This direct method for quantifying excessive urinary glycosaminoglycan excretion exploits the specific binding of 1,9-dimethylmethylene blue (DMB). The procedure obviates cumbersome and labor-intensive procedures for separating glycosaminoglycans from other constituents of urine. Pediatric pharmaceutical formulations (except heparin), in concentrations expected in urine, do not interfere with spectrophotometry, nor does protein. Results can be expressed in terms of urinary creatinine; thus the test is applicable to very small urine specimens (0.1 mL), such as those obtainable from neonates. In a pilot study, results of the direct DMB test for 48 urine specimens agreed with the clinical diagnosis, and quantitative measurements correlated moderately (r = 0.76) with results of a commonly used procedure (carbazole-borate reactivity after precipitation with cetylpyridinium chloride). The present method was also used to assess metabolic correction in a patient with Hurler's syndrome after treatment by bone-marrow transplantation. This quantitative method surmounts the major technical problems of developing mass screening programs for infants, thus offering the potential for earlier diagnosis and treatment of mucopolysaccharidosis diseases.

Fibroblasts are target cells for the autoimmune process in Graves' ophthalmopathy and pretibial dermopathy. Because the autoantigen involved in the hyperthyroidism of Graves' disease is the TSH receptor, we sought to determine whether RNA encoding this receptor might be present in retroocular and pretibial fibroblasts. RNA was reverse transcribed and the resulting cDNA was amplified by the polymerase chain reaction using primers spanning a region of the extracellular domain of the human TSH receptor. The predicted amplified product, verified by direct sequencing, was detected when RNA was derived from fibroblasts, but not from the nonfibroblast cells studied. The demonstration in fibroblasts of RNA encoding this important autoantigen in Graves' disease suggests that the TSH receptor might play a role in the pathogenesis of the connective tissue manifestations of this disease.

The thyrotropin receptor (TSHR) is the thyroid autoantigen against which stimulating autoantibodies are directed in Graves' hyperthyroidism. Recent evidence suggests that TSHR may also serve as an orbital autoantigen in Graves' ophthalmopathy (GO) and that expression of this protein is increased in the fatty connective tissues of the orbit in this condition. It has been shown that orbital fibroblasts from patients with GO increase thyrotropin (TSH)-dependent cyclic adenosine monophosphate (cAMP) production and TSHR gene expression when cultured under conditions known to stimulate adipocyte differentiation. In the current study, we wanted to determine whether treatment of these cells with particular cytokines (each 1 ng/mL) during differentiation might further augment TSHR expression. We found that exposure to interleukin (IL)-6 increased TSHR expression above control levels in cells from patients with GO. In contrast, this cytokine did not affect TSHR expression in normal orbital cells. Neither IL-4 nor IL-1alpha had a significant stimulatory effect in either normal or Graves' cultures. These findings suggest that IL-6 may play a role in the pathogenesis of GO by increasing expression of the putative autoantigen within the adipose/connective tissues of the orbit.

We and others have described previously a polymorphism at the first position of codon 52 (C52 --> A52) of the human thyrotropin receptor (hTSHr) gene. To determine its potential significance, we studied female (n = 100) and male (n = 25) patients with autoimmune thyroid disease (Graves' disease, n = 91; Hashimoto's thyroiditis, n = 34) and normal individuals [n = 121, female (n = 69), male (n = 52)]. Screening was performed using AciI restriction enzyme digestions of PCR-amplified genomic DNA. All codon 52 polymorphisms were verified by direct DNA sequencing. Data were analyzed using Chi-square or Fisher exact tests and p-values were corrected for multiple comparisons. Our studies demonstrated that this polymorphism is highly associated with autoimmune thyroid disease in the female population (corrected p = 0.008). We found no such association in the male population. Within females, there was a greater association between Graves' disease and the polymorphism (corrected p = 0.017) than between Hashimoto's thyroiditis and the polymorphism (corrected p = 0.090). The polymorphism was present in a higher proportion of Graves' disease patients with Graves' ophthalmopathy and pretibial dermopathy (40%) or Graves' ophthalmopathy, pretibial dermopathy, and acropachy (60%), than in patients with Graves' disease alone (15%), or Graves' disease and Graves' ophthalmopathy alone (17%). In conclusion, a polymorphism (C52 --> A52) of the hTSHr is associated with autoimmune thyroid disease in females.

Because the thyrotropin receptor (TSHR) has long been considered a thyroid-specific protein, its presence in extrathyroidal tissues has been controversial. In this study, we sought to detect and quantify this potentially low abundance mRNA in various extrathyroidal tissues using liquid hybridization analysis (LHA) and to detect protein with immunohistochemical studies. Strongly positive protected bands, indicating the presence of both intact (2.4 kb) and variant (1.3 kb) TSHr mRNA, were apparent in LHA gel lanes corresponding to normal thyroid, Graves' thyroid, and thymus. Less abundant protected bands of the same sizes were present in lanes corresponding to normal adrenal, and samples from normal kidney were faintly positive. The full-length transcript:variant transcript ratio was approximately 1:1 in all positive tissues. Immunohistochemical analysis of TSHR-like reactivity in paraffin-embedded thymus, adrenal, and kidney revealed specific staining in each of these tissues. No TSHR mRNA or TSHR-like immunoreactivity was detected in samples from several other normal human tissues. We conclude that measurable TSHR mRNA and protein expression is not restricted to the thyroid gland. Further study is warranted to determine whether these extrathyroidal receptors play a role in normal physiology or in disease.

An accumulation of glycosaminoglycans (GAG) is a feature characteristic of orbital connective tissues from patients with Graves' ophthalmopathy (GO) that leads directly to the clinical expressions of the disease. Interleukin-1 (IL-1), produced by macrophages and fibroblasts within the diseased orbit, stimulates GAG synthesis by orbital fibroblasts. We designed the current study to determine whether particular agents might block this effect and thus be useful in the treatment of GO. Orbital fibroblast cultures were grown to confluence and incubated for 48 h with IL-1 (1-10 U/mL) alone or IL-1 (10 U/mL) in combination with IL-1 receptor antagonist (IL-1ra; 1-40 ng/mL) or soluble IL-1 receptor (sIL-1R; 0.25-10 micrograms/mL). Cells were labeled with [3H]glucosamine and processed for GAG quantitation. The addition of IL-1 alone stimulated GAG synthesis by 73-176% (mean, 104%; P < 0.05). Significant inhibition of IL-1-stimulated GAG synthesis was observed after treatment of normal fibroblasts with IL-1ra at a concentration of 5 ng/mL (12.5-fold molar excess; mean, 33%; P < 0.05); essentially complete inhibition was achieved at 40 ng/mL (100-fold molar excess; mean, 86%; P < 0.05). Significant inhibition of GAG synthesis by sIL-1R was observed at a concentration of 0.5 microgram/mL (720-fold molar excess; mean, 79%; P < 0.05), and inhibition was essentially complete at 1 microgram/mL (1440-fold molar excess; mean, 89%; P < 0.05). IL-1ra and sIL-1R are potent inhibitors of IL-1-induced GAG production by cultured human orbital fibroblasts. Our results suggest that these two compounds, shown in early trials to be safe when administered parenterally, may be useful in the prevention or treatment of GO.

The thermal cycler can be used with maximal efficiency by multiple individuals with different experiments when all PCR amplifications are optimized for one cycling program. More than 150 regions of genomic DNA from human and other species were successfully amplified in our laboratory by using one basic protocol that includes Taq DNA polymerase, primers containing 14 to 17 bp of genomic sequence, and standard cycling conditions of one minute at 94°C, two minutes at 500C, and three minutes at 720C. Application of the standard three-step cycling protocol was unsuccessful for regions of the dopamine D4 receptor gene (D4DR), a gene with a very high G + C content (75%). In an effort to retain the standard cycling conditions, Taq, Tth, Pfif,

Previous work indicates that matrix metalloproteinase-1 (MMP-1) gene expression is a prognostic factor for local recurrence and metastasis in human chondrosarcoma. The current study evaluates the effects of MMP-1 knock-down via RNA interference on chondrosarcoma metastasis in vitro. Of nine siRNA oligos tested, one showed a 90% inhibitory effect on MMP-1 gene expression. Stable attenuation of MMP-1 in chondrosarcoma cells was achieved by using a plasmid vector-based siRNA system. Pronounced MMP-1 activity suppression was accompanied by marked inhibition of tumor cell invasion. Cells in which MMP-1 has been stably silenced are useful tools to elucidate the mechanism of MMP-1 mediated tumor metastasis.