Effects of a combination of 3,4-methylenedioxymeth amphetamine and caffeine on real time stimulated dopamine release in the rat striatum: Studies using fast cyclic voltammetry.

It is well documented that caffeine exacerbates the hyperthermia associated with acute exposure to 3,4-methylenedioxymethamphetamine (MDMA) in rats. Previous reports have also indicated that MDMA-related enhancement of dopamine release is exacerbated in the presence of caffeine. In the present study we have examined whether the effects of MDMA on real-time stimulated dopamine release, in the absence of uptake inhibition, are accentuated in the presence of caffeine. Isolated striatal slices from adult male Wistar rats were treated acutely with MDMA, caffeine, or a combination, and their effects on single and 5pulse stimulated dopamine release monitored using the technique of fast cyclic voltammetry. Caffeine at 10 or 100µM had no significant effect on single pulse stimulated dopamine release. However 100µM caffeine caused a significant peak increase in 5pulse stimulated dopamine release. Both 1 and 30µM MDMA gave rise to a significant increase in both single and 5-pulse dopamine release and reuptake. A combination of 100µM caffeine and 1 or 30µM MDMA did not significantly enhance the effects of MDMA on single or 5pulse dopamine release and reuptake when compared to that applied alone. Utilizing single action potential dependent dopamine release, these results do not demonstrate a caffeine-enhanced MDMA-induced dopamine release.

An in vitro approach to assessing a potential drug interaction between MDMA (ecstasy) and caffeine.

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is a popular recreational drug which causes long-term neurotoxicity and increased risk of fatality. In rats, MDMA toxicity is exacerbated by co-administration of caffeine. The aim of this study was to investigate whether caffeine altered the effects of MDMA in a battery of in vitro tests selected to model some of the known actions of MDMA in vivo. In cytotoxicity studies, caffeine modestly enhanced the effect of MDMA on neuronal N2a cell viability but not that of liver, intestinal or kidney cells. MDMA inhibited the formation of fluorescent metabolites by CYP2D6≫CYP3A4>CYP1A2 but this was not altered by caffeine. Similarly, the inhibition of synaptosomal [(3)H] 5-HT uptake by MDMA was not affected by the presence of caffeine. Thus, these in vitro tests failed to detect any substantial interaction between caffeine and MDMA, highlighting the difficulty of modelling in vivo drug interactions using in vitro tests. However, the results show that the inhibition of synaptosomal [(3)H] 5-HT uptake by MDMA was greater at 41°C and 25°C than at 37°C which raises the possibility that MDMA's effect on SERT in vivo may be increased as body temperature increases, contributing to its harmful effects in users.

Adenosine A1 receptor-mediated inhibition of dopamine release from rat striatal slices is modulated by D1 dopamine receptors.

Dopamine release is regulated by presynaptic dopamine receptors and interactions between adenosine and dopamine receptors have been well documented. In the present study, dopamine release from isolated striatal slices from Wistar rats was measured using fast cyclic voltammetry. Single-pulse stimulation (0.1 ms, 10 V) was applied every 5 min over a 2-h period. Superfusion with the adenosine (A)(1) receptor agonist N(6)-cyclopentyladenosine (CPA), but not the A(2) receptor agonist 3-[4-[2-[[6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-oxolan-2-yl]purin-2-yl]amino]ethyl] phenyl]propanoic acid (CGS 21680), inhibited dopamine release in a concentration-dependent manner (IC(50) 3.80 x 10(-7) m; n = 10). The dose-response curve to CPA was shifted to the right (IC(50) 6.57 x 10(-6) m; n = 6, P < 0.05 vs. control) by the A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). Neither the D(1) agonist 6-chloro-APB nor the D(1) antagonist R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3- benzazepine-7-ol (SCH 23390) altered dopamine release on their own. However, SCH 23390 (3 microm) significantly attenuated the response to CPA (IC(50) 1.44 x 10(-5) m; n = 6, P < 0.01 vs. control). Furthermore, the inhibitory effect of CPA was significantly increased in the presence of 6-chloro-APB (1 microm). In radioligand binding experiments, CPA interacted with high- and low-affinity states of [(3)H]DPCPX-lableled A(1) receptors. The high-affinity agonist binding to A(1) receptors was inhibited by the stable guanosine triphosphate analogue Gpp(NH)p. In contrast, neither the proportion nor the affinity of high-affinity A(1) receptors was altered by dopamine or SCH 23390. These results provide evidence that the inhibition of dopamine release by adenosine A(1) receptors is dependent, at least in part, on the simultaneous activation of D(1) dopamine receptors. While the mechanism underlying this interaction remains to be determined, it does not appear to involve an intramembrane interaction between A(1) and D(1) receptors.

Metastatic solid-pseudopapillary tumour of the pancreas: clinico-biological correlates and management.

Solid-pseudopapillary tumour of the pancreas is a rare neoplasm of young women, currently categorised in the World Health Organization classification under exocrine pancreatic tumours. Increased awareness of this condition correlated recently with an apparent rise in incidence as well as recognition of more aggressive clinical courses. We describe two patients with solid-pseudopapillary tumour of the pancreas. A smaller, localised tumour in an unusually young white man was surgically excised with no evidence of recurrence after 2 years. The other case also had an uncommon presentation, with an aggressive course resulting in vascular encasement of the superior mesenteric bundle and aorta, and local involvement of the mesenteric lymph nodes. A literature review was carried out, and the main clinico-pathological features and strategies of treatment of solid-pseudopapillary tumour of the pancreas are presented. Pathological, genetic and molecular features distinguish solid-pseudopapillary tumours from pancreatic ductal adenocarcinoma. Furthermore, neuroendocrine differentiation can be found focally in occasional cases of solid-pseudopapillary tumour. Patients with localised disease are usually cured by surgery. Prolonged survival can be seen in the presence of distant metastasis, if such lesions are resected surgically. Chemotherapy and radiation therapy are used in rare cases when resection is not possible. No current chemotherapy regimens are considered standard in the treatment of this tumour. A rational chemotherapy protocol for such a rare tumour needs to consider its origin and clinical behaviour. However, the indolent clinical progression of solid-pseudopapillary tumours is similar to that of pancreatic neuroendocrine tumour.

Behavioural, hyperthermic and neurotoxic effects of 3,4-methylenedioxymethamphetamine analogues in the Wistar rat.

1. The ability of N-ethyl (MDEA) and N-butyl (MDBA) analogues of 3,4-methylenedioxymethamphetamine (MDMA, 'Ecstasy') to induce acute behavioural changes and increases in body temperature, and to cause serotonergic neurotoxicity, was assessed in young adult male Wistar rats. The in vitro ability of MDMA analogues to evoke presynaptic monoamine release from crude rat forebrain synaptosomal preparations pre-labelled with [3H]5-HT or [3H]DA was also measured. 2. In behavioural experiments, acute MDMA and MDEA (20 mg/kg, i.p.) significantly increased rat open-field locomotion scores, decreased open-field rearing, and induced stereotypy, Straub tail and head weaving. MDBA did not produce any of these behaviours. 3. After repeated dosing (8 x 20 mg/kg, i.p., twice daily for 4 days), MDMA > MDEA >> MDBA > or = saline at decreasing forebrain [3H]paroxetine binding levels and concentrations of 5-HT and 5-HIAA at 14 days post-treatment. None of the analogues caused any long-term changes in dopamine or noradrenaline concentrations in the forebrain. 4. Acute MDMA and MDEA (20 mg/kg, i.p.) produced significant acute increases in rat aural temperature compared with saline-treated animals, while 20 mg/kg MDBA caused no significant effects. 5. MDA, MDMA and MDEA were equipotent at inducing [3H]5-HT release from frontal cortex/hippocampal synaptosomes, while MDBA only evoked a significant release at 100 microM concentrations. The potency order for inducing [3H]DA release from striatal synaptosomes was MDA > MDMA > MDEA = MDBA. 6. This study shows that large N-alkyl substitution decreases the ability of MDMA analogues to evoke presynaptic 5-HT and DA release, induce acute hyperthermia, hyperlocomotion and behavioural changes, and cause long-term serotonergic neurotoxicity. 7. The structure-activity relationship data presented here indicate that the neurotoxic damage caused by substituted amphetamines requires a combination of acute hyperthermia and increased neurotransmitter release. Induction of one of these effects in isolation is not sufficient to cause serotonergic nerve terminal degradation.

Synthesis of D-hexos-5-uloses by novel in situ hydrolysis of epoxides derived from 6-deoxyhex-5-enopyranosides.

[reaction: see text] Epoxides derived from 2,3, 4-tri-O-protected-6-deoxyhex-5-enopyranosides are hydrolyzed in situ to ultimately give novel protected-D-hexos-5-ulose derivatives (sugar 1,5-dicarbonyls, 5-ketohexoses) in moderate to high yields. The products adopt a bicyclic structure (1,6-anhydropyranos-5-ulose) in solution with the pyranose ring in (4)C(1) conformation. The methodology has been used to prepare D-xylo-hexos-5-ulose (5-ketoglucose), a synthetic precursor to 1-deoxynojirimycin and a possible intermediate in the biosynthesis of inositols.

20th-century advances in drug therapy in oncology--Part. II.

Ongoing research in cancer therapy has led to the development of antineoplastic agents which target specific components of the cell cycle. In Part II of this series, we discuss agents which target the mitotic mechanism by inhibiting microtubules. Although many of these agents are being shown to have multiple effects, the Vinca alkaloids and the taxanes are known as antimitotic drugs. They are among the most important anticancer agents currently available, and because of their unique mechanisms, can be combined with a wide variety of other antineoplastic agents in a spectrum of diseases. In addition, in part II, we are discussing agents that target DNA and prevent replication and thus cell growth by inhibiting the enzymes which protect DNA during replication, the topoisomerases. These drugs, too, have unique mechanisms of action and have become major components of combination regimens. The topoisomerase I inhibitors are new drugs derived from an older parent drug, and their full possibilities are still being explored.

Mucin inhibition of lymphocyte function does not require specific mucin-ligand interactions.

Mucins are large highly glycosylated molecules that have been postulated to interfere with certain cell-cell interactions. Steric, charge and specific signalling effects have been postulated for the inhibition by cell-surface mucin molecules. In this report we evaluate the inhibitory effects of bovine submaxillary mucin (BSM), a mucin without specific lymphocyte interactions, on lymphocyte function. BSM inhibits the adhesion of lymphocytes when coimmobilized with intercellular adhesion molecule-1 (ICAM-1) and blocks the activation of T lymphocytes when coimmobilized with anti-CD3. These data demonstrate a general mucin effect on lymphocyte adhesion and activation that is primarily steric in nature and implicates mucins as general barriers to lymphocyte-tumour cell interactions. Mucin blockade of cell-cell interactions may explain why mucinous tumours are often associated with a poor prognosis.

The isolated major histocompatibility complex class I alpha3 domain binds beta2m and CD8alphaalpha dimers.

The MHC class I molecule plays a crucial role in cytotoxic lymphocyte function. The heavy chain of the MHC class I molecule can form many non-covalent interactions with other molecules on multiple domains and surfaces. We have generated an isolated alpha3 domain of a murine MHC class I molecule and evaluated the contribution of this domain to binding with the MHC class I light chain, beta2m, and CD8. The alpha3 domain binds beta2m at a thousand-fold higher concentration than the whole MHC, and binds CD8alphaalpha with a dependence on the alpha3 CD loop. Our results are relevant for models of MHC folding and CD8-MHC function. The study of individual domains of complex molecules is an important strategy for understanding their dynamic structure and function.

ICAM-1 co-stimulation has differential effects on the activation of CD4+ and CD8+ T cells.

T cells play a central role in the initiation, maintenance and regulation of the immune response. Effector responses of T cells are controlled by complex combinations of lymphokines and adhesion/co-stimulatory molecule signals. To isolate the effects of the adhesion/co-stimulatory molecule ICAM-1, we have stimulated purified murine CD4+ and CD8+ T cells with plate-bound anti-CD3 in the presence or absence of plate-bound soluble ICAM-1. In this report, we demonstrate that the co-immobilization of soluble ICAM-1 and anti-CD3 leads to a much greater increase in IL-2 production by CD8+ T cells than CD4+ T cells. The ICAM-1-induced enhancement we observed has differential sensitivity to LFA-1 blockade, depending on the T cell subsets and cytokine evaluated. These effects may play an important role in the generation and modulation of immune responses.

Functional coupling of a recombinant human 5-HT5A receptor to G-proteins in HEK-293 cells.

1. We have cloned, expressed and pharmacologically characterized the Human 5-HT5A receptor. 2. We have shown that ligand activation of the Human 5-HT5A receptor results in functional coupling to G-proteins in HEK-293 cells. 3. Stimulation of the receptor with 5-CT (5-carboxamidotryptamine) resulted in a dose-dependent increase in the % [35S]-GTPgammaS binding over the basal level. This is the first study to describe such G-protein activation for the Human 5-HT5A receptor in any cell. 4. A dose-dependent inhibition of cyclic AMP accumulation was observed in the recombinant Human 5-HT5A receptor cell line, suggesting a functional coupling to a G alpha i, G-protein in the HEK-293 cell line. 5. A ligand-stimulated reduction in the detectable level of the catalytic domain of protein kinase A (PKA) in nuclear extracts isolated from Human 5-HT5A expressing cells was observed. This observation was consistent with the reduction in the level of cyclic AMP accumulation, in response to receptor activation.

Effects of a hybrid recombinant human alpha interferon (A/D) on in vitro cytotoxicity and in vivo localization of monoclonal antibody L6-cytosine deaminase conjugate in a colon cancer model.

L6 is an IgG2a murine monoclonal antibody which we have demonstrated binds well to HT29 human colon carcinoma cells by flow cytometry, whole cell ELISA, and mixed hemadsorption. In vitro cytotoxicity studies revealed that the monoclonal antibody L6-cytosine deaminase (L6-CD) immunoconjugate plus the nontoxic prodrug, 5-fluorocytosine (5-FC), is equivalent to 5-fluorouracil (5-FU) in its ability to kill HT29 cells. Human alpha-interferon (A/D) was able to enhance this cytotoxic effect. The I.C.50's revealed that very small amounts of L6-CD are needed for this cytotoxic effect (approximately, 5 pg/ml resulted in 50% viability). The limiting factor was the amount of 5-FC employed with L6-CD (3 microM yielded 50% cell viability). alpha-Interferon (A/D) lowered the requirement of 5-FC to 1 microM to achieve 50% cell lethality. In vivo biodistribution experiments indicated that 1 microgram of L6-CD is nonspecifically taken up by the liver and spleen and cleared rapidly from the blood. Significant localization of L6-CD to HT29 tumors occurred only when 99 micrograms of unlabeled L6-CD was added to 1 microgram of 125I-labeled immunoconjugate injected intravenously. Further augmentation of tumor/blood ratios without reduction in percent injected dose per gram of tumor was possible with the intravenous injection of 100 micrograms of anti-idiotypic monoclonal antibody 13B, 24 hours after L6-CD, which bound unreacted L6-CD and cleared it from the blood. The addition of 100,000 U of alpha-interferon (A/D) given intraperitoneally every day increased the clearance of L6-CD by the liver and spleen, but impaired tumor localization (percent injected dose per gram). These studies demonstrated that in vivo localization of the L6-CD conjugate to HT29 tumors could be optimized by injecting excess L6-CD followed by an equal amount of L6 anti-idiotype mAb 13B, 24 hours after L6-CD.

An investigation of new metal framework design for metal ceramic restorations.

STATEMENT OF PROBLEM: Metal ceramic restorations have been implicated in the discoloration of associated gingival tissues. Attempts to remedy this by altering the design of the metal frameworks for such restorations may lead to unacceptable decreases in fracture resistance. PURPOSE: This study evaluated a new metal framework design for metal-ceramic restorations. MATERIAL AND METHODS: Twenty artificial crowns were fabricated with various degrees of facial metal reduction; 0, 1, 2, and 3 mm. The study was conducted in two parts. The first part evaluated changes in light transmission into adjacent root tissue. A light box was fabricated so sample crowns could be illuminated on a mounted natural tooth. The root of the tooth remained outside the light box, and the light transmitted through the crowns into root tissue was measured with a light meter. The second part of the study evaluated changes in fracture strength. The sample crowns were subjected to a vertical load until fracture with use of an Instron machine at a crosshead speed of 1 mm per minute. The load at fracture was recorded. RESULTS: Results indicated a statistically significant increase in light transmission with 1 mm framework reduction or greater, and fracture strengths did not decrease with up to 1 mm of framework reduction. A 1 mm facial axial reduction of the metal framework may be indicated for anterior metal-ceramic restorations.

Prospective randomized trial of lisofylline for the prevention of toxicities of high-dose interleukin 2 therapy in advanced renal cancer and malignant melanoma.

The therapeutic application of high-dose interleukin (IL) 2 in human malignancy is limited by severe multiorgan toxicities that are mediated, in part, by tumor necrosis factor (TNF) and IL-1. CT1501R (lisofylline; LSF) is one of several methyl xanthine congeners that inhibit the effects of TNF by the interruption of specific signal transduction pathways. This randomized, placebo-controlled trial was designed to assess the activity of LSF in reducing the toxicities of high-dose IL-2 therapy. Fifty-three patients with metastatic renal cancer or malignant melanoma were treated with i.v. bolus IL-2, 600, 000 IU/kg every 8 h for 5 days (14 doses), followed by 9 days of rest and another 5-day course of IL-2. Patients were randomly assigned to LSF, 1.5 mg/kg i.v. bolus, or placebo every 6 h during IL-2 therapy. All patients were to be treated to individual maximum tolerance of IL-2 at the intensive care unit level of support. The end points for statistical analysis were the number of IL-2 doses administered during the first cycle of treatment (maximum, 28) and the toxicities experienced by each group after the first 8 planned IL-2 doses. There was no difference between the LSF and placebo groups in the mean number of IL-2 doses tolerated in the entire first cycle of therapy (19.6 +/- 5.4 versus 19.5 +/- 5.8, P = 0.86) or in the first or second 5-day course of IL-2. The only significant difference in toxicities occurring through the eighth dose of IL-2 was in the maximum elevation of serum creatinine (mean, 1.7 +/- 0.8 for placebo versus 1.5 +/- 0.6 mg/dl for LSF, P = 0.013). A Monte Carlo analysis of major toxicities over the first 14-dose course of therapy showed a statistically significant difference favoring the LSF-treated group (P = 0.025). LSF was well tolerated, associated only with mildly increased nausea (P = 0.006 after eight IL-2 doses, but not significant for the entire first cycle). The antitumor activity was comparable in both groups (objective responses, 2 of 28 with LSF versus 4 of 24 with placebo). The mean peak plasma concentrations of LSF on days 1, 5, and 19 were 6.24, 3.83, and 5.04 micromol/liter, respectively. In conclusion, with this dose and schedule, LSF did not alter the toxicities of high-dose i.v. IL-2 sufficiently to impact the overall dose intensity of IL-2. Successful IL-2 toxicity modulation may require the use of higher doses of LSF, the development of agents with more potent anti-TNF activity, and/or combined modulating agents that function via distinct mechanisms to interrupt cytokine-mediated signaling.

Development of two radioimmunoassays to detect paclitaxel in sera and in cerebrospinal, ascitic, and pleural fluids.

BACKGROUND: Paclitaxel is an antimitotic agent isolated from the Pacific yew tree. It has demonstrated antitumor activity in several cancers and is the first of a new class of antineoplastic agents containing a taxane ring system. Its levels in serum and urine have been measured previously by high performance liquid chromatography (HPLC). In this study, the authors developed two competitive radioimmunoassay methods to determine whether they could reliably be used to measure levels of paclitaxel in sera and in cerebrospinal, ascitic, and pleural fluids. METHODS: A monoclonal antibody prepared against paclitaxel was employed in an immunoradiometric assay (IRMA), in which 125I-labeled antibody was used, and in a more conventional tritiated radioimmunoassay (RIA),in which 3H-paclitaxel was used. RESULTS: Both radioimmunoassays detected levels of paclitaxel in sera that were comparable to those observed with HPLC. However, the IRMA was the most sensitive. Only the IRMA was able to detect low levels of paclitaxel in cerebrospinal fluid after paclitaxel infusion and in sera 3 weeks after infusion. Both the IRMA and RIA methods were able to detect paclitaxel in ascitic and pleural fluids. CONCLUSIONS: Monitoring paclitaxel levels reliably in sera and other bodily fluids is possible with these radioimmunoassays and may be of value in predicting and preventing toxicity and optimizing paclitaxel treatments.

Specificity analysis of murine monoclonal antibodies reactive with Tn, sialylated Tn, T, and monosialylated (2-->6) T antigens.

T, Tn, and sialyated Tn (sTn) are pancarcinoma antigens, and increased expression of these carbohydrate epitopes has been correlated with a poor prognosis in several epithelial malignancies. Ten murine monoclonal antibodies have been generated to these antigens, and compared by ELISA and immunohistochemistry to established mAbs reactive with these antigens. Nine mAbs (3 IgM and 6 IgG) reactive with synthetic T-human serum albumin (T-HSA) were produced after immunizing BALB/c mice with a synthetic T-keyhole limpet hemocyanin glycoconjugate (T-KLH). An additional IgM mAb (145.22) was produced in mice immunized with erythrocytes isolated from a patient with Tn syndrome. Three IgM and six IgG1 mAbs reactive with T-HSA did not react with natural T antigen present on desialyated glycophorin. All three IgM and several IgG1 mAbs, however, did react with LS-174T, a mucinous colon carcinoma cell line, 647V, a human bladder carcinoma cell line, and TA3Ha, a murine mammary carcinoma cell line as well as fresh frozen colon carcinomas. MAb 145.22 reacted with both natural and synthetic sources of sTn and Tn, as well as with LS-174T cells and mucin deposits in 10/11 colon carcinomas on fresh-frozen sections. MAb B72.3 reacted strongly with ovine submaxillary mucin (OSM) and sTn-HSA, while mAb CC49, a second-generation mAb to TAG-72 carcinoma mucin, reacted strongly with OSM, less strongly with desialyated OSM, and only weakly with sTn-HSA, suggesting that the epitope specificity for mAb CC49 is distinct from that of B72.3.

Nefiracetam (DM-9384) reverses apomorphine-induced amnesia of a passive avoidance response: delayed emergence of the memory retention effects.

Nefiracetam is a novel pyrrolidone derivative which attenuates scopolamine-induced learning and post-training consolidation deficits. Given that apomorphine inhibits passive avoidance retention when given during training or in a defined 10-12h post-training period, we evaluated the ability of nefiracetam to attenuate amnesia induced by dopaminergic agonism. A step-down passive avoidance paradigm was employed and nefiracetam (3 mg/kg) and apomorphine (0.5 mg/kg) were given alone or in combination during training and at the 10-12h post-training period of consolidation. Co-administration of nefiracetam and apomorphine during training or 10h thereafter produced no significant anti-amnesic effect. However, administration of nefiracetam during training completely reversed the amnesia induced by apomorphine at the 10h post-training time and the converse was also true. These effects were not mediated by a dopaminergic mechanism as nefiracetam, at millimolar concentrations, failed to displace either [3H]SCH 23390 or [3H]spiperone binding from D1 or D2 dopamine receptor subtypes, respectively. It is suggested that nefiracetam augments molecular processes in the early stages of events which ultimately lead to consolidation of memory.

The effect of posture change on the position of the skin marks for the transverse horizontal axis.

Marks are placed on facial skin in clinical dentistry to indicate the position of more deeply placed landmarks or reference points. In this study the movement of the transverse horizontal axis skin points overlying the craniomandibular articulation were observed and quantified. The time taken for the skin displacement to occur when the posture changed between upright and supine was also studied. The extent of displacement of the skin point was approximately 3 mm in the sagittal plane and 2 mm in the frontal plane when the posture was changed from upright to supine and vice versa. The displacement was complete after 30 seconds in 95% of subjects. The direction of the displacement was primarily cephalad but with a dorsal component of more than 10 degrees in 87% of subjects. The extent of the movement in the sagittal and frontal planes was correlated. There was no gender difference for the skin displacement. Awareness by clinicians of the extent and direction of such facial skin movements can help to prevent errors.