model system in C3H nu/nu mice. FRTL-5 tumors could be grown in nude mice provided serum TSH levels were elevated. Persistent TSH elevation was obtained by administration of Na131I, rendering the mice hypothyroid. After 4 weeks FRTL-5 cells were injected sc resulting in tumor growth within 2 weeks in eight out of eight mice. Although the tumors showed an apparently undifferentiated histology, lacking normal follicular structures, they were functional since the tumors were capable of concentrating [131]iodine, as demonstrated by nuclear imaging. From one of the tumors a new cell line was isolated (FRTL-5/T) that, like the parental FRTL-5 cell line, was TSH dependent for growth. In a control group of six euthyroid nude mice FRTL-5 tumor growth could not be obtained with one exception. After 3 months one animal developed a small tumor that grew rapidly thereafter. This tumor was easily transplantable in other euthyroid nude mice, showed an undifferentiated histology, and was nonfunctional, as it could not concentrate [131]iodine. From this tumor two cell lines were derived: one cultured in the presence of TSH (FRTL-5/TP) and one in the absence of TSH (FRTL-5/TA). Both cell lines were found to be TSH independent for growth. The cell lines were analyzed for TSH responsive functions and TSH receptor expression. Responsiveness to TSH in FRTL-5/T and the parental FRTL-5 cell line were similar for most thyroid specific functions tested. However, FRTL-5/T was less sensitive than FRTL-5 for TSH induced [3H]thymidine incorporation. Both cell lines had two classes of TSH binding sites with high and low affinity respectively, as determined by Scatchard analysis. FRTL-5/TP and FRTL-5/TA were both able to grow in TSH free medium and were nonresponsive to TSH in vitro, as tested for [3H]thymidine and [3H]uridine incorporation, iodine uptake, thyroglobulin iodination, and thyroglobulin secretion. This correlated with an approximately 100-fold decreased number of TSH binding sites compared to FRTL-5. The latter was caused by a complete absence of low affinity binding sites, whereas high affinity receptors were still detectable. The FRTL-5/TA cell line was the least differentiated one as thyroglobulin mRNA was detectable in only minute amounts and thyroid peroxidase expression could not be measured. These in vivo selected FRTL-5 cell lines offer a suitable model to investigate several aspects of TSH responsiveness, including signal transduction and postreceptor events, thyroid differentiation, and thyroid tumorigenesis.

Animals↗

PubMed1988

Graves' disease: changes in TSH receptor and anti-microsomal antibodies after thyroidectomy.

It has been suggested that intrathyroidal plasma cells are the major site of TSH receptor antibody synthesis in Graves' disease (GD). To investigate this hypothesis, serial serum levels of TSH receptor antibodies, as determined by a recently developed immunoprecipitation assay (IPA), were measured in 13 patients with GD before and after subtotal thyroidectomy (Tx). Pretreatment TSH receptor antibody levels (86.4 +/- 26.8 pM/ml; mean +/- s.d.) decreased significantly due to antithyroid drug therapy to 60.3 +/- 21.5 pM/ml (P less than 0.05) just before Tx. However, 4 h after Tx, there was a significant increase (84.9 +/- 35.5 pM/ml; P less than 0.05) due to leakage of antibodies from the manipulated thyroid gland. After Tx, no major decrease in autoantibody levels occurred; TSH receptor antibodies were still detectable 1 to 7 months after Tx (88.7 +/- 44.6 pM/ml). Neither an acute change nor an overall reduction in anti-microsomal (M) antibody titres was observed after Tx. Our conclusions are that serum TSH antibody levels exhibited a drug-induced decrease before Tx and an immediate, but transient, post-operative increase. The post-operative increase was caused by leakage of preformed TSH receptor antibodies from the manipulated thyroid gland. Several months after Tx, TSH receptor and anti-M antibodies were present in the same serum concentrations as before treatment. The present data does not support the hypothesis that plasma cells in the thyroid gland are the major site of autoantibody synthesis in GD at the time of Tx.

Adolescent↗

PubMed1986

Further evaluation of an immunoprecipitation assay for TSH-receptor autoantibodies in Graves' disease.

In ten patients with untreated Graves' disease, quantitative titers of TSH receptor antibodies, as measured by a recently developed immunoprecipitation assay (IPA), were correlated with results obtained in three other methods and with the severity of the hyperthyroidism, as assessed by thyroid function tests. Nine patients were positive in the IPA, six in the TBII (TSH-binding inhibitor immunoglobulins), six in the TSI (thyroid-stimulating immunoglobulins), and seven in the TGI (thyroid-growth stimulating immunoglobulins) assay. Three patients were positive in all four assays. No correlation was found between the IPA values and the results obtained in the TBII, TSI, or TGI assays. There was a modest correlation between the TBII and TSI assays (r = .74, P less than 0.02). There was a modest but significant correlation between the IPA titers of TSH receptor antibodies and serum T3 concentration, both before treatment (r = .63, P less than 0.05) and during treatment (n = 5; r = .84, P less than 0.05). No correlation between the severity of the hyperthyroidism and TBII, TSI, or TGI assays was observed. Finally, TGI did not correlate with goiter size as estimated by palpation. These results suggest that the IPA may be useful in monitoring the immunologic activity of TSH receptor antibodies in patients with Graves' disease. They suggest further that the IPA detects a number of different subpopulations of TSH receptor antibodies, including TSI, TBII, and perhaps TGI. This property may be particularly useful in screening for monoclonal TSH receptor antibodies.

Adult↗