Biomedical subjects

O Bakker

Publications and source records attributed to O Bakker.

At least 19 recordsLinked to original sources

PubMed2006

PGC-1alpha regulates the isoform mRNA ratio of the alternatively spliced thyroid hormone receptor alpha transcript.

Transcripts derived from the thyroid hormone receptor alpha (TRalpha) gene are alternatively spliced resulting in a functional receptor TRalpha1 and a non-T3-binding variant TRalpha2 that can exert a dominant negative effect on the transactivation functions of other TRs. There is evidence that the ratio of TRalpha isoform transcripts can be modulated and here, we investigate whether the PPARgamma co-activator alpha (PGC-1alpha) has an effect on this splicing process. PGC-1alpha was discovered not only as a transcriptional co-activator, but also has certain motifs characteristic of splicing factors. We demonstrate that PGC-1alpha alters the ratio of endogenously expressed TRalpha isoform transcripts in HepG2 cells, by decreasing TRalpha1 mRNA levels twofold. This change in isoform ratio is accompanied by a decrease in 5'-deiodinase expression, whereas no differences were found in TRbeta1 expression. Deletion of the RNA-processing domain of PGC-1alpha abrogated the effect on the TRalpha splicing, whereas expression of only the RNA-processing domain favored TRalpha1 expression. PGC-1alpha showed a similar effect on the splicing of a TRalpha minigene containing only the last four exons and introns of the TRalpha gene. These data suggest that PGC-1alpha is involved in the RNA processing of TRalpha transcripts.

Alternative Splicing↗

PubMed2006

Expression pattern and ontogenesis of thyroid hormone receptor isoforms in the mouse heart.

Nuclear thyroid hormone (T3) receptors (TR) play a critical role in mediating the effects of T3 on development, differentiation and normal physiology of many organs. The heart is a major target organ of T3, and recent studies in knockout mice demonstrated distinct effects of the different TR isoforms on cardiac function, but the specific actions of TR isoforms and their specific localization in the heart remain unclear. We therefore studied the expression of TRalpha1, TRalpha2 and TRbeta1 isoforms in the mouse heart at different stages of development, using monoclonal antibodies against TRalpha1, TRalpha2 and TRbeta1. In order to identify distinct components of the embryonic heart, in situ hybridization for cardiac-specific markers was used with the expression pattern of sarcoplasmic reticulum calcium-ATPase 2a as a marker of myocardial structures, while the pattern of expression of connexin40 was used to indicate the developing chamber myocardium and peripheral ventricular conduction system. Here we show that in the ventricles of the adult heart the TRbeta1 isoform is confined to the cells that form the peripheral ventricular conduction system. TRalpha1, on the other hand, is present in working myocardium as well as in the peripheral ventricular conduction system. In the atria and in the proximal conduction system (sinoatrial node, atrio-ventricular node), TRalpha1 and TRbeta1 isoforms are co-expressed. We also found the heterogeneous expression of the TRalpha1, TRalpha2 and TRbeta1 isoforms in the developing mouse heart, which, in the case of the TRbeta1 isoform, gradually revealed a dynamic expression pattern. It was present in all cardiomyocytes at the early stages of cardiogenesis, but from embryonic day 11.5 and into adulthood, TRbeta1 demonstrated a gradual confinement to the peripheral ventricular conduction system (PVCS), suggesting a specific role of this isoform in the formation of PVCS. Detailed knowledge of the distribution of TRalpha1 and TRbeta1 in the heart is of importance for understanding not only their mechanism of action in the heart but also the design and (clinical) use of TR isoform-specific agonists and antagonists.

Animals↗

PubMed2005

Functional thyrotropin receptor expression in the pituitary folliculo-stellate cell line TtT/GF.

Thyrotropin secretion from the anterior pituitary is regulated mainly through TRH and thyroid hormones. Recent findings of a TSH receptor (TSHR) on folliculo-stellate (FS) cells in the human anterior pituitary indicate that TSH secretion might, in addition, be regulated in a paracrine manner via FS cells. In order to elucidate the physiological relevance of TSHR expression in FS cells we evaluated the effects of TSH on a murine FS cell line, TtT/GF. First, Western blot analysis confirmed the expression of TSHR protein in these cells. Second, three potential second messenger pathways were studied. Last, cDNA array hybridization was used to evaluate the effect of TSH on gene expression levels. TSH failed to induce either the adenylate cyclase/cAMP pathway, the phosphatidylinositol/calcium pathway, or the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) 3 pathway. Most of the genes regulated by TSH were related to cell proliferation, cell differentiation, and apoptosis. Moreover, TSH induced STAT5a and TGFbeta2 expression. We report that TtT/GF cells express a functional TSHR that is not coupled to cAMP nor IP (3) but probably signals through the JAK/STAT5a pathway. Functional TSHR expression in this cell line offers an in vitro model to study the role of TSHR in FS cells.

Adenylyl Cyclases↗

PubMed2004

Hyper and hypothyroidism change the expression and diurnal variation of thyroid hormone receptor isoforms in rat liver without major changes in their zonal distribution.

We investigated the effect of hypothyroidism or hyperthyroidism on mRNA and protein expression, diurnal variation and zonal distribution of thyroid hormone receptor (TR) isoforms TRalpha1, TRalpha2 and TRbeta1 in rat liver. Hypothyroidism results in increased isoform mRNA and protein expression whereas hyperthyroidism shows a decreased TRalpha1 and TRalpha2 mRNA and protein expression. During hyperthyroidism no change is seen in TRbeta1 mRNA, but TRbeta1 protein is upregulated in the light period and downregulated in the dark period. Diurnal changes (measured at 13:30 and 19:30 h) in the TR isoform proteins are abolished in hypothyroidism and hyperthyroidism, with the exception of a reversal in diurnal changes of TRbeta1 in hyperthyroidism. Zonal distribution of the isoforms is not affected by hypo- or hyperthyroidism. We therefore conclude that thyroid hormone influences both the levels and the diurnal expression of its receptor isoforms but not the zonal distribution.

Animals↗

PubMed2004

Expression of adenohypophyseal-hormone receptors in a murine folliculo-stellate cell line.

Adenohypophyseal-hormone production is regulated by hypothalamic peptides and target-gland hormones. Additionally, paracrine regulation by folliculo-stellate cells within the pituitary has been suggested. We recently showed TSH receptor expression in human folliculo-stellate cells and speculated that receptors for other adenohypophyseal hormones might also be expressed by folliculo-stellate cells. Using RT-PCR, we evaluated the expression of receptors for TSH, GH, ACTH, LH, FSH and PRL in a murine folliculo-stellate cell line, TtT/GF. Transcripts of TSH receptor, GH receptor and ACTH receptor were detected in this cell line. LH receptor, FSH receptor and PRL receptor expression, however, could not be demonstrated. We conclude that the TtT/GF cells express some, but not all, receptors for anterior pituitary hormones. This indicates that folliculo-stellate cells might act as mediators in the paracrine regulation of at least some of the hormones secreted by the anterior pituitary.

Animals↗

PubMed2003

Diurnal variation in rat liver thyroid hormone receptor (TR)-alpha messenger ribonucleic acid (mRNA) is dependent on the biological clock in the suprachiasmatic nucleus, whereas diurnal variation of TR beta 1 mRNA is modified by food intake.

Previous studies have shown a diurnal variation of certain isoforms of thyroid hormone receptors (TR) in rat liver. The genesis of these diurnal changes is still unknown. To clarify whether the biological clock, located in the hypothalamic suprachiasmatic nucleus (SCN), is involved, we made selective SCN lesions. Rats with an SCN lesion lost their circadian rhythm of plasma corticosterone and TSH when compared with intact animals. TR alpha 1 and TR alpha 2 mRNA expression of control rats was higher in the light period than in the dark period; changes that were abolished in the rats with SCN lesions. In contrast, liver TR beta 1 mRNA of intact rats showed a diurnal variation that failed to reach statistical significance. To evaluate whether these effects could be explained indirectly by the disappearance of rhythmic feeding behavior in rats with SCN lesions, we performed a second experiment in which otherwise intact animals were subjected to a regular feeding (RF) schedule, with one meal every 4 h. When compared with rats with free access to food, RF only affected TR beta 1 mRNA expression and had no effect on the diurnal changes in TR alpha 1 and TR alpha 2. We conclude that liver TR beta 1 expression is most clearly affected by food intake. Diurnal changes in liver TR alpha 1 and TR alpha 2 are controlled by the biological clock in the SCN but not via changes in the daily rhythm of food intake. The findings may have physiological relevance for diurnal variation of T(3)-dependent gene expression, which is supported by a diurnal variation in the expression of the 5'-deiodinase gene.

Animals↗

PubMed2003

TSH-R expression and cytokine profile in orbital tissue of active vs. inactive Graves' ophthalmopathy patients.

OBJECTIVE: From in vitro studies using cultures of orbital fibroblasts, it has become clear that cytokines play an important role in the orbital inflammation in Graves' ophthalmopathy (GO). Orbital fibroblasts seem to be the key target cells of the autoimmune attack, and they are able to express the TSH receptor (TSH-R). In vivo data on the presence of cytokines in orbital tissues are sparse, and mostly limited to samples obtained from patients with endstage, inactive GO; the same holds true for the presence of the TSH-R. The aim of the present study was to determine whether the cytokine profile and TSH-R expression differ in the active vs. the inactive stage of GO. DESIGN AND MEASUREMENTS: Orbital fat/connective tissue was obtained from six patients with active, untreated GO undergoing emergency orbital decompression, and from 11 patients with inactive GO subjected to rehabilitative decompressive surgery. The mRNA levels of various cytokines and the TSH-R were assessed by real-time polymerase chain reaction (PCR) using the LightCycler. Data are expressed as ratios (unknown mRNA/beta-actin mRNA). RESULTS: Active GO patients had much higher TSH-R expression than inactive patients: 4/0-24 (median value/range) vs. 0/0-9, P = 0.01. TSH-R expression was related to the Clinical Activity Score (r = 0.595, P = 0.015). Patients with active GO compared to those with inactive GO had higher mRNA levels of the proinflammatory cytokines interleukin-1beta (IL-1beta) (445/153-877 vs. 0/0-455, P = 0.001), IL-6 (1583/968-18825 vs. 559/0-7181, P = 0.01), IL-8 (1422/38-7579 vs. 32/0-1081, P = 0.046) and IL-10 (145/58-318 vs. 27/0-189, P = 0.002). In active GO there also existed a trend towards a predominance of T helper 1 (Th1)-derived cytokines as evident from higher IL-2 (37/0-158 vs. 0/0-68, P = 0.043), interferon-gamma (IFN-gamma) (20/0-79 vs. 0/0-16, P = 0.12) and IL-12 (2.3/0-14.8 vs. 0/0-1.6, P = 0.10) mRNAs. IL-1 receptor agonist (IL-1RA), IL-2 receptor (IL-2R), IL-3, IL-4, IL-5, IL-13, IL-18 and tumour necrosis factor-alpha (TNF-alpha) mRNAs were similar in both groups. CONCLUSIONS: These data show that at the mRNA level, TSH-R expression is largely present only during the active stages of GO. The active phase is characterized by the presence of proinflammatory and Th1-derived cytokines, whereas other cytokines, among them Th2-derived cytokines, do not seem to be linked to a specific stage of GO.

Acute Disease↗

PubMed2003

Dronerarone acts as a selective inhibitor of 3,5,3'-triiodothyronine binding to thyroid hormone receptor-alpha1: in vitro and in vivo evidence.

Dronedarone (Dron), without iodine, was developed as an alternative to the iodine-containing antiarrhythmic drug amiodarone (AM). AM acts, via its major metabolite desethylamiodarone, in vitro and in vivo as a thyroid hormone receptor alpha(1) (TRalpha(1)) and TRbeta(1) antagonist. Here we investigate whether Dron and/or its metabolite debutyldronedarone inhibit T(3) binding to TRalpha(1) and TRbeta(1) in vitro and whether dronedarone behaves similarly to amiodarone in vivo. In vitro, Dron had a inhibitory effect of 14% on the binding of T(3) to TRalpha(1), but not on TRbeta(1). Desethylamiodarone inhibited T(3) binding to TRalpha(1) and TRbeta(1) equally. Debutyldronedarone inhibited T(3) binding to TRalpha(1) by 77%, but to TRbeta(1) by only 25%. In vivo, AM increased plasma TSH and rT(3), and decreased T(3). Dron decreased T(4) and T(3), rT(3) did not change, and TSH fell slightly. Plasma total cholesterol was increased by AM, but remained unchanged in Dron-treated animals. TRbeta(1)-dependent liver low density lipoprotein receptor protein and type 1 deiodinase activities decreased in AM-treated, but not in Dron-treated, animals. TRalpha(1)-mediated lengthening of the QTc interval was present in both AM- and Dron-treated animals. The in vitro and in vivo findings suggest that dronedarone via its metabolite debutyldronedarone acts as a TRalpha(1)-selective inhibitor.

Amiodarone↗

PubMed2003

Triiodothyronine affects the alternative splicing of thyroid hormone receptor alpha mRNA.

The c-erbAalpha gene encodes two thyroid hormone receptors, TRalpha1 and TRalpha2, that arise from alternative splicing of the TRalpha pre-mRNA. TRalpha2 is not able to bind triiodothyronine (T(3)) and acts as a weak antagonist of TRs. It has been suggested that the balance of TRalpha1 to TRalpha2 is important in maintaining homeostasis. Here, we study the effect of thyroid hormone on the splicing of TRalpha under various conditions in HepG2 cells. First, T(3) was added to HepG2 cells that endogenously express TRalpha. This resulted in a decrease in the TRalpha1:TRalpha2 mRNA ratio after the addition of 10(-)(8 )M or 10(-)(7 )M T(3). Then, HepG2 cells were incubated with sera from hypothyroid or hyperthyroid patients. Sera from hyperthyroid patients (n=6) decreased the TRalpha1:TRalpha2 ratio compared with HepG2 cells incubated with sera from euthyroid patients (n=8). Sera from hypothyroid patients (n=6) had no effect on the TRalpha1:TRalpha2 ratio but supplementation with T(3) caused a decrease in the ratio. Finally, we tested sera from patients with nonthyroidal illness (NTI; n=17) which showed no effect on TRalpha splicing when compared with controls. Free thyroxine levels in sera from hypo-, eu-, and hyperthyroid patients, but not that of NTI patients, were negatively correlated (P<0.01) to the TRalpha1:TRalpha2 ratio. We next studied the expression of the splicing factors hnRNP A1 and ASF/SF2 (SF2) in relation to the splicing of the TRalpha gene. In HepG2 cells incubated with NTI sera a negative relationship was found between the ratio of hnRNP A1:SF2 and the TRalpha1:TRalpha2 ratio. A high hnRNP A1:SF2 ratio is associated with the use of the distal 5'-splice site. The splicing direction should then change towards TRalpha2, which is indeed the case. Rev-ErbA, which is partly complementary to TRalpha2 and could therefore interfere in the splicing process, did not relate to the TRalpha1:TRalpha2 ratio. In conclusion, high T(3) levels induce a low TRalpha1:TRalpha2 ratio which could protect the cell from excessive T(3)-induced gene expression. In vivo, this might be a mechanism to keep tIssues relatively euthyroid during high serum T(3) levels.

Alternative Splicing↗

PubMed2003

Zonal expression of the thyroid hormone receptor alpha isoforms in rodent liver.

Many metabolic processes occur simultaneously in the liver in different locations along the porto-central axis of the liver units. These processes are often regulated by hormones, one of which is thyroid hormone which for its action depends on the presence of the different isoforms of the thyroid hormone receptor (TR). These are encoded by two genes: c-erbA-alpha encoding TRalpha1 and TRalpha2 and their respective Delta isoforms, and c-erbA-beta which encodes TRbeta1, TRbeta2 and TRbeta3. We recently found a zonal (pericentral) expression of and a diurnal variation in the TRbeta1 isoform in rat liver. We were therefore also interested to see whether TRalpha1 and TRalpha2 expression showed similar characteristics. For this reason we raised both polyclonal and monoclonal antibodies against TRalpha1 and TRalpha2 isoforms and characterised these. Antibody specificity was tested using Western blots and immunohistochemistry in liver of TR isoform-specific knockout animals. Using these antibodies we found that the TRalpha1 and TRalpha2 isoforms are zonally expressed around the central vein in rat liver. The experiments show that the portal to central gradient of TRalpha1 is broader than that of TRbeta1. Moreover, the expression of the TRalpha2 protein showed a diurnal variation with a peak in the afternoon when the animals are least active whereas no such variation was found for the TRalpha1 protein. From our data it appears that both the TRalpha1 and TRalpha2 isoforms show a zonal distribution in liver. This finding, together with the observed diurnal rhythm, has major implications for interpreting and timing experiments concerning the TR and its downstream actions in liver.

Animals↗

PubMed2002

Peri-ischemic aminoguanidine fails to ameliorate neurologic and histopathologic outcome after transient spinal cord ischemia.

Inhibition of neurotoxic events that lead to delayed cellular damage may prevent motor function loss after transient spinal cord ischemia. An important effect of the neuroprotective substance aminoguanidine (AG) is the inhibition of inducible nitric oxide synthase (iNOS), a perpetrator of focal ischemic damage. The authors studied the protective effects of AG on hind limb motor function and histopathologic outcome in an experimental model for spinal cord ischemia, and related these findings to the protein content of iNOS in the spinal cord. Temporary spinal cord ischemia was induced by 28 minutes of infrarenal balloon occlusion of the aorta in 40 anesthetized New Zealand White rabbits. Animals were assigned randomly to two treatments: saline (n = 20) or AG (n = 20; 100 mg/kg intravenously before occlusion). Postoperatively, treatment was continued with subcutaneous injections twice daily (saline or 100 mg/kg AG). Normothermia (38 degrees C) was maintained during ischemia, and rectal temperature was assessed before and after subcutaneous injections. Animals were observed for 96 hours for neurologic evaluation (Tarlov score), and the lumbosacral spinal cord was examined for ischemic damage after perfusion and fixation. Lastly, iNOS protein content was determined using Western blot analysis 48 hours after ischemia in five animals from each group. Neurologic outcome at 96 hours after reperfusion was the same in both groups. The incidence of paraplegia was 67% in the saline-treated group versus 53% in the AG-treated group. No differences in infarction volume, total number of viable motoneurons, or total number of eosinophilic neurons were present between the groups. At 48 hours after reperfusion, iNOS protein content in the spinal cord was increased in one animal in the AG-treated group and in three animals in the control group. The data indicate that peri-ischemic treatment with high-dose AG in rabbits offers no protection against a period of normothermic spinal cord ischemia. There was no conclusive evidence of spinal cord iNOS inhibition after treatment with AG.

Animals↗

PubMed2002

Specific detection of type III iodothyronine deiodinase protein in chicken cerebellar purkinje cells.

Because iodothyronine deiodinases play a crucial role in the regulation of the available intracellular T(3) concentration, it is important to determine their cellular localization. In brain, the presence of type III iodothyronine deiodinase (D3) seems to be important to maintain homeostasis of T(3) levels. Until now, no cellular localization pattern of the D3 protein was reported in chicken brain. In this study polyclonal antisera were produced against specific peptides corresponding to the D3 amino acid sequence. Their use in immunocytochemistry led to the localization of D3 in the Purkinje cells of the chicken cerebellum. Both preimmune serum as well as the primary antiserum exhausted with the peptide itself were used as negative controls. Extracts of chick cerebellum and liver were made in the presence of Triton X-100 to solubilize the membrane-bound deiodinases. Using these extracts in Western blot analysis, a band of the expected molecular weight ( approximately 30 kDa) could be detected in both tissues. Using a full-length (32)P-labeled type III deiodinase cRNA probe, we identified a single mRNA species in the cerebellum that was of the exact same size as the hepatic control mRNA (+/-2.4 kb). RT-PCR, followed by subcloning and sequence analysis, confirmed the expression of D3 mRNA in the chicken cerebellum. In this study we provide the first evidence of the presence of the D3 protein in a neuronal cell type, namely Purkinje cells, by means of immunocytochemical staining. We were able to detect a protein fragment corresponding to the expected molecular mass (30 kDa) for type III deiodinase by means of Western blot analysis. RT-PCR as well as Northern blot analysis confirmed the presence of D3 mRNA in the cerebellum.

Animals↗

PubMed2001

The suprachiasmatic nucleus generates the diurnal changes in plasma leptin levels.

At present it is not clear which factors are responsible for the diurnal pattern of plasma leptin levels, although the timing of food intake and circulating hormones such as glucocorticoids and insulin have both been proposed as independent determinants. In this study we show that ablation of the biological clock by thermal lesions of the hypothalamic suprachiasmatic nucleus (SCN) completely eliminates the diurnal pattern of plasma leptin levels. By contrast, removal of the diurnal corticosterone signal by adrenalectomy and corticosterone replacement did not affect diurnal plasma leptin levels. More importantly, removal of the nocturnal feeding signal by submitting the animals to a regular feeding schedule of six meals per day did not abolish the diurnal plasma leptin levels. However, both SCN lesions and the regular feeding schedule did cause an increase in the 24-h mean plasma leptin levels. As neither rhythmic feeding, insulin, or corticosterone signals can completely explain the diurnal plasma leptin rhythm, we conclude that biological clock control of the sympathetic input to the adipocyte is essential for regulation of the daily rhythm in leptin release.

Adipocytes↗

PubMed2001

The biological relevance of thyroid hormone receptors in immortalized human umbilical vein endothelial cells.

The gene expression of thyroid hormone receptors (TR) in ECRF24 immortalized human umbilical vein endothelial cells (HUVECs) was investigated at both the mRNA and the protein level. Endothelin-1 (ET-1) and von Willebrand factor (vWF) production were measured in response to triiodothyronine (T(3)) administration. A real-time PCR technique was used to quantify the presence of mRNAs encoding for the different isoforms of the TR. The binding of T(3) to nuclear TRs was studied in isolated endothelial cell nuclei by Scatchard analysis. Expression of TR at the protein level was investigated by immunocytochemistry and Western blotting using TR-isoform-specific polyclonal rabbit antisera. ET-1 and vWF were measured in cell supernatants with a two-site immunoenzymatic assay. Scatchard analysis yielded a maximum binding capacity of 55 fmol T(3)/mg DNA (+/-200 sites/cell) with a K(d) of 125 pmol/l. Messenger RNAs encoding for the TRalpha1 and the TRalpha2 and the TRbeta1 were observed. The approximate number of mRNA molecules per cell was at least 50 molecules per cell for TRalpha1, five for TRalpha2 and two for TRbeta1. Immunocytochemistry revealed (peri)nuclear staining for TRbeta1, TRalpha1 and TRalpha2. ET-1 and vWF secretion did not increase upon addition of T(3) (10(-10)-10(-6) M). Immortalized ECRF24 HUVECs express TR, but at low levels. The number of TRs per endothelial cell is probably too low to be functional and no change in ET-1 or vWF production was found after addition of T(3). Therefore we conclude that the genomic effects of T(3) are unlikely to occur in these immortalized HUVECs.

Blotting, Western↗

PubMed2000

Desethylamiodarone interferes with the binding of co-activator GRIP-1 to the beta 1-thyroid hormone receptor.

Ligand binding to the thyroid hormone nuclear receptor beta1 (TRbeta(1)) is inhibited by desethylamiodarone (DEA), the major metabolite of the widely used anti-arrhythmic drug amiodarone. Gene expression of thyroid hormone (triiodothyronine, T(3))-regulated genes can therefore be affected by amiodarone due to less ligand binding to the receptor. Previous studies have indicated the possibility of still other explanations for the inhibitory effects of amiodarone on T(3)-dependent gene expression, probably via interference with receptor/co-activator and co-repressor complex. The binding site of DEA is postulated to be on the outside surface of the receptor protein overlapping the regions where co-activator and co-repressor bind. Here we show the effect of a drug metabolite on the interaction of TRbeta(1) with the co-activator GRIP-1 (glucocorticoid receptor interacting protein-1). The T(3)-dependent binding of GRIP-1 to the TRbeta(1) is disrupted by DEA. A DEA dose experiment showed that the drug metabolite acts like an antagonist under 'normal' conditions (at 10(-7) M T(3) and 5x10(-6)-->10(-3) M DEA), but as an agonist under extreme conditions (at 0 and 10(-9) M T(3) and >10(-4) M DEA). To our knowledge, these results show for the first time that a metabolite of a drug which was not devised for this purpose can interfere with nuclear receptor/co-activator interaction.

Amiodarone↗

PubMed2000

Association of thyrotrophin receptor antibodies with the clinical features of Graves' ophthalmopathy.

OBJECTIVE: Graves' ophthalmopathy (GO) and Graves' hyperthyroidism are closely associated diseases and thought to be caused by the same autoimmune process. An obvious explanation for this would be the presence of autoantibodies reacting with an autoantigen present in the orbit and the thyroid gland. The TSH-Receptor (TSH-R) antibodies are a likely candidate, because they cause Graves' hyperthyroidism and the TSH-R appears to be present also in orbital tissues. If TSH-R antibodies are responsible for the ophthalmopathy one would expect their titres to correlate with clinical characteristics of the eye disease. The aim of the present study is to see whether TSH-R antibodies are related to the activity and severity of the thyroid-associated ophthalmopathy. DESIGN AND PATIENTS: TSH-R antibody levels were measured as TBII (TRAK assay), and TSI (cAMP response of a TSH-R transfected cell line) in serum of 63 patients with untreated moderately severe GO, accompanying Graves' thyroid disease; all patients had been euthyroid for > 2 months. RESULTS: TBII and TSI titres were strongly related to each other. TBII or TSI titres did not correlate with thyroidal or orbital disease duration, nor with TPO antibody levels. In contrast, we found a striking and highly significant correlation between the Clinical Activity Score (CAS) of the eye disease, and both TBII (r = 0.54; P < 0.0001) and TSI (r = 0.50; P < 0.0001). In addition, a weaker but significant relation was found between proptosis (in mm) and TBII (r = 0.36; P = 0.004) and TSI (r = 0.49; P = 0.0001). No correlation was found with eye muscle motility. CONCLUSION: TSH-R antibody levels correlate directly with clinical features of Graves' ophthalmopathy. The results support the hypothesis of a pathogenetic role of TSH-R antibodies and the TSH-R in the orbit of Graves' ophthalmopathy patients.

Antibodies↗

PubMed2000

Effect of ischemic pretreatment on heat shock protein 72, neurologic outcome, and histopathologic outcome in a rabbit model of spinal cord ischemia.

OBJECTIVE: In the present study, we investigated the effect of ischemic pretreatment on heat shock protein 72 concentration and neurologic and histopathologic outcome after transient spinal cord ischemia. METHODS: In 28 New Zealand White rabbits, an aortic occlusion device was placed infrarenally. The animals were randomly assigned to 2 groups: ischemic pretreatment (n = 14 animals) and control (n = 14 animals). The duration of ischemic pretreatment was 6 minutes. After 24 hours, the aorta was occluded for 26 minutes in both groups of animals. Neurologic function was assessed 24 and 48 hours after the definite ischemic insult. At 48 hours, the animals were killed for histopathologic evaluation of the spinal cord. In a separate set of animals, heat shock protein 72 levels were determined in the lumbar spinal cord after both a 6- and 10-minute ischemic period, with the use of a Western blot analysis. RESULTS: No significant difference in neurologic outcome between the groups was observed at 24 and 48 hours. The incidence of paraplegia and severe paresis at 48 hours was 79% in the control group and 92% in the ischemic pretreatment group. There was no difference in histopathologic scores between the groups. Heat shock protein 72 could be clearly detected 1 and 2 days after 6- or 10-minute periods of spinal cord ischemia. CONCLUSIONS: In the present rabbit study, ischemic pretreatment could not induce tolerance against a moderately severe spinal cord ischemic insult, despite increased heat shock protein 72 levels after the preconditioning stimulus.

Animals↗

PubMed2000

Altered gene expression in Staphylococcus aureus upon interaction with human endothelial cells.

Staphylococcus aureus is isolated from a substantial number of patients with infective endocarditis who are not known to have predisposing heart abnormalities. It has been suggested that the infection is initiated by the direct binding of S. aureus to human vascular endothelium. To determine the mutual response of the endothelial cells and the bacteria, we studied the interaction between S. aureus and human vascular endothelium. Scanning electron microscopic analyses showed that binding of S. aureus to human umbilical vein endothelial cells (HUVEC) mainly occurred via thread-like protrusions extending from the cell surface. Bound bacteria appeared to be internalized via retraction of the protrusions into newly formed invaginations of the endothelial cell surface. The growth phase of S. aureus had a major impact on the interaction with HUVEC. Logarithmically growing bacteria showed increased binding to, and were more readily internalized by, HUVEC compared to stationary-phase bacteria. To assess the bacterial response to the cellular environment, an expression library of S. aureus was used to identify genes whose expression was induced after 4 h of exposure to HUVEC. The identified genes could be divided into different categories based on the functions of the encoded proteins (transport, catabolism, biosynthesis, and DNA repair). Further analyses of five of the S. aureus transposon clones showed that HUVEC as well as human serum are stimuli for triggering gene expression in S. aureus.

Aspartic Acid↗