Tissue inhibitor of metalloproteinases-1 (TIMP-1) mRNA is elevated in advanced stages of thyroid carcinoma.

Tumour cell invasion and metastasis is a multistep process that involves the degradation of extracellular matrix proteins by matrix metalloproteinases (MMPs). Tissue inhibitors of metalloproteinases (TIMPs) act as negative regulators of MMPs and thus prevent tumour cell invasion and metastasis by preserving extracellular matrix (ECM) integrity. In the present study we examined the expression of one member of TIMPs, TIMP-1, in 39 thyroid tumour specimens and two thyroid carcinoma cell lines (NPA and SW579). We also investigated the effect of high TIMP-1 expression on the invasive potential of NPA cells. Northern blot analysis showed that TIMP-1 mRNA levels correlated directly with tumour aggressiveness: the highest number of TIMP-1 transcripts was found in stages III and IV vs benign goitre (P < 0.0001). However, TIMP-1 expression was not increased in NPA and SW579 cells, both of which are derived from poorly differentiated thyroid tumours. Immunohistochemical study showed strong TIMP-1 staining in the stroma cells of advanced stages of carcinomas. Overexpression of TIMP-1 by gene transfer resulted in a significant suppression of the malignant phenotype of NPA cells as judged by an in vitro tumour invasion assay. These results suggest that high levels of TIMP-1 transcripts in advanced stages of thyroid carcinoma likely come from stroma rather than thyroid cancer cells, and TIMP-1 may function as a thyroid tumour invasion/metastasis suppressor.

Modulation of the catabolic effects of interleukin-1 beta on human articular chondrocytes by transforming growth factor-beta.

The effects of IL-1 beta and TGF-beta on the biosynthesis of extracellular matrix structural components relative to the metalloproteinases and their inhibitor TIMP1 in human articular chondrocytes were investigated. It has been proposed that TGF-beta, acting as a positive regulator of matrix accretion, can counteract the increased loss of cartilage matrix induced by IL-1 beta. To allow a comparison of their effects on mRNA levels for these different components, quantitation by competitive RT/PCR was employed. This method was found to give reproducible estimates of mRNA levels and the observed effects of IL-1 beta and TGF-beta on individual components of this system agree with qualitative data obtained by northern blotting. IL-1 beta had a more pronounced effect on aggrecan mRNA levels than on those for type II collagen. Similar quantitative differences were observed between collagenase and stromelysin mRNA levels. TGF-beta generally counteracted the effects of IL-1 beta, and new steady state levels were attained within 24 h. However, the reversal of IL-1 beta induced suppression of matrix protein mRNA levels appeared more effective than its suppression of the increase in stromelysin and collagenase mRNA levels. Similarly TGF-beta did not reduce the extent of IL-1 beta induced secretion of stromelysin at the protein level. TIMP1 mRNA levels were only slightly reduced by IL-1 beta; however this cytokine effectively suppressed its induction by TGF-beta. The higher concentrations of TGF-beta and longer exposure times required to overcome the suppressive effects of IL-1 beta suggest that the interaction between IL-1 beta and TGF-beta in the regulation of TIMP1 expression follows a different mechanism to that operating for the metalloproteinases and matrix proteins. Thus the overall potential of TGF-beta to inhibit proteolysis is attenuated by its much slower effect on TIMP1 mRNA levels.

Xenografts of rat islets into diabetic mice. An evaluation of new smaller capsules.

Healthy rat islets were encapsulated in alginate-polylysine-alginate capsules measuring 0.25-0.35 mm in diameter using a modified encapsulation technique. The encapsulated islets were transplanted intraperitoneally in nonimmunosuppressed streptozotocin-induced diabetic BALB/c mice. The diabetic condition of the experimental animals was reversed within two days following the transplantation and the animals remained normoglycemic for up to 308 days, with a mean xenograft survival of 219.8 +/- 46.2 days. Four and six months posttransplant the capsules were removed from two recipients. This resulted in regression to a hyperglycemic state. After a second transplant of encapsulated islets, the animals returned to normoglycemia. In control mice that received free unencapsulated islets, the xenografts remained functional for no more than 12 days. Our study clearly demonstrates that the encapsulation of islets in the new smaller capsules can effectively prolong xenograft survival without immunosuppression.

Free versus microencapsulated pancreatic islet xenografts producing amelioration of streptozotocin toxicity.

This study examines the effect of pancreatic islet transplants on the streptozotocin(STZ)-associated toxicity in diabetic animals. Mice with STZ-induced diabetes were implanted with microencapsulated or free rat islets. The effectiveness of the transplant was evaluated in terms of: (A) blood glucose monitoring, (B) determination of subset levels of the helper and cytotoxic T-lymphocytes, and (C) STZ-associated mortality. The experimental results demonstrate that the transplanted islets can quickly restore normoglycemia. The restoration of normal blood glucose levels is accompanied by a significant increase in proportions of helper and cytotoxic T-cells. There was no mortality in the transplant recipients as a result of the STZ administrations, whereas a significant mortality was observed in the control group of mice. No significant differences between the encapsulated and free islet transplant recipients were observed.

Prolonged reversal of diabetic state in NOD mice by xenografts of microencapsulated rat islets.

Transplantation of the islets of Langerhans could be the most promising approach to the clinical treatment of insulin-dependent (type I) diabetes mellitus. In this study, we report on a modified encapsulation technique that produces small alginate-polylysine capsules (0.25-0.35 mm diam). In an in vitro study, both encapsulated and unencapsulated islets showed comparable responses to glucose challenge in terms of insulin secretion. With the new capsules, 16 spontaneously diabetic NOD mice received transplants of 800 encapsulated rat islets/animal. Nonfasting blood glucose concentration decreased from 24.4 +/- 1.4 to 4.0 +/- 1.3 mM. At 4 and 5 mo posttransplantation, the capsules were removed from 2 recipients. Both animals regressed to a hyperglycemic state after capsule removal. However, after another islet transplantation, normoglycemia was again restored in these 2 animals. In control mice, which received unencapsulated islets, the xenografts remained functional for less than 10 days. A high mortality rate was observed among these animals within 2 mo of the recurrence of the hyperglycemic state. Our results clearly indicate that encapsulation of pancreatic islets in the improved capsules can effectively prolong xenograft survival without immunosuppression in an animal model that mimics human type I diabetes mellitus.

Xenotransplantation of microencapsulated fetal rat islets.

Fetal pancreatic islets were isolated from 21-day pregnant Wistar rats and enclosed in semipermeable alginate-polylysine-alginate capsules. Encapsulated islets that had been previously cultured for eight days in vitro were shown to secrete insulin in response to glucose challenge: low-glucose, high-glucose, and high-glucose + 3-isobutyl-1-methyl-xanthine (IBMX). Transplants of 800-1000 encapsulated cultured fetal islets into the peritoneal cavities of BALB/c mice with streptozotocin-induced diabetes restored normoglycemia for up to 171 days without immunosuppression. When the capsules were removed from 2 of the recipients they both quickly regressed to a diabetic state. Control groups of diabetic mice received unencapsulated, uncultured islets or empty capsules. The mortality rate among these animals was high and none experienced relief from hyperglycemia for longer than 6 days. These results demonstrate that cultured microencapsulated fetal rat islets of Langerhans can release insulin in response to an in vitro glucose challenge, and that transplants of these islets into diabetic mice can restore normoglycemia without the need for immunosuppressive therapy.

Reversal of diabetes in BB rats by transplantation of encapsulated pancreatic islets.

Prolonged survival of pancreatic islet allografts implanted in diabetic BB rats was achieved by encapsulation of individual islets in a protective biocompatible alginate-polylysine-alginate membrane without immunosuppression. Intraperitoneal transplantation of the encapsulated islets reversed the diabetic state of the recipients within 3 days and maintained normoglycemia for 190 days. Normal body weight and urine volume were maintained during this period, and no cataracts were detected in the transplant recipients. In contrast, control rats receiving transplants of unencapsulated islets experienced normoglycemia for less than 2 wk. These results demonstrated that microencapsulation can protect allografted islets from both graft rejection and autoimmune destruction without immunosuppression in an animal model that mimics human insulin-dependent diabetes.