Microdialysis and response during regional chemotherapy by isolated limb infusion of melphalan for limb malignancies

This study sought to use a microdialysis technique to relate clinical and biochemical responses to the time course of melphalan concentrations in the subcutaneous interstitial space and in tumour tissue (melanoma, malignant fibrous histiocytoma, Merkel cell tumour and osteosarcoma) in patients undergoing regional chemotherapy by Isolated Limb Infusion (ILI). 19 patients undergoing ILI for treatment of various limb malignancies were monitored for intra-operative melphalan concentrations in plasma and, using microdialysis, in subcutaneous and tumour tissues. Peak and mean concentrations of melphalan were significantly higher in plasma than in subcutaneous or tumour microdialysate. There was no significant difference between drug peak and mean concentrations in interstitial and tumour tissue, indicating that there was no preferential uptake of melphalan into the tumours. The time course of melphalan in the microdialysate could be described by a pharmacokinetic model which assumed melphalan distributed from the plasma into the interstitial space. The model also accounted for the vascular dispersion of melphalan in the limb. Tumour response in the whole group to treatment was partial response: 53.8% (n = 7); complete response: 33.3% (n = 5); no responses 6.7% (n = 1). There was a significant association between tumour response and melphalan concentrations measured over time in subcutaneous microdialysate (P < 0.01). No significant relationship existed between the severity of toxic reactions in the limb or peak plasma creatine phosphokinase levels and peak melphalan microdialysate or plasma concentrations. It is concluded that microdialysis is a technique well suited for measuring concentrations of cytotoxic drug during ILI. The possibility of predicting actual concentrations of cytotoxic drug in the limb during ILI using our model opens an opportunity for improved drug dose calculation. The combination of predicting tissue concentrations and monitoring in microdialysate of subcutaneous tissue could help optimise ILI with regard to post-operative limb morbidity and tumour response. (C) 2001 Cancer Research Campaign http:,//www.bjcancer.com.

Arachidonic acid stimulates glucose uptake in 3T3-L1 adipocytes by increasing GLUT1 and GLUT4 levels at the plasma membrane: Evidence for involvement of lipoxygenase metabolites and peroxisome proliferator-activated receptor

Exposure of insulin-sensitive tissues to free fatty acids can impair glucose disposal through inhibition of carbohydrate oxidation and glucose transport. However, certain fatty acids and their derivatives can also act as endogenous ligands for peroxisome proliferator-activated receptor gamma (PPARgamma ), a nuclear receptor that positively modulates insulin sensitivity. To clarify the effects of externally delivered fatty acids on glucose uptake in an insulin-responsive cell type, we systematically examined the effects of a range of fatty acids on glucose uptake in 3T3-L1 adipocytes. Of the fatty acids examined, arachidonic acid (AA) had the greatest positive effects, significantly increasing basal and insulin-stimulated glucose uptake by 1.8- and 2-fold, respectively, with effects being maximal at 4 h at which time membrane phospholipid content of AA was markedly increased. The effects of AA were sensitive to the inhibition of protein synthesis but were unrelated to changes in membrane fluidity. AA had no effect on total cellular levels of glucose transporters, but significantly increased levels of GLUT1 and GLUT4 at the plasma membrane. While the effects of AA were insensitive to cyclooxygenase inhibition, the lipoxygenase inhibitor, nordihydroguaiaretic acid, substantially blocked the AA effect on basal glucose uptake. Furthermore, adenoviral expression of a dominant-negative PPARgamma mutant attenuated the AA potentiation of basal glucose uptake. Thus, AA potentiates basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes by a cyclooxygenase-independent mechanism that increases the levels of both GLUT1 and GLUT4 at the plasma membrane. These effects are at least partly dependent on de novo protein synthesis, an intact lipoxygenase pathway and the activation of PPARgamma with these pathways having a greater role in the absence than in the presence of insulin.

Glucose induced IEG expression in the thiamin-deficient rat brain

Glucose loading of rats made thiamin deficient by dietary deprivation of thiamin and the administration of pyrithiamin (40 mug/100 g, i.p.) precipitates an acute neuropathy, a model of Wernicke's encephalopathy in man (Zimitat and Nixon, Metab. Brain Dis. 1999;14:1-20). Immunohistochemical detection of Fos proteins was used as a marker to identify neuronal populations in the thiamin-deficient rat brain affected by glucose loading. As thiamin deficiency progressed, the extent and intensity of Fos-Like immunoreactivity (FLI) in brain structures typically affected by thiamin deficiency (the thalamus, mammillary bodies, inferior colliculus, vestibular nucleus and inferior olives) were markedly increased when compared to thiamin-replete controls. Glucose loading for 1-3 days further increased the intensity of FLI in these same regions, consistent with a dependence of Fos expression on carbohydrate metabolism as well as on thiamin deficiency. The timed acute changes that follow a bolus glucose load administered to thiamin-deficient animals may provide a sequential account of events in the pathogenesis of brain damage in this model of Wernicke's encephalopathy. (C) 2001 Elsevier Science B.V. All rights reserved.

Dipeptidyl-peptidase II and cathepsin B activities in amelogenesis of the rat incisor

A body of published evidence suggests that a significant portion of enamel matrix protein synthesized by ameloblasts localises in the lysosomal-endosomal organelles of these enamel organ cells. Little is known regarding the lysosomal proteolytic activities during amelogenesis. The aims of this study were to detect and measure the activities of lysosomal peptidases cathepsin B (E.C. 3.4.22.1) and dipeptidyl-peptidase II (E.C. 3.4.14.2) in the enamel organ of the rat incisor and to ascertain whether rat enamel matrix proteins are degraded by these peptidases in vitro. Whole enamel organs were dissected from rat mandibular incisors. Enamel protein was also collected from the rat teeth. Analysis indicated that the rat incisor enamel organs contained specific activities of both dipeptidyl-peptidase II and cathepsin B at levels comparable with those of kidney which is rich in both these lysosomal peptidases. Gel electrophoresis and immunoblotting demonstrated that both cathepsin B and dipeptidyl-peptidase II were able to substantially degrade the rat enamel proteins in vitro. Based on these observations, we propose that lysosomal proteases have roles in amelogenesis in the intracellular degradation of amelogenins.

Flaviviruses isolated from mosquitoes collected during the first recorded outbreak of Japanese encephalitis virus on Cape York Peninsula, Australia

In response to an outbreak of Japanese encephalitis (JE) virus on Cape York Peninsula, Australia, in 1998, mosquitoes were collected using CO2 and octenol-baited Centers for Disease Control and Prevention light traps. A total of 35,235 adult mosquitoes, comprising 31 species, were processed for virus isolation. No isolates of JE virus were recovered from these mosquitoes. However, 18 isolates of Kokobera virus, another flavivirus were obtained from Culex annulirostris. Twelve isolates were from western Cape York (minimum infection rate (MIR) of 0.61: 1,000 mosquitoes) and 6 were from the Northern Peninsula Area (MIR of 1.0:1,000). Potential explanations for the failure to detect JE virus in mosquitoes collected from Cape York Peninsula include the timing of collections, the presence of alternative bloodmeal hosts, differences in pig husbandry, asynchronous porcine seroconversion, and the presence of other flaviviruses.

Rearrangement of diflunisal acyl glucuronide into its beta-glucuronidase-resistant isomers facilitates transport through the small intestine to the colon of the rat

Many non-steroidal anti-inflammatory drugs (NSAIDs) which form acyl glucuronide conjugates as major metabolites have shown an antiproliferative effect on colorectal tumors. This study assesses the extent to which rearrangement of an acyl glucuronide metabolite of a model NSAID into beta -glucuronidase-resistant isomers facilitates its passage through the small intestine to reach the colon. Rats were dosed orally with diflunisal (DF), its acyl glucuronide (DAG) and a mixture of rearrangement isomers (iso-DAG) at 10 mg DF equivalents/kg. The parent drug DF appeared in plasma after all doses, with maximum concentrations of 20.5 +/- 2.5, 28.8 +/- 8.3 and 11.0 +/- 1.6 mug DF/ml respectively, obtained at 3.8 +/- 0.3, 3.6 +/- 1.8 and 7.5 +/- 0.9 hr after the DF, DAG and iso-DAG doses respectively. At 48 hr, 16.2 +/- 3.3, 19.8 +/- 0.8 and 42.9 +/- 10.1% of the doses respectively were recovered in feces, with less than or equal to 1% remaining in the intestine. About half of each dose was recovered as DF and metabolites in 48 hr urine: for DF and DAG doses, the majority was in the first 24 hr urine. whereas for iso-DAG doses, recoveries in the first and second 24 hr periods were similar. The results show that hydrolysis of both DAG and iso-DAG, and absorption of liberated DF, occur during passage through the gut, but that these processes occur more slowly and to a lesser degree for iso-DAG. The intrinsic hydrolytic capacities of various intestinal segments (including contents) towards DAG and iso-DAG were obtained by incubating homogenates under saturating concentrations of DAG/iso-DAG at 37 degreesC. Upper small intestine, lower small intestine, caecum and colon released 2400, 3200, 9200 and 22800 mug DF/hr/g tissue plus contents respectively from DAG substrate, and 18, 10, 140 and 120 mug DF/hr/g tissue plus contents respectively from iso-DAG substrate. The much greater resistance of iso-DAG to hydrolysis appears attributable to its resistance to beta -glucuronidases. The data suggest that in rats dosed with DF, DAG excreted in bile would be substantially hydrolysed in the small intestine and liberated DF reabsorbed, but that portion which rearranges to iso-DAG would likely reach the colon. (C) 2001 Elsevier Science Inc. All rights reserved.

Neonatal hepatic drug elimination

After the transition from in utero to newborn life, the neonate becomes solely reliant upon its own drug clearance processes to metabolise xenobiotics. Whilst most studies of neonatal hepatic drug elimination have focussed upon in vitro expression and activities of drug-metabolising enzymes, the rapid physiological changes in the early neonatal period of life also need to be considered. There are dramatic changes in neonatal liver blood how and hepatic oxygenation due to the loss of the umbilical blood supply, the increasing portal vein blood flow, and the gradual closure of the ductus venosus shunt during the first week of life. These changes which may well affect the capacity of neonatal hepatic drug metabolism. The hepatic expression of cytochromes P450 1A2, 2C, 2D6, 2E1 and 3A4 develop at different rates in the postnatal period, whilst 3A7 expression diminishes. Hepatic glucuronidation in the human neonate is relatively immature at birth, which contrasts with the considerably more mature neonatal hepatic sulfation activity. Limited in vivo studies show that the human neonate can significantly metabolise xenobiotics but clearance is considerably less compared with the older infant and adult. The neonatal population included in pharmacological studies is highly heterogeneous with respect to age, body weight, ductus venosus closure and disease processes, making it difficult to interpret data arising from human neonatal studies. Studies in the perfused foetal and neonatal sheep liver have demonstrated how the oxidative and conjugative hepatic elimination of drugs by the intact organ is significantly increased during the first week of life, highlighting that future studies will need to consider the profound physiological changes that may influence neonatal hepatic drug elimination shortly after birth.

Smooth muscle cells of injured rat and rabbit arteries in culture: contractile and cytoskeletal proteins

The aim of this study is to determine whether subpopulations of smooth muscle cells (SMC). as distinguished by variations in contractile and cytoskeletal proteins, appear in the neointima at different times after vascular injury, and/or whether subpopulations develop during serial passaging of these cells. Rat aortae and rabbit carotid arteries were injured with a 2F Fogarty balloon catheter and cultures established from the resulting neointima and the media 2, 6, 12, 16 and 24 weeks later. Cultures were examined at passages 1-5 and subpopulations of SMC categorised by intensity of staining for each protein by immunohistochemistry. Two populations of SMC with different staining intensities ('+ +', '+') were observed for each of the following proteins: alpha -SM actin, SM-myosin, desmin and vimentin. Populations without these proteins were also found. Changes in the percentages of cells expressing these proteins were transitory, indicating that the populations were not limited to a particular tissue (neointima or media), time after injury or passage number. One exception was found in rabbit cultures where the number of desmin-expressing cells quickly decreased with both time after injury and time in culture. Subpopulations of SMC were found at all times after injury in the media and neointima of rat and rabbit arteries, and after multiple passage of these cells. There was no pattern of development of one population suggesting that either no subpopulation has a proliferative or migratory advantage over others, or that only one population exists: that is capable of diverse phenotypic changes. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

Growth hormone receptor and IGF-1 receptor immunoreactivity during orthodontic tooth movement in the prednisolone-treated rat

Bone remodeling during tooth movement is regulated by local and systemic factors. Two regulators of bone metabolism are growth hormone (GH) and insulin-like growth factor-I (IGF-1). Their effects are mediated via binding to GH receptor (GHR) and IGF-I receptor (IGF-IR) in target tissues. Corticosteroids may affect the activity of these growth factors. This study examined the effect of prednisolone on GHR and IGF-IR expression in dental tissues following orthodontic tooth movement. The corti ticosteroid-treated group (N = 6) was administered prednisolone ( 1 mg/kg,) daily and the control group (N = 6) received equivalent volumes of saline. An orthodontic force (30 g) was applied to the maxillary first molar. Animals were sacrificed 12 days postappliance insertion. Sagittal sections of the first molar were stained for GHR and IGF-IR immunoreactivity. GHR and IGF-IR cell counts were elevated following appliance-treatment. Orthodontic tooth movement appeared to up-regulate GHR and IGF-IR immunoreactivity, but this up-regulation was reduced following prednisolone treatment. The suppression of GHR and IGF-I immunoreactivity in steroid-treated animals infers the mechanism whereby bone resorption and deposition, necessary for orthodontic tooth movement, may be inhibited by prednisolone. However, at 12 days postappliance insertion. no difference in orthodontic tooth movement was observed following low-dose prednisolone treatment.

Chemically and morphologically identifiable glomeruli in the rat olfactory bulb

Primary olfactory neurons that express the same odorant receptor are distributed mosaically throughout the olfactory neuroepithelium lining the nasal cavity, yet their axons converge and form discrete glomeruli in the olfactory bulb. We previously proposed that cell surface carbohydrates mediate the sorting out and selective fasciculation of primary olfactory axons en route to glomeruli. If this were the case, then axons that terminate in the same glomerulus would express the same complement of cell surface carbohydrates. In this study, we examined the expression of a novel carbohydrate (NOC-3) on neural cell adhesion molecule in the adult rat olfactory system. NOC-3 was expressed by a subset of neurons distributed throughout the olfactory neuroepithelium. The axons of these neurons entered the nerve fiber layer and terminated in a subset of glomeruli. It is interesting to note that we identified three unusually large glomeruli in the lateral, ventrolateral, and ventromedial olfactory bulb that were innervated by axons expressing NOC-3. NOC-3-expressing axons sorted out and fasciculated into discrete fascicles prior to entering these glomeruli. Each of these glomeruli was in a topographically fixed position in the olfactory bulbs of the same animal as well as in different animals, and their lengths were approximately 10% of the total length of the bulb. They could be identified reliably by both their topographical position and their unique morphology. These results reveal that axons expressing the same cell surface carbohydrates consistently target the same topographically fixed glomeruli, which supports a role for these molecules in axon navigation in the primary olfactory nerve pathway. J. Comp. Neurol. 436: 497-507, 2001. (C) 2001 Wiley-Liss, Inc.

Growth hormone is permissive for skeletal adaptation to mechanical loading

The Lewis dwarf (DW) rat was used as a model to test the hypothesis that growth hormone (GH) is permissive for new bone formation induced by mechanical loading in vivo. Adult female Lewis DW rats aged 6.2 +/- 0.1 months (187 +/- 18 g) were allocated to four vehicle groups (DW), four GH treatment groups at 32.5 mug/100 g body mass (DWGH1), and four GH treatment groups at 65 mug/100 g (DWGH2). Saline vehicle or GH was injected intraperitoneally (ip) at 6:30 p.m. and 6:30 a.m. before mechanical loading of tibias at 7:30 a.m. A single period of 300 cycles of four-point bending was applied to right tibias at 2.0 Hz, and magnitudes of 24, 29, 38, or 48N were applied. Separate strain gauge analyses in 5 DW rats validated the selection of loading magnitudes. After loading, double-label histomorphometry was used to assess bone formation at the periosteal surface (Ps.S) and endocortical surface (Ec.S) of tibias. Comparing left (unloaded) tibias among groups, GH treatment had no effect on bone formation. Bone formation in tibias in DW rats was insensitive to mechanical loading. At the Ec.S, mechanically induced lamellar bone formation increased in the DWGH2 group loaded at 48N (p < 0.05), and no significant increases in bone formation were observed among other groups. The percentage of tibias expressing woven bone formation (Wo.B) at the Ps.S was significantly greater in the DWGH groups compared with controls (p < 0.05). We concluded that GH influences loading-related bone formation in a permissive manner and modulates the responsiveness of bone tissue to mechanical stimuli by changing thresholds for bone formation.

Skeletal effects of low-intensity pulsed ultrasound on the ovariectomized rodent

Growing evidence supports low-intensity pulsed ultrasound (US) as an osteogenic mechanical stimulus. Its effects on isolated bone cells and on fractured bone are established. However, its effects on osteoporosis are not clear. This study examined US effects on ovariectomy (OVX) induced bone changes within the rodent hindlimb (distal femur and proximal tibia), and on normal bone in animals following sham-OVX. Animals were exposed to daily unilateral active-US and contralateral inactive-US for 12 weeks. Bone status was assessed using dual energy X-ray absorptiometry and histomorphometry. Ovariectomy resulted in significant bone changes. Low-intensity pulsed US did not influence these changes. These results suggest that the US dose introduced may not be a beneficial treatment for osteoporosis, and that intact bone may be less sensitive to US than fractured bone and isolated bone cells. This may relate to the biophysical mechanisms of action of US, US-bone interactions and tissue level processes taking place.