Effects of striatal injections of GABA(A) receptor agonists and antagonists in a genetic animal model of paroxysmal dystonia

The underlying mechanisms of idiopathic dystonias are poorly understood. The dystonic phenotype in the dt(sz) mutant hamster, a model of paroxysmal dystonia, has been suggested to be based on a deficit of gamma-aminobutyric acid (GABA)ergic interneurons and changes of the GABA(A)-benzodiazepine receptor complex in the striatum. In order to confirm and extend previous observations, the effects of compounds which bind to different sites of the GABA(A) receptor on the severity of dystonia were determined after striatal microinjections in comparison to systemic treatments in dt(sz) mutants. The GABA(A) receptor agonist (muscimol) and the benzodiazepine (flurazepam) reduced the severity of dystonia after striatal and systemic injections. The antidystonic effects of the barbiturate phenobarbital were less marked both after striatal and intraperitoneal administration of drugs. Intrastriatal injections of GABA delayed the onset of dystonic attacks. Striatal and systemic treatments with the GABA(A) receptor antagonist, bicuculline, and with pentylenetetrazole, which reduces GABAergic function, accelerated the onset of dystonia at subconvulsant doses. The benzodiazepine receptor antagonists flumazenil aggravated dystonia after systemic and intrastriatal injections. In all, the present data substantiate the relevance of striatal GABAergic disinhibition in the pathogenesis of paroxysmal dystonia in dt(sz) mutants.     

Motor disturbances in mice with deficiency of the sodium channel gene Scn8a show features of human dystonia

The med(J) mouse with twisting movements related to deficiency of the sodium channel Scn8a has been proposed as a model of kinesiogenic dystonia. This prompted us to examine the phenotype of these mice in more detail. By cortical electroencephalographic (EEG) recordings, we could not detect any changes, demonstrating that the motor disturbances are not epileptic in nature, an important similarity to human dystonia. The significantly decreased body weight of med(J) mice was related to reduced food intake. Observations in the open field and by video recordings revealed that the mice exhibit sustained abnormal postures and movements of limbs, trunk and tail not only during locomotor activity but also at rest. With the exception of the head tremor, the other motor impairments were persistent rather than paroxysmal. When several neurological reflexes were tested, alterations were restricted to the posture and righting reflexes. Results of the wire hang test confirmed the greatly reduced muscle strength in the med(J) mouse. In agreement with different types of human dystonia, biperiden, haloperidol and diazepam moderately reduced the severity of motor disturbances in med(J) mice. In view of the sodium channel deficiency in med(J) mice, the beneficial effects of the sodium channel blocker phenytoin was an unexpected finding. By immunohistochemical examinations, the density of nigral dopaminergic neurons was found to be unaltered, substantiating the absence of pathomorphological abnormalities within the brain of med(J) mice shown by previous studies. With the exception of muscle weakness, many of the features of the med(J) mouse are similar to human idiopathic dystonia.     

Clinical use of urinary markers for the detection and prognosis of bladder carcinoma: a comparison of immunocytology with monoclonal antibodies against Lewis X and 486p3/12 with the BTA STAT and NMP22 tests

PURPOSE: The noninvasive detection of urothelial carcinoma remains challenging. We prospectively evaluated urine markers for bladder carcinoma. We compared the NMP22 (Matritech, Cambridge, Massachusetts) and BTA Stat (Bard Diagnostics, Redmond, Washington) tests with immunocytology using mAbs 486p3/12 and BG7 against Lewis X antigen. MATERIALS AND METHODS: The NMP22 and BTA Stat tests were performed in urine samples and immunocytology with mAbs 486p3/12 and BG7 staining were performed in bladder washing specimens in 146 samples of 115 patients undergoing transurethral resection for suspected bladder carcinoma (70) or 45 undergoing followup cystoscopy for a history of bladder carcinoma (76). Bladder carcinoma was detected in 54 patients, including stages pTa in 25, pT1 in 20, pT2 in 8 and carcinoma in situ in 1, while 61 had no evidence of bladder carcinoma. The cutoff was 10 units per ml. for the NMP22, 30% positive cells for 486p3/12 and 5% positive cells for the Lewis X tests. RESULTS: BTA Stat was positive in 65 samples (44.5%) and NMP22 was positive in 69 (47.3%). Immunocytology with mAbs 486p3/12 and BG7 against Lewis X was positive in 52 (35.6%) and 109 (74.7%) samples, respectively. Sensitivity was 70.3% for BTA Stat, 68.5% for NMP22, 68.5% for 486p3/12 and 94.4% for Lewis X. Specificity was 70.6% for BTA Stat, 65.2% for NMP22, 83.6% for 486p3/12 and 36.9% for Lewis X. Area under the receiver operating characteristics curve was 0.6804 for NMP22, 0.7226 for Lewis X and 0.8002 for 486p3/12. False-positive results on BTA Stat in 2 of 22 patients (9%), on NMP22 in 2 of 25 (8%), on 486p3/12 in 3 of 11 (27%) and on Lewis X in 4 of 43 (9.3%) were associated with tumor recurrence. Furthermore, negative results on BTA Stat in 2 of 39 patients (2%), on NMP22 in 2 of 36 (0.5%), on Lewis X in 0 of 18 (0%) and on 486p3/12 in 1 of 50 (2%) was associated with tumor recurrence during followup. CONCLUSIONS: Immunocytology with mAbs against Lewis X showed higher sensitivity than all commercially available tests evaluated. Because of its high sensitivity and high negative predictive value, it may be useful for screening in a high risk population. Patients with a false-positive 486p3/12 test results are at increased risk for tumor recurrence compared with those with negative results.     

Effects of locally administered pentylenetetrazole on nigral single unit activity and severity of dystonia in a genetic model of paroxysmal dystonia

The dt(sz) hamster is a well-established animal model of idiopathic paroxysmal dystonia. Previous investigations of this mutant have indicated dysfunctions of the gamma-aminobutyric acid (GABA)-ergic system within the basal ganglia. Systemic administration of the central stimulant pentylenetetrazole (PTZ) aggravated dystonia at subconvulsant doses, whereas GABA-mimetic drugs have beneficial effects in dt(sz) hamsters. GABA mimetics also provide clinical benefit in humans with idiopathic paroxysmal dystonia. The spontaneous discharge rates of substantia nigra pars reticulata (SNr) neurons was unaltered in anesthetized dt(sz) hamsters, but systemic application of subconvulsant doses of PTZ caused significantly greater increases of discharge rates in dystonic hamsters compared with nondystonic controls. The present study tested the hypothesis that SNr neurons are more sensitive to local application of PTZ in dt(sz) hamsters than in nondystonic hamsters. PTZ applied locally by pressure injection at 2, 3, and 5 mM to the SNr during in vivo single unit recordings revealed a dose-dependent increase of SNr discharge rates in mutants and controls relative to predrug rates, with a significantly greater increase in mutants at 3 mM PTZ. To examine the functional relevance of the increased susceptibility of SNr neurons to PTZ in mutants, the effects of PTZ on severity of dystonia were investigated after microinjections into the SNr of freely moving dt(sz) hamsters. Bilateral nigral microinjection of 40 ng PTZ did not aggravate dystonia but exerted moderate antidystonic effects. Therefore, the previous findings of prodystonic effects of systemic administration of PTZ in dt(sz) hamsters are related to extranigral effects rather than to the elevation of nigral discharge rates in response to systemic, or locally applied, PTZ. The greater susceptibility of neurons within the SNr to PTZ suggests dysfunctions of the GABA(A) receptor in dt(sz) mutants.     

Risk factor for invasive zygomycosis in patients with hematologic malignancies

Zygomycosis (mucormycosis) is a relatively uncommon infection in immunocompromised patients most often diagnosed in patients with haematological malignancies and neutropenia. Postmortem series demonstrate a high mortality rate up to 80%. Pulmonary involvement mimicking the more frequently diagnosed invasive aspergillosis is the typical clinical presentation. Other risk factors for the development of zygomycosis that have been described in other patient populations include diabetic ketoacidosis, iron overload, use of deferoxamine and steroids. If these factors are also associated with zygomycosis in patients with haematological malignancies has not been described. In a retrospective case-control study including 13 patients with zygomycosis and 13 control patients with the same underlying diseases, without zygomycosis we determined the frequency of various risk factors. Patients with zygomycosis experienced a longer period of neutropenia (17 vs. 13 days) and lymphopenia (23 vs. 20 days). A relapse of their underlying disease was diagnosed more frequently in patients with zygomycosis (7/13 vs. 3/13) as were a diagnosis of diabetes mellitus (6/13 vs. 3/13) and a cardiovascular disease (6/13 vs. 1/13). The previous use of steroids was more frequent in patients with zygomycosis (8/13 vs. 4/13) as was a systemic antifungal prophylaxis with itraconazole (9/13 vs. 4/13). Knowledge of these risk factors may be of benefit in diagnosing and monitoring zygomycosis in patients with haematological malignancies.     

Unlabelled iododeoxyuridine increases the rate of uptake of [125I]iododeoxyuridine in human xenografted glioblastomas

5-Iodo-2'-deoxyuridine (IdUrd), a thymidine (TdR) analogue, can be radiolabelled with iodine-125, an Auger radiation emitter, to provoke double-strand breaks once incorporated into DNA of cancer cells. We have previously shown that co-incubation of [125I]IdUrd with unlabelled IdUrd provided an additive cytotoxicity in two human glioblastoma cell lines. This observation was unexpectedly correlated with an increase in the rate of DNA incorporation of [125I]IdUrd. Here, we further evaluated the effects of unlabelled IdUrd on the uptake of [125I]IdUrd in vitro and in vivo in mice xenografted with three human glioblastoma lines. The results showed that, in these three glioblastoma lines, unlabelled IdUrd increased the rate of uptake of [125I]IdUrd in vitro by 2- to 4.4-fold and in vivo by 1.5- to 2.8-fold. The rate of uptake of [125I]IdUrd in normal rapidly dividing tissues was also increased by 1.3- to 2.8-fold. TdR completely blocked [125I]IdUrd uptake in tumours and tissues. Analogues of IdUrd, such as deoxyuridine and 5-iodo-1,3-dimethyuracil, did not reproduce the effect of IdUrd on the uptake of [125I]IdUrd, suggesting that it is not related to protection against [125I]IdUrd degradation. It is concluded that combined administration of unlabelled IdUrd may improve the use of radiolabelled IdUrd for cancer diagnosis or therapy.     

Cultures of exfoliated epithelial cells from different locations of the human urinary tract and the renal tubular system

Exfoliated human urinary tract epithelial cells and renal tubular cells from urinary sediments of healthy adults, of urological patients and of internal patients were isolated and cultured. Cells started proliferating within 1 week after seeding a sediment. Proliferating cells formed colonies of different morphologies, designated as type-1 or type-2 cell colonies. Type-1 cell colonies showed irregular contours and spindle-like cells within the colonies. Subcultivation of type-1 cells for up to six passages was possible. Type-2 cell colonies showed smooth-edged contours and subcultivation was not possible. The epithelial character of type-1 cells was demonstrated by positive immunohistochemical staining for cytokeratin-7. In contrast to carbonic anhydrase-positive stained Madin Darby canine kidney cells (MDCK), which were used as positive controls for renal tubular cells, type-1 cells were carbonic anhydrase-negative on staining with the cobalt phosphate method. This indicates that type-1 cells were not of renal tubular origin. Type-2 cells were positively stained for carbonic anhydrase, indicating that type-2 cells were renal tubular cells. Type-2 cell colonies could be assigned to two subgroups with different cell forms. Colonies of cobblestone-like cells more often occurred than type-2 cell colonies with spindle-like cells, which are described in this study for the first time. Colonies with cobblestone-like cells formed domes (hemicysts), whereas spindle-like type-2 cell colonies did not. Cultures of urinary sediments from healthy adults, elderly multimorbid patients treated with furosemide, and urological patients with urolithiasis treated with sulfamethoxazole/trimethoprim and/or with a percutaneous nephrostomy catheter were compared. In 52% of all cultured sediments from healthy adults, in 30% of those from multimorbid patients, and in 75-80% of those from urological patients cells proliferated to colonies. The ratios of type-1 to type-2 cell colonies were 3.3:1 (healthy adults), 1.4:1 (urological patients with urolithiasis), and 1.8:1 (urological patients with urolithiasis, urine was directly collected from the renal pelvis with a percutaneous nephrostomy catheter). Successful cultures of the urinary sediments from these three groups revealed means of 3 or 4 colonies, 14 colonies, and 21 colonies, respectively. Differences in the number of colonies in relation to sex were observed only for the group of urological patients. It was shown that type-1 cells were urothelial cells, which did not show morphological differences due to their locations of origin within the urinary tract, whereas type-2 cells were probably renal tubular cells. These findings offer new aspects in the culturing of human urothelial or kidney epithelial cells with a method based on noninvasive collecting of specimens and requiring only minimal culture effort. The cultures obtained by this method can be used for in vitro studies in toxicological and clinical research.     

Isolation and characterization of endothelial cells from different organs of fetal pigs

The purpose of this study was to isolate endothelial cells from different organs of porcine fetuses and to examine the binding of endothelial markers including lectins. Endothelial cells were isolated from the aorta, cerebral cortex, myocardium, ovary and testis. Binding of the antibodies to von Willebrand factor (vWF) and angiotensin converting enzyme (ACE), the presence of Weibel Palade bodies (WPB), uptake of acetylated low density lipoprotein (acLDL), and labelling with the lectins Bandeiraea simplicifolia agglutinin I (BS I), Peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA) and Ulex europaeus agglutinin I (UEA I) were examined. Cell preparations displayed cobblestone-like morphology with the exception of testicular endothelium, which formed arcuate structures. Endothelium isolated from the brain labelled more strongly than any other cell line with the lectin PNA, but it did not express ACE. In contrast to other cell preparations, myocardial endothelium showed very low binding of anti-vWF. Ovarian endothelium was able to perform in vitro angiogenesis. Moreover, these endothelial cells possessed the largest number of WPB. Testicular endothelium displayed highest binding of vWF. Endothelium isolated from the aorta, in contrast to all other endothelial cells, did not take up acLDL. These results demonstrate that organ- and tissue-specific heterogeneity is already expressed in fetal endothelium.