Zbigniew L. Olkowski

Expression of a high-affinity serotonin transporter in human lymphocytes

This study was undertaken to assess the capability of lymphocytes to actively transport serotonin (5-HT). The data we obtained showed that lymphocytes isolated from the blood of normal human subjects contained a high-affinity uptake system for [3H]5-HT. Kinetic analysis of the uptake data as computed by regression analysis from Lineweaver--Burk plots, yielded a Km of 180 +/- 20 nM and Vmax of 94 +/- pmole/10(7) cells. The uptake of [3H]5-HT was temperature, sodium and chloride dependent and was potently inhibited by the antidepressants clomipramine, imipramine, fluoxetine and fluvoxamine, which are specific for the 5-HT transporter. Compounds that are more selective for norepinephrine and dopamine transporters such as mazindol, desipramine, and GBR 19209 had a lower inhibitory effect on the uptake of [3H]5-HT in human lymphocytes. The expression of a 5-HT transporter in human lymphocytes that resembles 5-HT uptake by platelets and brain synaptosomes may provide insights into the potential role of 5-HT in immune function and its relationship to the neurobiology of affective and addictive disorders.

VIRAL ONCOLYSATE IN THE MANAGEMENT OF MALIGNANT MELANOMA I. Preparation of the Oncolysate and Measurement of Immunologic Responses

Primary explants of human malignant melanoma were utilized in the preparation of oncolysates by Newcastle disease virus. The concentrated lysate, administered parenterally, was employed in an effort to augment antitumor immunologic responses in patients with metastatic melanoma. Observed cellular changes suggested a benefit, but humoral antibody measurements were not impressive. Cancer 40:672–679, 1977.

Biological responses of human apurinic endonuclease to radiation-induced DNA damage

Ionizing radiation produces a variety of DNA damage through active oxygen species such as the superoxide radical (Oi-), the hydroxyl radical (OH), and hydrogen peroxide (H202). Previous studies showed that human AP endonuclease, a counterpart of exonuclease 111, can functionally replace E. coli exonuclease 111 to repair methyl methanesulfonate (MMSFinduced but not H202-induced DNA damage.’,* Reduction of radiation-induced cytotoxicity by human AP endonuclease in a radiation-sensitive mutant of E. coli indicated that the AP site is one of the major forms of DNA damage produced by ionizing radiation.2 The removal of alkylation-induced AP sites and 3’-blocking deoxyribose fragments by human AP endonuclease indicates that this enzyme is playing a pivotal role in the repair of oxidative DNA damage.*,’ In the current study, we examined the biological roles of AP endonuclease as being responsible for the repair of radiation-induced DNA damage in human cells.

Active [3H]-Dopamine Uptake by Human Lymphocytes: Correlates with Serotonin Transporter Activity

The main objective of the present investigation was to determine whether the uptake of [3H]-dopamine in human lymphocytes is mediated through a serotonin transporter. This was examined by studying the effects of various monoamine uptake inhibitors on the uptake of [3H]-dopamine in human lymphocytes. Among the compounds tested, indatraline, imipramine and fluoxetine, selective inhibitors of neuronal serotonin transporter, were the most potent inhibitors of [3H]-dopamine uptake in lymphocytes. The 50% inhibiting concentration (IC50) for these inhibitors was in the range of 3.5-17 nmol/l. Bupropion, GBR 12909, nomifensine and xylamine, selective inhibitors of dopamine and norepinephrine transporters, had low affinity for the dopamine uptake system in human lymphocytes with IC50 values ranging between 1,000 and 40,000 nmol/l. These findings provide supportive evidence for the participation of a serotonin transporter in the uptake of [3H]-dopamine in human lymphocytes. The existence of a high affinity transport system for dopamine and serotonin in human lymphocytes may serve as a readily accessible model to detect changes in the neuronal uptake of dopamine and serotonin in addictive and psychiatric disorders.

Cytochemical response of kidney, liver and nervous system to fluoride ions in drinking water

SynopsisMorphological and cytochemical studies on the squirrel monkey have been made after maintaining the subjects on pure distilled water and fluoridated distilled water for 18 months with the objective of determining the effect of fluoride on the activity of some hydrolytic and oxidative enzymes in the kidney, liver and nervous system. Daily water intake by individual animals was measured over the final 10 months of the animals exposure to 0,1 and 5 ppm fluoride. Water consumption was considerably higher in the animals on higher fluoride intake. Whereas the nervous system remained totally unaffected by this experimental procedure, the liver showed a slightly enhanced activity of Krebs citric acid cycle enzymes. The kidneys, however, showed significant cytochemical changes, especially in the animals on 5 ppm fluoride intake in their drinking water. In these animals, the glomeruli showed an increase in the activity of acid phosphatase and the enzymes belonging to the citric acid cycle and the pentose shunt, whereas lactate dehydrogenase, a representative of the anaerobic glycolytic pathway, remained unchanged or only slightly changed. These observations suggest that fluoride in concentrations as low as 5 ppm interferes to some extent with the intracellular metabolism of the excretory system.

Binding of [3H]-dopamine to human lymphocytes: possible relationship to neurotransmitter uptake sites.

Freshly isolated human lymphocytes from 11 healthy subjects had specific binding sites for dopamine which were dependent on time, temperature and sodium, and appeared to follow Michaelis-Menten kinetics. The apparent affinity constant (KD) of human lymphocytes for dopamine and the maximal number of binding sites (Bmax) were 109 +/- 21 nM and 2.66 +/- 1.75 pmol/10(7) cells, respectively. Dopamine binding was markedly affected by cocaine (IC50 = 150 nM) and other inhibitors of biogenic amine uptake. The relatively high potency of cocaine in competing for dopamine binding suggested that human lymphocytes may serve as a readily accessible model to detect changes in the neuronal uptake of dopamine and perhaps other monoamine neurotransmitters.

Immunocompetence of cancer patients treated with levamisole

Nineteen outpatients with malignant melanoma and squamous cell carcinoma of the head and neck, who had surgical resection for complete removal of the tumor and no demonstrable metastases following surgery, were administered Levamisole (p.o., 150 mg per day, two days per week) and maintained on this dose for at least six months. Of this group, drug therapy was discontinued in four patients because of severe “flu‐like” syndromes leaving a group of 15 patients for detailed analysis. T‐lymphocyte percentages and levels, cAMP levels in the lymphocytes and a battery of skin tests for recall antigens were evaluated following surgery and at various intervals during immunotherapy. Patients who responded well to the treatment showed increased levels of T‐lymphocytes and increased cAMP levels, whereas non‐responders had low T‐cell levels and low cAMP levels. Also positive skin test reactions were observed in most patients who responded well to immunotherapy, although this was the least reliable indicator of patient response. Eight of the nine patients in the melanoma group have responded well clinically, whereas five of the six squamous cell carcinoma patients have developed recurrences.

Prevalence of high serotonin uptake in lymphocytes of abstinent alcoholics.

An impairment in serotonergic neurotransmission may be associated with alcoholism. We recently identified a high-affinity serotonin transporter (5-HTT) in human peripheral blood lymphocytes (PBLs). Moreover, molecular analysis of RNA samples of human lymphocytes using reverse transcription, coupled with polymerase chain reaction, enabled us to confirm the expression of a 5-HTT identical to the one reported in neuronal tissues, as evidenced by hybridization and sequence analysis. In this investigation, we measured the serotonin (5-HT) uptake in PBLs of recovering alcoholics (N = 10) with long-term abstinence (2-10 years) and non-alcoholic controls (N = 10). 5-HT uptake was measured by incubating 1 x 10(7) cells of PBLs with [3H]5-HT (3-1000 nM; sp. act. 23 Ci/mmol) for 10 min at 37 degrees. The results of this preliminary study revealed that abstinent alcoholics had significantly (P < 0.01) increased uptake of 5-HT (43.6 +/- 5.70 pmol/10(7) cells) as compared with controls (23.33 +/- 2.50 pmol/10(7) cells). An enhanced uptake of 5-HT in PBLs of abstinent alcoholics agrees with previously reported observations of increased 5-HT uptake in brain and platelets of former alcoholics and their descendants. This suggested that a serotonergic mechanism may be linked to the heredity of alcoholism.

A cocaine-sensitive active dopamine transport in human lymphocytes☆

Human lymphocytes possess a cocaine-sensitive high-affinity transport system for [3H]dopamine. [3H]Dopamine uptake was saturated with increasing dopamine concentrations and followed Michaelis-Menten kinetics. The uptake was temperature, sodium, and chloride dependent and was affected by the co-addition of ouabain, phloridzin, potassium cyanide, gramicidin, and other metabolic inhibitors. The uptake of dopamine was blocked significantly in a concentration-dependent manner by cocaine and its congeners. Furthermore, preliminary evidence is presented linking the possible relationship between decreased lymphocyte [3H]dopamine uptake and chronic cocaine abuse in humans.

Experimental protein malnutrition in primates: Cytochemical studies on the cerebellum of the squirrel monkey,Saimiri Sciureus

SynopsisThe cerebellum of healthy and malnourished squirrel monkeys was studied histopathologically and cytochemically for a number of important enzymes such as phosphatases (acid and alkaline phosphatase, ATPase, thiamine pyrophosphatase), esterases (simple esterase and acetylcholinesterase), dehydrogenases (succinate, malate and isocitrate dehydrogenase, lactate dehydrogenase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase), monoamine oxidase and phosphorylase. The Purkinje cells, stellate and basket cells were found to be more sensitive to protein malnutrition compared to the other types of cells in the cerebellum. An increase in the number of ‘dark cells’ with large amounts of ribonucleoprotein complex in the Purkinje cell layer of the extremely malnourished animals sacrificed after 15 weeks on a low protein diet may be significant and may reflect either an abnormal metabolic process or an interruption in the axonal transport of RNA complex. This may also be directly related to a significant reduction in the level of oxidative enzymes, especially those of the tricarboxylic acid cycle, these being the main source of energy stored in ATP. At the same time the level of lysosomal enzymes, which are responsible for the catalysis of the different degradation reactions, is greatly increased and indicates cellular catabolism. The present investigations point to the probability that the neurons adapt to the changed environment by beginning to utilize structural proteins for their basic metabolism.