Spectroscopic markers of bone quality in alendronate-treated postmenopausal women.

UNLABELLED: Comparison of infrared spectroscopic images of sections from biopsies of placebo-treated post-menopausal women and women treated for 3 years with 10 mg/day alendronate demonstrated significant increases in cortical bone mineral content, no alterations in other spectroscopic markers of "bone quality," but a decrease in tissue heterogeneity. METHODS: The material properties of thick sections from iliac crest biopsies of seven alendronate-treated women were compared to those from ten comparably aged post-menopausal women without bone disease, using infrared spectroscopic imaging at approximately 7 microm spatial resolution. Parameters evaluated were mineral/matrix ratio, crystallinity, carbonate/amide I ratio, and collagen maturity. The line widths at half maximum of the pixel histograms for each parameter were used as measures of heterogeneity. RESULTS: The mineral content (mineral/matrix ratio) in the cortical bone of the treated women's biopsies was higher than that in the untreated control women. Crystallinity, carbonate/protein, and collagen maturity indices were not significantly altered; however, the pixel distribution was significantly narrowed for all cortical and trabecular parameters with the exception of collagen maturity in the alendronate treatment group. CONCLUSIONS: The increases in mineral density and decreased fracture risk associated with bisphosphonate treatment may be counterbalanced by a decrease in tissue heterogeneity, which could impair tissue mechanical properties. These consistent data suggest that alendronate treatment, while increasing the bone mass, decreases the tissue heterogeneity.

The ultrastructure of the nexus. A correlated thin-section and freeze-cleave study.

A correlation is made between the appearances of the nexus ("gap junction") as revealed by thin-section and by freeze-cleave electron microscopy techniques. These methods reveal different aspects of a complex subunit assembly forming the nexus membranes. In thin sections, the nexus is formed by the very close apposition of two "unit" membranes. The electron-opaque tracer, colloidal lanthanum hydroxide, outlines an aspect of electron-lucent subunits that project into the central region of the nexus. The freeze-cleave technique demonstrates novel membrane faces that are generated from within the interior of plasma membranes by splitting them into two lamellae (Lm): Lm 1 adjacent to the cytoplasm, and Lm 2 adjacent to the extracellular space. Each of the two membranes forming the nexus can be split into these two lamellae. On the new face of Lm 1, particles approximately 50 A in diameter are closely packed in an array which is often hexagonal with a 90-100 A center-to-center spacing. The two apposed lamellae (Lm 2-Lm 2) of the nexus are constructed of sheets of subunits in a similar array. The Lm 1 particles appear to extend into the Lm 2 subunits to form macromolecular complexes. The Lm 2 subunits extend to the center of the nexus to form the contacts outlined by lanthanum in sections. It is postulated that central hydrophilic channels may extend through the subunit assembly to provide a direct route for intercellular communication.

Further observations on the occurrence of nexuses in benign and malignant human cervical epithelium.

An estimate is made of the frequency of occurrence of nexuses ("gap junctions") in a spectrum of human cervical epithelia, ranging from normal to malignant, since a deficiency of nexuses may be important in abnormal cell-to-cell communication in malignant tissues. The normal cervical epithelium has approximately ten nexuses per cell in the basal layer of proliferating cells and 200 nexuses per cell in the more differentiated intermediate zone. Nexuses are rare between invasive malignant epithelial cells (carcinoma cells). In many areas of cell proliferation near the edge of the tumor mass, fewer than one nexus per cell is present. However, up to four nexuses per cell can be found in some well differentiated regions of invasive carcinoma. Preinvasive malignant epithelia (severe dysplasia and carcinoma-in situ) have as few nexuses as invasive carcinoma. In abnormal but benign epithelia (squamous metaplasia and mild dysplasia), nexuses are abundant. The data indicate that a decrease in number of nexuses correlates with the severity of the morphological alteration in the dysplastic epithelium. Also the deficiency of nexuses in groups of carcinoma cells can occur many cell generations before the development of invasion of the malignant epithelium into the connective tissue. The diminution of nexuses before invasion suggests that a deficiency of nexuses may be one of the important factors in eventually permitting the development of the diffusely infiltrating type of invasion which is characteristic of highly malignant tumors such as squamous carcinomas.

Freeze-fracture of monolayer cultures.

This paper describes a simple method for the freeze-fracturing of cells in monolayers or multi-layer tissue cultures. The method produces high quality replicas and is applicable to the study of virtually any tissue culture or organ culture system. It uses standard materials and equipment for both tissue culture and freeze-fracturing.

Carcinoma of the cervix: deficiency of nexus intercellular junctions.

Intercellar junctions where cell membranes are in intimate contact (nexuses) are very abundant in the epithelium of normal human cervix. Squamous carcinoma cells are deficient in nexuses although a rare nexus is seen. Nexuses may be involved in normal growth regulation, while a deficiency of nexuses may be related to the invasive property of malignant growth.

Inside-out red cell membrane vesicles: preparation and purification.

Plasma membranes purified from human red cells were Converted into small vesicleS by disruption in alkaline buffer of low ionic strength. Most of these vesicles were inside-out. The presence of divalent cations prevented this inversion. The inside-out vesicles were separatcd from right-side-out vesicles by centrifugration to equilibrium in dextran density gradients.

Multiple immunoreactive forms of osteocalcin in uremic serum.

Circulating osteocalcin, which normally reflects the rate of bone formation, is elevated in uremia. In 18 patients receiving maintenance hemodialysis, serum osteocalcin levels were directly related to the bone formation rate (r = 0.88, P less than 0.001), osteoblastic osteoid surface density (r = 0.65, P less than 0.01), and osteoclastic resorptive surface density (r = 0.75, P less than 0.001). Multiple regression analysis showed that osteocalcin levels remained positively correlated with osteoclastic resorption when the bone formation rate was held constant (P less than 0.01). The intimation that the coupling of bone formation and resorption could not explain the relationship between osteocalcin and resorption led us to determine whether fragments of this abundant matrix protein are released by bone resorption and retained in uremia. Sera from dialysis patients with renal osteodystrophy were fractionated by sequential gel filtration and HPLC, and assayed for immunoreactive osteocalcin. When normal serum was analyzed, a single sharp peak was found. In pooled sera from patients with high osteoclastic resorptive surfaces identified by histomorphometry, we found five additional immunoreactive peaks, while three additional peaks were detected in sera from patients with lower osteoclastic surfaces. Bio-Gel P-10 chromatography showed that these multiple peaks were of lower molecular weight than intact osteocalcin. We suggest that the liberation of bone matrix by osteoclasts contributes to the circulating osteocalcin immunoreactivity in uremia.

Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. Potential mechanisms of their deleterious effects on bone.

Glucocorticoid-induced bone disease is characterized by decreased bone formation and in situ death of isolated segments of bone (osteonecrosis) suggesting that glucocorticoid excess, the third most common cause of osteoporosis, may affect the birth or death rate of bone cells, thus reducing their numbers. To test this hypothesis, we administered prednisolone to 7-mo-old mice for 27 d and found decreased bone density, serum osteocalcin, and cancellous bone area along with trabecular narrowing. These changes were accompanied by diminished bone formation and turnover, as determined by histomorphometric analysis of tetracycline-labeled vertebrae, and impaired osteoblastogenesis and osteoclastogenesis, as determined by ex vivo bone marrow cell cultures. In addition, the mice exhibited a threefold increase in osteoblast apoptosis in vertebrae and showed apoptosis in 28% of the osteocytes in metaphyseal cortical bone. As in mice, an increase in osteoblast and osteocyte apoptosis was documented in patients with glucocorticoid-induced osteoporosis. Decreased production of osteoclasts explains the reduction in bone turnover, whereas decreased production and apoptosis of osteoblasts would account for the decline in bone formation and trabecular width. Furthermore, accumulation of apoptotic osteocytes may contribute to osteonecrosis. These findings provide evidence that glucocorticoid-induced bone disease arises from changes in the numbers of bone cells.

Increased bone formation by prevention of osteoblast apoptosis with parathyroid hormone.

The mass of regenerating tissues, such as bone, is critically dependent on the number of executive cells, which in turn is determined by the rate of replication of progenitors and the life-span of mature cells, reflecting the timing of death by apoptosis. Bone mass can be increased by intermittent parathyroid hormone (PTH) administration, but the mechanism of this phenomenon has remained unknown. We report that daily PTH injections in mice with either normal bone mass or osteopenia due to defective osteoblastogenesis increased bone formation without affecting the generation of new osteoblasts. Instead, PTH increased the life-span of mature osteoblasts by preventing their apoptosis - the fate of the majority of these cells under normal conditions. The antiapoptotic effect of PTH was sufficient to account for the increase in bone mass, and was confirmed in vitro using rodent and human osteoblasts and osteocytes. This evidence provides proof of the basic principle that the work performed by a cell population can be increased by suppression of apoptosis. Moreover, it suggests novel pharmacotherapeutic strategies for osteoporosis and, perhaps, other pathologic conditions in which tissue mass diminution has compromised functional integrity.

Linkage of decreased bone mass with impaired osteoblastogenesis in a murine model of accelerated senescence.

Bone marrow is the principal site for osteoclastogenesis and osteoblastogenesis; and an increase in the former has been linked with bone loss caused by acute loss of gonadal steroids. We have now used an established murine model of accelerated senescence and osteopenia (SAMP6) to test the hypothesis that reduced osteoblastogenesis is linked with decreased bone mass. At 1 mo of age, the number of osteoblast progenitors in SAMP6 marrow was indistinguishable from controls; however a threefold decrease was found at 3-4 mo of age. Impaired osteoblast formation was temporally associated with decreased bone formation and decreased bone mineral density, as determined by histomorphometric analysis of tetracycline-labeled cancellous bone and dual-energy x-ray absorptiometry, respectively. Osteoclastogenesis determined in ex vivo bone marrow cultures was also decreased in these mice, as was the number of osteoclasts in histologic sections. Moreover, unlike controls, senescence-accelerated mice failed to increase osteoclast development after gonadectomy. The osteoclastogenesis defeat was secondary to impaired osteoblast formation as evidenced by the fact that osteoclastogenesis could be restored by addition of osteoblastic cells from normal mice. These findings provide the first demonstration of a link between low bone mineral density and decreased osteoblastogenesis in the bone marrow and validate the senescence-accelerated mouse as a model of involutional osteopenia.

Prevention of osteocyte and osteoblast apoptosis by bisphosphonates and calcitonin.

Glucocorticoid-induced osteoporosis may be due, in part, to increased apoptosis of osteocytes and osteoblasts, and bisphosphonates (BPs) are effective in the management of this condition. We have tested the hypothesis that BPs suppress apoptosis in these cell types. Etidronate, alendronate, pamidronate, olpadronate, or amino-olpadronate (IG9402, a bisphosphonate that lacks antiresorptive activity) at 10(-9) to 10(-6) M prevented apoptosis of murine osteocytic MLO-Y4 cells, whether it was induced by etoposide, TNF-alpha, or the synthetic glucocorticoid dexamethasone. BPs also inhibited apoptosis of primary murine osteoblastic cells isolated from calvaria. Similar antiapoptotic effects on MLO-Y4 and osteoblastic cells were seen with nanomolar concentrations of the peptide hormone calcitonin. The antiapoptotic effect of BPs and calcitonin was associated with a rapid increase in the phosphorylated fraction of extracellular signal regulated kinases (ERKs) and was blocked by specific inhibitors of ERK activation. Consistent with these in vitro results, alendronate abolished the increased prevalence of apoptosis in vertebral cancellous bone osteocytes and osteoblasts that follows prednisolone administration to mice. These results suggest that the therapeutic efficacy of BPs or calcitonin in diseases such as glucocorticoid-induced osteoporosis may be due, in part, to their ability to prevent osteocyte and osteoblast apoptosis.

Telemedicine and telepresence for trauma and emergency care management.

The use of telemedicine is long-standing, but only in recent years has it been applied to the specialities of trauma, emergency care, and surgery. Despite being relatively new, the concept of teletrauma, telepresence, and telesurgery is evolving and is being integrated into modern care of trauma and surgical patients. This paper will address the current applications of telemedicine and telepresence to trauma and emergency care as the new frontiers of telemedicine application. The University Medical Center and the Arizona Telemedicine Program (ATP) in Tucson, Arizona have two functional teletrauma and emergency telemedicine programs and one ad-hoc program, the mobile telemedicine program. The Southern Arizona Telemedicine and Telepresence (SATT) program is an inter-hospital telemedicine program, while the Tucson ER-link is a link between prehospital and emergency room system, and both are built upon a successful existing award winning ATP and the technical infrastructure of the city of Tucson. These two programs represent examples of integrated and collaborative community approaches to solving the lack of trauma and emergency care issue in the region. These networks will not only be used by trauma, but also by all other medical disciplines, and as such have become an example of innovation and dedication to trauma care. The first case of trauma managed over the telemedicine trauma program or "teletrauma" was that of an 18-month-old girl who was the only survival of a car crash with three fatalities. The success of this case and the pilot project of SATT that ensued led to the development of a regional teletrauma program serving close to 1.5 million people. The telepresence of the trauma surgeon, through teletrauma, has infused confidence among local doctors and communities and is being used to identify knowledge gaps of rural health care providers and the needs for instituting new outreach educational programs.

Glucocorticoids act directly on osteoclasts to increase their life span and reduce bone density.

Glucocorticoid administration to mice results in a rapid loss of bone mineral density due to an imbalance in osteoblast and osteoclast numbers. Whereas excess glucocorticoids reduce both osteoblast and osteoclast precursors, cancellous osteoclast number surprisingly does not decrease as does osteoblast number, presumably due to the ability of glucocorticoids to promote osteoclast life span. Whether glucocorticoids act directly on osteoclasts in vivo to promote their life span and whether this contributes to the rapid loss of bone with glucocorticoid excess remains unknown. To determine the direct effects of glucocorticoids on osteoclasts in vivo, we expressed 11beta-hydroxysteroid dehydrogenase type 2, an enzyme that inactivates glucocorticoids, specifically in the osteoclasts of transgenic mice using the tartrate-resistant acid phosphatase promoter. Bone mass, geometry, and histomorphometry were similar in untreated wild-type and transgenic animals. Glucocorticoid administration for 7 d caused equivalent increases in cancellous osteoblast apoptosis, and equivalent decreases in osteoblasts, osteoid, and bone formation, in wild-type and transgenic mice. In contrast, glucocorticoids stimulated expression of the mRNA for calcitonin receptor, an osteoclast product, in wild-type but not transgenic mice. Consistent with the previous finding that glucocorticoids decrease osteoclast precursors and prolong osteoclast life span, glucocorticoids decreased cancellous osteoclast number in the transgenic mice but not wild-type mice. In accord with this decrease in osteoclast number, the loss of bone density observed in wild-type mice was strikingly prevented in transgenic mice. These results demonstrate for the first time that the early, rapid loss of bone caused by glucocorticoid excess results from direct actions on osteoclasts.

Unusual cell junctional complexes in canine mammary adenoacanthomas.

Unusual cell junctional complexes were described in spontaneously arising adenoacanthomas of canine mammary glands. These junctional complexes were a manifestation of bidirectional differentiation of tumor cell membranes.

Modulation of the metastatic phenotype by 13-cis-retinoic acid.

KLN 205 murine squamous carcinoma cells were grown in medium supplemented with the retinoid 13-cis-retinoic acid (RA) to study the relationship between RA-induced cell surface changes and alterations of the metastatic phenotype. Modulation of the cell surface glycoconjugate expression was measured by flow cytometric analysis of the RA-treated tumor cells stained with fluoresceinated lectins. RA treatment (5 X 10(-6) and 5 X 10(-7) M) altered the glycoconjugate expression of KLN 205 cells in a selective, dose-dependent fashion. Tumor cells grown in RA-supplemented medium for more than 4 days demonstrated greatly increased binding of fluoresceinated Griffonia simplicifolia I lectin, peanut lectin, wheat-germ lectin, concanavalin A, and soybean lectin (P less than .001), but the increased binding of Ulex europaeus lectin was of a much smaller magnitude (P = .02). After 15 days of growth in these noncytotoxic or cytostatic concentrations of RA, malignant KLN 205 cells had a greatly decreased proclivity to metastasize, as measured by the lung colony assay (P = .0003). The RA-induced cell surface glycoconjugate changes preceded the decrease in experimental metastatic potential. Since enzymatic (neuraminidase) alteration of the tumor cell surface to produce glycoconjugate expression similar to that seen in RA-treated cells also reduced the ability of the KLN 205 cells to form lung colonies (P = .0022), it is suggested that RA-induced alteration of the cell surface carbohydrate antigens is related to the decreased experimental metastatic potential seen in tumor cells treated with RA.

Changes in plasma membrane structure associated with malignant transformation in human urinary bladder epithelium.

Integral membrane proteins are visualized as intramembrane particles (IMP; also called membrane-associated particles) at the cleaved surfaces of freeze-fractured plasma membranes. Topographical distributions of the IMP of urothelial cell membranes in normal human bladder and for a small series of low-grade noninvasive transitional cell carcinomas and invasive transtional cell carcinomas are shown to be significantly different. Using several statistical methods that test IMP topography via à vis the random (Poisson) hypothesis, it is demonstrated that IMP are mildly aggregated in plasma membranes of normal human urothelial cells and that, in noninvasive carcinomas, IMP aggregation is increased. In invasive transitional cell carcinomas, IMP are statistically nonaggregated and are in a random distribution in the plane of the membrane. IMP numerical densities are also altered in the course of neoplastic transformation. IMP are significantly increased in number in plasma membranes in human noninvasive transitional cell carcinomas but are similar to control values in invasive tumors. Loss of IMP and changes in IMP topography may be related to tumor invasiveness or they may represent an epiphenomenon.

Correlations between cell surface protease activities and abnormalities of occludens junctions in rat bladder carcinoma in vitro.

Microenvironmental alterations, i.e., proteolytic enzymes, may play a causative role in abnormalities of zonulae occludentes. To test this hypothesis, we compared in vitro the ultrastructure of three carcinoma cell lines which were derived from N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide-induced tumors of the rat urinary bladder. One of these lines had a high cell surface protease activity; the other two lines exhibited relatively low activities. Quantitative electron microscopy data revealed differences in configuration and distribution of zonula occludens-intramembrane fibrils among these cell lines, as indicated by means and standard deviations of zonulae occludens widths, and numbers of intramembrane fibrils. Although the total length of the intramembranous fibrils per square micrometer of occludens junction area was not statistically different in the three lines, junctional morphology varied greatly. Thus, carcinoma cells with high surface protease activities are able to synthesize near-normal amounts of intramembrane fibrils but are unable to assemble normal zonulae occludentes. This indicates that alterations in zonula occludens morphology, which have been induced by exogenous proteolytic enzymes, are identical to those observed in a cell line with high cell surface protease activity.

Solutol HS 15, nontoxic polyoxyethylene esters of 12-hydroxystearic acid, reverses multidrug resistance.

A recently developed non-ionic surfactant called Solutol HS 15 (poly-oxyethylene esters of 12-hydroxystearic acid), with low toxicity in vivo, was shown to reverse completely the multidrug resistance of KB 8-5 and KB 8-5-11 human epidermoid carcinoma cells in vitro but did not potentiate drug toxicity in drug-sensitive KB 3-1 cells. At a concentration of 10% of its own IC50 (mean concentration of drug that causes 50% inhibition of cell growth compared to controls), Solutol HS 15 produced a 35-, 28-, and 42-fold reduction in the resistance of KB 8-5-11 cells to colchicine, vinblastine, and doxorubicin, respectively. Solutol HS 15 was relatively much more potent than the prototypic reversing agent, verapamil, for reversing colchicine resistance, compared to the ability of each agent to reverse colchicine resistance, compared to the ability of each agent to reverse vinblastine resistance. Like verapamil, Solutol HS 15 promoted a 50-fold accumulation of rhodamine 123 in KB 8-5-11 cells, as measured by flow cytometry. Also, Solutol HS 15 and verapamil reduced the efflux of rhodamine 123 from KB 8-5-11 cells previously loaded with rhodamine 123 to a similar low rate. Solutol HS 15 did not affect the transport of alanine or glucose into KB 8-5-11 cells, indicating that its effect upon membrane active transport is not entirely nonspecific. Considering their different structure and different relative potency for reversing colchicine resistance, Solutol HS 15 and verapamil probably reverse multidrug resistance by different mechanisms. Solutol HS 15 merits consideration as a potential therapeutic agent because of its effectiveness for reversing multidrug resistance in vitro and its low toxicity in vivo.

Desmosome ultrastructure and biological behavior of chemical carcinogen-induced urinary bladder carcinomas.

In the quantitative electron microscopic study, we examined the relationship of desmosomes to tumor invasiveness in chemical carcinogen (N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide)-induced urinary bladder transitional cell carcinomas in the Fischer rat. The number of a desmosomes per unit area of plasma membrane was significantly reduced in carcinomas. However, the percentage of cell surface area occupied by desmosomes was greater in carcinomas than in controls. This was accounted for on the basis of increases in desmosomal size, which result from squamous differentiation within the tumors. Areas of transitional cell differentiation and squamous differentiation demonstrated an equal capacity for invasiveness. Desmosomes were abundant in invading nests of tumor cells. These findings cast doubt on the validity of the concept of decreased intercellular adhesion as a prerequisite for tumor invasion, since strong interadhesion is probably a function of the area occupied by the intercellular junctions.

Flow cytometric analysis of heterogeneity in blood group-related antigen expression in a human urinary bladder carcinoma cell line, 647V.

Previous immunohistological studies showed a relationship between expression of blood group-related antigens (BG-Ag) and invasive potential in human urinary bladder carcinoma, but the marked variability of antigen staining within many individual tumors has obscured the biological basis of this finding. We studied the expression of the A, H, and T (Thomsen-Friedenreich) BG-Ag by flow cytometry in a human bladder carcinoma cell line (647V) using fluoresceinated BG-Ag-specific lectins (Dolichos bifloris, Ulex europaeus, and Arachis hypogaea). Cell cycle compartments were quantitated by flow cytometry using propidium iodide staining. Expression of all three antigens was highly variable, but staining for each antigen produced a distinct profile. T antigen expression appeared independent of A or H antigen expression. Cell populations sorted by T antigen expression showed heritable antigenic differences persistent over many weeks in culture. However, much of the T antigen variability was nonheritable, since the stable staining profiles of the sorted cells were intermediate between the parental and the profiles obtained immediately after sorting. The nonheritable antigenic variation did not appear entirely explainable by cell size or cell cycle fluctuation. These results were confirmed by isolating 64 clones from the dim part of the T antigen staining profile, 19 of which had a persistently dim phenotype. The variability of BG-Ag expression by human bladder carcinoma cells in vitro may explain the staining patterns observed in the study of antigen expression in resected human bladder carcinomas.