Rapid modulation of lipogenesis by clofibrate in rat and monkey hepatocytes.

Previous studies have proposed various possible mechanisms for the hypolipidemic actions of clofibrate. In the present study, isolated liver cells freshly prepared from rats and squirrel monkeys were used to investigate the acute effects of sodium clofibrate (sodium chlorophenoxyisobutyrate) on hepatic lipogenesis. Incubation of liver cells with levels of the drug (1 mM) known to lower plasma lipid level in vivo produced a rapid inhibition of [14C]acetate incorporation into cellular lipids. The degree of inhibition caused by the drug was not diminished by preincubation with an excess of unlabeled acetate. Conversion of [14C]acetate to 14CO2 was not significantly altered by sodium clofibrate at 1--3 mM levels, but was decreased at 5--10 mM levels. Incorporation of [14C]acetate into all lipid classes was suppressed by this agent, but inhibition of sterol synthesis was more pronounced than that of fatty acids. Sodium clofibrate (1 mM) reduced the synthesis of sterols from either labeled acetate or labeled mevalonate to a similar degree, but the decline was augmented in each case by a 30-min preincubation of the cells with sodium clofibrate. Addition of fatty acids to cell incubations stimulated sterol generation from [14C] acetate or from [3H] mevalonate, but did not reverse the inhibitory effects of the drug. These results suggest (a) the sodium clofibrate rapidly interferes with hepatic fatty acid and sterol synthesis by direct or indirect actions at diverse loci; (b) that these effects probably occur after acetyl-CoA formation and do not stem from precursor pool dilution; (c) that sodium clofibrate diminishes sterol production at post-mevalonic and perhaps also at pre-mevalonic sites; (d) that a similar pattern of inhibition occurs with sodium clofibrate in the presence of added fatty acids; and (e) that rat and primate livers demonstrate similar metabolic responses to the drug.

Purification and characterization of fully functional human osteoclast precursors.

The identification and purification of human osteoclast precursors is essential to further our understanding of the mechanisms that control human osteoclast differentiation. Osteoclastoma tissue potentially provides a rich source of human osteoclast precursors, and in previous studies we have demonstrated the existence of a population of mononuclear cells within this tissue that is reactive with osteoclast-selective vitronectin receptor monoclonal antibodies. In this study, mononuclear cells expressing the vitronectin receptor, as defined by their ability to react with a murine monoclonal antibody to the beta 3 chain of the vitronectin receptor (87MEM1), were isolated from collagenase digests of osteoclastoma tissue using a fluorescence activated cell sorter. Based on their fluorescence signal and size, approximately 2-3% of the viable cells (typically 2 x 10(5)) were obtained and prepared for further phenotyping. The isolated cells demonstrated a number of phenotypic characteristics of osteoclasts: positive tartrate-resistant acid phosphatase (TRAP) activity, reactivity with human osteoclast-selective antibodies, expression of calcitonin receptors, cathepsin K (a novel osteoclast-selective cysteine proteinase) mRNA, and osteopontin mRNA and protein. These phenotypic characteristics were also detected in mononuclear cells within cryostat sections of the native osteoclastoma tissue as well as in resorption lacunae of sections of human bone. In contrast, isolated peripheral blood monocytes were negative for TRAP activity and osteopontin expression and, unlike the osteoclastoma-derived cells, demonstrated strong nonspecific esterase activity. Significantly, when the osteoclastoma-derived 87MEM1 positive cells were cocultured on whale dentine for 1-3 weeks with stromal cells, extensive resorption of the dentine surface was observed. This is the first demonstration of the purification of human osteoclast precursors. These cells provide an homogeneous cell population for studying cellular events that occur during human osteoclast differentiation.

Stimulation of fatty acid utilization by sodium clofibrate in rat and monkey hepatocytes.

The acute effects of sodium clofibrate (NaCPIB) on the metabolism of [1-14C]palmitate, [1-14C]octanoate, [1-14C]butyrate, and [2-3H]glycerol by freshly isolated hepatocytes were tested to explore its mechanism of action. Labeled long-, medium-, and short-chain fatty acids were incorporated into all the major lipid classes and were oxidized to 14CO2 by the liver cells. The partitioning of labeled fatty acids from lipogenic towards oxidative pathways was inversely related to fatty acid chain length. [1-14C]Palmitate was incorporated mainly into cellular triglycerides and phospholipids; [1-14C]octanoate, mainly into triglycerides and free cholesterol; and [1-14C]butyrate, mainly into free cholesterol and phospholipids of the cells. NaCPIB (1-3 mM) rapidly stimulated the esterification of labeled palmitate or glycerol to triglycerides, but drug levels greater than 5 mM were inhibitory to esterification. NaCPIB (1 mM) increased the oxidation of [1-14C]palmitate to 14CO2 by either rat or monkey hepatocytes and enhanced the release of labeled lipids from [2-3H]glycerol-prelabeled cells into the extracellular medium. Accelerated [1-14C]octanoate incorporation into glycerolipids and sterols and increased [1-14C]octanoate conversion to 14CO2 were observed in rat liver cells incubated with 1 mM NaCPIB. In contrast, the same drug level stimulated the oxidation of [1-14C]butyrate to 14CO2 but greatly diminished its incorporation into hepatocellular sterols or glycerolipids. These results indicate that (a) NaCPIB acutely alters hepatic ultilization of fatty acids by actions at diverse loci; (b) these metabolic alterations vary with fatty acid chain length; and (c) these effects are probably due to rapid changes in biochemical regulatory mechanism and/or in substrate channelling within the cells. These data further suggest that the early hypolipidemic effect of the drug in rats and primates may be related to an enhanced hepatic oxidation of long-chain fatty acids, but cannot be attributed simply to a reduction in their esterification to complex lipids.

Concurrent administration of vinorelbine with human recombinant granulocyte-macrocyte stimulating factor.

Thirty-three patients with incurable neoplasms resistant to standard therapy received vinorelbine 10 mg/m(2)/day by continuous infusion with concurrent administration of rHGM-CSF 4 microg/m(2)/day. The duration of the vinorelbine infusion was individualized; the infusion was continued until early evidence of hematopoietic toxicity was noted. The concurrent administration of GM-CSF permitted a substantial increase in dose intensity of the anti-cancer agent without a corresponding increase in drug toxicity. There was no evidence that the anti-tumor effect of vinorelbine was compromised by the concurrent administration of GM-CSF.

Absence of the glycerol phosphate shuttle in the various zones of the growth plate.

UNLABELLED: The various zones of the growth plate of the rib of six-week-old male New Zealand White rabbits were obtained by means of a specially devised guillotine slicing apparatus. Cartilage slices from each zone were assayed enzymatically for activity of glycerol phosphate dehydrogenase by an adaptation of a fluorimetric technique based on the conversion of non-fluorescent resazurin to the highly fluorescent resorufin in the presence of nicotinamide adenine dinucleotide and the enzyme. No glycerol phosphate dehydrogenase activity was detectable in any zone of the growth plate, whereas control liver slices exhibited abundant enzyme activity. Thus the glycerol phosphate shuttle, one pathway whereby reducing equivalents are carried or shuttled from cytoplasmic nicotinamide adenine dinucleotide to the intramitochondrial respiratory chain, is entirely lacking in growth-plate chondrocytes. The lack of this enzyme, and the absence of the glycerol phosphate shuttle, may explain the high rate of lactate accumulation in the presence of ample oxygen concentration (aerobic accumulation) even though the Krebs cycle (tricarboxylic acid cycle) and electron transport are proceeding at normal rates. CLINICAL RELEVANCE: The growth plate is a complex structure whose metabolism is still not well understood. This study demonstrates that, in at least one metabolic pathway, the growth-plate chondrocyte does not resemble other normal cells. Only as we are able to formulate a more complete picture of the metabolism of the normal growth plate will be able to understand, and perhaps correct or prevent, those disease states in which growth-plate metabolism has bone away.

Fluoroquinolone's effect on growth of human chondrocytes and chondrosarcomas. In vitro and in vivo correlation.

Clinical and in vitro studies have demonstrated that fluoroquinolones are toxic to chondrocytes; however, the exact mechanism of fluoroquinolone arthropathy is unknown. We investigated the toxicity of ciprofloxacin on normal cartilage and on cartilaginous tumors. Normal human cartilage, enchondroma, and chondrosarcoma explants were cultured either alone or with the addition of ciprofloxacin at 1, 10, or 20 mg/L of medium. Samples were collected up to twenty-one days after treatment and were processed for electron microscopy and conventional light microscopy. The specimens were characterized morphologically with use of conventional light microscopy, electron microscopy, and immunohistochemistry to identify extracellular matrix, cell proliferation, and apoptosis. Cultures of normal chondrocytes expressed type-II collagen. Electron microscopy revealed a large amount of glycogen in the cells; the presence of fat droplets, rough endoplasmic reticulum, and prominent Golgi apparatus; and a proteoglycan layer surrounding the cells. With prolonged ciprofloxacin treatment and with increased doses, there was an increase in dilated rough endoplasmic reticulum, the appearance of phagosomes, and disintegrated bundles of vimentin filaments. The treated chondrocytes showed a decrease in cell proliferation, but there was no induction of apoptosis or effect on the expression of extracellular matrix proteins. Ciprofloxacin-treated chondrosarcoma cultures and tissue samples showed changes in cartilage matrix composition. Ultrastructural analysis demonstrated clumped glycogen, dilation of endoplasmic reticulum, numerous abnormal lysosomes containing degeneration products, and a decreased proteoglycan deposit surrounding the tumor cells. Treated chondrosarcoma cells and tissue specimens did not proliferate, and apoptosis was induced. In contrast, the in vitro growth of other noncartilaginous malignant tumors like osteosarcoma and liposarcoma was unaffected by ciprofloxacin. Our results indicate that ciprofloxacin is toxic to chondrocytes. In vitro and in vivo treated chondrosarcomas are the most affected.

Infusional chemotherapy combined with recombinant human granulocyte colony stimulating factor: advantages and limitations.

We have studied the simultaneous administration of granulocyte colony stimulating factor (G-CSF), given concomitantly with an infusion of either doxorubicin or ifosphamide--the former, both intraarterially (i.a.) and intravenously (i.v.), the latter, intravenously--to a group of patients with various malignancies. Such simultaneous administration enabled us to substantially increase the dosage intensity of both, thereby increasing the effectiveness of each drug. No untoward effects have been noted to date.

Concurrent administration of vinorelbine with recombinant human granulocyte colony-stimulating factor: an effective method of increasing dose intensity.

Thirty-three patients with incurable neoplasms resistant to standard therapy received vinorelbine 8 mg/m2 to 10 mg/m2 per day by continuous infusion with concurrent administration of recombinant human granulocyte colony-stimulating factor 5 microm/m2 per day. The duration of the vinorelbine infusion was individualized; the infusion was continued until early evidence of hematopoietic toxicity was noted. The concurrent administration of recombinant human granulocyte colony-stimulating factor permitted a substantial increase in dose intensity of the anticancer agent without a corresponding increase in drug toxicity. There was no evidence that the antitumor effect of vinorelbine was compromised by the concurrent administration of recombinant human granulocyte colony-stimulating factor.

Therapy of desmoid tumors and fibromatosis using vinorelbine.

In patients with either desmoids or fibromatosis who do not tolerate vinblastine and methotrexate because of neurotoxicity, the combination of vinorelbine and methotrexate can be substituted in most. Patients with the same condition who had not been previously treated with a combination of vinblastine and methotrexate responded well to the combination of vinorelbine and methotrexate, with significantly less neurotoxicity and a similar objective and subjective response rate. Sixty percent of patients had either a substantial partial remission or a complete remission. In no patients did the disease progress while they were receiving this therapy. Symptomatic relief, primarily of pain, occurred in 80% of patients. While minimal neurotoxicity was seen in 16% of these patients, it did not interfere with the completion of therapy. The combination of vinorelbine and methotrexate appears to be active in the treatment of both desmoid tumors and fibromatosis and is associated with significantly less neurotoxicity then that seen with the combination of vinblastine and methotrexate. No long-term toxicity was seen in any patient in this series.

The treatment of sacral giant-cell tumours by serial arterial embolisation.

Giant-cell tumours of the sacrum are difficult to treat. Surgery carries a high risk of morbidity, local recurrence and mortality. Radiation is effective in some patients, but has a risk of malignant change. We evaluated the effectiveness of serial arterial embolisation as an alternative to surgery. Five patients with giant-cell tumours of the sacrum which had been primarily treated by serial embolisation were retrospectively reviewed for changes in the size of the tumour. In four the symptoms resolved with full return of function and arrest in the growth of the tumour. They remained free from growth, recurrence, or metastases at follow-up (4 to 17 years). One patient died from metastatic disease within 18 months of the initial diagnosis.

Plantar fibromatosis: a review of primary and recurrent surgical treatment.

Plantar fibromatosis can be quite disabling to the patient, as well as a technical challenge to the surgeon. Patients who undergo previous local excisions and in whom aggressive recurrences develop are difficult to manage successfully. We present a consecutive series of five primary procedures on patients with painful plantar fibroma and seven revision operations on patients with recurrent plantar fibroma. The average follow-up was 47 months (range, 22-66 months) in the primary group and 40 months (range, 21-78 months) in the revision group. The overall results were satisfactory in four of the five primary operations, with only one recurrence. In the revision group, five of seven results were satisfactory with no recurrences. The major complication that led to unsatisfactory results was the development of a postoperative neuroma. In this article, we outline our present surgical techniques of wide primary excision and a staged revision procedure with delayed split-thickness skin graft closure. These techniques can be used successfully to manage this disabling, progressive disease.

Lymphoma masquerading as infection.

During surgical training, medical students and residents constantly are reminded to culture every suspected tumor and send tissue for pathologic evaluation for every suspected abscess. A diagnosis of cancer can be missed easily if this procedure is not followed, delaying the diagnosis and possibly adversely affecting the patient's prognosis. The confusion also may be compounded by a sterile abscess, positive culture results or a negative biopsy specimen. Therefore it is imperative to do a biopsy and a culture on any suspect lesion. An additional workup and possible biopsy may be warranted for a nonhealing wound that has been treated appropriately. The cases of three patients with lymphoma that were treated as infectious processes are presented. In all three instances, the appropriate treatment was delayed because of a delay in diagnosis.

Isolated ophthalmic migraine in the differential diagnosis of cerebro-ocular ischemia.

Thirty-three of 129 patients who incurred isolated ophthalmic migraine had monocular attacks of scotomatous visual field loss. Fifteen of 33 patients with monocular attacks had immediate or remote evidence of vascular disease. Four patients had carotid bruits on the same side as the monocular attacks and low ophthalmodynamometer readings. One patient had ischemic optic neuropathy and two had atheromatous disease (advanced stage in one patient). Forty-five percent of the patients with monocular attacks and only 13% of the remaining patients with homonymous attacks had vascular complications. This represents an important finding even in such a small group of patients. It is felt that, whether the vascular problems are trigger mechanisms or coexistence pathology to the migraine-type attack, one should strongly suspect such an association when a patient describes a monocular attack and one should look for a possible vascular explanation other than migraine.

Low-dose chemotherapy of desmoid tumors.

Eight patients with desmoid tumors, symptomatic, and none a candidate for conservative surgery, were treated with weekly vinblastine, maximum dose 10 mg/week, and methotrexate, maximum dose 50 mg/week. Symptomatic relief was obtained in all patients. Using Eastern Cooperative Oncology Group (ECOG) criteria, two patients had a complete remission, one of which has lasted for 30 months, four patients have had partial remissions, one patient has had a mixed response, and one patient who has been treated for only 4 weeks, a minimal response. Toxicity has been minor and transient. Chemotherapy appears to be an acceptable alternative to radical surgery in selected patients with desmoid tumors.

Stimulated secretion of esterified lipids and VLDL by perifused hepatocytes.

This report describes a new method for investigation of hepatic metabolism by perifusion of medium through a batch of hepatocytes isolated from fed rats. Oxygenated medium flows by gravity through a hepatocyte-containing glass column that is immersed in 37 degrees C water bath. The effluent medium is then collected in consecutive aliquots in test tubes on ice. The pattern of export of esterified lipids, glucose, and VLDL by isolated liver cells into the perifusate was examined under both basal conditions and response to the infusion of certain metabolic stimulants. Perifusion of medium containing sodium clofibrate (1 mM and 10 mM levels) through lipid-prelabeled cells augmented the secretion of radioactive triacylglycerols that reached a maximal rate by about 30 min after exposure to this agent. Measurement of effluent glucose levels after perifusion of hepatocytes with media lacking glucose but containing a gluconeogenic precursor revealed steadily declining concentrations despite the addition of glucagon or epinephrine. Concomitantly, glycogen granules disappeared from the cytoplasm, but the cells retained intact ultrastructure after the course of perifusion. Protein-prelabeled hepatocytes released labeled VLDL into the perifusate, and this release was enhanced by prolonged exposure of the cells to medium containing palmitate (0.80 mM). The hepatocyte perifusion system thus offers a simple, reproducible method whereby hepatocellular secretory processes can be sequentially examined under carefully controlled basal and stimulated conditions.