Retinal pigment epithelial cells from Royal College of Surgeons dystrophic rats can take up melanin granules

· Background: Many successful pigment epithelium transplantation studies involving pink-eyed Royal College of Surgeons (RCS) dystrophic rats showed highly pigmented transplanted cells forming a double layer with slightly pigmented cells, attached to Bruch’s membrane. Since it is not clear whether transplanted pigmented cells can displace retinal pigment epithelial (RPE) host cells from Bruch’s membrane, we suggested that RPE cells of RCS dystrophic rats can phagocytize melanin granules, possibly derived from perished transplanted cells. · Methods: In a series of three experiments, RPE cells of nine pink-eyed, 2-month-old RCS dystrophic rats were isolated by trypsinization and mechanical dissection and cultivated in Dulbecco’s modified Eagles’ medium. These cells were then fed with melanin granules, isolated from bovine RPE cells, double-trypsinized after phagocytosis and viewed by light and electron microscopy. We also transplanted iris pigment epithelial (IPE) cells of 20-day-old Long-Evans rats into the subretinal space of pink-eyed RCS dystrophic rats of the same age, shown in light-microscopic photography after 42 days. · Results: Living RPE cells were heavily pigmented after feeding with isolated melanin granules in all three experiments as viewed by light microscopy. In addition, we identified melanin granules phagocytized by dystrophic RPE cells in electron microscopy. After transplantation of pigmented IPE cells into the subretinal space of pink-eyed RCS dystrophic rats’ eyes, a layer of slightly pigmented cells was seen on Bruch’s membrane below the transplanted IPE cells, shown in light microscopy. · Conclusion: We have shown by phagocytosis assay that dystrophic RPE cells can take up melanin granules in vitro. Our results assume that pigmented cells in transplantation studies, found as a monolayer, attached to Bruch’s membrane, cannot automatically be identified as transplanted cells. Instead, the possibility of perished transplanted cells serving as melanin donors for RPE host cells must be taken into consideration. Received: 11 March 1998 Revised version received: 5 May 1998 Accepted: 26 May 1998     

Activation of 5-HT1B receptors in the nucleus accumbens reduces amphetamine-induced enhancement of responding for conditioned reward

Previously, we have demonstrated that 5-hydroxytryptamine (5-HT) injected into the nucleus accumbens attenuates the potentiating effects of d-amphetamine on responding for conditioned reward (CR). The present studies examined the 5-HT receptor involved in this effect by investigating the effects of 5-HT agonists with differing affinities for 5-HT₁ and 5-HT₂ receptors on d-amphetamine-induced potentiation of responding for CR. Rats were trained to associate a light/tone stimulus (subsequently the CR) with water delivery. In a test phase, they were allowed access to a lever delivering the CR, and an inactive (NCR) lever. Responding on the CR lever was greater than responding on the NCR lever, indicating that the light/tone stimulus functioned as a CR. Responding for the CR was selectively potentiated by injections of d-amphetamine (10 μg) into the nucleus accumbens. This effect was reduced by injections into the nucleus accumbens of 5-CT (0.5 and 1 μg), RU24969 (10 μg), CP93,129 (1.25 and 2.5 μg) but not by DOI (10 μg) or 8-OH-DPAT (5 μg). The lower doses of 5-CT and CP93,129 did not reduce baseline responding for CR, or responding for water in a separate group of animals, indicating that the effects of these drugs were behaviourally selective. The higher doses abolished the CR effect, and in the case of 5-CT and RU24969 also reduced responding for water. All of the effective drugs share in common the ability to stimulate 5-HT_1B receptors, albeit with differing selectivities. The effect of CP93,129, the most selective of the 5-HT_1B agonists, to inhibit the response-potentiating effect of d-amphetamine was reversed by the5-HT_1B/1D antagonist GR127935 (3 mg/kg). The results indicate that activation of 5-HT_1B receptors within the nucleus accumbens attenuates the effects of a dopamine-dependent behaviour, and that activation of these receptors can oppose the behavioural effects of elevated mesolimbic dopamine transmission. Received: 22 April 1998/Final version: 28 July 1998     

Automatic decomposition electromyography in idiopathic inflammatory myopathies

Automatic decomposition electromyography (ADEMG) is a commercially available software package with installed reference values that enables the objective measurement of motor unit action potentials (MUAPs). To assess the diagnostic yield of this package in idiopathic inflammatory myopathies (IIM) we performed biceps brachii ADEMG in 17 patients with polymyositis, dermatomyositis and inclusion body myositis. Results were compared with those in 12 controls, and with the results of conventional EMG of the biceps and other muscles. Decreased mean values for MUAP duration occurred significantly more frequently in IIM patients than in controls; other MUAP characteristics did not differ. In IIM patients, decreased mean amplitude and increased mean number of turns occurred significantly less frequently on ADEMG than did corresponding abnormalities on conventional biceps EMG. Decreased mean values for duration and amplitude, and increased mean values for number of turns were seen significantly less often on ADEMG than corresponding abnormalities on conventional EMG of four different, individually chosen muscles. Overall evaluation of ADEMG resulted in a diagnosis of possible myopathy in 1 and probable myopathy in 8 patients, whereas overall evaluation of conventional EMG led to a diagnosis suggestive of IIM in 13 patients. We conclude that, although measurement of mean MUAP duration might be valuable in IIM diagnosis, our results do not favour the use of biceps brachii ADEMG and the installed reference values for the diagnosis of IIM. We suggest modifications to improve ADEMG's applicability.     

Endothelin-induced inositol phosphate formation in rat kidney. Studies on receptor subtypes, G-proteins and regulation during ontogenesis

The aim of this study was to characterize the properties of endothelin (ET)-receptor subtypes mediating inositol phosphate (IP)-formation in rat kidney and their regulation during ontogenesis. In renal cortical slices of adult rats (12–16 weeks old) ET's concentration-dependently increased IP-formation with an order of potency ET -1 ET 3. While the non-selective ET receptor antagonist bosentan (10 M) completely suppressed ET-induced IP-formation, the ETA-receptor antagonist BQ-123 (10 M) inhibited it only by 70%, the ET_B-receptor antagonist IRL 1038 (1 M) by 25%; combined application of BQ-123 + IRL 1038 caused complete inhibition of ET-1-induced IP-formation. Pretreatment of isolated renal cells with pertussis toxin (PTX, 500 ng/ml) overnight did not attenuate but significantly increased ET-1-induced IP-formation. Ontogenetic studies in renal slices from neonatal, 1, 2, 3, 6, 12 and 24 weeks old rats revealed that ET-1-induced IP-formation maturation-dependently declined being highest in neonatal rats (increase: 169% over basal) and lowest in 24 weeks old rats (increase: 47% over basal). This decline in ET-induced IP-formation was accompanied by a decrease in renal ET receptor number and the amount of immunodetectable G_q/11 (assessed by Western-blotting using the QL-antiserum). Moreover, ET receptor subtypes changed during the maturation process: from neonates to 12 weeks old rats number and functional responsiveness of ET_A-receptors declined, while that of ET_B-receptors increased. We conclude that in adult rat renal cortex ET-induced IP-formation is mediated by activation of both ET_A- and ET_B-receptors and does not involve a PTX-sensitive G-protein. ET-induced IP-formation declines during the maturation process; this is associated with a decrease in ET-receptor number and the immunodetectable amount of G_q/11.     

Primary vitrectomy without scleral buckling for rhegmatogenous retinal detachment

Background: Pars planta vitrectomy has evolved as an alternative method in the treatment of more complicated rhegmatogenous retinal detachments. We report a series of patients who underwent primary vitrectomy with gas tamponade without the use of additional scleral buckling. Methods: A retrospective study of 53 patients with a follow-up of 6–45 months (mean 17.8 months) was carried out. Preoperative findings included unusual, multiple or large breaks, vitreous haemorrhage, proliferative vitreoretinopathy and bullous retinal detachment. Preoperative visual acuity was between light perception and 1.0, with 30% (16/53) of patients with 0.4 or better. Results: Retinal reattachment was achieved in 64% of cases (34/53) with one and in 92% (49/53) with one or more operations. Final visual acuity was between light perception and 1.0, with 41% (22/53) of patients with 0.4 or better. Cataract formation occurred in 86% (37/43) of all patients with a clear lens preoperatively. Macular pucker was noted in 11 % (6/53) and postoperative proliferative vitreoretinopathy causing redetachment in 6% (3/53). Conclusion: With primary vitrectomy, a high final anatomical success rate with few intraoperative complications can be achieved in more complicated forms of rhegmatogenous retinal detachment. The major drawback of the procedure is the high incidence of post-operative cataract formation.     

Prolongation of the ejaculation latency in the male rat by thioridazine and chlorimipramine

Thioridazine (3 mg/kg) and chlorimipramine (1.5–6.0 mg/kg) prolonged the ejaculation latency and increased the number of mounts but did not change the number of intromissions preceding ejaculation. Blockade of peripheral and central noradrenaline receptors by phentolamine and phenoxybenzamine respectively resulted in a suppression of all aspects of the sexual behavior with increasing doses. dl-5-HTP (25–100 mg/kg) in combination with an inhibitor of peripheral 5-HTP decarboxylase (benserazide, 25 mg/kg) produced, like chlorimipramine and thioridazine, a prolongation of ejaculation latency and an increase in the number of mounts preceding ejaculation. Selective inhibition of 5-HT reuptake however, by zimelidine (0–20 mg/kg) or alaproclate (0–20 mg/kg) did not affect the mating behavior. At higher doses of these drugs some animals failed to initiate sexual activities. There was an increase in the postejaculatory interval but no change in the ejaculatory latency.It is concluded that the prolonged ejaculation latencies observed following treatment with thioridazine or chlorimipramine is not due to a blockade of central or peripheral adrenergic -receptors.     

Expression pattern of the cell cycle promoter cyclin e in benign extravillous trophoblast and gestational trophoblastic lesions: correlation with expression of Ki-67.

In this study, a specific monoclonal antibody was used to immunohistochemically investigate correlated expression of the cell cycle promoter cyclin E and the proliferation marker Ki-67 in benign extravillous trophoblast and gestational trophoblastic lesions. Our data show that cyclin E is expressed in the normal extravillous trophoblast, with strongest levels of expression in the cell columns of anchoring villi. Differences could be observed in expression of Ki-67 in both normal extravillous trophoblast and gestational trophoblastic lesions. In the extravillous trophoblast of the cell columns, expression of cyclin E started more distal compared with Ki-67 and was maintained (with less intensity) into the deeper layers of interstitial trophoblast. In the benign trophoblastic lesions (exaggerated placental site [EPS] and placental site nodule [PSN]) and in the trophoblast proliferations on the surface of hydropic villi of hydatidiform moles (HM), the percentage of cells expressing cyclin E was higher than of those expressing Ki-67. The same observation could be made for a case of placental site trophoblastic tumor (PSTT). In contrast, choriocarcinomas (N=8), which are definitely malignant tumors, showed an opposite pattern, with a much higher percentage of strongly Ki-67-positive cells compared with cyclin E-positive cells. We conclude that cyclin E is expressed in benign extravillous trophoblast and gestational trophoblastic lesions, where a ratio cyclin E/Ki-67<1 characterizes choriocarcinomas, whereas PSTT and the benign lesions (HM, EPS, PSN) show expression of cyclin E in a higher percentage of cells than Ki-67 (cyclin E/Ki-67 ratio >1).     

Effects of a high-selenium yeast supplement on celecoxib plasma levels: a randomized phase II trial.

A combination of celecoxib and selenium was used in a randomized double-blind Phase II trial as a preliminary study to a multicenter Phase III colorectal cancer chemoprevention trial using these two agents together. The purpose of this trial was to determine whether high-selenium baker's yeast [(Saccharomyces cerevisiae) 200 microg once daily] in combination with celecoxib (400 mg once daily) altered the steady-state plasma concentration of celecoxib or produced clinically significant toxicities. Seventy-three healthy subjects (ages 40-75 years) were recruited to the 6-week study from the general local population and were randomized to either the celecoxib plus selenized baker's yeast group or the celecoxib plus placebo group after a 2-week run in period of celecoxib only. Blood samples were taken at baseline (to document that there was no evidence of celecoxib intake), after the 2-week run-in period on celecoxib to verify steady-state blood levels of this agent, and at end of study (4 weeks postrandomization). Toxicities were monitored at 2 weeks after initiation of celecoxib, at 4 weeks after initiation, and at the end of the study. Blood level concentrations of celecoxib did not differ between the two groups as determined by high-performance liquid chromatography analysis nor were there significant differences in blood chemistry values between the two groups. Subjects' self-report of general physical toxicities was uncommon and limited to National Cancer Institute toxicity grade 2 or less; however, 2 female participants (3%) were removed from the study medications because of grade 2 edema and significant weight gain after 2 and 2.5 weeks of celecoxib administration. In conclusion, high-selenium yeast and celecoxib can be taken at the described doses with minimum short-term negative effects. In future Phase III chemoprevention trials of celecoxib, weight gain should be carefully monitored, and participants should be made aware of this potential side effect before study entry.     

Transendothelial migration of myeloma cells is increased by tumor necrosis factor (TNF)-alpha via TNF receptor 2 and autocrine up-regulation of MCP-1.

The proinflammatory cytokine tumor necrosis factor (TNF)-alpha has been shown to facilitate leukocyte transendothelial migration. In multiple myeloma, TNF-alpha is an important factor in the promotion of growth and survival of the malignant cells. Studies have shown that enhanced TNF-alpha levels in myeloma patients correlated with aggressive disease. Therefore, we investigated the effect of recombinant human TNF-alpha on the migrational behavior of myeloma cells across the physiological barrier of the major disease compartment, i.e., human bone marrow endothelial cells. In the presence of TNF-alpha, we observed significantly increased migration both in established myeloma cell lines and in plasma cells from myeloma patients. Expression of TNF-receptor 2 (TNF-R2) but not TNF-receptor 1 (TNF-R1) was detected in myeloma cell lines. Myeloma cells of patients also showed expression of TNF-R2 but not TNF-R1. The effect of TNF-alpha could not be explained by altered expression of adhesion molecules or metalloproteases. Instead, we found an up-regulation of monocyte chemoattractant protein (MCP)-1 and confirmed that myeloma cells express the relevant receptor C-C chemokine receptor 2. Preincubation of myeloma cells with recombinant human MCP-1 also enhanced cell migration, and this effect, as well as the effect of TNF-alpha, was abolished by treatment with anti-MCP-1 antibody. In contrast, migration of myeloma cells in the direction of an MCP-1 gradient, i.e., chemotaxis, could not be observed in the cell lines investigated. Additionally, the mRNA level of TNF-alpha was up-regulated by the cytokine treatment, which points to an autocrine loop augmenting and/or stabilizing the TNF-alpha-MCP-1 pathway. In summary, our data clearly support additional investigations using anti-MCP-1 antibodies in myeloma progression.     

Serologic evidence of human papillomavirus 16 and 18 infections and risk of prostate cancer.

Human papillomavirus (HPV) subtypes 16 and 18 are sexually transmitted and have been associated with an increased incidence of several anogenital tumors. Although previous epidemiological studies have suggested that sexual behaviors such as an early age at first intercourse and larger numbers of sexual partners are also related to an increased risk of prostate cancer, seroepidemiological studies of these infectious agents in relation to prostate cancer have produced differing results. To further evaluate this potential relationship, we completed a population-based control study in King County, Washington. Middle-aged (40-64 years) men diagnosed with prostate cancer (n = 642) were ascertained through the Seattle-Puget Sound Surveillance, Epidemiology, and End Results cancer registry between January 1993 and December 1996. Controls (n = 570) of similar age were selected from the same population as the cases by random digit dialing. Overall, there was no association between serological evidence of prior HPV-16 (adjusted odds ratio, 1.06; 95% confidence interval, 0.71-1.57) or HPV-18 (adjusted odds ratio, 1.36; 95% confidence interval, 0.69-2.69) infection and the risk of prostate cancer. Analyses of clinical features demonstrated no relationship between HPV infection status and Gleason score, stage of disease, or a combined measure of disease aggressiveness. Our findings indicate that HPV-16 and HPV-18 are not associated with prostate cancer risk.