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Biomedical subjects

M L Brandi

Publications and source records attributed to M L Brandi.

At least 127 records · Page 7Linked to original sources
PubMed1994

Role for autocrine TGF-beta 1 in regulating differentiation of a human leukemic cell line toward osteoclast-like cells.

Increasing evidence suggests that transforming growth factor-beta (TGF-beta) is involved in bone formation during remodeling. Using a recently cloned human leukemic cell line (FLG 29.1 cells) we demonstrate that these cells synthesize and secrete TGF-beta 1 and that exogenous or autocrine TGF-beta 1 can induce the same features of osteoclastic-like cells, exerting its effects through the binding to TGF-beta specific receptors. Scatchard analysis of 125I-labeled TGF-beta 1 to FLG 29.1 cells revealed the presence of a single high affinity binding site with a Kd value of approximately 25 pM and a binding capacity of approximately 900 sites/cell. Affinity labeling experiments showed that FLG 29.1 cells express type I and type II TGF-beta receptors. Stimulation of FLG 29.1 cells with low TGF-beta 1 doses reduced cell proliferation and increased cell adhesion and tartrate resistant acid phosphatase (TRAcP) activity. Pretreatment of FLG 29.1 cells with TGF-beta 1 caused a significant and dose-dependent response to calcitonin. Northern blot of total mRNA and analysis of the conditioned media (CM) showed that TGF-beta 1 was synthesized by FLG 29.1 cells. TPA treatment, which induces partial differentiation of these cells, markedly increased TGF-beta 1 mRNA expression and growth factor release. The majority of TGF-beta 1 secreted by TPA-treated cells was in its latent form. However, anti-TGF-beta antibodies inhibited TGF-beta 1 and TPA-induced growth inhibition, calcitonin responsiveness, and TRAcP activity, suggesting that the TPA effect is mediated in part by autocrine TGF-beta 1 and indicating that the cells can activate and respond to the TGF-beta that they secrete. These findings support a potential autocrine role for TGF-beta 1 in osteoclast differentiation.

Acid Phosphatase↗
PubMed1994

Prostaglandin-stimulated second messenger signaling in bone-derived endothelial cells is dependent on confluency in culture.

New bone formation is associated with an increase in blood flow by the invasion of capillaries. Endothelial cells that line the capillaries can produce paracrine factors that affect bone growth and development, and in turn, could be affected by products produced by bone cells, in particular the osteoblasts. Since osteoblasts produce prostaglandins E2 and F2 alpha (PGE2, PGF2 alpha), it was investigated if these PGs were agonists to bone-derived endothelial cells (BBE) by assessing changes in cAMP and free cytosolic calcium concentration ([Ca2+]i) second messenger generation. We found that confluent cultures of BBE cells, a clonal endothelial cell line derived from bovine sternal bone, responded to 1 microM PGE2 by an increase in cAMP. PGF2 alpha at the same concentration was less potent in stimulating an increase in cAMP production in confluent BBE cells. Subconfluent cells with a morphology similar to that of fibroblastic cells were not as sensitive to PGE2-stimulated cAMP generation. PGF2 alpha failed to elicit any cAMP production in subconfluent cultures. PGE2 and PGF2 alpha both stimulated an increase in [Ca2+]i concentration in a dose-dependent manner. The potency of PGE2 was similar to that of PGF2 alpha in stimulating an increase in [Ca2+]i. The Ca2+ response was mostly independent of extracellular Ca+, was unchanged even with prior indomethacin treatment, was unaffected by caffeine pretreatment, but was abolished subsequent to thapsigargin pretreatment. The PG-induced increase in [Ca2+]i was also dependent on the confluency of the cells. In a subconfluent state, the responses to PGE2, or PGF2 alpha were either negligible, or only small increases in [Ca2+]i were noted with high concentrations of these two PGs. Consistent, dose-dependent increases in [Ca2+]i were stimulated by these PGs only when the cells were confluent and had a cobblestoned appearance. Since it was previously demonstrated that BBE cells respond to parathyroid hormone (PTH) by the production of cAMP, we tested if bovine PTH(1-34) amide ]bPTH(1-34) also increased [Ca2+]i in these cells. No change in [Ca2+]i was found in response to bPTH (1-34), although bPTH (1-34) stimulated a nine to tenfold increase in cAMP. We conclude that BBE cells respond to PGE2 and PGF2 alpha but not to bPTH(1-34) by an increase in [Ca2+]i probably secondary to stimulation of phospholipase C and that the cAMP and [Ca2+]i second messenger responses in BBE cells are dependent on the state of confluency of the cells.

Animals↗
PubMed1994

Comparison of immuno- and HPLC-assays for the measurement of urinary collagen cross-links.

Pyridinoline (Pyr) and deoxypyridinoline (D-Pyr) are two cross-links of collagen molecules, that are present in the extracellular matrix and released during its degradation. Pyr is present in bone and cartilage, but not in significant amounts in other connective tissues and D-Pyr appears to be specific for bone tissue. Therefore, the urinary excretion of Pyr and D-Pyr might be a sensitive marker of bone matrix degradation. For the determination of urinary Pyr and D-Pyr two methods are available: a chromatographic method (HPLC) by which it is possible to measure separately Pyr and D-Pyr, and a new immunoassay which measures total free and low molecular weight pyridinoline released in the urine. We compared the results obtained by HPLC analysis of 205 urinary samples from normal subjects and patients affected by various bone disorders with those obtained by the immunoassay. The overall correlation coefficient between the results obtained by the two methods was 0.34. When calculated in a range of pyridinoline concentrations from 0 to 30, 30 to 60, and over 60 pmol/mumol creatinine the correlation coefficient was respectively -0.094, 0.38, and 0.12. The two methods yielded variable profiles in the detection of circadian rhythms and these differences did not segregate with normal or pathological conditions. We conclude that the immunoassay proposed for the determination of urinary collagen cross-links is not immediately applicable to clinical use. The improvement of the antibody specificity will probably contribute to replace the HPLC method with the immunoassay.

Adult↗
PubMed1994

Production of basic fibroblast growth factor by gastric carcinoid tumors and their putative cells of origin.

Using immunohistochemical techniques a subpopulation of endocrine cells in the human oxyntic mucosa was found to react with antibodies against basic fibroblast growth factor (bFGF). These cells were identified as histamine-producing enterochromaffin-like (ECL) cells and, to a minor extent, serotonin-producing enterochromaffin cells. Basic fibroblast growth factor immunoreactive cells were most frequently found in hyperplastic lesions of ECL cells occurring in hypergastrinemic patients (20 of 27 cases) and in ECL cell carcinoid tumors (10 of 17 cases). In addition, bFGF mRNA was demonstrated by Northern blot analysis of homogenates from two gastric carcinoids cytologically characterized as pure ECL cell tumors. Although the function of bFGF in normal cells remains unknown, its production in neoplastic conditions may be responsible for the associated desmoplastic and angioblastic proliferations. Moreover, secretion of bFGF by hyperplastic or neoplastic ECL cells may contribute to the circulating levels of the bFGF-like mitogenic factor identified in patients affected by multiple endocrine neoplasia type 1 syndrome.

Carcinoid Tumor↗
PubMed1994

Familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Effects of mutant gene dosage on phenotype.

Neonatal severe hyperparathyroidism is a rare life-threatening disorder characterized by very high serum calcium concentrations (> 15 mg/dl). Many cases have occurred in families with familial hypocalciuric hypercalcemia, a benign condition transmitted as a dominant trait. Among several hypothesized relationships between the two syndromes is the suggestion that neonatal severe hyperparathyroidism is the homozygous form of familial hypocalciuric hypercalcemia. To test this hypothesis, we refined the map location of the gene responsible for familial hypocalciuric hypercalcemia on chromosome 3q. Analyses in 11 families defined marker loci closely linked to the gene responsible for familial hypocalciuric hypercalcemia. These loci were then analyzed in four families with parental consanguinity and offspring with neonatal severe hyperparathyroidism. Each individual who was homozygous for loci that are closely linked to the gene responsible for familial hypocalciuric hypercalcemia had neonatal severe hyperparathyroidism. The calculated odds of linkage between these disorders of > 350,000:1 (lod score = 5.56). We conclude that dosage of the gene defect accounts for these widely disparate clinical phenotypes; a single defective allele causes familial hypocalciuric hypercalcemia, while two defective alleles causes neonatal severe hyperparathyroidism.

Chromosome Mapping↗
PubMed1994

Frequent RET protooncogene mutations in multiple endocrine neoplasia type 2A.

The occurrence of mutations in the RET protooncogene has been investigated in 12 multiple endocrine neoplasia type 2A families and 18 cases of sporadic thyroid medullary carcinomas and pheochromocytomas. Ten of 12 families showed single base substitutions in the RET protooncogene exons 10 and 11, coding for the extracellular domain of the protein. Tumor tissues from 2 multiple endocrine neoplasia type 2A patients were analyzed at the DNA and ribonucleic acid levels and revealed the same heterozygous mutations found in the peripheral blood lymphocytes. This demonstrates that both the normal and mutant alleles are expressed. No mutations in these exons were detected in the 18 cases of sporadic tumors investigated. These data provided further evidence that the mutated RET protooncogene acts in a dominant fashion and is responsible for the pathogenesis of this syndrome.

Adrenal Gland Neoplasms↗
PubMed1994

Circulating fibroblast growth factor-like autoantibodies in two patients with multiple endocrine neoplasia type 1 and prolactinoma.

Basic fibroblast growth factor (bFGF) is a potent endothelial cell mitogen found in a variety of normal and tumor tissues. bFGF lacks a classical amino-terminal signal sequence and is not readily detectable in plasma from normal subjects. In earlier studies we showed increased bFGF-like mitogenic activity for parathyroid-derived endothelial cells and (increased) bFGF immunoreactivity (0.24-1.28 ng/mL) in plasma of subjects with multiple endocrine neoplasia type 1 (MEN-1). In the present study we examined the proliferative activity of MEN-1 and normal plasmas (applied to protein-A columns) in calf pulmonary artery endothelial cells. Protein-A-eluted activity in plasma from MEN-1 prolactinoma plasma exceeded activity from normal and MEN-1 nonprolactinoma plasma in three of eight MEN-1 subjects with untreated or recurrent prolactinoma. Protein-A-eluted active fractions from MEN-1 prolactinoma plasma had several properties of an immunoglobulin G, including affinity for antihuman immunoglobulin G (IgG) agarose, sensitivity to thiols, and (prepared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions) apparent mol wt corresponding to those of the heavy and light chains of IgG. The IgG fraction of MEN-1 prolactinoma plasma had far more activity in endothelial cells than did optimal concentrations of known growth factors or conditioned medium from prolactinoma cells. Endothelial cell bioactivity in protein-A-eluted fractions from MEN-1 prolactinoma plasma was neutralized 70% by rabbit antibodies to intact bFGF. These results imply novel growth stimulatory bFGF-like autoantibodies in a subset of MEN-1 patients with prolactinoma.

Adult↗
PubMed1994

Pharmacologic treatment of desmoid tumors in familial adenomatous polyposis: results of an in vitro study.

BACKGROUND: Desmoid tumors (DTs), the result of an abnormal proliferation of connective tissue, occur frequently in familial adenomatous polyposis. Treatment of DT is difficult because of the high rate of recurrence after operation. Recently, antiestrogens and nonsteroidal antiinflammatory drugs have been used with good results as inhibitors of DT cell proliferation. METHODS: In this report we performed in vitro studies on cultured desmoid cells and skin fibroblasts of four patients who underwent surgical resection of DT and normal skin biopsy. We evaluated the expression of estrogen receptors and the mitogenic effect of 17 beta-estradiol and sulindac, a nonsteroidal antiinflammatory compound, on cell proliferation and collagen synthesis of desmoid cells. RESULTS: Proliferation and collagen synthesis of desmoid cells were stimulated by 17 beta-estradiol, and tamoxifen, an antiestrogenic compound, inhibited this effect. Desmoid cells also expressed estrogen receptors. Moreover, growth of desmoid cells from one of the patients was inhibited by sulindac. CONCLUSIONS: The in vitro evaluation of drug responsiveness in patients operated on for DT could be used as both a prognostic tool in the natural history of DT and in addressing pharmacologic therapy in this disorder.

Adenomatous Polyposis Coli↗
PubMed1993

New treatment strategies: ipriflavone, strontium, vitamin D metabolites and analogs.

Drugs used to treat osteoporosis can be grouped in two main categories: those that decrease bone resorption and those that increase bone formation. Antiresorptive drugs are active in preventing bone fractures in patients characterized by a negative calcium balance. However, because antiresorptive agents are often coupled to inhibition of bone formation, inhibitors of bone resorption may not be candidates as potential curative drugs in osteoporosis. Conversely, drugs that act by increasing bone formation produce an increase in bone mass above the fracture threshold; therefore, these agents are good candidates for the treatment of osteoporosis. The ideal curative drug in osteoporosis should have the ability both to decrease bone resorption and to maintain a relatively high rate of bone formation, thus inducing a favorable uncoupling of bone remodeling.

Aged↗
PubMed1993

Cytogenetics of multiple endocrine neoplasia syndromes. III. Analysis of an insulinoma from a subject with MEN 1 by chromosome painting.

The cytogenetics of an insulinoma from a subject with MEN 1 characterized by the consistent presence of double minute chromosomes (dmins) and by five characteristic marker chromosomes was investigated with fluorescence in situ hybridization after labeling with a chromosome 11 library. The dmins were consistently negative for 11q material, with the exception of one metaphase which had two positive dmins. This indicated that the dmins are not derived massively from chromosome 11 and that they can be heterogeneous in their origin. One of the marker chromosomes, tentatively identified as a del (7), turned out to be the product of a 7;11 translocation.

Chromosome Aberrations↗
PubMed1993

Effects of endothelin-1 on bovine parathyroid cells.

Endothelin-1 (ET-1) is synthesized and released by parathyroid epithelium. The effects of endothelin isopeptides were studied in clonal bovine parathyroid endothelial (BPE) cells. BPE cells did not produce ET-1, but showed ETA receptors (Kd = 0.1 +/- 0.02 nM, mean +/- SE). ET-1 (10(-8)10(-11)M) increased the intracellular calcium ion concentration ([Ca2+]i) in BPE cells, while endothelin-3 (ET-3) was ineffective. The increase in [Ca2+]i was less sustained in the absence of extracellular Ca2+ ions. Moreover ET-1 induced phospholipase C (PLC) activation, as demonstrated by the increase in inositol trisphosphate. Cell growth was not affected by ET-1 in a wide range of concentrations. The present findings demonstrate: 1) BPE cells possess ETA receptors; 2) the peptide activates PLC and increases cytosolic [Ca2+]i via both a release of Ca2+ ions from intracellular calcium pool(s) and an influx of the cation from the extracellular milieu. A possible role of ET-1 as a paracrine factor in parathyroid tissue can be hypothesized.

Animals↗
PubMed1993

Release of extracellular matrix components by bovine bone endothelial cells in continuous culture.

Production of collagen type I (pIp), type III (pIIIp), type IV (CIV), type VI (CVI), fibronectin (FN), laminin and glycosaminoglycans (GAGs) has been investigated in a clonal line of Bovine Bone Endothelial (BBE) cells continuously growing in culture. BBE cells produced pIp, pIIIp and FN, whereas they did not synthetize significant amounts of CIV, CVI and laminin. pIp, pIIIp and FN syntheses were influenced by cell density:pIp secretory activity reached its maximum just after cell seeding, whereas pIIIp and FN production increased later, when cells were subconfluent. Furthermore, BBE cells produced and released significant amounts of GAGs into medium.

Animals↗
PubMed1993

Bone endothelial cells as estrogen targets.

In the present study, we investigated the effects of estrogens on bone endothelial cell metabolism and the presence of estrogen binding sites in the same cells. For these studies, we have used a continuous cell line of clonal bovine bone endothelial cells for evidence of a direct response to estrogens in vitro. Receptor analysis to intact viable cells was steroid specific and saturable, with an apparent dissociation constant of 17.2 nM and a Bmax of 3.2 x 10(4) sites/cell. Northern blot analysis revealed a 6.5-kilobase mRNA that hybridized with a cDNA to human estrogen receptor. The 6.5-kilobase size is in close agreement with the reported size of the human estrogen receptor mRNA. In vitro estrogen responses of bone endothelial cells included a stimulation of cell proliferation as well as an inhibition of parathyroid hormone responsiveness. These findings clearly demonstrate the presence of functional estrogen receptors in bone endothelial cells in vitro, suggesting a role of estrogens in bone angiogenesis and in the entire process of bone remodeling.

Animals↗