Reduced growth of beard as the only diagnostic sign in a patient with macroprolactinoma.
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Adenoma↗
Biomedical subjects
J Georgieva
Publications and source records attributed to J Georgieva.
Detection of malignant melanoma in a giant congenital naevocytic naevus by positron emission tomography.
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Humans↗
Magnetic bead RT-PCR: establishment of a new method for detecting circulating melanoma cells.
This study was performed to detect circulating melanoma cells in peripheral blood using a novel method based on magnetic-activated cell separation (MACS) followed by a nested reverse transcriptase-polymerase chain reaction (RT-PCR) for tyrosinase and MART-1 mRNA. Samples to be tested were enriched for tumour cells either by isolating melanoma cells using two anti-melanoma antibodies (MART-1 and HMB-45) or by CD45 depletion of the non-melanoma cell fraction. The tumour cell-enriched fractions were subjected to mRNA isolation using oligo-deoxythymidylate (oligo-dT) magnetic beads followed by a nested RT-PCR. Sensitivity was assessed by spiking experiments and compared with a commonly used total RNA isolation system previously established in our department. Positive isolation of melanoma cells showed insufficient sensitivity, whereas negative isolation by depletion of leukocytes showed a detection limit of at least one melanoma cell per millilitre of whole blood. In further experiments, the depletion assay was applied to 25 peripheral blood samples of melanoma patients. The preliminary data obtained from the new method indicate a comparable detection rate to the established total RNA extraction method. However, not all the results were concordant. Therefore, future experiments need to be performed with a statistically greater number of patients.
Adult↗
Ethanol administration potentiates cocaine-induced dopamine levels in the rat nucleus accumbens.
Ethanol and cocaine are frequently co-abused, and the drug combination has been reported to produce an increased and prolonged subjective euphoria as compared to when either drug is administered alone. Acute administration of ethanol or cocaine increases the extracellular dopamine (DA) concentration in the nucleus accumbens (NAcc), a terminal region of the mesolimbic dopaminergic pathway. In the present study, the effects of separate and concurrent administration of cocaine and ethanol on DA concentrations in the NAcc were studied in rats pretreated with ethanol. Four groups of rats received either ethanol (2 g/kg, i.p.) or saline twice daily for 6 consecutive days. Thereafter, rats were given injections of saline or cocaine for another 2 days (i.e. treatment days 7 and 8) using a 'binge' administration pattern (three i.p. injections of 15 mg/kg each with 1-h interval starting 40 min after the first of the two daily doses of ethanol/saline). Stereotypic behavior was scored after each 'binge' of cocaine or saline on days 7 and 8. The DA and DA metabolite concentrations were measured using microdialysis on day 8. Ethanol enhanced the effect of cocaine on DA concentration in the NAcc as compared to a single administration of cocaine. The DA levels increased and reached their maximum values within 20-40 min after the cocaine administration, then gradually declined until the next injection 1-h later. Cocaine-induced stereotypic behavior was significantly increased in both saline and ethanol pretreated groups, though there was no significant difference between the two groups. The results of this study suggest that the enhanced DA transmission may be related to the experience produced by concurrent abuse of ethanol and cocaine in humans.
Animals↗
Expression of cyclins and cyclin dependent kinases in human benign and malignant melanocytic lesions.
AIMS: The regulation of cell proliferation is a key event in normal development, pathophysiological responses to injury, and tumorigenesis. The orderly progression of cells through the cell cycle depends on a finely tuned balance between the concentrations of activated cyclins and cyclin dependent kinases. This study was undertaken to compare the expression of cell cycle regulators in benign and malignant melanocytic lesions during tumour progression. METHODS: Immunohistochemistry was used to analyse 49 primary cutaneous malignant melanomas, 18 metastatic melanomas, and 12 histologically confirmed naevus cell naevi for their expression of cyclins (A, B1, D1, D2, D3, and E) and cyclin dependent kinases (CDK1, CDK2, and CDK4). RESULTS: Cyclin E and CDK2 had the highest expression patterns in human cutaneous melanomas and metastases and correlated positively with histological type and tumour stage. Cyclins B1, D2, and D3 had significantly increased expression in metastases, but normal or even decreased expression in primary melanomas. However, cyclins A and D1, and CDK1 and CDK4 were expressed very weakly in situ with no significant differences between naevi, melanomas, or metastases, and there was no correlation with histopathological staging. The specificity of recognition by the antibodies used was confirmed by western blotting on a panel of seven human melanoma cell lines. Cyclins A, B, and E were expressed by all seven, whereas cyclin D1 was detectable in six of seven and CDK2 and cdc2 were present in five of seven lines analysed. CONCLUSIONS: Taken together, this study demonstrated a significant increase of cyclin E and CDK2 expression during tumour progression in malignant melanomas.
Biomarkers, Tumor↗
Basal and cocaine-induced opioid receptor gene expression in the rat CNS analyzed by competitive reverse transcription PCR.
The basal mRNA levels of kappa and mu opioid receptors, as well as their regulation after 'binge' cocaine administration, were determined with competitive reverse transcription polymerase chain reaction (RT-PCR) in brain regions of male Sprague-Dawley rats. The procedure proved to be a reliable method to quantify the relative opioid receptor gene expression. The highest basal mRNA levels for the kappa opioid receptor were found in the nucleus accumbens and hypothalamus, whereas the highest basal mRNA expression for the mu opioid receptor was observed in the hypothalamus. Rats were separately treated with 'binge' (three hourly injections) cocaine HCl (45 mg/kg/day i.p.) or saline (1 ml/kg i.p.) for 2 days. A significant down-regulation of the kappa opioid receptor mRNA was detected in the nucleus accumbens. The mu opioid receptor mRNA was not affected. The data suggest a selective effect on kappa receptor expression in the nucleus accumbens as a consequence of 'binge' cocaine use.
Animals↗
Differential effects of intrastriatally infused fully and endcap phosphorothioate antisense oligonucleotides on morphology, histochemistry and prodynorphin expression in rat brain.
In the present study, we investigated the selectivity and specificity associated with continuous intrastriatal treatment with antisense oligonucleotides. Rats were given intrastriatal infusions for 72 h with phosphodiester, and fully and endcap phosphorothioated oligonucleotide probes complementary to prodynorphin mRNA. Dynorphin (Dyn) peptide levels were measured by radioimmunoassay. The integrity of three other striatal transmitter systems, the neuropeptide Y (NPY)-ergic interneurons, the cholinergic interneurons and the dopaminergic afferent innervation, was assessed histochemically. The gross morphology of the striatum and the distribution of fluorescently labelled antisense probes were also investigated. Brains infused with phosphodiester probes had tissue Dyn levels not different from control. They also showed little or no change in staining for NPY, acetylcholinesterase (AChE) and tyrosine hydroxylase (TH) and essentially normal striatal gross morphology. In contrast, brains treated with fully phosphorothioated oligonucleotides showed significant decreases in striatal Dyn levels but also severe tissue damage accompanied by massive cell infiltration and decreases in immunoreactivities for the striatal neurochemical markers. Fluorescently labelled phosphorothioate probes were observed widely in the striatum and adjacent structures and, presumably retrogradely transported, in the dopamine cell bodies in the substantia nigra, also revealing the presence of abnormal cellular structures within the striatum. By comparison, endcap probes significantly reduced striatal Dyn levels and showed good tissue penetration without inducing major changes in tissue morphology or histochemistry of non-dynorphinergic systems, except for cell infiltration. The deleterious tissue effects of fully phosphorothioated oligonucleotides and the ineffectiveness of phosphodiester oligonucleotides in inhibiting protein synthesis suggest that, of the probes examined in this study, endcap oligonucleotides are the most useful for in vivo studies in the central nervous system.
Acetylcholinesterase↗
Downregulation of kappa opioid receptor mRNA levels by chronic ethanol and repetitive cocaine in rat ventral tegmentum and nucleus accumbens.
The combination of ethanol and cocaine is commonly abused by human addicts which has serious clinical consequences. Here, the effects of separately and concurrently administered ethanol and 'binge' cocaine on kappa opioid receptor (KOR) mRNA in the ventral tegmental area (VTA) and nucleus accumbens (NAc) of rats were studied. KOR mRNA was down-regulated in both brain regions during concurrent as well as separate treatment with these drugs. In the VTA, the most pronounced decrease was obtained following combined treatment with ethanol and 'binge' cocaine. In the NAc, the strongest decrease was observed in the 'binge' cocaine group. This profound decrease of KOR mRNA in regions important for brain reward suggests a potential role of the KOR system in the abuse of cocaine and ethanol.
Animals↗
In vivo metabolism of nociceptin/orphanin FQ in rat hippocampus.
The in vivo metabolism of the newly identified endogenous ligand for the ORL1 receptor, the opioid-like peptide nociceptin (orphanin FQ) in rat hippocampus was studied using size-exclusion chromatography linked to electrospray ionization mass spectrometry. The results show that nociceptin is metabolized step-wise in vivo into fragments (1-13) and (14-17) as well as (1-9) and (10-13), respectively. Interestingly, the (1-13) and (1-9) fragments have the same C-terminus, Arg-Ala-Lys, suggesting that this is a motif recognized by an enzyme which fragments the peptide in two consecutive steps. Injection of the (1-13) fragment into rat hippocampus had no effect on spatial learning or motor function under conditions where nociceptin is active, showing that this metabolic conversion reduces affinity for the ORL-1-receptor.
Amino Acid Sequence↗
Hippocampal dynorphin B injections impair spatial learning in rats: a kappa-opioid receptor-mediated effect.
The hippocampus plays a central role in the acquisition and storage of information. Long-term potentiation in the mossy fibre pathway to the CA3 region in the hippocampus, an animal model of memory acquisition, is modulated by dynorphin peptides. This study investigated the possible role of hippocampal dynorphin in spatial learning. Male rats were trained in the Morris Water Task after microinjection with different doses of dynorphin B (1, 3.3 or 10 nmol/rat) or artificial cerebrospinal fluid (as control) into the CA3 region of the dorsal hippocampus. Dynorphin B was found to impair spatial learning at all tested doses. The synthetic kappa1-selective opiate receptor antagonist nor-binaltorphimine (2 nmol) also given into the hippocampus fully blocked the acquisition impairment caused by dynorphin B (10 nmol), while nor-binaltorphimine alone did not affect learning performance. These findings suggest that dynorphin peptides could play a modulatory role in hippocampal plasticity by acting on hippocampal kappa-receptors and thereby impair spatial learning.
Animals↗
Vaccination with IL-12 gene-modified autologous melanoma cells: preclinical results and a first clinical phase I study.
Cytokine gene transfer into tumor cells has been shown to mediate tumor regression and antimetastatic effects in several animal models via immunomodulation. Therefore, clinical protocols have been developed to treat cancer patients with cytokine gene-modified tumor cells. We inserted the genes coding for the p35 and p40 chain of interleukin-12 (IL-12) in two independent eukaryotic expression vectors and transduced melanoma cells of 15 different primary tumor cultures with both plasmids by a ballistic gene transfer approach. Secreted IL-12 demonstrated strong bioactivity by inducing interferon-gamma release from peripheral blood lymphocytes upon coculture with cell culture supernatants after IL-12 gene transfer which could at least partly be blocked by IL-12-specific antisera. Further enrichment of transduced tumor cells by magnetic separation directly after gene transfer increased cytokine secretion from a mean of 119 pg in the unsorted to 507 pg IL-12 (24 h/10(8) cells) in the magnetically enriched cell fraction. Irradiation of these cells led to a further elevation of secreted IL-12 (mean 987 pg). Elevated IL-12 levels were detected over 7 days after irradiation in vitro. In a subsequent first clinical phase I study six patients with metastatic melanoma were vaccinated with autologous, interleukin-12 gene-modified tumor cells. Melanoma cells were expanded in vitro from surgically removed metastases, transduced by ballistic gene transfer, irradiated and were then injected subcutaneously (s.c.) at weekly intervals. Clinically, there was no major toxicity except for mild fever. All patients completed more than four s.c. vaccinations over 6 weeks and were eligible for immunological evaluation. Post-vaccination, peripheral mononuclear cells were found to contain an increased number of tumor-reactive proliferative as well as cytolytic cells as determined by a limiting dilution analysis in two patients. Two patients developed DTH reactivity against autologous melanoma cells and one had a minor clinical response. Biopsies taken from that patient's metastases revealed a heavy infiltration of CD4+ and CD8+ T lymphocytes. In conclusion, vaccination induced immunological changes even in a group of advanced, terminally ill patients. These changes can be interpreted as an increased antitumor immune response.
Adult↗
Nociceptin/orphanin FQ microinjected into hippocampus impairs spatial learning in rats.
The newly discovered peptide nociceptin/orphanin FQ has been found to increase reactivity to pain and to influence locomotor activity after intracerebroventricular administration. This study investigated the possible role of hippocampal nociceptin/orphanin FQ in spatial learning and in spontaneous locomotion. Male rats were trained in the Morris water task after microinjection of 10 nmol nociceptin/orphanin FQ or artificial cerebrospinal fluid (as control) into the CA3 region of the dorsal hippocampus. Nociceptin/orphanin FQ was found to severely impair spatial learning without interfering with swimming performance. Intrahippocampal injection of nociceptin/ orphanin FQ markedly decreased exploratory locomotor activity including vertical movements (rearing). The data suggest that nociceptin/orphanin FQ is a potent modulator of synaptic plasticity within the hippocampus.
Animals↗
Involvement of a c-fos-dependent mechanism in caffeine-induced expression of the preprotachykinin A and neurotensin/neuromedin N genes in rat striatum.
Striatal c-fos induction was blocked by local administration of phosphorothioated c-fos antisense oligonucleotides (AS-ODN) to examine the possible role of caffeine-induced c-fos expression in transcriptional regulation of striatal preproenkephalin, prodynorphin, preprotachykinin A and neurotensin/neuromedin N. Caffeine (100 mg/kg i.p.) induced both c-fos mRNA and Fos-protein, and this induction was significantly attenuated by intrastriatal injection of 4 (but not 1) nmol c-fos AS-ODN. This suggests that, in addition to translational arrest, other mechanisms may be involved in the mediation of antisense action. The action of the AS-ODN was sequence specific. The antisense blockade of c-fos reduced the effect of caffeine on the expression of mRNAs for preprotachykinin A and neurotensin/neuromedin N in the ventrolateral caudate-putamen. Levels of preproenkephalin and prodynorphin transcripts were unaffected. Thus caffeine induction of striatal preprotachykinin A mRNA and neurotensin/neuromedin N mRNA, but not of preproenkephalin mRNA or prodynorphin mRNA, may at least in part be mediated by a pathway involving Fos protein. The findings illustrate the utility of blockade of gene expression with antisense oligonucleotides for in vivo studies of drug actions.
Animals↗
Neurochemical effects of prolonged treatment with remoxipride as assessed by intracerebral microdialysis in freely moving rats.
1. At three microdialysis sessions, dialysates were collected from the striatum of the same rats. 2. Microdialysis session 1. A single s.c. injection of remoxipride (40 mumol/kg), resulted in increased dialysate concentrations of dopamine, DOPAC and HVA. 3. Microdialysis session 2. Continuous administration of remoxipride (8.6 mumol/rat/day) for 14 days, using mini-osmotic pumps, produced maintained elevated levels of dopamine, DOPAC and HVA. 4. Microdialysis session 3. A challenge dose of remoxipride (40 mumol/kg s.c.), given to the rats after a 48-hour wash-out period following the continuous remoxipride treatment, increased the dialysate concentrations of dopamine, DOPAC and HVA to similar extent as at dialysis session 1. 5. It is concluded that after long-term treatment of remoxipride, an adaptation of the basal state of the DA system appears to take place, implying a lowering of basal DA release and DA metabolism. However, the capacity to respond with increased DA release and DA metabolism to renewed remoxipride treatment is retained, indicating little, if any, tolerance.
3,4-Dihydroxyphenylacetic Acid↗
Tissue and microdialysate changes after repeated and permanent probe implantation in the striatum of freely moving rats.
Neurochemical and morphological effects of repeated microdialysis or permanent microdialysis probe implantations in striatum were studied. The extracellular levels of dopamine did not change between a first and a second probe insertion separated by 2 weeks or at a third dialysis session 2 days later. The 3,4-dihydroxyphenylacetic acid and homovanillic acid levels were similar at the first and second microdialysis session, but decreased at the third. Probes implanted permanently for 2 weeks clogged, and the recovery varied markedly after insertion of new probes. Tyrosine hydroxylase-stained dopamine fibers appeared unaffected after all dialysis sessions, although some swollen fibers were observed surrounding the probes. No change in the glial fibrillary acidic protein staining was seen immediately after the first dialysis session, although 2 weeks later gliosis was observed. After the second and third dialysis a diffuse gliosis was observed, while a glial barrier was seen surrounding the permanently implanted probes. Immediately after the first dialysis session enlarged laminin-stained blood vessels were seen, whereas repeated probe implantation also increased the blood vessel density. Thus, chronic in vivo microdialysis with permanently implanted probes is limited by severe technical problems and marked tissue changes. On the other hand, repeated probe insertion in the same brain site appears to be acceptable for performing chronic microdialysis studies in the same subject, provided neurochemical and morphological changes are taken into consideration.
3,4-Dihydroxyphenylacetic Acid↗
The effects of the diterpene sclareol glycol on seizures do not depend on central benzodiazepine receptors.
Using pentylenetetrazol (PTZ) in subconvulsant doses, it was found that the semisynthetic diterpene sclareol glycol (SG) from the labdane family of diterpenes at a dose well below the lethal dose, although not being convulsant, had a proconvulsant action in mice. This action is indicated by the number of mice undergoing convulsions and the reduction of latency. Specific binding of [3H]-diazepam in vitro to whole homogenate fractions prepared from hippocampus of rats was not inhibited by SG. At the same time comparatively investigated Ro 15-1788 inhibited [3H]-diazepam binding. It is suggested that the proconvulsant effect of SG is realized not through interactions with central benzodiazepine receptors as Ro 15-1788 does, but perhaps through interactions with adenylate cyclase (stimulating its catalytic subunit and thus increasing brain 3',5'-AMP availability). Other brain neurotransmitter systems (e.g., GABA) should also participate in realization of the proconvulsant effect of SG.
Animals↗
The response of diterpene sclareol glycol to acute hypoxia in mice.
The effects of a reversible activator of adenylate cyclase sclareol glycol (SG), a semisynthetic diterpene of the labdane family, on acute hypoxia (asphyctic, hemic-induced by 300 mg/kg of sodium nitrite injected subcutaneously, and histotoxic-induced by 20 mg/kg sodium nitroprusside injected intraperitoneally) in mice were studied. SG was applied at doses well below the lethal dose. SG increased the latency to convulsions and the survival time to death. It is suggested that the antihypoxic effects of SG on these three models of acute hypoxia are induced mainly via its effects on adenylate cyclase and perhaps its involvement in synaptic transmitter action.
Animals↗