Sándor Czirják

Downregulation of the CB1 Cannabinoid Receptor and Related Molecular Elements of the Endocannabinoid System in Epileptic Human Hippocampus

Endocannabinoid signaling is a key regulator of synaptic neurotransmission throughout the brain. Compelling evidence shows that its perturbation leads to development of epileptic seizures, thus indicating that endocannabinoids play an intrinsic protective role in suppressing pathologic neuronal excitability. To elucidate whether long-term reorganization of endocannabinoid signaling occurs in epileptic patients, we performed comparative expression profiling along with quantitative electron microscopic analysis in control (postmortem samples from subjects with no signs of neurological disorders) and epileptic (surgically removed from patients with intractable temporal lobe epilepsy) hippocampal tissue. Quantitative PCR measurements revealed that CB1 cannabinoid receptor mRNA was downregulated to one-third of its control value in epileptic hippocampus. Likewise, the cannabinoid receptor-interacting protein-1a mRNA was decreased, whereas 1b isoform levels were unaltered. Expression of diacylglycerol lipase-α, an enzyme responsible for 2-arachidonoylglycerol synthesis, was also reduced by ∼60%, whereas its related β isoform levels were unchanged. Expression level of N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D and fatty acid amide hydrolase, metabolic enzymes of anandamide, and 2-arachidonoylglycerols degrading enzyme monoacylglycerol lipase did not change. The density of CB1 immunolabeling was also decreased in epileptic hippocampus, predominantly in the dentate gyrus, where quantitative electron microscopic analysis did not reveal changes in the ratio of CB1-positive GABAergic boutons, but uncovered robust reduction in the fraction of CB1-positive glutamatergic axon terminals. These findings show that a neuroprotective machinery involving endocannabinoids is impaired in epileptic human hippocampus and imply that downregulation of CB1 receptors and related molecular components of the endocannabinoid system may facilitate the deleterious effects of increased network excitability.

Down-Regulation of Wee1 Kinase by a Specific Subset of microRNA in Human Sporadic Pituitary Adenomas

CONTEXTnThe tumorigenic mechanisms involved in pituitary adenomas, especially of nonfunctional pituitary adenomas (NFAs), remains unclear. Various cell cycle inhibitors have been found to be underexpressed in pituitary tumors; however, Wee1 kinase, a nuclear protein that delays mitosis and was recently recognized as a tumor suppressor gene, has not been previously investigated in pituitary tumors.nnnOBJECTIVEnOur objective was to examine the expression of Wee1 in pituitary tumors and to identify microRNAs (miRs) that can regulate its expression.nnnDESIGNnExpression of Wee1 was examined by immunohistochemistry and quantitative real-time PCR (qRT-PCR). Identification of miRs targeting the Wee1 3-untranslated region was performed by miR array followed by expression analysis of identified miRs using qRT-PCR. Dual-luciferase assay and transient transfection of miRs into Hela cells followed by immunoblot analysis of Wee1 protein and cell proliferation analysis were carried out.nnnPATIENTSnA total of 57 pituitary tissue samples including 27 NFAs, 15 GH-producing adenomas with or without prolactin overproduction, and 15 normal pituitary glands were analyzed.nnnRESULTSnWee1 protein expression was decreased in NFAs and GH-producing tumors with or without prolactin production, but no change in mRNA expression was observed with qRT-PCR. A specific subset of five miRNAs revealed by in silico target prediction was significantly overexpressed in NFA samples; three miRs (miR-128a, miR-155, and miR-516a-3p) targeted the 3-untranslated region of the Wee1 transcript, and exogenous overexpression of these miRs inhibited Wee1 protein expression and HeLa cell proliferation.nnnCONCLUSIONSnTo our knowledge, this is the first report suggesting that regulation of Wee1 kinase by miRs may be linked to pituitary tumorigenesis.

Sequence analysis of the PRKAR1A gene in sporadic somatotroph and other pituitary tumours

objective Carney complex (CNC) is an autosomal dominant multiple neoplasia syndrome featuring cardiac, endocrine, cutaneous and neural tumours, as well as a variety of pigmented lesions of the skin and mucosa. Pituitary GH‐secreting tumours are found in approximately 10% of patients with CNC. One of the genes responsible for CNC, the PRKAR1A gene located on human chromosome 17q22‐24, has recently been cloned. This represents a putative tumour suppressor gene, coding for the type 1α regulatory subunit of protein kinase A (PKA), which is found to be mutated in approximately half of the patients with CNC. However, it is currently unclear as to whether similar mutations occur in sporadic pituitary tumours. We have therefore investigated a series of GH‐secreting and other pituitary tumours for sequence abnormalities in the PRKAR1A gene. The mRNA produced by the PRKAR1A undergoes decay if it codes for a truncated protein; we therefore also determined PRKAR1A mRNA levels in the tumours, and compared them with known mutant PRKAR1A‐carrying lymphocyte samples.

MicroRNA profile indicates downregulation of the TGFβ pathway in sporadic non-functioning pituitary adenomas

MicroRNAs (miRs) are small, 16–29 nucleotide long, non-coding RNA molecules which regulate the stability or translational efficiency of targeted mRNAs via RNA interference. MiRs participate in the control of cell proliferation, cell differentiation, signal transduction, cell death, and they play a role in carcinogenesis. The aims of our study were to analyse the expression profile of miRs in sporadic clinically non-functioning pituitary adenomas (NFPA) and in normal pituitary tissues, and to identify biological pathways altered in these pituitary tumors. MiR expression profiles of 12 pituitary tissue specimens (8 NFPA and 4 normal pituitary tissues) were determined using miR array based on quantitative real-time PCR with 678 different primers. Five overexpressed miRs and mRNA expression of Smads (Smad1-9), MEG and DLK1 genes were evaluated with individual Taqman assays in 10 NFPA and 10 normal pituitary tissues. Pathway analysis was performed by the DIANA-mirPath tool. Complex bioinformatical analysis by multiple algorithms and association studies between miRs, Smad3 and tumor size was performed. Of the 457 miRs expressed in both NFPA and normal tissues, 162 were significantly under- or overexpressed in NFPA compared to normal pituitary tissues Expression of Smad3, Smad6, Smad9, MEG and DLK1 was significantly lower in NFPA than in normal tissues. Pathway analysis together with in silico target prediction analysis indicated possible downregulation of the TGFβ signaling pathway in NFPA by a specific subset of miRs. Five miRs predicted to target Smad3 (miR-135a, miR-140-5p, miR-582-3p, miR-582-5p and miR-938) were overexpressed. Correlation was observed between the expression of seven overexpressed miRs and tumor size. Downregulation of the TGFβ signaling through Smad3 via miRs may have a possible role in the complex regulation of signaling pathways involved in the tumorigenesis process of NFPA.

Long-term outcome after temporal lobe surgery—Prediction of late worsening of seizure control

We analyzed possible predictors of late worsening of seizure control in 94 adult patients who had anterior temporal lobectomy (ATL) from the Epilepsy Center of the National Institute of Psychiatry and Neurology, Budapest between 1985 and 2001. We evaluated data regarding epilepsy, presurgical evaluation, pre- and postoperative EEG, structural imaging, histology and operative complications. The mean follow-up was 6.1 years (range: 2-17 years). The outcome was measured as Engel class, the time to the first seizure and the longest seizure free period. Multiple regression analysis was used to assess predictors. Seizure free outcome was achieved in 72% of the patients 1-year after surgery. Eighty-seven percent of them remained seizure free at the second year of follow-up, 74% at the fifth, and 67% at the tenth year of follow-up. After 2 years of follow-up improvement was present in 3%, worsening in 18% of the patients. Factors associated with long-term worsening were: postoperative ipsilateral EEG spikes over the resected side, preoperative bilateral interictal discharges, cortical dysplasia of Taylors type, and ictal contralateral propagation. In these patients, even in seizure free state, therapy reduction might be inappropriate.

Dynamic changes of CB1-receptor expression in hippocampi of epileptic mice and humans

The endocannabinoid system plays a central role in retrograde synaptic communication, and controls both glutamatergic and γ‐aminobutyric acid (GABA)ergic transmission via type 1 cannabinoid (CB1) receptor. Both in sclerotic human hippocampi and in the chronic phase of pilocarpine‐induced epilepsy in mice with sclerosis, CB1‐receptor–positive interneuron somata were preserved both in the dentate gyrus and in the CA1 area, and the density of CB1‐immunostained fibers increased considerably in the dentate molecular layer. This suggests that, although CB1 receptors are known to be reduced in density on glutamatergic axons, the CB1‐receptor–expressing GABAergic axons sprout, or there is an increase of CB1‐receptor levels on these fibers. The changes of CB1 immunostaining in association with the GABAergic inhibitory system appear to correlate with the severity of pyramidal cell loss in the CA1 subfield. These results confirm the involvement of the endocannabinoid system associated with GABAergic transmission in human temporal lobe epilepsy (TLE), as well as in the chronic phase of the pilocarpine model in mice. Pharmacotherapy aimed at the modulation of endocannabinoid‐mediated retrograde synaptic signaling should take into account the opposite change in CB1‐receptor expression observed on glutamatergic versus GABAergic axon terminals.

A mutation and expression analysis of the oncogene BRAF in pituitary adenomas

Objectiveu2002 BRAF is an oncogene that is commonly mutated in both melanomas and papillary thyroid carcinomas, usually at position V600E that leads to constitutive activity in the Ras–mitogen‐activated protein kinase (MAPK) pathway. We speculated that this same gene may be either mutated at this site, or overexpressed, in pituitary adenomas.

Bilateral supraorbital keyhole approach for multiple aneurysms via superciliary skin incisions

BACKGROUNDnConsidering that multiple aneurysms carry a high risk for fatal rupture, there is a need for complete treatment of all lesions in one surgical session using either unilateral-contralateral or bilateral approaches. Contralateral approaches have been used mainly for small anteriorly projecting middle cerebral and medially expanding ophthalmic types of aneurysms. They are limited by the narrow space for surgical manipulation, forced elevation of frontal lobes, and stretching of the olfactory nerves. These problems might result in damage to structures along the unusually long intracranial way of the approach. The complications associated with the unnecessarily large conventional fronto-temporal and bifrontal craniotomies, and the developments in visualization, neuroanaesthesia, microneurosurgery, cerebrospinal fluid (CSF) drainage, and brain protection have led to less invasive methods in cerebral base surgery. These achievements have supplied the background for the supraorbital keyhole approach to aneurysms of the anterior circulation or basilar tip. Because the supraorbital keyhole approach offers several advantages over the classic fronto-temporal craniotomies to the anterior skull base, it was extended for both sides in one surgical session to treat bilateral multiple aneurysms as well.nnnMETHODSnOut of a series of 150 patients harboring 188 saccular aneurysms operated on via a supraorbital keyhole approach with a superciliar skin incision, 36 had multiple aneurysms. Thirty patients with multiple aneurysms underwent surgery for their ruptured aneurysms (17 cases in the acute phase and 13 patients during the chronic stage); in 6 cases silent aneurysms were operated on. The multiple aneurysms were managed from one side in 18 cases. A bilateral supraorbital keyhole approach was performed during one surgical session in 11 patients, and in 7 cases the unilateral supraorbital keyhole approach was combined with contralateral fronto-temporal (3 cases), suboccipital (2 cases), or frontal-parasagittal (2 cases) exploration. The operations were carried out through an approximately 2.5 x 3 cm supraorbital keyhole craniotomy following a skin incision just above the eyebrow. The roughly 4 cm superciliar skin incision begins medial to the supraorbital nerve and ends 3 to 10 mm beyond the lateral edge of the eyebrow. The technical details of the method are presented, and the benefits, limitations, and complications are discussed.nnnRESULTSnIn the 36 patients operated on via the supraorbital keyhole approach 74 aneurysms were clipped successfully. In 2 cases premature intraoperative rupture of the aneurysms occurred, but these events were managed successfully. Despite the small size of the craniotomy the approach allows enough room for intracranial manipulation with maximal protection of the brain and other intracranial structures. One patient died because of pulmonary embolism. There were no craniotomy-related complications in the present series.nnnCONCLUSIONnThe supraorbital keyhole approach together with the advent of the modern neuroanaesthesia, CSF drainage, and microsurgical techniques is a safe approach in the hands of experienced neurosurgeons for the treatment of supratentorial or basilar tip aneurysms. Because the approach is simple and swift, the bilateral single-session craniotomy does not have any disadvantages compared to two-stage procedures. However, the one-sitting surgery reduces the high risk of fatal rupture in the perioperative period associated with multiple aneurysms.

Reduced expression of the growth hormone and type 1 insulin‐like growth factor receptors in human somatotroph tumours and an analysis of possible mutations of the growth hormone receptor

objective Clinical acromegaly is characterized by elevated GH secretion in the presence of high circulating IGF‐I levels. We hypothesized that the physiological IGF‐I/GH negative feedback loop may be reset in somatotroph adenomas, specifically in terms of the level of expression of these receptors or mutations of the GH receptor (GH‐R) in such tumours.

Diagnostic performance of salivary cortisol and serum osteocalcin measurements in patients with overt and subclinical Cushing's syndrome

OBJECTIVEnThe cut-off value for salivary cortisol measurement for the diagnosis of Cushings syndrome (CS) may depend both on the severity of the disease and the composition of control group. Therefore, we examined the utility of midnight salivary cortisol measurements in patients who were evaluated for signs and symptoms of CS or because they had adrenal incidentalomas. Because serum osteocalcin (OC) is considered as a sensitive marker of hypercortisolism, we also investigated whether OC could have a role in the diagnosis of CS.nnnPATIENTS AND METHODSnEach of the 151 patients was included into one of the following groups: (A) overt CS (n=23), (B) subclinical CS (n=18), (C) inactive adrenal adenomas (n=40), (D) patients without HPA disturbances (n=70). Patients (C+D) were used as controls. Serum, salivary and urinary cortisol, and OC were measured by electrochemiluminescence immunoassay.nnnRESULTSnGroup A had suppressed OC as compared to both group B and group (C+D). Serum and salivary cortisol concentrations showed strong negative correlations with OC in patients with overt CS. The areas under the curves of salivary and serum cortisol at 24:00 h (0.9790 and 0.9940, respectively) serum cortisol after low dose dexamethasone test (0.9930) and OC (0.9220) obtained from ROC analysis for the diagnosis of overt CS were not statistically different.nnnCONCLUSIONnThis study confirms the usefulness of midnight salivary cortisol measurements in the diagnosis of overt CS in the everyday endocrinological praxis. Our results suggest that OC may have a role in the diagnosis of overt CS.