Peritoneal macrophages express the serotonin transporter.

Although it is known that macrophages take up serotonin, a specific monoamine transporter has not been identified in macrophages. In this study, mRNA coding for the serotonin transporter (SERT) was detected with the reverse transcription-polymerase chain reaction (RT-PCR) in recruited mouse peritoneal macrophages. Sequencing confirmed the identity of the RT-PCR product to mouse SERT mRNA. SERT protein was detected by Western blotting. Macrophage activation with lipopolysaccharide had no effect on expression of SERT mRNA or protein. Consistent with expression of a functional SERT, specific uptake of (3)H-serotonin in macrophages was sodium dependent and inhibited by fluoxetine (IC(50) 6.9 nM) and desipramine (IC(50) 32 nM) but not by nisoxetine or reserpine.

Phenylethanolamine N-methyltransferase in the brains of streptozotocin diabetic rats.

Recent evidence suggests that adrenergic neurons in the brainstem and hypothalamus are important for regulation of endocrine and cardiovascular function and the response to stress. Since abnormalities in several of these functions are observed in diabetic subjects, we investigated the effects of streptozotocin-induced diabetes on the activity of the enzyme that converts norepinephrine to epinephrine [phenylethanolamine N-methyltransferase (PNMT)] in the brainstem and hypothalamus of the rat. Enzyme activity was measured in the supernatant of sonicated brain tissues of saline-treated control, diabetic, and insulin-treated diabetic rats. One month after streptozotocin treatment, increases in plasma glucose and food intake occurred; these were restored to control levels by insulin treatment. PNMT activity was approximately 2-fold higher in the brainstem of diabetic rats than in controls (P less than 0.0001), and administration of insulin partially prevented the effects of diabetes on PNMT activity (P less than 0.01 compared to diabetics and P less than 0.05 compared to controls). Brainstem enzyme activity measured in all animals (n = 53) exhibited a significant correlation with plasma glucose concentrations (r = 0.51; P less than 0.001). Diabetes had no apparent effect on PNMT activity in the hypothalamus. These findings are the first to suggest that experimental diabetes alters PNMT activity in the medulla/pons.

Correlation between the biological and immunological activities of growth hormone circulating in rats bearing MtTW15 tumors.

Growth hormone-like activity was demonstrated in diluted plasma of rats bearing the GH-secreting tumor, MtTW15, using an in vitro bioassay. The bioassay used depends upon the ability of GH in vitro to stimulate the uptake of 3-0-methyl glucose (3-OMG) into the isolated diaphragm of the hypophysectomized rat. It was found that the effect of the diluted plasma on this system was qualitatively like that of rat pituitary GH. Theophylline, a drug which blocks the action of GH on 3-OMG uptake by the diaphragm but has no effect on the actions of insulin or somatomedin on this process, completely abolished the stimulatory action of the plasma on the transport of the sugar. Furthermore, antibodies against rat pituitary GH (ArGH) completely abolished the biological activity of the diluted plasma. When plasma was diluted to give specific concentrations of rat GH (rGH), as determined by radioimmunoassay (RIA), the plasma produced responses in the 3-OMG assay equivalent to those obtained with similar concentrations of pituitary rGH. A good correlation was also observed between the concentration of rGH in the plasma of tumor-bearing rats measured by RIA and by an in vivo bioassay in which the effects were determined of injections of diluted plasma into hypophysectomized rats on the subsequent incorporation of [3H]thymidine into costal cartilage. This in vivo biological activity was abolished by incubation of the plasma with ArGH prior to its injection into the test animals. Thus, these studies indicate that in rats bearing the MtTW15 tumor there is a good correlation between the biological and immunological activities of the rGH circulating the bloodstream.

Effects of branched chain amino acids on spontaneous growth hormone secretion in the baboon.

Blood concentrations of the branched chain amino acids (BCAAs) are elevated during fasting in healthy subjects and are abnormally high both postprandially and during fasting in diabetic patients. Despite evidence that these amino acids influence brain metabolism and neurotransmitter synthesis, there is little information on the neuroendocrine effects of the BCAAs. This study provides evidence that elevation of postprandial blood levels of the BCAAs alters the ultradian rhythm of GH secretion observed in the baboon during daylight hours. To mimic the postprandial rise in the BCAAs that occurs in diabetic patients, we infused either saline or a mixture of valine, leucine, and isoleucine into six conscious male baboons from 1530-1900 h daily for 4 days during and after the normal feeding time. On the last day of the infusions, blood samples were collected at 20-min intervals from 0800-1500 h and at 30-min intervals from 1500-2000 h. The amino acid infusions increased postprandial blood concentrations of the BCAAs 2- to 5-fold over control levels and lowered the blood concentrations of tyrosine, phenylalanine, and lysine compared to concentrations observed during control infusions. A significant elevation in GH levels occurred in association with BCAA treatment in each animal between 0800 and 1100 h, 13 h after the previous day's infusion. Average +/- SE maximum GH levels observed between 0800 and 1100 h were 11.6 +/- 2.9 ng/ml under experimental conditions compared to a control value of 3.8 +/- 1.2 (P less than 0.02). Whether the increased GH levels represented the generation of a new peak or a phase shift in a nocturnal peak was not determined. Combined with evidence that spontaneous release of GH is neurally regulated in the baboon, this study suggests that changes in the blood levels of the BCAAs modulate neural mechanisms that regulate GH rhythmicity.

Effects of fasting on growth hormone secretion in the male baboon.

The effects of prolonged fasting on the quantity and pattern of spontaneous GH secretion in 5 adolescent male baboons were investigated. Serum GH concentrations were measured in blood samples taken at 20-min intervals over 12 daytime hours after an overnight fast (control period) and during 84-96 h of fasting. Rhythmic GH secretion, with a mean (+/- SE) period of 5.4 +/- 0.2 h occurred in 4 of the 5 animals in 11 control experiments, and GH peaks occurred randomly in the fifth animal. In response to prolonged fasting, the percent half-amplitude of daytime GH peaks decreased from control values of 144 +/- 12% to 105 +/- 17%. The period of the GH rhythm in 4 animals decreased during fasting, but the change was not statistically significant, and the episodic pattern of GH release in animal 5 was apparently unaffected by fasting. After 84 h of fasting, the mean and integrated concentrations of GH released over 12 daytime hours in the 5 animals were not significantly different from control values. In summary, despite a reduction in GH peak amplitude, the quantity and rhythm of GH secretion were maintained in baboons fasted for 84 h. The observed decrease in GH maxima may play a role in the metabolic adaptation to fasting in the baboon.

Pulsatile release of growth hormone and prolactin from the primate pituitary in vitro.

Perifused anterior hemipituitaries from one male and 4 female monkeys released GH and PRL in a pulsatile pattern, with mean +/- SE interpulse intervals of 8.2 +/- 0.4 and 8.5 +/- 0.3 min, as determined by a cycle detection computer algorithm. Mean hormone concentrations in the perifusate fractions collected at 2-min intervals were 435 +/- 89 (GH) and 515 +/- 262 (PRL) ng/ml. Pulse amplitudes averaged 74 +/- 16 ng/ml for GH and 189 +/- 89 ng/ml for PRL. These findings suggest the presence of a high frequency pulsatile secretory mechanism within the primate pituitary.

Catecholamines in murine bone marrow derived mast cells.

Cultured murine bone marrow derived mast cells (BMMC) were found to store high levels of dopamine (3753+/-844 pg/10(7) cells) and occasionally produce norepinephrine and epinephrine. The catecholamine synthesis inhibitor, alpha-methyl-para-tyrosine, decreased intracellular catecholamine concentrations, and activation with ionomycin stimulated dopamine release. Neither dopaminergic receptor antagonists nor exogenous dopamine < or =10 microM affected IL-3-induced cell proliferation. High exogenous dopamine (20-100 microM) decreased proliferation and increased apoptosis, and the anti-oxidant ascorbic acid prevented these effects. Increased expression of the anti-apoptotic factor Bcl-2 or loss of pro-apoptotic Bax expression attenuated dopamine-induced apoptosis, suggesting the apoptosis proceeds through a mitochondrial pathway.

Cardiovascular responses and central catecholamines in streptozocin-diabetic rats.

Brain catecholamine levels, spinal cord levels of the norepinephrine metabolite methoxy-hydroxy-phenylglycol (MHPG), and heart rate were measured in nondiabetic and streptozocin-diabetic rats after sham surgery or bilateral carotid ligation. Although carotid ligation increased heart rate in both diabetic and nondiabetic rats, in diabetic animals the response did not differ from the response to sham surgery. Carotid ligation increased epinephrine concentrations in the medulla/pons of diabetic animals but was not associated with alterations in other central catecholamines. In all diabetic rats spinal ratios of MHPG/norepinephrine (an index of noradrenergic activity) were higher than in nondiabetics, and the change in heart rate (post-surgical-pre-surgical rate) correlated inversely with hypothalamic dopamine (R = -0.60). In sham-operated diabetic rats there were high inverse correlations of the change in heart rate with medullary epinephrine and of pre- and post-surgical heart rate with spinal MHPG/NE (R = -0.87 to -0.95). Central catecholamines and heart rate were not correlated in nondiabetic animals and correlated only weakly when nondiabetic and diabetic animals were pooled. Correlations in diabetic animals were usually abolished or reduced by carotid ligation. These findings suggest a link between central catecholamines and heart rate in diabetic rats subjected to surgical stress. Whether catecholaminergic neurons contribute to abnormal chronotropic responses in diabetic rats or respond to stimuli that affect both heart rate and neural function remains to be determined.

Effects of branched-chain amino acids on postprandial 3-OH butyrate and glucagon in the baboon.

Although chronic postprandial elevation of branched-chain amino acids (BCAAs) occurs in diabetic subjects and in subjects consuming high-protein diets, the metabolic effects of simultaneously increasing levels of these three amino acids are unclear. In this study, a mixture of the BCAAs was infused intravenously into baboons, beginning 30 minutes after the daily meal and continuing for 200 minutes on four consecutive days. Blood samples were collected on the last day of treatment. Infusion of the BCAAs into fed baboons promoted an increase in peak levels of glucagon, a decrease in postprandial levels of seven amino acids, and an increase in plasma levels of 3-OH butyrate. The ketone body response occurred despite an increase in the plasma ratio of insulin/glucagon in four of the five animals and was not associated with a change in the rate of lipolysis as indicated by plasma glycerol measurements. These findings raise the possibility that ketone bodies are one of the metabolic products of BCAA metabolism induced by high concentrations of leucine or ketoisocaproate. The observation that chronic elevation of BCAAs augments glucagon secretion may explain the parallel increases in plasma glucagon and plasma BCAAs observed in subjects fed high protein diets.

Impaired distribution of alpha-methyl-L-p-tyrosine in diabetic rats.

Although alpha-methyl-L-p-tyrosine (alpha-MPT), an inhibitor of catecholamine synthesis, has been used to study catecholamine turnover in diabetic animals, effects of diabetes on metabolism of the drug have not been investigated. In this study, administration of a standard dose of alpha-MPT (250 mg/kg initially and 125 mg/kg at 2 h intervals) resulted in lower plasma and tissue levels of alpha-MPT and its metabolites in streptozocin-diabetic rats than in controls. Two to six hours after the initial dose of alpha-MPT, concentrations of alpha-MPT were 2-8-fold lower in the hypothalamus, medulla/pons, and plasma of diabetic animals than in controls. Brain and plasma levels of the alpha-MPT metabolite, alpha-methyl DOPA (alpha-MD) were 2-10-fold lower in tissues of diabetic animals. Levels of the alpha-MPT metabolite alpha-methyl norepinephrine (alpha-MNE), measured only in the hypothalamus, were 4-fold lower in diabetic rats than in controls. There were no differences in the ratio of free/conjugated alpha-MPT in plasma. Treatment of diabetic rats with insulin restored alpha-MPT and alpha-MD to control levels. These findings indicate that i.p. administration of alpha-MPT does not result in equivalent levels of the drug in diabetic and control rats and suggest caution in the use of alpha-MPT to compare catecholamine turnover in diabetic and healthy animals.

Phenylethanolamine N-methyltransferase mRNA in rat hypothalamus and cerebellum.

Using the reverse transcription polymerase chain reaction, we detected a single form of phenylethanolamine N-methyltransferase (PNMT) mRNA in hypothalamus and medulla/pons and two forms in cerebellum. These findings indicate that the PNMT gene is expressed in these brain areas and suggest that tissue specific splicing of PNMT mRNA may occur.

Effects of branched-chain amino acids on beta-endorphin in the pituitary of the rat.

Despite evidence that the branched-chain amino acids (BCAAs) influence brain metabolism and neurotransmitter synthesis, there is little information on the neuroendocrine effects of the BCAAs. We now report that administration of a mixture of the BCAAs 3 times daily for 12 days to Sprague-Dawley rats decreased the concentration of beta-endorphin immunoreactivity (BEI) in the neurointermediate lobe of the pituitary. In BCAA-treated rats, BEI was 21 +/- 4 compared to control levels of 57 +/- 12 (ng/microgram of protein). The BCAAs had no effect on the level of BEI in the adenohypophysis. These findings suggest that changes in availability of leucine, isoleucine, and valine mediate specific neuroendocrine responses to metabolic perturbations in the rat.

Phenylethanolamine N-methyltransferase mRNA in rat spleen and thymus.

Phenylethanolamine N-methyltransferase (PNMT), the final enzyme in the biosynthesis of epinephrine, has been detected in rat and human spleen with radioenzymatic assays, but the presence of PNMT has not been examined in other lymphoid tissues. Using the reverse transcription polymerase chain reaction (RT-PCR) and Southern blot analysis, we tested for PNMT mRNA in rat spleen and thymus. A single PCR fragment from spleen, thymus, adrenal and brainstem gave a strong hybridization signal with a PNMT cDNA probe, whereas a PCR fragment from liver was only faintly visible on Southern blots. These findings indicate that the PNMT gene is expressed in spleen and thymus and raise the possibility that lymphoid organs synthesize epinephrine as an intrinsic regulator.

Effects of gonadal steroids on catecholamine levels in the brain of the oyster toadfish.

We studied the effects of gonadol steroid implants on catecholamine levels in the brain of ovariectomized female toadfish (Opsanus tau L.). Control values for dopamine (DA), norepinephrine (NE) and epinephrine (EPI) in the brain were 2181 +/- 226, 1112 +/- 80 and 6.5 +/- 2.02 ng/g, respectively. Compared to controls, 17 beta-estradiol decreased brain levels of DA to 547 +/- 261, and dihydrotestosterone decreased DA to 444 +/- 290 ng/g (P less than 0.001). Furthermore, dihydrotestosterone increased brain levels of EPI to 15.0 +/- 4.7 ng/g. The steroids had no effect on brain levels of NE. These results suggest a role for both androgens and estrogens in regulating brain levels of DA and EPI in toadfish.

Some applications of near-ultraviolet laser-induced fluorescence detection in nanomolar- and subnanomolar-range high-performance liquid chromatography or micro-high-performance liquid chromatography.

In this work we present some applications of near-UV laser-induced fluorescence (LIF) with micro-HPLC (microHPLC) and HPLC. To test the sensitivity of the detection, we used pyrene and aflatoxins, because both of these molecules exhibit native fluorescence. Then we studied catecholamines derivatized with 1,2-diphenylethylenediamine. The results show that we were able to reach better sensitivity levels than previously described in LIF studies. For catecholamines, a 50-fold increase in sensitivity compared to conventional fluorescence was obtained. These results indicate that LIF detection associated with HPLC or microHPLC can be used to detect very low concentrations of substances that can be excited in the near-UV range after labeling at nanomolar concentrations.

Soluble and particulate phenylethanolamine N-methyltransferase in hypothalamus of diabetic rats.

Experimental diabetes increases total phenylethanolamine N-methyltransferase (PNMT) activity in the medulla-pons but not in the hypothalamus. In this study diabetes was induced with streptozotocin (65 mg/kg) in male Sprague-Dawley rats. Twenty-eight days after treatment there were no differences in soluble PNMT activity in the hypothalamus of diabetics and controls, but PNMT activity in a membrane-associated (particulate) fraction of hypothalamus was evaluated approximately twofold in tissues of diabetic animals compared with controls. A specific PNMT inhibitor, incubated with tissue extracts of control rats, abolished greater than 90% of particulate PNMT activity in the hypothalamus but reduced soluble PNMT activity in the hypothalamus by only 47%. These findings indicate that membrane-associated PNMT activity in rat hypothalamus differs from soluble hypothalamic PNMT in the in vitro response to an inhibitor and the in vivo response to diabetes and suggest the importance of separating subcellular hypothalamic fractions prior to assay of PNMT.

Failure of branched-chain amino acids to affect synaptosomal uptake of norepinephrine and dopamine.

Despite evidence for neuroendocrine actions of the branched-chain amino acids (BCAAs) and implications that these amino acids alter the turnover of central catecholamines, the mechanism of these actions of the BCAAs is uncertain. In this study, we tested the effects of the BCAAs on uptake of catecholamines by axon terminals (synaptosomes) isolated from whole mouse brain minus the cerebellum. Neither high physiological (1-2 mM) nor pharmacological levels (10 mM) of an equimolar mixture of the BCAAs altered the uptake of 3H-dopamine and 3H-norepinephrine by synaptosomes. These findings suggest that the BCAAs do not influence the reuptake mechanism for termination of catecholamine-mediated neurotransmission.

Overview of a quality assurance/quality control compliance program consistent with FDA regulations and policies for somatic cell and gene therapies: a four year experience.

Somatic cell and gene therapy involve the application of biological technologies to an individual patient through the use of living cells which provide a therapeutic benefit (Aliski, 1991). Various forms of cellular and gene therapies are being developed and evaluated in an increasing number of clinical trials for congenital and acquired disorders. The potential and progress of these therapeutic applications have resulted in an increasing effort by the Food and Drug Administration (FDA) to develop the regulatory framework under which these therapeutic approaches would insure safety and efficacy, the primary mandate of the FDA. Over five years ago Cellcor began to define the parameters, specifications, and conditions relevant to a Quality Assurance/Quality Control (QA/QC) program that has evolved to insure safety and maximize the efficacy of applications of the company's ex vivo technology, autolymphocyte therapy. Autolymphocyte therapy is an outpatient form of somatic cell immunotherapy based upon the infusion of T cells that have been activated ex vivo using a combination of previously generated autologous cytokines and an anti-CD3 monoclonal antibody. We have been able to demonstrate the feasibility for the safe, controlled, and consistent preparation and delivery of a cellular therapy by application of relevant GMP regulations. This presentation reviews aspects of this program and chronicles our experience which at present amounts to over 4400 in fusions for over 700 patients. This program provides a high degree of assurance that a cellular therapy program can be carried out in a multisite mode involving hundreds of patients through the strict adherence to cGMP as set forth in existing regulations.(ABSTRACT TRUNCATED AT 250 WORDS)