Bone metastasis and rehabilitation.

Patients with cancer that has metastasized to bone will frequently develop functional problems that may respond to rehabilitative treatment. Many rehabilitation professionals, however, are concerned about the possibility of producing pathologic fracture with their treatment. Several methods have been proposed for identifying which malignant lesions in bone are at risk of fracture. In this article, these methods are reviewed and statistical analyses of them are presented. The risk of rehabilitating patients with bony metastases is also reviewed, as are the reported outcomes of these rehabilitation efforts. Standard approaches to the rehabilitation of these patients have evolved, although most of them have not been rigorously validated, and these are discussed. None of the methods for identifying lesions at risk of pathologic fracture are useful in other than long bones, and they are limited even there. The risk of producing pathologic fractures in cancer patients by increasing mobility and function, however, is low. Satisfactory outcomes have been demonstrated in attempting to rehabilitate patients who have had recent surgical repair of pathologic or impending fractures. Rehabilitation of cancer patients with bony metastases can be safely and effectively accomplished using standard approaches to the treatment of these patients.

Cytokines increase neonatal cardiac myocyte calcium concentrations: the involvement of nitric oxide and cyclic nucleotides.

Neonatal rat cardiac myocytes were treated with cytokines, with or without the nitric oxide synthase (NOS) inhibitors N-monomethyl-L-arginine (LNMMA) and N-nitro-L-arginine methyl ester (LNAME), and systolic and diastolic calcium levels were measured by fluorescence spectrophotometry and confocal microscopy. Time-dependent changes following interferon-gamma (IFN-gamma) treatment revealed a continuing increase in intracellular calcium, which was reduced with LNMMA, but not with LNAME. Increases in calcium also occurred with interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), but not to the extent seen with IFN-gamma. Increased cyclic guanosine monophosphate (cGMP) was involved in the results described with short-term (2 hr) TNF-alpha and long-term (18 hr) IFN-gamma treatments. Short-term exposure to IFN-gamma produced an increase in cyclic adenosine monophosphate (cAMP) and also an initial increase in the myocyte-bearing rate, with calcium levels either (i) subsequently returning to control levels while maintaining a fast beating rate or (ii), retaining a high systolic calcium level, but beating at control rates. Treatment with both IL-1beta and IFN-gamma stabilized the beating rate of the cells on some occasions. Shortening of myocytes increased with isoproterenol and following treatment with IFN-gamma, while isoproterenol stimulation of IFN-gamma-treated cells revealed increased contractile activity after short, but not long, treatment. LNMMA, but not reduced the increased contractile response with short-term IFN-gamma treatment. Our findings suggest that TNF-alpha acts via a cGMP-dependent pathway, whereas the actions of IFN-gamma involve adenylate cyclase, and possibly a NO-forming mechanism and cGMP pathway as well. It is also apparent that the two NO inhibitors function via different mechanisms or that LNMMA has a direct effect on the calcium-signaling pathway.

Indium-111 DTPA-heparin: radiolabeling, pharmacokinetics, and biodistribution following intravenous administration in rat and rabbit.

Heparin was coupled to DTPA using the bicyclic anhydride and labeled with Indium-111. This resulted in a radiochemically pure preparation (greater than 95% activity in one peak) as determined by high pressure liquid radiochromatography and did not affect the anticoagulant properties of heparin. Biodistribution in the rat at 1, 20, and 60 minutes after intravenous injection showed rapid blood clearance with uptake in the liver followed by bone and kidney when expressed as percent injected total dose per organ and liver followed by kidney and spleen when expressed as percent injected total dose per gram. Blood elimination in the rabbit was 18.5 minutes which decreased to 7.5 minutes when followed by the injection of protamine. Radioactivity cleared from the liver and lungs as a single exponential with a half-time of 30 minutes, but there was very rapid increase of radioactivity in the lungs, peaking at 1-2 minutes, following the injection of protamine. Indium-111 DTPA-heparin may be used to study in vivo pharmacokinetics and biodistribution of heparin.

Practical risk management principles for physicians.

Most medical schools and postgraduate residency programs do not focus adequate attention on risk management and quality management issues. This article will prepare physicians with an adequate working knowledge of risk management and quality management information, which will enable them to practice more effectively in today's litigious and regulatory climate.

Apoptotic cells in peripheral blood from patients with low serum cobalamin.

Apoptotic leukocytes were found by using ultrastructural and light microscopic techniques to examine peripheral blood in ten of twelve patients with low serum cobalamin (vitamin B12) and rarely in normal controls. A total of 88 apoptotic cells (.14% of total leukocytes) were identified in all the patients. One patient also had apoptotic cells found on a routine blood smear. There was no correlation between the finding of these cells and nuclear hypersegmentation, serum cobalamin levels, serum intrinsic factor antibody, serum methylmalonic acid and homocysteine or the Schilling test. Two patients, however, with the most severe vitamin deficiency did not have increased numbers of apoptotic cells suggesting that these patients had lost the ability to initiate the cell death program.

Most lactotrophs from lactating rats are able to respond to both thyrotropin-releasing hormone and dopamine.

Intracellular free calcium concentration ([Ca2+]i) was measured with video imaging in lactotrophs from lactating rats. The median resting [Ca2+]i was 24 nM (85 cells). The great majority of cells responded to thyrotropin-releasing hormone (TRH) with an increase in [Ca2+]i, (median peak [Ca2+]i after TRH = 298 nM; n = 73). In 77% of these cells this [Ca2+]i increase was biphasic, with [Ca2+]i remaining high after the initial peak (median [Ca2+]i 90 s after TRH application = 104 nM; n = 56); the second phase depended on calcium influx. Most cells also responded to dopamine (DA), after TRH had been applied. DA reduced or abolished TRH-induced calcium influx and also reduced resting [Ca2+]i if this was above its initial value. A few lactotrophs responded to TRH only after DA application and withdrawal. We conclude that the population of lactotrophs in lactating rats is heterogeneous, but is not composed of two distinct sub-groups defined by their responsiveness to TRH or DA.

Ca2+ entry in gonadotrophs and alpha T3-1 cells: does store-dependent Ca2+ influx mediate gonadotrophin-releasing hormone action?

In pituitary gonadotrophs GnRH causes biphasic (spike and plateau) increases in cytosolic Ca2+ ([Ca2+]i) and gonadotrophin release. The spike phases reflect mobilization of stored Ca2+ and the plateau responses are attributed, in part, to Ca2+ influx via voltage-sensitive Ca2+ channels. In recent years, store-dependent Ca2+ influx (SDCI), in which depletion of the intracellular inositol 1,4,5-trisphosphate-mobilizable pool stimulates Ca2+ influx, has emerged as a major form of Ca2+ entry activated by phosphoinositidase C-coupled receptors in non-excitable cells. More recent evidence also indicates a role for SDCI in excitable cells. We have used dynamic video imaging of [Ca2+]i in alpha T3-1 cells (a gonadotroph-derived cell line) and manipulation of the filling state of the GnRH-mobilizable Ca2+ pool to test the possible role of SDCI in GnRH action. In Ca(2+)-containing medium, GnRH caused a biphasic increase in [Ca2+]i whereas in Ca(2+)-free medium only a transient increase occurred. The response to a second stimulation with GnRH in Ca(2+)-free medium was reduced by > 95% (demonstrating that Ca2+ pool depletion had occurred) and was recovered after brief exposure to Ca(2+)-containing medium (which enables refilling of the pool). Ionomycin (a Ca2+ ionophore) and thapsigargin (which inhibits the Ca(2+)-sequestering ATPase of the endoplasmic reticulum) also transiently increased [Ca2+]i in Ca(2+)-free medium and depleted the GnRH-mobilizable pool as indicated by greatly reduced subsequent responses to GnRH. Pool depletion also occurs on stimulation with GnRH in Ca(2+)-containing medium because addition of ionomycin and Ca(2+)-free medium during the plateau phase of the GnRH response caused only a reduction in [Ca2+]i rather than the transient increase seen without GnRH. To deplete intracellular Ca2+ pools, cells were pretreated in Ca(2+)-free medium with thapsigargin or GnRH and then, after extensive washing, returned to Ca(2+)-containing medium. Pretreatment with thapsigargin augmented the increase in [Ca2+]i seen on return to Ca(2+)-containing medium (to two- to threefold higher than that seen in control cells) indicating the activation of SDCI, whereas pool depletion by GnRH pretreatment had no such effect. To ensure maintained pool depletion after Ca2+ re-addition, similar studies were performed in which the thapsigargin and GnRH treatments were not washed off, but were retained through the period of return to Ca(2+)-containing medium. Return of GnRH-treated cells to Ca(2+)-containing medium caused an increase in [Ca2+]i which was inhibited by nicardipine, whereas the increase seen on return of thapsigargin-treated cells to Ca(2+)-containing medium was not reduced by nicardipine. The quench of fura-2 fluorescence by MnCl2 (used as a reporter of Ca2+ influx) was increased by GnRH and thapsigargin, indicating that both stimulate Ca2+ influx via Mn2+ permeant channels. The GnRH effect was abolished by nicardipine whereas that of thapsigargin was not. Finally, depletion of intracellular Ca2+ pools by pretreatment of superfused rat pituitary cells with GnRH or thapsigargin in Ca(2+)-free medium did not enhance LH release on return to Ca(2+)-containing medium. The results indicate that (a) thapsigargin stimulates SDCI in alpha T3-1 cells via nicardipine-insensitive Ca2+ channels, (b) in spite of the fact that GnRH depletes the hormone-mobilizable Ca2+ pool, it fails to stimulate SDCI, (c) GnRH stimulates Ca2+ entry predominantly via nicardipine-sensitive channels, a route not activated by SDCI and (d) in rat gonadotrophs, GnRH-stimulated LH release is not mediated by SDCI.

Ultrastructure of peripheral blood granulocytes from patients with low serum cobalamin.

Peripheral blood granulocytes from ten patients with low serum cobalamin were studied using light and electron microscopic techniques. Six patients had classic neutrophil hypersegmentation using light microscopy, but three patients had an increase in band forms and rare hypersegmented cells. The electron microscope revealed nuclear appendages consisting of nuclear stalks, rings and blebs in neutrophils from all patients regardless of the degree of hypersegmentation but not from normal controls; these did not appear to result from sectioning artifact and their formation may be interrelated. Numerous cytoplasmic empty granules were seen in neutrophils from three patients. Another three showed what appeared to be granules in the shape of water-wings under low power, but on higher power were unique mitochondria. Some disorder of the eosinophil granule centrum was seen in all patients but was extreme in four patients; lipid cytoplasmic inclusions were also seen in these four and one other patient.

Functional expression of a cloned Drosophila muscarinic acetylcholine receptor in a stable Drosophila cell line.

A cloned Drosophila muscarinic acetylcholine receptor (mAChR) has been stably expressed in a Drosophila cell line (S2) under the control of an inducible Drosophila metallothionein promoter. A clonal cell line (S2-Dm1-1) has been isolated which, after induction of mAChR expression with CuSO4, exhibits high-affinity, saturable, specific binding of the muscarinic antagonist N-methyl scopolamine (NMS). The apparent molecular mass of the expressed protein, calculated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), is in good agreement with the apparent molecular mass of mAChRs purified from Drosophila brain. Functional expression of the cloned mAChR in this stable cell line has been demonstrated by quantitative fluorescence ratio-imaging of Fura-2-loaded cells. We have observed transient, agonist-induced elevations in intracellular Ca2+ levels which can be completely blocked by atropine, whereas AFDX-116, a muscarinic antagonist which binds preferentially to the vertebrate mAChR M2 subtype, has little effect at 100 mumol l-1. The suitability of this stable Drosophila expression system for the characterization of neurotransmitter receptors is discussed.

Rehabilitation of cancer patients with skeletal metastases.

Complications of cancer that are amenable to treatment with rehabilitation techniques will develop in many patients with skeletal metastases. This treatment can be given safely, with a low prevalence of pathologic fracture. Many patients with skeletal metastases and pathologic fracture have been shown to be good candidates for intensive rehabilitation programs if they do not have hypercalcemia caused by lytic metastases or pain severe enough to require parenteral narcotics.

Effects of pituitary adenylate cyclase-activating polypeptide in the pituitary: activation of two signal transduction pathways in the gonadotrope-derived alpha T3-1 cell line.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is thought to play a hypophysiotropic role, but little is known of the identity of PACAP-stimulated cells in the pituitary, the nature of the PACAP receptors on specific cell types, and the effector systems for these receptors. Here we describe the effects of PACAP in alpha T3-1 cells, a gonadotrope-derived cell line. In these cells, PACAP38 causes concentration-dependent increases in cAMP accumulation (EC50, 3 nM), [3H]inositol phosphate ([3H]IP) production (EC50, 20 nM), and the cytosolic Ca2+ concentration. The Ca2+ response is biphasic and is sustained only in Ca(2+)-containing medium. Intact alpha T3-1 cells possess a single class of [125I]PACAP27-binding sites (Kd, 3.3 nM; binding capacity, 35 fmol/10(6) cells). The rank orders of potencies for stimulation of cAMP and [3H]IP production and for inhibition of [125I] PACAP27 binding by three related peptides are identical (PACAP38 = PACAP27 > > vasoactive intestinal peptide). In addition to stimulation of LH release from primary cultures of rat pituitary cells and [3H] IP accumulation in alpha T3-1 cells, PACAP38 synergizes with low GnRH concentrations in the production of these effects. Moreover, long term exposure to PACAP38 stimulates [3H]thymidine incorporation and increases steady state levels of the gonadotropin alpha-subunit in alpha T3-1 cells. We conclude that alpha T3-1 cells possess type I PACAP receptors which mediate the observed effector system responses, and demonstration of the effects of PACAP on this gonadotrope-derived cell line provides further evidence that gonadotropes are direct targets for PACAP action. The data imply that stimulation of phospholipase-C by PACAP is responsible (at least in part) for the observed increase in cytosolic Ca2+, which, in turn, probably mediates the effects of PACAP on LH release. We suggest, however, that in gonadotropes, the effects of PACAP on cell replication and gonadotropin synthesis may prove more important than the peptide's modest effects on LH release.

Arginine vasopressin mobilises intracellular calcium via V1-receptor activation in astrocytes (pituicytes) cultured from adult rat neural lobes.

An extremely close association exists between the membranes of the neurosecretory endings and the resident astrocytes (pituicytes) of the neurohypophysis. Indeed, synaptoid contacts involving neurosecretory vesicle-containing axons contacting pituicytes have been observed, suggesting pituicytes as targets of the products released from neurosecretory axons. We have investigated the effects of various neural lobe peptides on pituicytes in primary culture from adult neurohypophyses. Using Fura-2 loaded cells and dynamic ratio imaging, we have determined that arginine vasopressin (AVP) or V1- but not V2-receptor agonists, mobilise pituicyte intracellular Ca2+ ([Ca2+]i) in the absence of extracellular Ca2+. AVP was consistently effective at concentrations of 10 nM or higher in elevating [Ca2+]i by 200-1000 nM. These responses could be blocked by V1-antagonists and were shown to be associated with accumulation of phosphoinositides. Oxytocin was also found to mobilise [Ca2+]i but was effective only at higher concentrations than for AVP. Oxytocin-evoked [Ca2+]i elevations were also blocked by V1-antagonists. Raising [K+]0 was ineffective in changing [Ca2+]i suggesting that these cells lack voltage-gated Ca2+ channels. We conclude that pituicytes possess V1-receptors, activation of which mobilises [Ca2+]i, possibly functioning to initiate a Ca(2+)-activated K+ conductance which could contribute to further depolarisation of secretory terminals and facilitate exocytosis.

Functional outcome of pathologic fracture secondary to malignant disease in a rehabilitation hospital.

Fifty-eight patients with 62 pathologic fractures secondary to metastatic disease were admitted to a rehabilitation hospital during a 5-year period. Thirty-four patients were discharged home, 7 were transferred to other facilities, and 17 died. The average hospital stay for the patients who went home (37 days) was only 3 days longer than for patients with nonpathologic fractures. No patient could transfer independently or ambulate at the time of admission, but 26 and 23, respectively, could do so by the time of discharge; 27 patients showed significant improvement in their ability to perform activities of daily living as measured by Kenny scores. All 11 patients who had hypercalcemia died. Eleven of 13 patients requiring parenteral narcotics died. Patients with pathologic fractures secondary to metastatic disease are excellent candidates for intensive rehabilitation programs, but hypercalcemia and administration of parenteral narcotics suggest a poor rehabilitation outcome.

Dynamic video imaging of cystolic Ca(2+) in the alphaT3-1, gonadotrope-derived cell line.

Studies with pituitary cell cultures have revealed Ca(2+) as a mediator of gonadotropin-releasing hormone (GnRH) action. Here we have used dynamic video imaging to characterize Ca(2+) metabolism in the gonadotrope-derived, alphaT3-1 cell line focusing on stimuli that influence Ca(2+) metabolism of gonadotropes in primary culture. At least 90% of the cells imaged responded to 100 nMGnRH with an increase in cytosolic Ca(2+) (typically 10- to 20-fold). GnRH (10-1000 nM) caused a biphasic "spikeplateau" increase, whereas the spike phase was not seen with 0.01-1 nM GnRH. The plateau response was blocked by Ca(2+)-free medium and inhibited by 10 muM nifedipine (a dihydropyridine Ca(2+) channel antagonist) but not by blockade of voltage-sensitive Na(+) channels with 10 muM tetrodotoxin. In Ca(2+)-free medium GnRH caused only a transient Ca(2+) increase. Endothelin-1 (400 nM) also increased Ca(2+) although only 30-50% of the cells responded and the average response was less than that with 100 nM GnRH. Depolarization with 30 mM KCl increased Ca(2+) in >90% of the cells with an average effect comparable to the 100 nM GnRH effect. Protein kinase C activation approximately doubled the Ca(2+) concentration and induced Ca(2+) oscillations in some cells. The close parallels between responses of these cells and those observed with primary cultures suggests that alphaT3-1 cells will provide a valuable model for further studies of the involvement of Ca(2+) in GnRH action.

Prevalence of intrinsic factor antibodies and vitamin B12 malabsorption in older patients admitted to a rehabilitation hospital.

It is possible that the commonly measured serum level of vitamin B12 may miss some cases when used to detect vitamin B12 malabsorption and deficiency in older persons. Serum levels of vitamin B12 and intrinsic factor antibody (IFAB) were determined on 250 consecutive patients over the age of 70 admitted to a rehabilitation hospital. Patients with abnormal results on either test were given the standard Schilling test when possible. Eight patients had documented B12 malabsorption. Of these, five had a low serum B12 level alone and one had a low serum B12 level and a positive IFAB level; however, two patients had positive IFAB and normal serum B12 levels. Serum IFAB level may serve as a useful adjunct to serum B12 level in detecting vitamin B12 malabsorption in older patients.