Real-world EGFR testing practices for non-small-cell lung cancer by thoracic pathology laboratories across Europe.

BACKGROUND: Testing for epidermal growth factor receptor (EGFR) mutations is an essential recommendation in guidelines for metastatic non-squamous non-small-cell lung cancer, and is considered mandatory in European countries. However, in practice, challenges are often faced when carrying out routine biomarker testing, including access to testing, inadequate tissue samples and long turnaround times (TATs). MATERIALS AND METHODS: To evaluate the real-world EGFR testing practices of European pathology laboratories, an online survey was set up and validated by the Pulmonary Pathology Working Group of the European Society of Pathology and distributed to 64 expert testing laboratories. The retrospective survey focussed on laboratory organisation and daily EGFR testing practice of pathologists and molecular biologists between 2018 and 2021. RESULTS: TATs varied greatly both between and within countries. These discrepancies may be partly due to reflex testing practices, as 20.8% of laboratories carried out EGFR testing only at the request of the clinician. Many laboratories across Europe still favour single-test sequencing as a primary method of EGFR mutation identification; 32.7% indicated that they only used targeted techniques and 45.1% used single-gene testing followed by next-generation sequencing (NGS), depending on the case. Reported testing rates were consistent over time with no significant decrease in the number of EGFR tests carried out in 2020, despite the increased pressure faced by testing facilities during the COVID-19 pandemic. ISO 15189 accreditation was reported by 42.0% of molecular biology laboratories for single-test sequencing, and by 42.3% for NGS. 92.5% of laboratories indicated they regularly participate in an external quality assessment scheme. CONCLUSIONS: These results highlight the strong heterogeneity of EGFR testing that still occurs within thoracic pathology and molecular biology laboratories across Europe. Even among expert testing facilities there is variability in testing capabilities, TAT, reflex testing practice and laboratory accreditation, stressing the need to harmonise reimbursement technologies and decision-making algorithms in Europe.

Effect of malnutrition and body composition on the quality of life of COPD patients.

BACKGROUND: Pathological alterations in nutritional status may develop in Chronic Obstructive Pulmonary Disease (COPD) patients through production of inflammatory cytokines and inadequate diet. OBJECTIVE: The aim of our study was to determine the correlation between nutritional status and quality of life of COPD patients. METHODS: We evaluated the nutritional status of COPD patients of Hungarian National Koranyi Institute for Pulmonology using the Malnutrition Universal Screening Tool (MUST) and bioelectrical impedance analysis (BIA) between January 1 and June 1, 2019. Lung function, physical fitness, and respiratory muscle strength were included in the assessment. RESULTS: Fifty patients (mean age was 66.3 ± 9.6 years) participated in our study. Mean body mass index (BMI) was 26.2 ± 6.1 kg/m2 and mean fat-free mass index (FFMI) was 16.8 ± 2.4 kg/m2. Overweight patients had better lung function values (FEV1ref%: 46.3 ± 15.2) than normal (FEV1ref%: 45.1 ± 20.9) and underweight patients (FEV1ref%: 43.8 ± 16.0). The Modified Medical Research Council Dyspnea Scale (mMRC) was significantly associated with various parameters; strongest correlation was found with FFMI (r = -0.537, P < 0.001), skeletal muscle mass index (SMMI) (r = -0.530, P < 0.001), and 6-minute walking distance (6MWD) (r = -0.481, P < 0.001). CONCLUSIONS: Our results indicate that malnourished COPD patients may have reduced lung function and lower quality of life compared to normal weight patients. Thus, our findings suggest that nutritional therapy be included in the treatment of COPD patients combined with nutritional risk screening and BIA during the follow-up.

Health-related quality of life of COPD patients aged over 40 years.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is the fourth most frequent disease globally, and its worldwide prevalence is projected to increase in the following decades. Health-related quality of life (HRQOL) of COPD patients depends on multiple factors. OBJECTIVE: The aim of this study was to identify the most important risk factors affecting HRQOL of COPD patients and to measure how specific clinical parameters can predict HRQOL. METHODS: A questionnaire-based cross-sectional study combined with clinical data was conducted among patients diagnosed with COPD (n = 321, 52.6% females, mean age 66.4 ± 9.5) at the National Koranyi Institute for Pulmonology, Budapest in 2019-2020. The inclusion criteria were age ≥40 years and existing COPD. Multivariate linear regression analyses were conducted on three components of the COPD-specific Saint George's Respiratory Questionnaire (SGRQ-C) and on the physical (PCS) and mental component scales (MCS) of the 36-Item Short Form Health Survey (SF-36). Multiple linear regression analysis was performed to evaluate the effects of patient and disease characteristics on COPD Assessment Test (CAT) scores. RESULTS: We found that frequent exacerbations, multiple comorbidities and tobacco smoking were associated with worse HRQOL. Engaging in more frequent physical activity and better 6-minute walking distance results were associated with better HRQOL. CONCLUSIONS: Our results indicate that the complex therapy of COPD should focus not only on improving lung functions and preventing exacerbation, but also on treating comorbidities, encouraging increased physical activity, and supporting smoking cessation to assure better HRQOL for patients.

Dynamics of High Risk Clinical Target Volume reduction during Brachytherapy and impact on its coverage in patients with inoperable cervical cancer.

Cervical cancer is the third most common cancer in women worldwide. Standard of care for patients with node positive or locally advanced tumors >4 cm is definitive radiotherapy and concurrent chemotherapy. Brachytherapy is an integral part of definitive radiotherapy for cervical cancer. The aim of the study was to show a dynamics of High Risk Clinical Target Volume (HR-CTV) reduction during Brachytherapy (BT) as a part of definitive treatment (External Beam Radiotherapy /EBRT/ +/- Chemotherapy /ChT/) depending on the initial Gross Tumor Volume (GTV) and its impact on HR-CTV coverage in patients with inoperable cervical cancer. We analyzed the dosimetric data for BT of 54 patients who have had Three Dimensional Planning of BT (3D BT). The Gross Tumor Volume, HR-CTV and organs at risk (OARs) were contoured on the magnetic resonance imaging (MRI), subsequently on the co-registered MRI images with computed tomography (CT). Point A and ICRU 38 rectal and bladder points were defined on reconstructed CT images. Patients were categorized on the basis of whether the 100% isodose line of the point-A prescription dose encompassed the HR-CTV (1st group) or not (2nd group). The 30cc volume has been determined as a cut-off value, which represented the most acceptable value of intermediate size of volumes. The initial mean value of GTV was 42cc. After completion of EBRT/ChT, the mean GTV was 3.24cc what was 91% reduction rate in relation to the initial value. We followed the dynamics of HR-CTV reduction during BT and have noted its minimal reduction from 24.3cc (mean value) at the time of the first fraction to the 24.1cc before fourth fraction. The mean V100 was 98% and increased with decreasing of the volume size (p=0.0063, Fisher's exact test). D90 (mean value was 96.3 Gy10) has been correlated with V100 and also, it increased with decreasing of the volume size (p=0.0003). The mean D0.1cc and D2cc of rectum doses were 80 Gy3 and 65.6 Gy3, respectively. The mean ICRU rectal dose for all patients was 67.2 Gy3. The mean D0.1cc (99.5 Gy3), D2cc (79.5 Gy3) and ICRU (75.2 Gy3) of bladder doses were acceptable. Dynamics of HR-CTV reduction during BT was minimal, although, significant reduction of the GTV was achieved after EBRT/ChT. This study revealed that the dose prescription of 7 Gy × 4 fractions to point A was not sufficient indicator for dose coverage of the HR-CTV. However, dosimetric parameters as V100 and D90 were strong indicators for coverage of HR-CTV which was inversely related to the volume of the target and the extension of tumor. However, dosimetric parameters for rectum and bladder (D0.1cc, D2cc and ICRU) did not show dependence on the target volumes.

Utilisation of water soluble iridium catalysts for signal amplification by reversible exchange.

The catalytic hyperpolarisation of pyridine, 3-hydroxypyridine and oxazole by the Signal Amplification By Reversible Exchange (SABRE) process is achieved by a series of water soluble iridium phosphine and N-heterocyclic carbene dihydride complexes. While the efficiency of the SABRE process in methanol-d4 solution or ethanol-d6 solution is high, with over 400-fold (1)H polarisation of pyridine being produced by [Ir(H)2(NCMe)(py)(IMes)(monosulfonated-triphenylphosphine)]BF4, changing to a D2O or a D2O-ethanol solvent mixture leads to dramatically reduced activity which is rationalised in terms of low H2 solubility.

Multiple metachronous malignant fibrous histiocytomas of the upper limbs -  a case report.

BACKGROUND: Soft-tissue sarcomas are rare tumors with the incidence of multiple metachronous or synchronous lesions in the extremities being even more uncommon. In effort to preserve the function of upper extremities, limb-salvage surgery became the treatment of choice for soft-tissue sarcomas. Subsequent adjuvant chemotherapy, as well as radiotherapy, is believed to decrease local recurrence rates, however, their effect on overall survival remains unclear. CASE: We report herein a case of symmetrical bilateral metachronous malignant fibrous histiocytomas of the shoulder. A 19-year-old patient presented with stiffness and pain in the right shoulder. The same symptoms developed 1.5 years later in the other shoulder. The culprit tumors are reported metachronous with regard to the succession in the onset of symptoms. Wide tumor resection was performed in both shoulders, and postoperative radiotherapy was then conducted. Chemotherapy was not indicated after the first surgery; whereas, in the second case it was the patient who refused the recommended adjuvant chemotherapy. CONCLUSION: The phenomenon of either metachronous or synchronous incidence of multiple soft tissue sarcomas is very rare and systematic reporting of every new case in the literature could contribute to further knowledge of tumors unique behavior.Key words: malignant fibrous histiocytoma - radiotherapy - upper extremity - neoplasms - multiple primary.

Therapy-related myelodysplastic syndrome and acute myeloid leukemia in patients with chronic lymphocytic leukemia treated with fludarabine and cyclophosphamide.

We retrospectively studied four cases of t-MDS/AML among 210 (1.9%) consecutive patients with CLL treated at a single center with fludarabine and cyclophosphamide (FC) either as the first- or second-line therapy. The median follow-up of the whole cohort of patients was 46months (range: 7-60). Two of these patients (2/130, 1.7%) had been treated with FC only, and two more (2/80, 2.3%) with CHOP and CHOP+FND, respectively, prior to FC. The median age was 61.5years (range: 49-71); three were male. They developed t-MDS/AML after a median latency period of 41months (range: 7-56) from the FC completion. Chromosomal aberrations with an adverse prognostic impact were present in the karyotype of all four patients, including abnormalities of chromosome 5 in three of them, and a rare chromosomal translocation in one patient. Median survival after t-MDS/AML diagnosis was 4months (range: 2-8). Although the agents administered prior to FC make it difficult to assess the risk of t-MDS/AML attributable to FC, this report might be a valuable addition to the literature.

The role of catecholamines in the prolactin release induced by salsolinol.

Salsolinol (1,2,3,4-tetrahydro-6,7-dihydroxy-1-methylisoquinoline) is an endogenous prolactin releasing agent. Its action can be inhibited by another isoquinoline, 1-methyl-3,4-dihydroisoquinoline (1MeDIQ), which has a strong norepinephrine releasing activity. Salsolinol does not alter the dopamine release in median eminence in vitro, providing evidence for the lack of interaction with presynaptic D2 dopamine receptors. At the same time, lack of norepinephrine transporter abolishes salsolinol's action. Salsolinol decreases tissue level of dopamine and increases norepinephrine to dopamine ratio in organs innervated by the sympathetic nervous system indicating a possible decrease of norepinephrine release. Enzymes of catecholamine synthesis and metabolism are probably also not the site of action of salsolinol. In summary, based upon all of these observations a physiologically relevant interplay might exist between the sympatho-neuronal system and the regulation of prolactin release.

The peripheral noradrenergic terminal as possible site of action of salsolinol as prolactoliberin.

Salsolinol, an endogenous isoquinoline, induces selective prolactin release in rats [Tóth, B.E., Homicskó, K., Radnai, B., Maruyama, W., DeMaria, J.E., Vecsernyés, M., Fekete, M.I.K., Fülöp, F., Naoi, M., Freeman, M.E., Nagy, G.M., 2001. Salsolinol is a putative neurointermediate lobe prolactin releasing factor. J. Neuroendocrinol. 13, 1042-1050]. The possible role of dopaminergic and adrenergic signal transduction was investigated to learn the mechanism of this action. The effect of salsolinol (10mg/kg i.v.) was inhibited by reserpine treatment (2.5mg/kg i.p.) and reinstated by pretreatment with monoamine oxidase inhibitor (pargyline 75 mg/kg i.p.). Salsolinol did not affect the in vitro release of dopamine (DA) in the median eminence, and did not inhibit the L-DOPA induced increase of DA level in the median eminence. 1-Methyl dihydroisoquinoline (1MeDIQ) is an antagonist of salsolinol induced prolactin release and causes increase in plasma NE level [Mravec, B., Bodnár, I., Fekete, M.I.K., Nagy, G.M., Kvetnansky, R., 2004. An antagonist of prolactoliberine induces an increase in plasma catecholamine levels in the rat. Autonom. Neurosci. 115, 35-40]. Using tissue catecholamine contents as indicators of the interaction between salsolinol and 1MeDIQ we found no interaction between these two agents to explain the changes in prolactin release in the median eminence, lobes of the pituitary, superior cervical and stellate ganglion. Increasing doses of salsolinol caused a dose dependent decrease of tissue dopamine concentration and increase of NE/DA ratio in the salivary gland, atrium and spleen. These changes of DA level and NE/DA ratio run parallel in time with the increase of prolactin release. 1MeDIQ antagonized the increase of prolactin release and decrease of tissue DA content caused by salsolinol. Neither this increase of prolactin secretion nor the decrease of DA level in spleen could be demonstrated in NE transporter (NET) knock out mice. The results presented argue for the possible role of peripheral norepinephrine release as a target for salsolinol in its action releasing prolactin. The dominant role of norepinephrine transporter may be suggested.

Methylnaltrexone inhibits opiate and VEGF-induced angiogenesis: role of receptor transactivation.

Angiogenesis or the formation of new blood vessels is important in the growth and metastatic potential of various cancers. Therefore, agents that inhibit angiogenesis have important therapeutic implications in numerous malignancies. We examined the effects of methylnaltrexone (MNTX), a peripheral mu opioid receptor antagonist, on agonist-induced human EC proliferation and migration, two key components in angiogenesis. Using human dermal microvascular EC, we observed that morphine sulfate (MS), the active metabolite, morphine-6-glucuronide (M6G), DAMGO ([d-Ala(2), N-Me-Phe(4), Gly(5)-ol]enkaphalin) and VEGF induced migration which were inhibited by pretreatment with MNTX at therapeutically relevant concentration (0.1 microM). The biologically inactive metabolite morphine-3-glucuronide (M3G) did not affect EC migration. We next examined the mechanism(s) by which MNTX inhibits opioid and VEGF-induced angiogenesis using human pulmonary microvascular EC. MS and DAMGO induced Src activation which was required for VEGF receptor transactivation and opioid-induced EC proliferation and migration. MNTX inhibited MS, DAMGO and VEGF induced tyrosine phosphorylation (transactivation) of VEGF receptors 1 and 2. Furthermore, MS, DAMGO and VEGF induced RhoA activation which was inhibited by MNTX or VEGF receptor tyrosine kinase inhibition. Finally, MNTX or silencing RhoA expression (siRNA) blocked MS, DAMGO and VEGF-induced EC proliferation and migration. Taken together, these results indicate that MNTX inhibits opioid-induced EC proliferation and migration via inhibition of VEGF receptor phosphorylation/transactivation with subsequent inhibition of RhoA activation. These results suggest that MNTX inhibition of angiogenesis can be a useful therapeutic intervention for cancer treatment.

NOL7 is a nucleolar candidate tumor suppressor gene in cervical cancer that modulates the angiogenic phenotype.

Cervical cancer is associated with human papilloma virus infection. However, this infection is insufficient to induce transformation and progression. Loss of heterozygosity analyses suggest the presence of a tumor suppressor gene (TSG) on chromosome 6p21.3-p25. Here we report the cloning NOL7, its mapping to chromosome band 6p23, and localization of the protein to the nucleolus. Fluorescence in situ hybridization analysis demonstrated an allelic loss of an NOL7 in cultured tumor cells and human tumor samples. Transfection of NOL7 into cervical carcinoma cells inhibited their growth in mouse xenografts, confirming its in vivo tumor suppressor activity. The induction of tumor dormancy correlated with an angiogenic switch caused by a decreased production of vascular endothelial growth factor and an increase in the production of the angiogenesis inhibitor thrombospondin-1. These data suggest that NOL7 may function as a TSG in part by modulating the expression of the angiogenic phenotype.

Spinal extradural arachnoid cyst causing cord compression in a 15-year-old girl: a case report.

The authors describe the case of a fifteen-year-old girl with progressive paraparesis of the lower limbs that was caused by an intraspinal extradural dorsal arachnoid cyst at the level of Th 3-6. Diagnosis was established with MRI and MRI myelography. The latter revealed the CSF-like content of the cyst. The patient underwent laminotomy and en bloc resection of the cyst. Ligation of the pedicle of the cyst was done with laminoplasty. Quick and complete recovery was observed after surgery.

Pivotal role of an endogenous tetrahydroisoquinoline, salsolinol, in stress- and suckling-induced release of prolactin.

In mammals, the role of a prolactin-releasing factor (PRF) in the acute changes of prolactin (PRL) secretion that usually occur after challenges (e.g., suckling stimulus or stress) of homeostasis has been suspected for a long time. We have recently observed that 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, salsolinol (SAL), produced by the hypothalamus and the neuro-intermediate lobe (NIL) of the pituitary gland, can selectively release PRL from the anterior lobe (AL). Moreover, binding sites for SAL have been detected in areas like median eminence, NIL, and AL. It has been proposed that SAL is a putative endogenous PRF. We have also found that a structural analogue of SAL, 1-methyl-3,4-dihydroisoquinoline (1MeDIQ), is able to block dose-dependently SAL-, suckling-, and immobilization (IMO) stress-induced release of PRL without having any influence on alpha-methyl-p-tyrosine (alphaMpT)-induced PRL responses. Neither SAL nor 1MeDIQ has any effect on alpha-melanocyte-stimulating hormone (alphaMSH), adrenocorticotrophic hormone (ACTH), beta-endorphin (beta-END) and arginine-vasopressin (AVP) secretion. Moreover, SAL-induced PRL response was attenuated in male rats pretreated with dexamethasone (DEX). These results strongly suggest that SAL has an important role in the regulation of PRL release induced by physiologic and environmental stimuli; therefore, it can be considered as the strongest candidate for being the PRF in the hypothalamo-hypophysial system. Our findings also indicate that the adrenal steroids may play an inhibitory feedback role in SAL-mediated PRL response.

Stress- as well as suckling-induced prolactin release is blocked by a structural analogue of the putative hypophysiotrophic prolactin-releasing factor, salsolinol.

Prolactin is secreted from the anterior lobe of the pituitary gland in response both to suckling and to stress. We recently observed that 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol), produced in the neurointermediate lobe of the pituitary gland, as well as in the medial basal hypothalamus, can selectively release prolactin from the anterior pituitary. Therefore, it has been proposed that salsolinol is a putative endogenous prolactin-releasing factor (PRF). Here, we report that one structural analogue of salsolinol, 1-methyl-3,4-dihydroisoquinoline (1MeDIQ), can block salsolinol-induced release of prolactin, but does not affect prolactin release in response to thyrotropin releasing hormone (TRH), alpha-methyl-p-tyrosine (alpha MpT) (an inhibitor of tyrosine hydroxylase), domperidone (a D(2) dopamine receptor antagonist), or 5-hydroxytryptophan (5-HTP), a precursor of serotonin). 1MeDIQ profoundly inhibited suckling-, immobilization-, as well as formalin-stress induced prolactin release without any influence on corticosterone secretion. The 1MeDIQ-induced reduction in prolactin response to immobilization stress was dose-dependent. These results suggest that salsolinol can play a pivotal role in the regulation of prolactin release induced by either physiological (suckling) or environmental (stress) stimuli.

Salsolinol is a putative endogenous neuro-intermediate lobe prolactin-releasing factor.

The isolation and identification of a prolactin-releasing factor (PRF) from the neuro-intermediate lobe of the pituitary gland has been pursued for over a decade. Using high-pressure liquid chromatography with electrochemical detection (HPLC-ECD) and gas chromatography/mass spectrometry (GC/MS) (R)-salsolinol (SAL) (a dopamine-related stereo-specific tetrahydroisoquinoline) was found to be present in neuro-intermediate lobe as well as median eminence extracts of male, intact-, and ovariectomized female rats. Moreover, analysis of SAL concentrations in neuro-intermediate lobe revealed parallel increases with plasma prolactin in lactating rats exposed to a brief (10 min) suckling stimulus following 4-h separation. SAL appears to be a selective and potent stimulator of prolactin secretion in vivo and it was without effect on the secretion of other pituitary hormones. We have also found that SAL can elevate prolactin release, although to a lesser extent, in pituitary cell cultures as well as in hypophysectomized rats bearing anterior lobe transplants under the kidney capsule. Lack of interference of SAL with [3H]-spiperone binding to AP homogenates indicates that SAL does not act at the dopamine D2 receptor. Moreover, [3H]-SAL binds specifically to homogenate of AL as well as neuro-intermediate lobe obtained from lactating rats. Taken together, our data clearly suggest that SAL is synthesized in situ and this compound can play a role in the regulation of pituitary prolactin secretion.

Paracrine angiogenic loop between head-and-neck squamous-cell carcinomas and macrophages.

Angiogenesis, an essential step in the development of neoplasia, is a complex process that involves the interaction of tumor cells with stromal cells. Tumor-associated macrophages (TAMs) can participate in the induction of tumor angiogenesis and are thought to be of prognostic value in some neoplasms. We have investigated how macrophages contribute to angiogenesis in head-and-neck squamous-cell carcinoma (HNSCC) and have found that tumor cells attract monocytes and activate them to secrete angiogenic factors. The attraction of macrophages was due to the secretion of monocyte chemotactic protein-1 and TGF-beta1 by tumor cells, while tumor production of TGF-beta1 was responsible for activating macrophages. In addition, activated macrophages produced cytokines that acted in a paracrine fashion by secreting both TNF-alpha and IL-1, which in turn stimulated tumor cells to secrete increased levels of IL-8 and VEGF. These data demonstrate that TAMs play an important role in the in vivo induction of angiogenesis in HNSCC and suggest that anti-angiogenic therapies for HNSCC and perhaps other neoplasms must include strategies that will block the ability of tumor cells to recruit macrophages into the tumor micro-environment.

Prediction of RNA base pairing probabilities on massively parallel computers.

We present an implementation of McCaskill's algorithm for computing the base pair probabilities of an RNA molecule for massively parallel message passing architectures. The program can be used to routinely fold RNA sequences of more than 10,000 nucleotides. Applications to complete viral genomes are discussed.

Anxiolytic 2,3-benzodiazepines, their specific binding to the basal ganglia.

Over the past 20 years, several members of the 2,3-benzodiazepine family have been synthesized. Some of these compounds--tofisopam (Grandaxin), girisopam, nerisopam--exert significant anxiolytic and antipsychotic activities. Sites where actions of 2,3-benzodiazepines are mediated differ from those of 1,4-benzodiazepines. Binding of 2,3-benzodiazepines to neuronal cells in the central nervous system shows a unique and specific distribution pattern: their binding sites are located exclusively to the basal ganglia. Chemical lesioning of the striato-pallido-nigral system, surgical transections of the striato nigral pathway and the activation of c-fos expression in the basal ganglia after application of 2,3-benzodiazepines suggest that these compounds mainly bind to projecting neurons of the striatum. The binding sites are transported from the striatum to the substantia nigra and the entopeduncular nucleus. Recent studies on mechanism of action of 2,3-benzodiazepines indicate their possible role in opioid signal transduction since 2,3-benzodiazepines augment the agonist potency of morphine to induce catalepsy and analgesia, and their action is diminished in morphine tolerant animals. The possible biochemical target of 2,3-benzodiazepines is an alteration in the phosphorylation of protein(s) important in the signal transduction process. Agents affecting emotional responses evoked by endogenous opioids without danger of tolerance and dependence may represent a new therapeutic tool in the treatment of addiction and affective disorders.

Dopamine transporters participate in the physiological regulation of prolactin.

Three populations of hypothalamic neuroendocrine dopaminergic (NEDA) neurons, arising from the arcuate and periventricular nuclei of the hypothalamus release dopamine (DA) that acts at the pituitary gland to regulate the secretion of PRL. It is generally accepted that NEDA neurons lack functional DA transporters (DATs), which are responsible for uptake of DA from the synaptic cleft into the presynaptic axon terminal. This study localized DATs to the hypothalamo-pituitary axis and evaluated the effect of DAT blockade on the hypothalamo-pituitary regulation of PRL. After 7 days of treatment with cocaine (a nonspecific amine transporter blocker) or mazindol (a specific DAT blocker), the relative abundance of PRL messenger RNA (mRNA) in the anterior lobe (AL) of OVX rats was significantly decreased, whereas the relative abundance of tyrosine hydroxylase mRNA in the hypothalamus was significantly increased. The effect of cocaine or mazindol administration on DA turnover and serum PRL concentration was examined in estradiol (E2)-treated OVX rats. E2 administration (i.v.) resulted in a significant increase in serum PRL within 4 h; however, cocaine or mazindol administration abolished the E2-induced increase of PRL. Cocaine or mazindol significantly increased the concentration of DA at the site of the axon terminals within the median eminence (ME), intermediate lobe (IL) and neural lobe (NL), indicating blockade of uptake. Because formation of DOPAC requires uptake of DA, concentrations of DOPAC in the ME, IL and NL decreased following treatment with either cocaine or mazindol. These data, together with the presence of immunopositive DAT in the ME, pituitary stalk, IL, and NL, suggest that a functional DAT system is present within all three populations of NEDA neurons. Moreover, similarity between the effects of cocaine and mazindol treatment indicate that blockade of the DAT, but not other amine transporters, is responsible for suppression of PRL gene expression and secretion. Blockade of DATs prevent uptake of DA into NEDA neurons and consequently increases the amount of DA that diffuses into the portal vasculature and reaches the AL. These data provide evidence that DATs play a physiological role in the regulation of DA release from and TH expression in NEDA neurons and consequently PRL secretion and PRL gene expression and further support our previous observation that the regulation of PRL secretion involves all three populations of NEDA neurons.