Three dimensional reconstruction of a human breast carcinoma using routine laboratory equipment and immunohistochemistry.

AIMS: To establish a three dimensional reconstruction of an invasive breast carcinoma using basic laboratory equipment to evaluate and characterise the spatial arrangement of the parenchymal cells of the breast. METHODS: One hundred and twenty eight sequential 4 microm sections (20 microm apart) of the tumour were stained immunohistochemically with an epithelial specific marker (AE1/AE3) or tumour specific marker (c-erbB-2) to reconstruct two different three dimensional images of the normal and malignant parenchymal cells. Sections were digitally imaged using a microscope, scanner, and digital camera linked to a conventional personal computer. Accurate alignment of the digitalised images was carried out using a semiautomatic graphical method of manual interaction, using the cross correlation coefficient as a goodness of fit measure, and an automatic search algorithm using the Fibonacci search algorithm for automatic alignment. The volume was reconstructed using maximum, minimum point projection and "back to front" opacity blending. RESULTS: The quality of the reconstructed images was distinct and perfect, providing a comprehensive and explicit view of the normal and malignant parenchymal tissues of the breast that is not possible by viewing two dimensional histological sections. Specifically, this approach showed the spatial arrangement of the tumour cells and their relation to the surrounding tissues at a high resolution. CONCLUSION: This simple and reproducible approach enables the spread and infiltration of invasive carcinoma to be understood and could also be used to analyse the spatial relation between atypical hyperplastic and malignant in situ lesions of the breast.

Intervention assessment in an Indian health service pharmacy.

OBJECTIVE: To document and evaluate pharmacists' interventions in a setting that has complete and immediate access to patient information. DESIGN: Descriptive report evaluating self-reported interventions made by pharmacists during the conduct of routine dispensing activities. The data collection period was from February 15 to April 1, 1994. SETTING: Ambulatory care facility offering medical and dental care to high school residents, Native Americans, and Alaska Natives in Northwestern Oregon. MAIN OUTCOME MEASURES: Intervention rate per 100 new prescriptions dispensed. Each intervention was evaluated with regard to the information used to initiate it, when during the dispensing process it was initiated, and the intervention type. Outside evaluators determined the clinical significance of the interventions, including potential adverse health consequences, the likelihood of their occurrence, and the level of medical care that would have been required to treat the problem. RESULTS: Of 2,535 orders screened, 104 interventions (4.1%) were collected; 71% of these occurred during chart screening. Pharmacists most often used the medication order itself (60.6%) to detect prescribing problems, followed by other records in the patient's chart (29.8%). Outside evaluators identified 47.1% of the 104 interventions as clinically significant. The most common adverse health consequence prevented was inadequate control of the patient's condition. Outside evaluators also found that the most common level of corrective care that would have been needed if the intervention had not occurred, was a scheduled physician office visit (59.2%). CONCLUSION: This information suggests that pharmacists who have access to patient information may intervene at higher rates and that more of their interventions may be deemed clinically significant. However, larger, double-blinded, case-controlled studies are needed to definitively draw these conclusions.

An essential role for ectodomain shedding in mammalian development.

The ectodomains of numerous proteins are released from cells by proteolysis to yield soluble intercellular regulators. The responsible protease, tumor necrosis factor-alpha converting enzyme (TACE), has been identified only in the case when tumor necrosis factor-alpha (TNFalpha) is released. Analyses of cells lacking this metalloproteinase-disintegrin revealed an expanded role for TACE in the processing of other cell surface proteins, including a TNF receptor, the L-selectin adhesion molecule, and transforming growth factor-alpha (TGFalpha). The phenotype of mice lacking TACE suggests an essential role for soluble TGFalpha in normal development and emphasizes the importance of protein ectodomain shedding in vivo.

Effects of intermittent hPTH(1-34) alone and in combination with 1,25(OH)(2)D(3) or risedronate on endosteal bone remodeling in canine cancellous and cortical bone.

Therapies utilizing intermittent human parathyroid hormone(1-34) (hPTH[1-34]) in combination with other agents have recently been proposed as possible anabolic regimens for the treatment of osteoporosis. We conducted a 24 week study in aged beagle dogs to determine the effects of intermittent hPTH(1-34) administered alone or in combination with 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) on the endosteal remodeling in cancellous and cortical bone. Additionally, we tested the interaction between hPTH(1-34) and a new potent bisphosphonate, risedronate. The three treatment groups were compared with a vehicle control group. Kinetic reconstruction of the remodeling unit revealed substantial differences between the groups in resorption and formation at the basic multicellular unit level. Although the estimates of final erosion depth were unaffected by treatment, tunneling resorption was noted in six of the eight dogs administered hPTH(1-34) alone. These qualitative morphological changes in the resorption lacunae were attenuated or absent in dogs administered hPTH(1-34) in combination with either 1,25(OH)(2)D(3) or risedronate. Functional periods for resorption were significantly increased, and the resorption rates were significantly decreased in the hPTH(1-34) + risedronate group. Analyses of the formative site demonstrated that the wall thickness was significantly increased and the bone balance significantly more positive in all three hPTH(1-34) treatment groups. The most positive bone balance was achieved in the combined hPTH(1-34) + risedronate group (+ 15.6 + or - 14.2 mm, p <0.05). Increases in the mineral apposition rate in the early phases of the formative period suggest that an increase in osteoblastic activity (number or function) may contribute to the increase in wall thickness. Treatment with hPTH(1-34) alone or in combination with 1,25(OH)(2)D(3) caused an approximately 2-fold increase in the activation frequency in cancellous bone, which was essentially normalized to control values by the coadministration of risedronate. The impact of these changes on the cancellous bone microstructure was significant only in the combined hPTH(1-34) + risedronate group where normalized bone turnover in the face of a positive bone balance effected a significant increase in the trabecular thickness. Analyses of sequential fluorochrome labels, administered to reconstruct the temporal changes in intracortical activation, demonstrated the presence of an apparent cyclic pattern of activation in the cortex of placebo-treated dogs. Generally, activation was increased throughout the study in dogs administered hPTH(1-34) alone or in combination. However, in the hPTH(1-34) + risedronate group, activation was significantly blunted toward the end of the study, and the cyclic pattern of activation was modulated. These data suggest that intermittent hPTH(1-34) in combination with risedronate may be superior to hPTH(1-34) in combination with 1,25(OH)(2)D(3) as a therapeutic regimen for osteoporosis due to the protective effect of this bisphosphonate on the cortical and endocortical envelope.

Histologic and immunocytochemical characterization of canine distemper-associated metaphyseal bone lesions in young dogs following experimental infection.

The proximal metaphyses of the humerus of weanling gnotobiotic dogs experimentally infected with canine distemper virus (CDV) were investigated histologically and immunocytochemically between 4 and 41 days after infection. Viral antigen was demonstrated in hematopoietic marrow and bone cells at postinfection day (PID) 5 and PID 7, respectively. Between PID 8 and 27, CDV antigen was abundantly present in marrow cells, osteoclasts, and osteoblasts and less frequently in osteocytes. Immunopositive cells in both osseous tissues and bone marrow declined between PID 29 and PID 36 and were absent by PID 41. Chondrocytes of the growth plate were negative for viral antigen throughout the observation period. In bone, viral antigen was more frequently observed in bone cells of the primary spongiosa than in the secondary spongiosa. There was a strong correlation between occurrence of CDV antigen and osseous changes. Associated metaphyseal bone lesions were mild and most prominent between PID 8 and PID 32. Lesions consisted of necrosis of osteoclasts, which was associated with subsequent persistence of the primary spongiosa (growth retardation lattice). Atrophy and necrosis of osteoblasts and marrow cells were also noted. Infection of metaphyseal bone cells appears to be common in young dogs with experimental systemic distemper. Bone cell infection is preceded by infection of marrow cells, and infected bone cells may experience degeneration and necrosis. This subtle viral effect may result in defects in bone modeling in CDV-infected dogs.

Unbiased estimation of vertebral trabecular connectivity in calcium-restricted ovariectomized minipigs.

The topological changes in vertebral cancellous bone were estimated in vertebrae from calcium-restricted ovariectomized Sinclair S-1 minipigs, a recently described animal model of cancellous osteopenia. Connectivity was estimated using unbiased stereological principles in disector pairs of sections from the first lumbar vertebrae. Connectivity density was increased approximately twofold when compared with sham-operated minipigs fed a standard diet. These alterations in topology occurred coincident with a 25% increase in final resorption depth and a 150% increase in vertebral marrow star volume. Taken together, these changes suggest that in calcium-restricted ovariectomized minipigs, trabecular plates are transformed into rods by perforation. These changes in topology appear to be due, at least in part, to excessive resorptive cell function at the level of the bone remodeling unit. Conventional two-dimensional estimators of structural parameters of cancellous bone were not only less sensitive to these changes in topology but, in some cases, the estimates were directionally reversed.

Metaphyseal bone lesions in young dogs with systemic canine distemper virus infection.

Bone lesions, restricted to the metaphyses of long bones, were observed in young dogs with systemic distemper following experimental and spontaneous infection. Canine distemper virus (CDV) antigen was found immunocytochemically in hematopoietic marrow cells, osteoclasts, osteoblasts and rarely in osteocytes. In experimentally infected dogs, viral antigen was demonstrated in the metaphysis between 5 and 36 days after infection. Associated lesions, characterized by necrosis of osteoclasts, persistence of primary spongiosa and atrophy and necrosis of osteoblasts and marrow cells, were mild and most prominent between 8 and 32 days postinfection. Metaphyseal osteosclerosis (MO) of the long bones, varying from mild to severe, was observed macroscopically in 8 (19%) out of 42 dogs with spontaneous distemper. Affected animals were between 3 and 6 months of age and belonged mainly to the large breeds. In these animals, MO was characterized histologically by persistence of primary spongiosa, loss of bone marrow cells and necrosis of osteoclasts and bone marrow cells varying from mild to severe. Summarized, CDV-associated bone lesions were only transient and there were no indications of viral persistence in bones of dogs experimentally infected with CDV. Although no clinical signs related to the bones were observed, the present study reveals that infection of metaphyseal bone cells is common in young dogs with systemic distemper and occurrence of viral antigen in these cells results in defects in bone modelling.

Establishing an active patient partnership.

Pharmacists face many changes in the coming decade, some of which threaten their professional survival. Although uncertainty may currently prevail, one of these changes, the shift in the patient-health care professional relationship from the patient taking a passive role to an active partnering role, provides pharmacists with many opportunities to realize the vision of patient-centered care that has been advocated by pharmacy innovators and leaders for almost three decades. To take advantage of these changes, pharmacists must modify their practice paradigms and use their existing strengths, such as easy patient access and high levels of patient trust, to help develop a new model of pharmaceutical care. The concern that the magnitude of these changes will prevent successful practice transformations may be exaggerated. In reality, these proposed "new" roles have been in existence for much of this century. Most pharmacists can expand and enhance their traditional roles as self-care advisors and patient educators simply by incremental improvements in interpersonal and clinical skills. Rather than a Star Trek approach to "go where no man has gone before," the profession needs only a pharmaceutical sequel to Back to the Future.

Direct stereological estimation of three-dimensional connectivity in rat vertebrae: effect of estrogen, etidronate and risedronate following ovariectomy.

Newly developed unbiased stereological methods were employed to investigate the effects of estrogen deficiency on the three-dimensional connectivity of vertebral cancellous bone from ovariectomized (OVX) rats. The effects of two classes of antiresorptive agents, estrogen and bisphosphonates, on changes in connectivity in this animal model were also evaluated. Female rats were either sham-operated (sham-op) or surgically OVX at 90 days of age. OVX rats were administered either vehicle, estrogen (10 micrograms/kg 17-beta estradiol, 5 days/week subcutaneously [SC], etidronate disodium (5 mg/kg SC) or risedronate (5 micrograms/kg SC). The bisphosphonates were administered daily for 1 week followed by 3 weeks with no treatment. Treatment duration was 360 days. Systematic random sections, 30-microns thick, were prepared from methylmethacrylate-embedded decalcified second lumbar vertebrae. Total trabecular number and connectivity density were estimated using the ConnEulor principle. Vertebral cancellous bone volume was estimated on undecalcified sections from the first lumbar vertebrae. Connectivity density and cancellous bone volume were significantly reduced (approximately 25% and 40%, respectively) in the OVX group compared with the sham-op group. Estrogen treatment essentially maintained connectivity and cancellous bone volume at the level of the sham-op rats. Connectivity density and total trabecular number were significantly increased in the etidronate- and risedronate-treated rats compared with both the sham-op and OVX rats. These data demonstrate that reduction in the three-dimensional connectivity of vertebral cancellous bone is a long-term consequence of ovariectomy in the rat. This reduction in connectivity can be effectively prevented by the administration of antiresorptive agents such as estrogen, etidronate and risedronate.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of risedronate on canine cancellous bone remodeling: three-dimensional kinetic reconstruction of the remodeling site.

To investigate the dose-dependent effects of risedronate on cancellous bone remodeling, adult female beagle dogs were treated with either placebo, 0.1, 0.5, or 2.5 mg/kg/day of risedronate orally in an intermittent cyclic regimen (7 days on 21 days off), repeated three times. Iliac cancellous bone samples were subjected to histomorphometric analysis and three-dimensional (3-D) kinetic reconstruction of the remodeling site was performed. In the 0.1 mg/kg dose group, resorption and activation indices were no different from the placebo group. However, wall thickness was increased resulting in a positive bone balance at the level of the remodeling unit. In the 0.5 and 2.5 mg/kg dose groups, a dose-dependent reduction in activation frequency and tissue level bone formation was observed. Resorption rates were also significantly decreased, 60% and 80% for the 0.5- and 2.5-mg/kg groups, respectively. An approximate 25% reduction in final erosion depth was noted in both these groups. Analyses of the growth curves of the bone packet confirmed that the kinetics of the growth of a completed packet were different in the 0.5- and 2.5-mg/kg dose groups compared with placebo. These changes were associated with a significant increase in the final wall thickness in both groups indicating no net impairment of osteoblast function. These increases in wall thickness in combination with the reductions in final erosion depth in the 0.5 and 2.5 mg/kg groups resulted in a significant dose-dependent positive bone balance. This pharmacological profile suggests that risedronate may be of therapeutic utility in the treatment of metabolic bone diseases where reductions in activation frequency and resorptive cell activity at the level of the remodeling unit are a therapeutic goal.

Stochastic simulation of vertebral trabecular bone remodeling.

Bone remodeling changes bone mass, architecture, and thereby bone strength, during normal aging. These changes seem to be accelerated during the menopause. Several therapeutic agents have been used in order to delay the onset of the menopause-related changes. The effects of these agents on the remodeling process have been determined histomorphometrically in several short-term clinical studies, but data from long-term clinical studies are difficult to achieve, as are data on the influence on bone strength. The aim of this study was to develop a computer stimulation model that could assist in predicting the long-term effects of changes in the remodeling process on bone mass, trabecular thickness, and perforations. The paper presents such a stochastic model of the remodeling process in human vertebral trabecular bone. The computer model is based on histomorphometric and structural data from human studies. It is presented in terms of flow charts, and simulations performed with the model are discussed in relation to measurements on human vertebral bone samples. The results show that a menopause-related doubling of the activation frequency causes a transient, mainly reversible bone loss. If the menopause is accompanied by an increase in both activation frequency and resorption depth, then the resulting bone loss will be more pronounced and with a larger part being irreversible bone loss (perforations). The two antiresorptive agents. Etidronate and estrogen both cause a slight increase in bone mass (reducing remodeling space), and Etidronate also seems capable of preventing perforations. During fluoride therapy, an initial increase in remodeling space followed by a reduction is seen. Very few perforations are found to take place during fluoride therapy. The present model has been validated by assessing the effects of the menopause and treatment with antiresorptive or anabolic agents. It was found that the results mirrored or anabolic agents. It was found that the results mirrored very closely the results (bone mass measurements) from short-term clinical studies. It is therefore concluded that the model provides a tool for evaluating existing and new therapeutic regimens.

Antiresorptive drugs and trabecular bone turnover: validation and testing of a computer model.

A computer model of trabecular bone turnover has been developed, based on concepts of Jonathan Reeve [1]. This model predicts changes in bone volume by summing bone resorption and formation over a large number of remodeling sites. Clinical data [histomorphometry and bone mineral content (BMC)] from two clinical studies using an antiresorptive drug (etidronate disodium, EHDP) in postmenopausal osteoporosis were used to test the model. The results for BMC obtained from the EHDP and placebo groups in each study at 60 and 120 weeks were correctly predicted by the model from the histomorphometric data obtained from baseline and week 60 biopsies. The parameter in this model having the greatest influence on predicted changes in bone volume was found by sensitivity analysis to be activation frequency. These results suggest that the contribution of bone turnover to BMC can be predicted solely by considering the cell kinetics of the basic multicellular unit (BMU), and that, in the case of antiresorptive drugs, maximal effects on bone volume may be achieved by pharmacological reduction of activation frequency. The results also suggest that the present model may be useful in predicting in clinical studies the effects of EHDP and similar drugs on bone turnover.

The Conneulor: unbiased estimation of connectivity using physical disectors under projection.

The Euler number and the connectivity of an arbitrary object is defined, and it is illustrated why the connectivity of an n-dimensional object cannot be estimated in an (n-1)-dimensional section. The disector--principle for 3-D counting of the Euler--events is illustrated in cancellous bone. The correct handling for unbiased counting of events at artificial edges is outlined. A nomogram for predicting the precision of an estimate is provided.