Design and methods of the REMOVAL-HD study: a tRial Evaluating Mid cut-Off Value membrane clearance of Albumin and Light chains in HaemoDialysis patients.

BACKGROUND: Removal of uraemic toxins is inadequate using current dialysis strategies. A new class of dialysis membranes have been developed that allow clearance of larger middle molecules. The REMOVAL-HD study (a tRial Evaluating Mid cut-Off Value membrane clearance of Albumin and Light chains in HaemoDialysis patients) will address safety, efficacy and the impact on patient-centred outcomes with the use of a mid cut-off (MCO) dialyser in a chronic haemodialysis (HD) population. METHODS: REMOVAL-HD is an open label, prospective, non-randomised, single-arm, multi-centre device study in 85 chronic HD participants. All visits will be conducted during regular HD sessions and participants will undergo a 1 month wash-in period using a standardised high flux dialyser, 6 months of intervention with a MCO dialyser and 1 month of wash-out using a high flux dialyser. The primary endpoint is change in pre-dialysis concentrations of serum albumin, with secondary endpoints including the efficacy of clearance of free light chains and ß-2 microglobulin, and patient-centred outcomes including quality of life, symptom burden, functional status, nutritional status, hospitalisation and death. DISCUSSION: MCO dialysers are a novel form of HD membrane. The REMOVAL-HD study is a pivotal study designed to monitor the immediate and medium-term effects following exposure to this dialyser. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry Number (ANZCTRN) 12616000804482 . Date of registration - 21/06/2016.

Attribution of mental illness to work: a Delphi study.

BACKGROUND: Clinicians may be asked whether mental ill-health has been caused by work but there is no guidance on how this judgement should be made. AIMS: To seek a consensus on the factors that should be considered and how they should be sought when attributing mental ill-health to work. METHODS: A three-round Delphi study involving expert academics, occupational physicians, psychiatrists and psychologists. We deemed consensus had been reached when 66% or more of the experts were in agreement. RESULTS: Of 54 invited experts, 35 (65%) took part in the first round, 30 of these 35 (86%) in the second and 29 of these 30 (97%) in the final round. Consensus was reached for 11 workplace stressors: high job strain; effort-reward imbalance; major trauma; interpersonal conflict; inadequate support; role ambiguity; person-job mismatch; organizational injustice; organizational culture; work scheduling and threats to job security. Seven personal factors were identified as being important: previous mental illness; personality traits of neuroticism; adverse life events or social circumstances; resilience; a family history of mental illness and secondary gain. The worker, manager and co-workers were thought to be the most useful sources of workplace information. Consensus was reached for a definition of occupational mental illness but not for a threshold of work-relatedness. CONCLUSIONS: The attribution of mental ill-health to work is complex and involves the consideration of both workplace stressors and personal factors of vulnerability. Clinical consultation with an occupational physician who is familiar with the workplace is central to the process.

Role of the EGF receptor in PPARγ-mediated sodium and water transport in human proximal tubule cells.

AIM/HYPOTHESIS: This study aimed to determine the interaction between the EGF receptor (EGFR) and peroxisome proliferator-activated receptor γ (PPARγ) and the role of EGFR in sodium and water transport in the proximal tubule. METHODS: Primary human proximal tubule cells (PTCs) were exposed to high glucose in the presence and absence of pioglitazone. Total and phospho-EGFR levels and EGFR mRNA expression were determined by western blot and real-time PCR, respectively. Sodium-hydrogen exchanger-3 (NHE3), PPARγ and aquaporin 1 (AQP1) levels were determined by western blot. The role of EGFR was elucidated using the EGFR tyrosine kinase inhibitor, PKI166. The role of PPARγ in high-glucose conditions was determined using specific PPARγ small interfering (si)RNA. P-EGFR, PPARγ, AQP1 and NHE3 production in a rat model of diabetes (streptozotocin-induced hypertensive Ren-2 transgenic [mRen2]27 rats) and controls, with or without pioglitazone treatment, was determined by immunohistochemistry. The PPARγ and EGFR interaction was determined by chromatin immunoprecipitation assay, and the effect of pioglitazone on EGFR activation by luciferase assay. RESULTS: PTCs exposed to both high glucose and pioglitazone increased protein abundance of P-EGFR, NHE3, AQP1 and PPARγ. Pioglitazone-induced upregulation of NHE3 and AQP1 was abolished by PKI166. High-glucose-induced increases in P-EGFR, NHE3 and AQP1 were decreased with PPARγ siRNA. AQP1 and NHE3 but not PPARγ were increased in a diabetic rat model and further increased by pioglitazone treatment. Pioglitazone induced PPARγ binding to the EGFR promoter and subsequent downstream activation. CONCLUSIONS/INTERPRETATION: Our data suggest that EGFR activation mediates PPARγ-induced sodium and water reabsorption via upregulation of NHE3 and AQP1 channels in the proximal tubule. EGFR inhibition may be a therapeutic strategy in the treatment of diabetic nephropathy and in limiting salt and water retention, which currently restricts the use of PPARγ agonists.

Parents of children with cystic fibrosis: how they hope, cope and despair.

BACKGROUND: Cystic fibrosis is a chronic, life-threatening illness. Coping, vicarious hope and vicarious despair are constructs that may explain why some children and parents adjust well to cystic fibrosis, while others adjust poorly. Vicarious hope refers to parent expectations that desirable things will occur in their child's future, whereas vicarious despair refers to parent expectations that undesirable things will occur in their child's future. The aims of this study were: (1) to examine parent coping strategies and associations with child and parent adjustment to cystic fibrosis; (2) to investigate the effects of vicarious hope and vicarious despair on coping, parent adjustment and child adjustment; and (3) to examine distinctions between coping, vicarious hope and vicarious despair. METHODS: Participants were 35 parents of children with cystic fibrosis. RESULTS: Self-blame and behavioural disengagement were coping strategies associated with child and parent maladjustment. Social support predicted less parental emotional impact. Vicarious hope and vicarious despair predicted child mental health, parent anxiety and parent emotional impact. CONCLUSIONS: Results indicate that vicarious hope and vicarious despair are distinct constructs from coping. Interventions directed at parent coping, vicarious hope and vicarious despair are implicated.

Rational design of alpha-helical antifreeze peptides.

The alanine-rich alpha-helical antifreeze protein from the winter flounder Pseudopleuronectes americanus adsorbs to specific planes of ice guided by an ice lattice match to threonine residues regularly spaced 16.6 A apart. We report here that by redesigning the winter flounder antifreeze peptide to incorporate a 27.1-A spacing between putative 'ice-binding' threonines, the deduced binding alignment of the helical molecule on the ice lattice is changed from the Miller indices directional vector [1102 ] to [2203 ]. Subsequent ice-binding characteristics are altered, including changes in adsorption specificity, decreases in thermal hysteresis activity and the formation of rotated hexagonal bipyramid ice crystal morphology.

Phenylacetate enhances the antiproliferative effect of retinoic acid in follicular thyroid cancer.

BACKGROUND: Retinoic acid (RA) has antiproliferative as well as redifferentiating effects in thyroid cancers. Similar effects have been seen with phenylacetate (PA) therapy. These observations prompted us to evaluate the potential antiproliferative effects of the combination of RA and PA in follicular thyroid cancer. METHODS: Three follicular cell lines were treated in vitro with varying concentrations of all-trans RA or PA alone or in combination. Growth was measured by dimethyl-thiazol-diphenyltetrazolium bromide assays. RESULTS: RA (1-2.5 micromol/L) and PA (1-10 mmol/L) alone inhibited cell growth in a time- and dose-dependent manner, with maximum effect at 5 days. The combination of RA and PA had synergistic antiproliferative effects. In the FTC-133 cell line, RA alone (2.5 micromol/L) inhibited growth 16% and PA alone (10 mmol/L) inhibited growth 35% versus controls, whereas the combination of the 2 inhibited growth by 60% at 5 days (P < .005). Similar results were seen with FTC-236 and FTC-238 cell lines. CONCLUSIONS: Our results support that RA and PA have antiproliferative effects in follicular thyroid cancer and are synergistic. The combination of RA and PA may be beneficial in the treatment of advanced thyroid cancers for which conventional therapy fails or as an adjuvant to radioactive iodine therapy in aggressive tumors.

Molecular field analysis of clozapine analogs in the development of a pharmacophore model of antipsychotic drug action.

In an attempt to elucidate some aspects of clozapine's favorable receptor binding profile, we modeled a series of 30 clozapine analogs using a pharmacophore based on the ligands octoclothepin and tefludazine. Molecular field analysis using CoMFA combined with HINT was carried out on published D2 receptor binding affinities. Several alternative alignments of the analogs gave r2 values in the range of 0.8-0.95. The final model had good predictive abilities with q2 > 0.6 and r2 > 0.9. This provides an excellent framework to aid in the design of novel antipsychotics with diminished propensity to produce clinically limiting side effects.

Neutralizing vascular endothelial growth factor activity inhibits thyroid cancer growth in vivo.

BACKGROUND: Without angiogenesis, tumor growth is limited to a few millimeters, the limit of diffusion. Vascular endothelial growth factor (VEGF) is an endothelial-specific mitogen and a major regulator of angiogenesis. METHODS: To investigate the relationship between VEGF and thyroid tumor angiogenesis, we xenografted human dermal matrix inoculated with FTC-133 cells into nude mice or directly injected FTC-133 cells subcutaneously. To block the function of VEGF, the neutralizing anti-VEGF monoclonal antibody A.4.6.1 (mAb A.4.6.1) was injected intraperitoneally twice weekly. As control, an antibody of the same isotype (Ab 5B6) or phosphate buffer saline solution (PBS) was used. To evaluate the dermal matrix as a model for angiogenesis studies, recombinant human VEGF was inoculated into the dermal matrix pocket and xenografted into mice. RESULTS: In the dermal matrix angiogenesis model, the number of blood vessels paralleled the concentration of recombinant human VEGF and was highest at 100 ng/mL. Mice that were treated with the mAb A4.6.1 developed fewer blood vessels (mean, 6.6 per HPF) than control mice (18 per HPF in Ab 5B6 and 22 per HPF in PBS; P <.01). Tumors from mice that were treated with mAb A.4.6.1 were much smaller (mean +/- SD, 0.09 +/- 0.02 gm) at 5 weeks, compared with the tumors treated with Ab 5B6 (5.38 +/- 1.15 gm) or PBS (4.0 +/- 0.72 gm; P <.001). CONCLUSIONS: VEGF is produced by the follicular thyroid cancer cell line and stimulates angiogenesis and growth of thyroid cancer. This stimulation can be blocked by mAb A.4.6.1.

Phenylacetate inhibits growth and vascular endothelial growth factor secretion in human thyroid carcinoma cells and modulates their differentiated function.

There is increasing evidence that phenylacetate inhibits growth and modulates differentiation in a variety of tumors with effects on gene expression, and protein prenylation and glycosylation at concentrations that have been safely used in humans. We evaluated the antineoplastic effects of phenylacetate in five thyroid cancer cell lines of follicular cell origin in vitro. We found early growth inhibition occurred with phenylacetate treatment at a dose of 2.5-10 mmol/L. The growth inhibition was cytostatic with the thyroid carcinoma cells arrested in the G0-1 cell phase. When evaluating the effect of phenylacetate on the differentiated functions of thyroid carcinoma cells, phenylacetate exposure: 1) decreased the TSH (10 mU/mL) growth response; 2) increased radioactive iodine (125I) uptake in two out of five cell lines; and 3) inhibited thyroglobulin secretion. Phenylacetate also inhibited the secretion of vascular endothelial growth factor (a glycoprotein dependent on glycosylation for efficient cellular excretion) from the thyroid cancer cell lines. Our results support that phenylacetate has an antiproliferative effect in many cell types, but the differentiating effects were not uniform. Importantly, we have identified that phenylacetate inhibits the secretion of vascular endothelial growth factor, which possibly mediates the antiangiogenic effects observed in vivo. Because of the minimal toxicity associated with phenylacetate treatment in humans, at concentrations we show to have a significant antineoplastic effect in thyroid carcinoma cells, phenylacetate could be useful in patients with differentiated thyroid cancer who fail conventional therapy or as an adjuvant to radioactive iodine therapy in patients with aggressive tumors.

Development of reverse transcription-competitive polymerase chain reaction method to quantitate the expression levels of human sodium iodide symporter.

The sodium iodide symporter (NIS) is the plasma membrane protein that mediates active iodide uptake into thyroid follicular cells. To investigate whether human NIS (hNIS) mRNA levels in papillary thyroid carcinomas (PCs) correlate with the ability of tumors to concentrate radioiodide, we developed a reverse transcription-competitive polymerase chain reaction (RT-cPCR) method to quantify the hNIS mRNA levels in thyroid tissues. We studied 7 normal thyroid tissues, 8 PCs, and 1 follicular adenoma. hNIS mRNA levels in PCs were generally lower than those found in normal thyroid tissues. The reduced radioiodide concentrating activity of PCs is due, at least in part, to the reduced expression and/or the decreased stability of hNIS mRNA.

Glutathione S-transferases--a review.

The Glutathione S-transferases (GSTs) form a group of multi-gene isoenzymes involved in the cellular detoxification of both xenobiotic and endobiotic compounds. GSTs have been divided into a number of subclasses, alpha, mu, pi, and theta. The classification was made on the basis of sequence similarity and immunological cross-reactivity. GSTs show a high level of specificity toward GSH but the electrophilic second substrate can vary significantly both between and within the classes in spite of their sequence similarity. X-ray crystallography and site-directed mutagenesis studies have together elucidated the structure and mechanism of GSTs. Catalysis occurs by conjugation with glutathione (GSH) and the less toxic and more hydrophilic products can then be partially metabolised and excreted. This invaluable service is however disadvantageous during chemotherapy where GSTs have been associated with multi-drug resistance of tumour cells. Levels of expression of different isoforms of GSTs are tissue specific. The variations in expression between normal and tumour cells are of interest and in most cases the levels of GSTs are increased, especially p-GST. Understanding the complex role that GSTs play in drug resistance begins with determining the pattern of isoform expression and the substrate specificities of each isoform. The use of isozyme-specific, GSH analogues as inhibitors to modulate GST activity during chemotherapy is a promising strategy in the battle against cancer. This review attempts to provide a detailed overview of the literature concerning the different classes of GSTs, their function and mechanism and the use of GSTs as therapeutic targets for disease as current at the time of submission.

A Val 677 activating mutation of the thyrotropin receptor in a Hürthle cell thyroid carcinoma associated with thyrotoxicosis.

Thyroid nodules presenting as hot at 131I-scintigraphy are usually benign follicular adenomas. We report a 42-year-old female patient with an autonomously functioning Hürthle cell thyroid carcinoma causing thyrotoxicosis. Genetic analysis of her thyroid tumoral DNA revealed a heterozygotic activating mutation of the thyrotropin receptor (TSHR) gene that was located downstream to all of the other genetic alterations currently identified, and is due to a base substitution at codon 677 (normal cytosine replaced by guanine, CTG for GTG causing leucine substitution by valine in the seventh transmembrane domain of the receptor). This mutation was detected in the tumor, but not in the leucocytes from the same patient. The Val 677-TSHR mutant showed constitutive activity, in terms of cyclic adenosine monophosphate (cAMP) production, when permanently transfected in Chinese hamster ovary (CHO) cells. Gsp and ras oncogenes and the p53 tumor suppressor gene were not present in the Hürthle cell cancer. The TSHR mutation in this Hürthle cell carcinoma may be responsible for maintaining differentiated thyroid function and hyperthyroidism.

Heterologous desensitization in neoplastic thyroid cells: influence of the phospholipase C signal transduction system on the thyrotropin-adenylate cyclase signal transduction system.

Desensitization is defined as a decreased functional response after continuous or repetitive stimulation of a receptor with its agonist. Thyrotropin (TSH) increases cAMP levels in normal and neoplastic thyroid tissue. The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) activates protein kinase C (PKC). The aim was to determine whether TPA induces heterologous desensitization of the TSH-adenylate cyclase (AC) signal transduction system. Three human thyroid neoplasms in culture for 6 months or longer (one papillary carcinoma, one Hurthle cell carcinoma, one follicular adenoma) were incubated with TSH (10 mU/ml) and TPA (1.6 x 10(-8) M) separately and together for various time periods (from 10 minutes to 24 hours). The mixture was subsequently incubated for 30 minutes with TSH. TPA alone had no effect on cAMP levels, but co-incubation of TPA and TSH caused a significant reduction in cAMP response when compared to the cAMP response that resulted after stimulation with only TSH (p < 0.001). cAMP levels in response to TSH decreased by 31%, 44%, and 57% after preincubation with TSH for 10 minutes, 4 hours, and 24 hours, respectively (p < 0.01; ANOVA). Co-incubation of cells with TPA and staurosporine (10 ng/ml), a PKC inhibitor, prevented the effect of TPA on desensitization at 10 minutes and blunted the effect at 4 hours. This is the first demonstration in human neoplastic thyroid cells that TPA induced heterologous desensitization of the cAMP response to TSH. This TPA-induced effect appears to involve PKC activation, as it can be blocked by staurosporine.

Desensitization in normal and neoplastic human thyroid cell lines.

Because some papillary thyroid cancers continue to grow when thyroid-stimulating hormone (TSH) levels are suppressed, we questioned whether desensitization (i.e., a decreased cAMP response to repeat stimulation with TSH) occurs in normal and neoplastic thyroid tissue. If desensitization does occur, is it similar or different in these human thyroid cells? Normal and papillary thyroid cancer cells from the same patient were cultured as we have previously described. Normal and neoplastic thyroid tissues responded to TSH (0.01-10.0 mU/ml) by increasing cAMP production and growth in a dose-dependent manner. In normal cells there was an 11-fold mean increase in cAMP production at 4 hours, and all thyroid cultures responded. In neoplastic cells cAMP production increased from 1.5-fold to 3.0-fold with a mean 2.0-fold increase at 4 hours. In normal thyroid cells the cAMP response to a second TSH stimulus (desensitization) decreased up to 75% (range 25-75%), and desensitization occurred in all normal thyroid cell cultures. In neoplastic thyroid cells, however, the cAMP response to a second TSH stimulus decreased up to 17% (range 0-17%); and desensitization occurred in only two of the five neoplastic thyroid cell cultures. Thus when normal thyroid and neoplastic cells from the same patients were studied, greater desensitization occurred in the normal cells (75% vs. 17%). These studies document that there is greater desensitization in normal tissue than in neoplastic thyroid tissue, which may account for the increased growth of thyroid neoplasms in the presence of ever-changing low levels of TSH.

Vascular endothelial growth factor expression is higher in differentiated thyroid cancer than in normal or benign thyroid.

Vascular endothelial growth factor (VEGF) is an angiogenic factor, and its expression has been rarely demonstrated in thyroid tumors. We, therefore, investigated the expression of VEGF messenger RNA (mRNA) and production of VEGF protein in cell lines from human primary and metastatic follicular (FTC-133, FTC-236, and FTC-238), papillary (TPC-1), Hürthle cell (XTC-1), and medullary thyroid cancers (MTC-1.1 and MTC-2.2), and in human thyroid tissues (papillary, follicular, medullary, and Hürthle cell cancers, follicular adenomas, and Graves' thyroid tissue) by Northern blot, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) studies. All thyroid cell lines expressed a 4.2-kilobase VEGF mRNA. The VEGF mRNA levels were higher in the thyroid cancer cell lines than in primary cultures of normal thyroid cells, and higher in thyroid cancers of follicular than those of parafollicular cell origin. The VEGF mRNA levels were similar in primary and metastatic thyroid tumors. Immunohistochemical staining and Northern blot analysis of the cell lines correlated positively, thus thyroid cancer cell lines stained more intensely than normal thyroid cells and follicular tumor cells more intensely than parafollicular tumor cells. Again, no difference was noted in VEGF staining between primary and metastatic thyroid tumors. Deparafinized sections of papillary, follicular, and Hürthle cell cancers also stained much stronger than those of medullary thyroid cancers, benign, or hyperplastic (Graves' disease) thyroid tissue. Thyroid cancer cell lines (XTC-1 > TPC-1 > FTC-133 > MTC-1.1) also secreted more VEGF protein as measured by ELISA than did normal thyroid cells. VEGF secretion of cell lines derived from primary and metastatic thyroid tumors were similar. VEGF mRNA is therefore expressed, and VEGF protein is secreted by normal, hyperplastic, and neoplastic thyroid tissues. The higher levels of VEGF expression in differentiated thyroid cancers of follicular cell origin suggests a role in oncogenesis.

Antiproliferative effect of suramin on primary cultures of human pheochromocytomas and rat PC12 pheochromocytoma cells.

BACKGROUND: Suramin inhibits growth of neural crest-derived cells and is used to treat adrenocortical cancer and neuroblastoma in clinical trials. The antiproliferative effect of suramin was evaluated in primary cultures of human pheochromocytoma and the PC12 rat pheochromocytoma cell line in vitro and in vivo. METHODS: Human pheochromocytoma and PC12 rat pheochromocytoma cells were grown in medium supplemented with suramin at concentrations of 1-1,000 micrograms/ml (1.43-1.43 mM) for up to five generations. Suramin did not induce neuronal differentiation, but inhibited growth of cultured human pheochromocytoma cells with IC50 (inhibitory concentration at which a 50% reduction of proliferation is observed) of 50-250 micrograms/ml. Also, suramin inhibited proliferation of PC12 cells with IC50 of 228 micrograms/ml after 5 days and 161 micrograms/ml at 10 days of treatment. Colony formation assays demonstrated these effects to be cytotoxic rather than cytostatic. Thus when reproductive integrity of PC12 cells was taken into account, IC50 was calculated with 118 micrograms/ml and 129 micrograms/ml, respectively. In vivo experiments were performed with subcutaneously xenotransplanted PC12 cells (BALB/c NCR-NU mice). Suramin did not alter tumorigenicity and did not inhibit local tumor growth. RESULTS: These data determine for the first time an antiproliferative effect of suramin in pheochromocytoma cells. Suramin is cytotoxic to pheochromocytoma cells in vitro at levels that are clinically achievable. CONCLUSIONS: Suramin may have potential as an antiproliferative drug in nonresectable pheochromocytoma.

Desensitization of cyclic adenosine 3,5'-monophosphate response to thyrotropin in normal and primary or metastatic papillary thyroid cancer cells in vitro.

BACKGROUND: Desensitization is an important physiologic process resulting in a decreased functional response after continuous or repeated stimulation. Prior exposure of normal human thyroid tissue to thyrotropin either in vivo or in vitro causes desensitization of adenylate cyclase. Little is known, however, about whether the thyrotropin-adenylate cyclase-cyclic adenosine 3,5'-monophosphate (cAMP) signal transduction system desensitizes in human thyroid carcinomas. Failure to desensitize could result in increased growth or metastases. METHODS: Cell cultures from Chinese hamster ovary (CHO) cells transfected with normal human thyrotropin receptor (hTSHr) and thyroid neoplasms including one papillary carcinoma and one papillary lymph node metastases were evaluated for desensitization. CHO cells were stably transfected with plasmid DNA containing hTSHr. Cells were incubated with thyrotropin (10 mU/ml) for different periods (1 to 24 hours). A second incubation (30 minutes) was done with and without thyrotropin in medium including 3-isobutyl-1-methylxanthine (1 mmol/L). Intracellular cAMP accumulation was determined by means of radioimmunoassay. RESULTS: Maximal stimulation and desensitization to thyrotropin were observed at 30 minutes and 4 hours, respectively. The cAMP response to a second incubation with thyrotropin was 52% and 48% lower than the initial response to thyrotropin in CHO-hTSHr and papillary thyroid cancer cells, respectively (p < 0.001). A papillary carcinoma lymph node metastases had an increased basal cAMP level and also an increased cAMP level in response to thyrotropin stimulation (227%) but failed to desensitize (p < 0.001). CONCLUSIONS: Desensitization of the cAMP response to thyrotropin occurred in a papillary thyroid cancer but not in a metastatic thyroid cancer. Failure to desensitize might play a role in tumor progression.

Thyroid-stimulating hormone promotes the secretion of vascular endothelial growth factor in thyroid cancer cell lines.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a vascular endothelial cell-specific mitogen secreted by some cancer cells and is a major regulator of angiogenesis. Because thyroid-stimulating hormone (TSH) promotes growth and progression of thyroid cancers, we postulated that TSH may increase the production and secretion of VEGF by thyroid cancer cells. METHODS: We examined primary cultures of normal human thyroid (NT 1.0), medullary thyroid cancer (MTC 1.1), and cell lines derived from the papillary (TPC-1), follicular (FTC-133), and Hürthle cell (XTC-1) thyroid cancer. We quantified the concentration of VEGF in conditioned medium by means of enzyme-linked immunosorbent assay. RESULTS: Cell lines derived from thyroid secrete VEGF. Basal VEGF secretion was similar in normal and thyroid cancer cells, except XTC-1, which has high basal secretion (p < 0.01). All thyroid cancer cells secrete significantly more VEGF than normal thyroid cells after TSH (10 mIU/ml) stimulation (p < 0.05). TSH stimulated secretion of VEGF in FTC-133 (8.2 ng/dl versus 18.8 ng/dl), TPC-1 (5.5 ng/dl versus 26.9 ng/dl), and MTC 1.1 (5.9 ng/dl versus 13.4 ng/dl) cell lines (p < 0.01), but not in NT 1.0 (8.4 ng/dl versus 9.9 ng/dl) and XTC-1 (25.4 ng/dl versus 31.2 ng/dl) cells. CONCLUSIONS: These results suggest that VEGF secretion is constitutively activated in some thyroid cancers and that VEGF secretion is stimulated by TSH; thus TSH may promote growth in some thyroid cancers by stimulating VEGF secretion and angiogenesis.

Structure-activity relationships of competitive NMDA receptor antagonists.

The interaction of structurally constrained competitive NMDA receptor antagonists, (+/-)-cis-4-phosphonomethyl-2-piperidine carboxylic acid (CGS 19755), (2-amino-4,5-(1,2-cyclohexyl))-7-phosphonoheptanoic acid (NPC 12626), (+/-)-6-phosphonomethyl-de-cahydroisoquinoline-3-carboxylic acid (LY 274614), (S)-alpha-amino-5-phosphonomethyl[1,1'-biphenyl]-3-propanoic acid (SDZ EAB-515) and (S)-alpha-amino-5-phosphonomethyl[1,1':4',1"-terphenyl]-3-propa noi c acid (SDZ 215-439), with their receptor was assessed using radioligand binding, protection against neurotoxicity in cortical neuronal cultures and computerised molecular modelling. All compounds inhibited the specific binding of [3H]CGS 19755 and/or [3H]CGP 39653 (inhibition constants 40-2000 nM), and protected neuronal cultures from NMDA-mediated injury (IC50 values 1.3-5.6 microM). Quantitative conformational analyses indicated that the molecules fitted well to a NMDA receptor model. Our results draw attention to a deep hydrophobic pocket, defined by the bi- and terphenyl containing antagonists (SDZ EAB-515, SDZ 215-439), which may influence potency and selectivity.

A sensitive and specific polymerase chain reaction-based assay for the diagnosis of cytomegalovirus retinitis.

PURPOSE: To develop a sensitive and specific laboratory assay for the diagnosis of cytomegalovirus retinitis. METHOD: We used a polymerase chain reaction-based assay for detection of cytomegalovirus DNA in vitreous samples. We attempted to detect cytomegalovirus DNA in 19 vitreous samples from patients with the acquired immunodeficiency syndrome (AIDS) who had untreated cytomegalovirus retinitis and in 40 vitreous samples from patients with AIDS who had been treated with systemic ganciclovir or foscarnet, or both. We also attempted to detect cytomegalovirus DNA in vitreous samples from 54 immunocompetent patients, including 32 with retinal detachment or macular hole, 11 with vitreous inflammation, and 11 with vitreous hemorrhage. Additionally, we attempted to detect cytomegalovirus DNA in 15 vitreous samples from patients with AIDS who had vitreoretinal inflammation not caused by cytomegalovirus. RESULTS: Cytomegalovirus DNA was detected in 18 of 19 eyes with untreated cytomegalovirus retinitis. We detected cytomegalovirus DNA in 19 of 40 vitreous samples from patients with previously treated cytomegalovirus retinitis. Cytomegalovirus DNA was not detected in any of 69 patients who did not have a clinical diagnosis of cytomegalovirus retinitis. Thus, the assay had an estimated sensitivity of 95% in detecting untreated cytomegalovirus retinitis and a sensitivity of 48% in detecting cytomegalovirus retinitis that had been treated with systemic ganciclovir or foscarnet, or both. The assay did not give false-positive results in patients with vitreous hemorrhage or vitreous inflammation. Most important, the assay did not give false-positive results in AIDS patients with vitreous inflammation from causes other than cytomegalovirus retinitis. CONCLUSION: We have developed a sensitive and specific diagnostic assay that will assist in the diagnosis of cytomegalovirus retinitis.