Revascularisation of human parathyroid tissue transplanted to athymic mice

The revascularisation process of transplanted human normal, hyperplastic and adenomatous parathyroid tissue was analysed at 2 and 4 days and 1, 2, 4, 7 and 12 weeks after transplantation to athymic mice. The transplants were examined by light and electron microscopy, immunohistochemistry and autoradiography. Vessels were detected by monoclonal antibodies specific for mouse and human endothelial cells. Immunohistochemistry demonstrated ingrowth of vessels from the host into the transplant and at one week numerous capillary sprouts were observed in the peripheral parts of the transplants. During the first week, peak levels of proliferation (labelling index) were observed in endothelial cells and capsular fibroblasts, and the proliferative capacity of endothelial cells was most pronounced in adenoma transplants. Fenestrated capillaries were observed in hyperplastic and adenomatous transplants, but not in transplants of normal tissue. In conclusion, revascularisation of transplanted human parathyroid tissue is enabled by ingrowth of vessels from the host into the transplant. The proliferative capacity of endothelial cells is higher and the process of maturation is faster in hyperplastic and adenomatous tissue compared to normal tissue.     

Survival and histopathologic characteristics of human melanocytic nevi transplanted to athymic (nude) mice

Melanocytic nevi (n = 406) covering a range of sizes and gross morphologic features were excised from human donors, sampled for histologic diagnosis, and transplanted to athymic (nude) mice. Ninety percent of these xenografts survived transplantation, of which a subset was irradiated daily with ultraviolet light to promote neoplastic transformation. Over 16 weeks of observation, nearly all grafts histologically showed focal inflammatory cell infiltration and fibrosis, progressing in approximately 30% of grafts to complete regression at final observation. During the inflammatory phase, the nevi often had junctional intraepidermal melanocytic hyperplasia in a lentiginous pattern, with cytologic hypertrophy, dendritic morphology, and hypermelaninization. These changes were evident in approximately 20-30% of nevi where they were absent before transplantation, suggesting that host factors, such as those related to the immune response, had stimulated growth. Graft survival was independent of nevus size and initial histologic diagnosis. No melanomas developed in any of the grafts, either spontaneously or with ultraviolet irradiation. These results indicate that successful transplantation can be achieved in a high proportion of human nevus xenografts and that the majority survive for a period of time that would be sufficient for experimental studies. The host response, however, has effects on intraepidermal melanocytic growth that lead to progressive fibrous replacement of the nevus, introducing significant artifacts that compromise the model. Furthermore, malignant transformation of engrafted melanocytes seems to be a rare event, which would limit studies of neoplastic progression in the transplanted melanocytes. Nonetheless, the intraepidermal melanocytic pattern described here evidently constitutes one pattern of melanocyte growth that could be exploited experimentally for studies of growth and differentiation control in melanocytes.     

Growth of transplanted tumours in mice treated with macrophages and their products

The antitumour activity of peritoneal macrophages, added to tumour inocula (Winn test), was determined. Macrophages harvested from mice immunised with tumour, or from tumour-bearing mice, suppressed or inhibited tumour growth when in direct contact with tumour cells. Incubation of macrophages exhibiting antitumour activity with tumour cells yielded supernatant fluids capable of interfering with tumour growth. Consistent induction of antitumor activity required either addition of indomethacin to the incubation mixture, or collection of macrophages from immunised or tumour-bearing donors that had ingested indomethacin in the drinking water. Tumour growth was significantly inhibited when tumour inocula were suspended in supernatant fluid, or when mice were given several subcutaneous injections of supernatant prior to tumour transplantation.     

Transplantation of parathyroid tissue in experimental hypoparathyroidism: in vitro and in vivo function of parathyroid tissue microencapsulated with a novel amitogenic alginate

Microencapsulation of tissues is an alternative to postoperative immunosuppression in transplantation. In 1994 iso-, allo- and xenotransplantation of microencapsulated parathyroid tissue was achieved in vivo. However, continued analysis of the coating substance (an alginate) determined mitogenic properties. Here, we report on the in vitro and in vivo function of parathyroid tissue microencapsulated with a novel amitogenic alginate suitable for use in humans. To assess in vitro function, parathyroid tissue encapsulated with mitogenic and amitogenic alginate was exposed to rising concentrations of calcium. For in vivo experiments, it was isotransplanted into parathyroidectomized rats. PTH release into medium and PTH serum levels as well as calcium levels of recipient rats were analyzed and compared to native (non-microencapsulated) tissue and empty capsules, respectively. In vivo, transplants were excised and subjected to histologic examination six months after trans-plantation. In vitro, parathyroid tissue encapsulated with amitogenic alginate releases approximately half of the PTH of the native tissue, not different from tissue encapsulated with the mitogenic alginate. In vivo, the novel alginate preserved parathyroid function similar to that of native tissue over the six month period resulting in complete reversal of hypoparathyroidism. Correspondingly, histologic examination revealed vital parathyroid tissue in intact microcapsules. By establishing in vitro function and successful long-term transplantation, we have documented the principle of microencapsulation of parathyroid tissue to be effective also with the novel amitogenic alginate, which is suitable for clinical use.     

High phosphate level directly stimulates parathyroid hormone secretion and synthesis by human parathyroid tissue in vitro

Phosphate retention plays an important role in the pathogenesis of secondary hyperparathyroidism in patients with renal failure. In in vitro studies, high extracellular phosphate levels directly stimulate PTH secretion in rat and bovine parathyroid tissue. The present study evaluates the effect of high phosphate levels on the secretion of PTH and the production of prepro PTH mRNA in human hyperplastic parathyroid glands. The study includes parathyroid glands obtained from patients with primary adenomas and from hemodialysis and kidney-transplant patients with diffuse and nodular secondary hyperplasia. The experiments were performed in vitro using small pieces of parathyroid tissue. The ability of high calcium levels to decrease PTH secretion was less in adenomas than in secondary hyperplasia; among the secondary hyperplasia, nodular was less responsive to an increase in calcium than diffuse hyperplasia. In diffuse hyperplasia, PTH secretion was increased in response to 3 and 4 mM phosphate compared with 2 mM phosphate, despite a high calcium concentration in the medium; prepro PTH mRNA levels increased after incubation in 4 mM phosphate. Similar results were obtained with nodular hyperplasia, except that the elevation of PTH secretion in response to 3 mM phosphate did not attain statistical significance. In adenomas, high calcium concentrations (1.5 mM) did not result in inhibition of PTH secretion, independent of the phosphate concentration, and the prepro PTH mRNA was not significantly increased by high phosphate levels. In conclusion, first, the PTH secretory response to an increase in calcium concentration is less in nodular than diffuse hyperplasia; second, high phosphate levels directly affect PTH secretion and gene expression in patients with advanced secondary hyperparathyroidism.     

Antiproliferative effect of methyl-beta-cyclodextrin in vitro and in human tumour xenografted athymic nude mice

A nondestructive leak detection method developed at Technical Research Centre of Finland (VTT) was tested for both gas-flushed and vacuum flexible packages. In the method, a gas package containing 0.5 to 5.0% (vol/vol) hydrogen in nitrogen was positioned in a test chamber, a controlled vacuum was pulled in the chamber through a pipe connected to a hydrogen sensor, and leaking packages were detected by the sensor as increased H2 concentration. The H2 tracer gas (0.5 to 5.0%) was introduced into leaking finished vacuum packages at 200 kPa pressure. Within 1 to 4 s the developed test method was able to detect leaks down to 10 to 15 microns and 20 to 30 microns in diameter in commercially manufactured gas-flushed packages filled with roasted meat balls and vacuum packages filled with ground coffee, respectively. Before leak testing, the vacuum packages were charged with H2 for 30 s. The sensitivity and leak detection time of the test method were improved when the H2 concentration in the package was increased and when the free space in the test chamber was decreased. The evaluated H2 concentrations did not affect the sensory or microbiological quality of the roasted meat balls. This study clearly demonstrated that the hydrogen tracer gas leak detection method has potential to be further developed as a fast, nondestructive, on-line leak testing apparatus for flexible packages with or without a headspace.     

ND-2001 suppresses lung metastasis of human renal cancer cells in athymic mice

Explants of highly metastatic human renal cell carcinoma SN12Cpm6 cells in athymic mice were treated with sodium D-glucaro-delta-lactam (sodium 5-amino-5-deoxy-D-glucosaccharic acid-delta-lactam; ND-2001). ND-2001 (50 micrograms/ml) caused 78% inhibition of lung metastasis of SN12Cpm6 cells (two of five animals remaining metastasis free). The in vitro tumor cell invasion assay showed that ND-2001 (100 micrograms/ml) suppressed the invasive activity of SN12Cpm6 cells to Matrigel matrix at an inhibition rate of 72%. These results suggest that ND-2001 may be a new anti-metastatic drug against human cancer cells.     

Role of glycosylation in expression and function of the human parathyroid hormone/parathyroid hormone-related protein receptor

Parathyroid hormone (PTH) regulates calcium metabolism through a specific G protein-coupled, seven-transmembrane helix-containing receptor. This receptor also binds and is activated by PTH-related protein (PTHrP). The human (h) PTH/PTHrP receptor is a membrane glycoprotein with an apparent molecular weight of approximately 85000 which contains four putative N-glycosylation sites. To elucidate the functional role of receptor glycosylation, if any, we studied hormone binding and signal transduction in human embryonic kidney cells transfected with hPTH/PTHrP receptor (HEK-293/C-21). These cells stably express 300000-400000 receptors per cell. Inhibition of N-glycosylation with an optimized concentration of tunicamycin yielded completely nonglycosylated hPTH/PTHrP receptor (approximately 60 kDa). This receptor form is fully functional; it maintains nanomolar binding affinity for PTH- and PTHrP-derived agonists and antagonists. PTH and PTHrP agonists stimulate cyclic AMP accumulation and increases in cytosolic calcium levels. In addition, the highly potent benzophenone (pBz2)-containing PTH-derived radioligand [Nle8,18,Lys13(epsilon-pBz2),L-2-Nal23,Tyr34 3-125I)]bPTH(1-34)NH2 can photoaffinity cross-link specifically to the nonglycosylated receptor. The molecular weight (approximately 60000) of the band representing the photo-cross-linked, nonglycosylated receptor (obtained from the tunicamycin-treated HEK-293/C-21 cells) was similar to that of the deglycosylated photo-cross-linked receptor (obtained by enzymatic treatment with Endoglycosidase-F/N-glycosidase-F). Our findings indicate that glycosylation of the hPTH/PTHrP receptor is not essential for its effective expression on the plasma membrane or for the binding of ligands known to interact with the native receptor. The nonglycosylated hPTH/PTHrP receptor remains fully functional with regard to both of its known signal transduction pathways: cAMP-protein kinase A and phospholipase C-cytosolic calcium.     

Myelination by transplanted human and mouse central nervous system tissue after long-term cryopreservation

BACKGROUND: There is limited information available regarding the effect of catheter ablation of the antioventricular (AV) junction on left ventricular (LV) function. Both deterioration and improvement in LV function have been reported following direct current (DC) ablation of the AV junction. The deterioration of LV function following DC ablation of the AV junction may be due to the accompanying barotrauma, DC arcing and direct coagulation, or even the effects of chronic ventricular pacing. If this deterioration of LV function was a result of the accompanying effects of DC shock, the use of radiofrequency ablation (RF) should not result in deterioration of LV function. AIM: To study LV function before and after different methods of AV junction ablation and in patients with chronic ventricular pacing without AV junction ablation. MATERIAL: This study assessed LV function in patients following RF ablation, low energy DC ablation of the AV junction and compared the results with our previously reported finding in patients who had AV junction ablation using high energy DC shock. A group of patients undergoing permanent single chamber ventricular pacemaker implantation without AV junction ablation were selected as controls. METHODS: All patients were paced in the ventricle at 110 beats/minute during LV function assessment by radionuclide angiography. Global LV function and segmental wall motion abnormalities were assessed before, immediately following and three months after ablation. RESULTS: In the high energy DC ablation group, a fall in global LV function (50 +/- 3.0% to 43 +/- 3.0%, p = 0.02) and impairment of segmental wall motion were detected. Low energy DC ablation resulted in segmental wall motion impairment similar to high energy DC but without affecting global ejection fraction (47.0% +/- 6.7 to 45.5% +/- 3.1, p > 0.05). Neither RF ablation (44.0% +/- 3.3 to 45.3% +/- 3.5, p > 0.05), nor chronic pacing (46.7% +/- 4.9 to 47.0% +/- 2.9 p > 0.05) had any effect on global or segmental LV function. CONCLUSIONS: Low energy DC or RF ablation of the AV junction does not affect global LV ejection fraction. The deterioration of global LV function after high energy DC shock ablation appears to be related to the accompanying effects of DC energy and not to the effects of chronic ventricular pacing.     

Reduced engraftment and wound closure of cryopreserved cultured skin substitutes grafted to athymic mice

A simulation model was developed to test the accuracy of indirect estimates of maternal mortality (the sisterhood method). The model generated a first generation of grandmothers, a second generation of mothers (with brothers and sisters), and a third generation of children (births). In the second generation, maternal mortality was introduced. Empirical values for the parameters of fertility and mortality were taken from a prospective survey in Senegal (Niakhar). Results based on 100 simulations of the same situation revealed several limitations of the sisterhood method: The indirect estimates could fall as far as 33 percent from the true values on individual cases; the indirect estimates tended to be systematically higher than the direct estimates; their range was wider, as were their confidence intervals; and biases were particularly strong for the younger age groups of respondents. Reasons for these biases are explored.     

Clonal growth of colorectal-carcinoma cell lines transplanted to nude mice

It is generally assumed that tumor progression is a microevolutionary process in which increasingly aggressive clones, generated through genetic instability, emerge in an initially monoclonal lesion. The present study was undertaken to determine how rapidly a dominant clone will emerge from an initial polyclonal situation, and whether dominance of these clones is a prerequisite for the onset of metastasis. To this end, colon-carcinoma cells were infected in culture with an amphotropic retroviral vector containing the neomycin-phosphotransferase gene, which makes cells resistant to neomycin. A heterogeneous population of neomycin-resistant cells carrying random retroviral integrations was xenografted to the subcutis and to the cecum of nude mice. The xenografts obtained, as well as the available metastases, were analyzed as to viral integrations by Southern blotting. The results show that, (i) clonal selection already takes place during growth of the primary tumor; (ii) dominant clones also generate metastases. The retroviral integration pattern of metastases turned out to be identical to that found in the primary xenografts. This pattern remained unchanged in tumors obtained after serial transplantations of cells cultured from metastases.     

Ia-bearing macrophages in athymic mice: antigen presentation and regulation

Ia-bearing macrophages from spleens and peritoneal cavities of athymic (nu/nu) and euthymic (nu/+) were compared. Macrophages from both strains of mice had equal antigen-presenting ability. The basal numbers of unstimulated Ia-bearing peritoneal macrophages were equal, but only euthymic mice recruited large numbers of Ia-bearing macrophages after Listeria infection. The i.p. injections of a "macrophage Ia-recruiting factor" induced exudates rich in Ia-bearing macrophages in both athymic and euthymic mice. These data suggest 2 levels of control of Ia-bearing macrophages: a basal T cell-independent level and a stimulated level dependent on mature T cells.     

Developmental features of human striatal tissue transplanted in a rat model of Huntington''s disease

The actions of the novel calcium (Ca2+) channel antagonist mibefradil (Ro 40-5967), a selective T-type channel blocker in myocardium, were investigated in embryonic rat spinal motoneurones maintained in culture. Whole-cell currents were recorded with the patch-clamp technique. Motoneurones displayed transient, low-voltage-activated (LVA) and, more sustained, high-voltage-activated (HVA) Ca2+ currents. The LVA currents were small and preferentially blocked by amiloride and low doses of nickel. Most of the HVA Ca2+ current flowed through N-type Ca2+ channels, while L-, and P/Q-type channels represented a smaller fraction. Mibefradil caused a rapid and reversible dose-dependent block of inward Ca2+ channel currents. Inhibition was nearly complete at 10 microM, suggesting mibefradil blockade of all subclasses of Ca2+ channels. The IC50 was approximately 1.4 microM on currents measured at 0 mV, from a holding potential of -90 mV. Inhibition of LVA Ca2+ current occurred over the same contraction range. Slow tail currents induced by the dihydropyridine agonist Bay K 8644 were also blocked by mibefradil, although with a slightly lower potency (IC50 = 3.4 microM). These broad inhibitory effects of mibefradil on Ca2+ influx were also supported by the strong inhibition of depolarization-induced intracellular calcium transients, measured from Indo-1 loaded motoneurones imaged with confocal microscopy. We conclude that mibefradil has potent blocking effects on Ca2+ channels in mammalian motoneurones. We hypothesize that therapeutic and pharmacological effects of mibefradil may involve actions on Ca2+ channels other than type T.     

Influence of dietary linoleic acid on experimental human breast cancer cell metastasis in athymic nude mice

The effects of linoleic acid (LA), an omega-6 fatty acid precursor for prostaglandin biosynthesis, on the later stages of human breast cancer cell metastasis were studied by the intravenous injection of tumor cells into nude mice (). MDA-MB-435 cells were grown as solid tumors in donor mice fed a 12% or 2% LA-containing diet. These cells were harvested, and injected via a tail vein into recipient mice also fed a 12% LA (Group 1) or 2% LA (Group 2) diet. Other groups were fed 12% LA (Group 3) or 2% LA (Group 4), but injected with the cells grown in vitro in a low-LA culture medium. At necropsy 8 weeks later, the incidence of metastatic lung nodules was higher in Group 1 high LA donor/high LA recipient mice (p<0.001), and, to a lesser degree, Group 2 low LA donor/low LA recipient mice (p<0.05) compared with Groups 3 or 4. The extent of metastasis was significantly higher in Group 1 compared with any of the other groups, including metastasis to the ovaries, which occurred in 27% of the Group 1 mice. These findings show that LA, most likely by increased synthesis of cyclooxygenase products, stimulates metastasis, at least in part, by direct effects on the tumor cells, rather than on potential metastatic sites in the host.     

Magnesium enhances function of postischaemic human myocardial tissue

OBJECTIVES: The effect of Mg2+ on the developed force and concentrations of high energy phosphate metabolites in isolated human atrial trabeculae has been investigated. METHODS: Human atrial trabeculae, obtained from right atrial appendages of patients undergoing cardiac surgery requiring cardiopulmonary bypass, were dissected at room temperature in modified Krebs-Henseleit buffer containing 1.2 or 16 mM Mg2+, mounted on muscle stands, and rewarmed to 34 degrees C in the same buffer. After 30 minutes, their mechanical function was assessed. At the end of the protocol, trabeculae were fast frozen for measurement of concentrations of metabolites of high energy phosphates. RESULTS: Trabeculae collected and rewarmed in 16 mM Mg2+ Krebs-Henseleit buffer showed significantly higher mean developed force (0.59(SEM 0.10) g, p < 0.01) than those rewarmed in 1.2 mM Mg2+ Krebs-Henseleit buffer (0.32(0.03) g). Trabeculae that had a developed force > or = 0.8 g, a resting force < or = 0.7 g, and a cross sectional area < or = 1 mm2 ("functional" trabeculae) were selected for further comparison. New reverse phase high performance liquid chromatography techniques developed for the analysis of small samples (0.5-5 mg dry weight) were used to measure nucleotide, nucleoside, and creatine compounds. Total adenylate (ATP+ADP+AMP) concentrations in trabeculae revived in the presence of 16 mM Mg2+ (15.4(1.1) mumol.g-1 dry weight) were significantly higher (p < 0.01) than in those revived with 1.2 mM Mg2+ (11.8(1.0) mumol.g-1), but lower (p < 0.01) than in trabeculae fast frozen immediately after removal from the patient (22.6(1.0) mumol.g-1). There were no significant differences in NAD and total creatine (phosphocreatine+creatine) concentrations between the three groups. CONCLUSIONS: The presence of high Mg2+ during the rewarming of human atrial trabecular preparations maintains a significantly higher developed force and a significantly higher total adenylate pool than does collection and rewarming with normal concentrations of Mg2+.     

A pilot experiment to compare regression of transplants of two different human tumors in athymic nude mice following single exposures to 60co radiation

Human tumor transplants were grown in a thymus defective nude mouse mutant (nu/nu BALB/c/A/Bom). Procedures for local irradiation of the tumor with 60Co-radiation are described. An introductory test of the usefulness of these transplants in studies of radiation effects on human tumors is performed by investigation of the time course of regression of adenocarcinoma transplants in six mice following single exposures of 375, 750, 1180 and 1575 rd, respectively, and malignant melanoma transplants in two mice following single exposures to 1475 and 2420 rd, respectively. The higher radioresistance of the malignant melanomas relative to that of the ovarian tumor is expected on the basis of clinical experience and indicates that radiation effects on human tissue implants in nude mice mirror some of the fundamental features of the in situ situation.     

Transforming growth factor beta 1 can induce estrogen-independent tumorigenicity of human breast cancer cells in athymic mice

We have examined the effect of transforming growth factor beta 1 (TGF-beta 1) overexpression in human breast cancer cell tumorigenicity in athymic mice. Estrogen-dependent MCF-7 cells were stably transfected with pSVTGF beta 1. A clone was isolated which overexpressed TGF-beta 1 mRNA and secreted > 10-fold more TGF-beta activity into the tissue culture medium. Similar to the parent line, the MCF-7/TGF-beta 1 cells were relatively insensitive to exogenous TGF-beta 1 and exhibited low levels of TGF-beta receptors. Clonogenicity in soft agarose, doubling time, morphology, and sensitivity to 17 beta-estradiol and the antiestrogen tamoxifen were not altered in the transfected cells. Inoculation s.c. of MCF-7/TGF-beta 1 cells in ovariectomized nude mice resulted in 100% tumor formation which was totally abrogated by i.p. administration of the neutralizing anti-TGF-beta 2G7 IgG2B. The parent cells formed tumors only after estrogen supplementation. By immunohistochemistry, higher levels of TGF-beta 1 protein were detected in MCF-7/TGF-beta 1 tumors than in estrogen-induced parent MCF-7 tumors. Administration of 1 microgram TGF-beta 1 i.p. daily for 3 weeks after tumor cell inoculation transiently supported estrogen-independent growth of parent MCF-7 tumors in castrated nude mice. These data indicate that overexpression of TGF-beta 1 in human breast cancer cells can contribute to their escape from hormone dependence.     

Growth hormone inhibits lipoprotein lipase activity in human adipose tissue

The in vitro effects of GH on human adipose tissue lipoprotein lipase (LPL) activity and messenger ribonucleic acid (mRNA) levels were studied using a tissue incubation technique. After preincubation for 3 days, abdominal sc adipose tissue pieces were exposed to cortisol (1000 nmol/L) for 3 days to induce LPL activity. Addition of GH (50 micrograms/L) to the cortisol-containing medium during the last 24 h (day 6) caused a decrease by 84 +/- 4% (P < 0.01) in heparin-releasable LPL activity and by 65 +/- 4% (P < 0.01) in total LPL activity. Moreover, the heparin-releasable fraction was reduced from 42% of the total LPL activity with cortisol alone to 17% when both GH and cortisol were present in the incubation medium during the last 24 h (P < 0.01). The reduction in LPL activity in response to GH was not accompanied by a decrease in the level of LPL mRNA measured by a solution hybridization ribonuclease protection assay. In adipose tissue incubated in the control medium for 6 days, the addition of GH alone during the last 24 h caused an insignificant decrease in heparin-releasable LPL activity. Low control activities limited the scope for further decrease. It is concluded that GH counteracts the potent stimulatory effect of glucocorticoids on LPL activity without affecting LPL mRNA levels. Therefore, the inhibition of LPL activity by GH probably occurs during translation and/or posttranslational processing of the enzyme, and the mechanism may involve a decreased channeling of the lipase to the cell surface.