[Periodontitis - infection or inflammation?] / Parodontitis; infectie of ontsteking?

Periodontitis is a complex, multifactorial disease. Multiple factors such as (epi)genetic factors, environmental factors (microbial biofilm), lifestyle (smoking, stress) and general health (diabetes mellitus) contribute to the development of periodontitis. A healthy subgingival microbiome is in balance with its host, is very stable and diverse, and keeps the host healthy. Changes, such as declining oral hygiene, lead to changes in the microbial composition in the subgingival sulcus: anaerobic and protein-degrading microorganisms with pro-inflammatory properties increase in number and disturb the proportions, leading in turn to changes in the subgingival environment and an increase in pro-inflammatory microorganisms. If the first line of the immune system is unable to restore subgingival equilibrium, microorganisms and their products invade the periodontal soft tissues, resulting in activation of osteoclasts and, ultimately, in the destruction of the periodontium and the onset of periodontitis.

Gingival epithelium attachment to well- or partially cured resin composites.

Ideal restoration material for caries would allow attachment of gingival epithelia. The attachment of epithelial cells to specimens of the 4 most commercially used well- or partially-cured resin composites, with and without TEGDMA, was assessed. Effects of resin composite on the Ca9-22 gingival epithelial cell-line were assessed by measuring the cytotoxicity, viability and gene expression for attachment, apoptosis, ROS-production, pro-inflammatory cytokines, and matrix metalloproteinases. As controls, cells on tissue culture plastic or bovine tooth enamel specimens were used. Significantly less cell attachment was measured on freshly made resin-composite specimens. Concomitantly, significantly higher cytotoxicity was measured in the presence of freshly made resin-composite specimens. However, after 8 d of leakage, the cell attachment to and cytotoxicity of the resin composite was comparable to bovine tooth enamel. Significantly higher expressions of IL6, MMP2, BCL6 and ITGA4 were measured in cells attached to resin-composite surfaces than controls. There were no significant differences between the results using different conditions of resin composite, with or without TEGDMA and well or partially cured. Less cell attachment and presence of more inflammatory markers were observed on all freshly-made resin-composite surfaces. However, after a leakage period attachment of cells to the resin composite improved to the level of natural tooth materials such as enamel. This indicated that the negative effects of resin composites on epithelial cells might be transient.

Tumor necrosis factor-α antagonist infliximab inhibits osteoclast formation of peripheral blood mononuclear cells but does not affect periodontal ligament fibroblast-mediated osteoclast formation.

BACKGROUND AND OBJECTIVE: The inflammatory cytokine tumor necrosis factor-alpha (TNF-α) is elevated in inflamed periodontal tissues and may contribute to periodontitis progression. TNF-α stimulates formation and activity of osteoclasts, the cells that are recruited in periodontitis, that cause alveolar bone degradation and subsequent tooth loss. We previously showed that TNF-α is elevated in co-cultures of periodontal ligament fibroblast (PDLF) and peripheral blood mononuclear cells (PBMC). Hence, TNF-α could be a determining factor in osteoclast formation in these cultures, as osteoclasts are formed despite the fact that prototypical osteoclast differentiation factor receptor activator of nuclear factor kappa-B ligand is outnumbered at least 100-fold by its inhibitor osteoprotegerin in these cultures. MATERIAL AND METHODS: To assess the role of TNF-α in periodontitis-associated osteoclast formation in vitro, osteoclast formation was analyzed in the presence of the anti-TNF-α therapeutic agent infliximab in two culture systems: (i) PBMC in co-culture with PDLFs from controls and patients with periodontitis, or (ii) with PBMC only. PDLFs from control and patients with periodontitis were exposed to infliximab, PBMCs were added and the formation of osteoclast-like cells was assessed. RESULTS: TNF-α was highest levels in supernatants at 7 d in co-cultures and declined at 14 and 21 d. TNF-α was undetectable in cultures that received infliximab. The formation and activity of osteoclasts in co-cultures was not affected by infliximab. In contrast, infliximab in cultures of only PBMC significantly reduced the formation of osteoclasts. This reduction was accompanied by a decreased number and size of cell clusters, a step that precedes the formation of osteoclasts. TNF-α was again undetectable in the supernatant of infliximab-treated cultures, but was detectable at similar levels in cell lysates of control and infliximab-treated PBMC cultures. CONCLUSION: Our study shows that the contribution of TNF-α to osteoclast formation is cell system dependent. It contributes to PBMC-induced osteoclast formation, possibly by establishing stronger cell-cell interactions that precede osteoclast formation.

Clonidine increases bone resorption in humans.

SUMMARY: Inhibition of sympathetic signaling to bone reduces bone resorption in rodents. In contrast, we show that pharmacological reduction of the sympathetic tone increases bone resorption in humans in vivo. This effect does not appear to be mediated via a direct pharmacological effect on the osteoclast. INTRODUCTION: Inhibition of sympathetic signaling to bone reduces bone resorption in rodents. It is uncertain whether a similar role for the sympathetic nervous system exists in humans. The sympathetic tone can be reduced by clonidine, which acts via alpha-2-adrenergic receptors in the brainstem. Our objective was to determine the effect of clonidine on bone turnover in humans. METHODS: The acute effect of a single oral dose of 0.3 mg clonidine on serum bone turnover markers (C-terminal cross-linking telopeptides of collagen type I (CTx), a marker for bone resorption, and procollagen type 1 N propeptide (P1NP), a marker for bone formation) was determined in a randomized crossover design in 12 healthy volunteers, aged 18-70 years. In addition, we assessed the effect of clonidine on the number of tartrate-resistant acid phosphatase-positive multinucleated cells (TRAcP(+) MNCs) and bone resorption. RESULTS: CTx concentrations increased after clonidine treatment compared to the control condition (p = 0.035). P1NP concentrations were not affected by clonidine (p = 0.520). In vitro, clonidine had no effect on the number of TRAcP(+) MNCs (p = 0.513) or on bone resorption (p = 0.996). CONCLUSIONS: We demonstrated that clonidine increases bone resorption in humans in vivo. This effect does not appear to be mediated via a direct effect on the osteoclast.

Role of periodontal ligament fibroblasts in osteoclastogenesis: a review.

During the last decade it has become clear that periodontal ligament fibroblasts may contribute to the in vitro differentiation of osteoclasts. We surveyed the current findings regarding their osteoclastogenesis potential. Periodontal ligament fibroblasts have the capacity to select and attract osteoclast precursors and subsequently to retract and enable migration of osteoclast precursors to the bone surface. There, fusion of precursors takes place, giving rise to osteoclasts. The RANKL-RANK-osteoprotegerin (OPG) axis is considered crucial in this process. Periodontal ligament fibroblasts produce primarily OPG, an osteoclastogenesis-inhibitory molecule. However, they may be influenced in vivo by direct or indirect interactions with bacteria or by mechanical loading. Incubation of periodontal ligament fibroblasts with bacteria or bacterial components causes an increased expression of RANKL and other osteoclastogenesis-stimulating molecules, such as tumor necrosis factor-α and macrophage-colony stimulating factor. Similar results are observed after the application of mechanical loading to these fibroblasts. Periodontal ligament fibroblasts may be considered to play an important role in the remodelling of alveolar bone. In vitro experiments have demonstrated that periodontal ligament fibroblasts adapt to bacterial and mechanical stimuli by synthesizing higher levels of osteoclastogenesis-stimulating molecules. Therefore, they probably contribute to the enhanced osteoclast formation observed during periodontitis and to orthodontic tooth movement.

A novel highly selective 5-HT6 receptor antagonist attenuates ethanol and nicotine seeking but does not affect inhibitory response control in Wistar rats.

Recent studies suggest a potential role for 5-hydroxytryptamine(6) (5-HT(6)) receptors in the regulation of addictive behavior. In the present study, our aim was to investigate whether the novel highly selective 5-HT(6) receptor antagonist compound (CMP) 42 affected nicotine and ethanol seeking behavior in Wistar rats. We have also studied whether CMP 42 had beneficial effects in a model of impulse control, as measured in the 5-choice serial reaction time task (5-CSRTT). Rats were trained to nose poke to receive intravenous infusions of nicotine or an ethanol drop. CMP 42 (3-30 mg/kg intraperitoneally, i.p.) was administered to investigate the effects on nicotine self-administration. Rats were also tested for cue-induced reinstatement of nicotine and ethanol seeking. In addition, the effects of CMP 42 were studied on the number of anticipatory responses in the 5-CSRTT. CMP 42 was effective in reducing nicotine self-administration and reinstatement of nicotine seeking at a dose of 30 mg/kg (i.p.). CMP 42 was also effective in reducing reinstatement of ethanol seeking (30 mg/kg i.p.). In contrast, CMP 42 did not affect anticipatory responding at doses tested, indicating no effects on impulse control. These results add to a body of evidence implicating the 5-HT(6) receptor as a viable target for the control of drug abuse. Specifically, we demonstrated for the first time effects on nicotine self-administration and on nicotine and ethanol reinstatement. Further, these effects are probably not mediated by effects on impulse control.

Osteoclast progenitors from cats with and without tooth resorption respond differently to 1,25-dihydroxyvitamin D and interleukin-6.

Both vitamin D and inflammatory cytokines can stimulate osteoclast formation and activity. We studied the effect of 1,25-dihydroxycholecalciferol (1,25(OH)(2)D), and interleukin-6 (IL-6), on the formation and activity of feline osteoclasts, using peripheral blood mononuclear cells (PBMCs) from cats with and without tooth resorption (TR(+) and TR(-)) as a source of osteoclast precursors. The formation of osteoclast-like cells (defined as multinucleated, tartrate-resistant acid phosphatase-positive cells) was assessed at 7 and 14 days. In the presence of M-CSF and RANKL, with and without IL-6, more osteoclasts were formed from TR(-) PBMCs than from TR(+) PBMCs on plastic. More osteoclasts were formed from TR(+) PBMCs on bone slices in the presence of M-CSF/RANKL with 1,25(OH)(2)D. This opposite effect may be due to a higher expression of the vitamin D receptor (VDR) in TR(+) osteoclasts and precursors on bone. Formation of resorption pits was analyzed and confirmed with scanning electron microscopy. In conclusion, we propose that TR(+) PBMCs when cultured on bone are sensitive to 1,25(OH)(2)D, whereas the differentiation of TR(-) PMBCs on bone seem more sensitive to IL-6, suggesting that osteoclast precursors from cats with and without tooth resorption respond differently to osteoclast stimulating factors.

Periodontal ligament and gingival fibroblasts from periodontitis patients are more active in interaction with Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Inflammatory responses of host cells to oral pathogenic bacteria, such as Porphyromonas gingivalis, are crucial in the development of periodontitis. Host cells, such as periodontal ligament and gingival fibroblasts, from periodontitis patients may respond to P. gingivalis in a different manner compared with cells from healthy persons. The aim of this study was to investigate inflammatory responses to viable P. gingivalis by periodontal ligament and gingival fibroblasts from periodontitis patients and healthy control subjects. MATERIAL AND METHODS: Primary periodontal ligament and gingival fibroblasts from periodontitis patients (n=14) and healthy control subjects (n=8) were challenged in vitro with viable P. gingivalis. Gene expression of Toll-like receptors (TLRs) 1, 2, 4, 6, 7 and 9, CD14, nuclear factor-κB1 and its putative inhibitor NF-κB inhibitor-like protein1, and of interleukin-1ß, interleukin-6, interleukin-8, tumour necrosis factor-α, monocyte chemotactic protein-1 and regulated upon activation, normal T-cel expressed, and secreted, were assessed by real-time PCR. RESULTS: Periodontal ligament fibroblasts from periodontitis patients had a higher mRNA expression of TLR1, TLR4, TLR7 and CD14, and a lower expression of NFKBIL1, both before and after P. gingivalis challenge. In contrast, gingival fibroblasts from periodontitis patients had stronger induction of TLR1, TLR2 and TLR7 by P. gingivalis. Cytokine responses were not different between patients and control subjects. Interestingly, periodontal ligament, but not gingival, fibroblasts from P. gingivalis culture-positive persons responded more strongly to P. gingivalis than periodontal ligament fibroblasts from P. gingivalis-negative persons. CONCLUSION: Periodontal ligament and gingival fibroblasts respond to P. gingivalis in a different manner and may play different roles in periodontitis. Both subsets of fibroblasts from patients appear more active in interaction with P. gingivalis. Moreover, periodontal ligament fibroblasts from P. gingivalis-positive donors are more responsive to an in vitro P. gingivalis challenge.

SLV330, a cannabinoid CB(1) receptor antagonist, attenuates ethanol and nicotine seeking and improves inhibitory response control in rats.

Cannabinoid CB(1) receptor (CB(1)R) signaling has been shown to play a role in the regulation of addictive behavior. In the present study, our aim was to investigate whether the CB(1)R antagonist SLV330 could reduce ethanol and nicotine self-administration and cue-induced reinstatement of ethanol and nicotine seeking behavior in Wistar rats. In operant chambers, rats were learned to emit a specific response (nose poke) in order to receive an ethanol solution or intravenous injections of nicotine. Discrete light and tone cues were presented during ethanol and nicotine delivery. These cues are particularly important for drug self-administration behavior and, through Pavlovian conditioning, acquire conditioned reinforcing and motivational properties and are therefore able to generate and maintain drug-seeking behavior. Subsequently, the CB(1)R antagonist SLV330 (doses ranging from 1 to 10mg/kg, given orally, p.o.) was administered to investigate the effects on drug self-administration. In addition, responding for ethanol and nicotine was extinguished. Then, the animals were tested for cue-induced reinstatement of ethanol and nicotine seeking and treated with vehicle or SLV330. Finally, the effects of SLV330 were studied on the number of anticipatory responses in the 5-choice serial reaction time task (5-CSRTT) in order to determine whether this compound could also increase impulse control in Wistar rats. The CB(1) antagonist SLV330 was effective in reducing ethanol self-administration at a lowest effective dose (LED) of 10mg/kg (p.o.) and reinstatement of ethanol seeking at a LED of 3mg/kg (p.o.). SLV330 was also effective in reducing nicotine self-administration and reinstatement of nicotine seeking, although at a LED of 10mg/kg (p.o.). Finally, SLV330 decreased time delay-dependent anticipatory responding (LED of 3.0mg/kg, p.o.), indicating an increased inhibitory control. These findings are in agreement with results reported with other CB(1) antagonists. The combined action of reducing the reinforcing and motivational properties of nicotine and alcohol and the improvement of impulse control supports the idea that the cannabinoid system is a promising target for anti-relapse medication.

Gingival and periodontal ligament fibroblasts differ in their inflammatory response to viable Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis is an oral pathogen strongly associated with destruction of the tooth-supporting tissues in human periodontitis. Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDLF) are functionally different cell types in the periodontium that can participate in the host immune response in periodontitis. This study aimed to investigate the effects of viable P. gingivalis on the expression of genes associated with inflammation and bone degradation by these fibroblast subsets. MATERIAL AND METHODS: Primary human GF and PDLF from six healthy donors were challenged in vitro with viable P. gingivalis W83 for 6 h. Gene expression of inflammatory cytokines in GF and PDLF was analyzed using real-time PCR, and protein expression was analyzed using ELISA. RESULTS: Viable P. gingivalis induced a strong in vitro inflammatory response in both GF and PDLF. We found increased gene expression of interleukin (IL)-1beta, IL-6, IL-8, tumor necrosis factor-alpha, monocyte chemotactic protein-1 and regulated upon activation, normal T-cell expressed and secreted (RANTES). Macrophage colony-stimulating factor was induced and the expression of osteoprotegerin was decreased in GF, but not in PDLF. In nonchallenged cells, a higher level of expression of IL-6 was observed in GF than in PDLF. Between individual donors there was large heterogeneity in responsiveness to P. gingivalis. Also, in each individual, either GF or PDLF was more responsive to P. gingivalis. CONCLUSION: Considerable heterogeneity in responsiveness to P. gingivalis exists both between GF and PDLF and between individuals, which may be crucial determinants for the susceptibility to develop periodontitis.

Contextual renewal of nicotine seeking in rats and its suppression by the cannabinoid-1 receptor antagonist Rimonabant (SR141716A).

Nicotine-associated paraphernalia such as cigarettes and ashtrays are potent smoking relapse triggers. In addition to these discrete cues, environmental contexts previously associated with smoking elicit strong cigarette craving, indicating that contextual stimuli also contribute to high smoking relapse rates. Nonetheless, little is known about the precise role of these stimuli in smoking relapse and the neuropharmacological mechanisms implicated herein. To address this issue, we determined whether re-exposure to the nicotine self-administration context after long-term extinction reinstates nicotine seeking behavior in rats. Further, we examined the effects of SR141716A (Rimonabant), a selective cannabinoid CB1 receptor antagonist which has been shown to attenuate cue-induced relapse to nicotine seeking, on context-induced reinstatement of nicotine seeking. Rats were trained to self-administer nicotine intravenously (30microg/kg/infusion). Nicotine infusions were paired with an audiovisual compound stimulus. Subsequently, nose poking behavior was extinguished in the presence of this discrete cue in a context different from the self-administration context. Hereafter, rats were injected with 0, 1, or 3mg/kg Rimonabant (i.p.) prior to re-exposure to either the self-administration or the extinction context. Re-exposure to the self-administration context, but not to the extinction context robustly reinstated responding for the discrete nicotine cues, an effect that was dose-dependently attenuated by Rimonabant. This is the first demonstration of contextual renewal of nicotine seeking in rodents after prolonged withdrawal. Further, our results indicate that the endocannabinoid system is involved in context-induced relapse to nicotine seeking, and as such these data provide further evidence for the use of CB1 antagonists in smoking cessation.

Expression of amphetamine sensitization is associated with recruitment of a reactive neuronal population in the nucleus accumbens core.

RATIONALE: Repeated exposure to psychostimulant drugs causes a long-lasting increase in the psychomotor and reinforcing effects of these drugs and an array of neuroadaptations. One such alteration is a hypersensitivity of striatal activity such that a low dose of amphetamine in sensitized animals produces dorsal striatal activation patterns similar to acute treatment with a high dose of amphetamine. OBJECTIVES: To extend previous findings of striatal hypersensitivity with behavioral observations and with cellular activity in the nucleus accumbens and prefrontal cortex in sensitized animals. MATERIALS AND METHODS: Rats treated acutely with 0, 1, 2.5, or 5 mg/kg i.p. amphetamine and sensitized rats challenged with 1 mg/kg i.p. amphetamine were scored for stereotypy, rearing, and grooming, and locomotor activity recorded. c-fos positive nuclei were quantified in the nucleus accumbens and prefrontal cortex after expression of sensitization with 1 mg/kg i.p. amphetamine. RESULTS: Intense stereotypy was seen in animals treated acutely with 5 mg/kg amphetamine, but not in the sensitized group treated with 1 mg/kg amphetamine. The c-fos response to amphetamine in the accumbens core was augmented in amphetamine-pretreated animals with a shift in the distribution of optical density, while no effect of sensitization was seen in the nucleus accumbens shell or prefrontal cortex. CONCLUSIONS: A lack of stereotypy in the sensitized group indicates a dissociation of behavioral responses to amphetamine and striatal immediate-early gene activation patterns. The increase in c-fos positive nuclei and shift in the distribution of optical density observed in the nucleus accumbens core suggests recruitment of a new population of neurons during expression of sensitization.

Expression of gp100, MART-1, tyrosinase, and S100 in paraffin-embedded primary melanomas and locoregional, lymph node, and visceral metastases: implications for diagnosis and immunotherapy. A study conducted by the EORTC Melanoma Cooperative Group.

With the recent availability of novel antibodies against melanoma antigens tyrosinase and MART-1, it is important to validate their usefulness in pathology practice and in screening patients for immunotherapy treatment. In the present study conducted by the Melanoma Cooperative Group of the European Organization for Research and Treatment of Cancer (EORTC-MCG), immunohistochemical staining for gp100 (antibodies NKI-beteb and HMB-45), MART-1 (A103), tyrosinase (T311), and S100 (S100) was compared on formalin-fixed and paraffin-embedded tumour lesions from 80 patients with 130 malignant melanoma lesions, comprising 44 primary tumours, 18 locoregional metastases, 41 lymph node metastases, and 27 visceral metastases from the lung, liver, and brain. A score between 0 and 5 was allocated to each immunohistochemically stained section. These scores were evaluated in a statistical analysis. S100 was by far the most sensitive marker in all four types of lesions tested. Apart from a significantly better performance for T311 in primary melanomas compared with HMB-45, no significant differences were observed between the four remaining antigens tested. Three settings were next investigated to determine whether the expression of melanoma antigens decreases with tumour progression. First, within the primary melanomas, only NKI-beteb and A103 staining showed a nearly significant negative correlation with Clark's level of invasion and a similar tendency was observed for these antibodies with Breslow thickness. Second, when comparing primary melanoma-metastasis pairs from the same patient, lymph node metastases showed less staining with NKI-beteb, HMB-45, A103, and T311, at a level near significance. This difference was not significant when comparing the primary tumour with visceral metastases, probably due to the lower numbers of pairs. Third, regarding tumour progression from primary melanoma to locoregional, to lymph node, to visceral metastasis, a significant decrease with progression was found only for T311. The apparently stable expression of most of the melanoma antigens, and the small contribution of decreased expression in melanoma tumour progression, supports the rationale for immunotherapy based on the melanoma immunogens gp100, MART-1, and tyrosinase.

Analysis of melanoma cells in peripheral blood by reverse transcription-polymerase chain reaction for tyrosinase and MART-1 after mononuclear cell collection with cell preparation tubes: a comparison with the whole blood guanidinium isothiocyanate RNA isolation method.

Melanoma cell detection in peripheral blood by tyrosinase reverse transcription-polymerase chain reaction (RT-PCR) is usually performed on RNA isolated from whole blood using a guanidinium isothiocyanate (GITC)/phenol extraction method or from Ficoll Hypaque isolated mononuclear cells. The first method contains environmentally harmful reagents, and the second is laborious in the preanalytical steps. Cell preparation tubes (CPTs) are ready-to-use Ficoll Hypaque-based tubes that avoid the time-consuming and critical loading on Ficoll Hypaque. We examined whether CPTs can be used to determine melanoma cell dissemination in peripheral blood. We first investigated whether melanoma cells were retained in the mononuclear cell layer. All six morphologically different melanoma cell lines studied in the spiking experiments were retained in the upper layer. In further experiments, we were able to detect low dilutions of added SK-MEL-28 cells more consistently after nested RT-PCR for tyrosinase or MART-1 in the RNA isolated from mononuclear cells from CPTs than from RNA isolated with the GITC method. In addition, RNA was extracted from paired blood samples from 24 analysable stage III and stage IV melanoma patients and analysed for the presence of tyrosinase and MART-1 RNA using both the CPT/RNeasy and the whole blood/GITC method. The quality of the CPT/RNeasy RNA was better than the RNA isolated from whole blood with GITC/phenol. However, the RT-PCR results were less unequivocal: MART-1 mRNA was more often detected with CPTIRNeasy compared with whole blood/GITC (six versus three), whereas tyrosinase mRNA was found less often in CPT/RNeasy RNA (two versus eight). Taken together these results suggest that the CPT isolation method is suitable for the isolation of mononuclear cells, including melanoma cells.

Synergistically interacting dopamine D1 and NMDA receptors mediate nonvesicular transporter-dependent GABA release from rat striatal medium spiny neurons.

Given the complex interactions between dopamine D1 and glutamate NMDA receptors in the striatum, we investigated the role of these receptors in transporter-mediated GABA release from cultured medium spiny neurons of rat striatum. Like NMDA receptor-mediated [(3)H]-GABA release, that induced by prolonged (20 min) dopamine D1 receptor activation was enhanced on omission of external calcium, was action potential-independent (tetrodotoxin-insensitive), and was diminished by the GABA transporter blocker nipecotic acid, indicating the involvement of transporter-mediated release. Interestingly, lowering the external sodium concentration only reduced the stimulatory effect of NMDA. Blockade of Na(+)/K(+)-ATPase by ouabain enhanced NMDA-induced but abolished dopamine-induced release. Moreover, dopamine appeared to potentiate the effect of NMDA on [(3)H]-GABA release. These effects of dopamine were mimicked by forskolin. mu-Opioid receptor-mediated inhibition of adenylyl cyclase by morphine reduced dopamine- and NMDA-induced release. These results confirm previous studies indicating that NMDA receptor activation causes a slow action potential-independent efflux of GABA by reversal of the sodium-dependent GABA transporter on sodium entry through the NMDA receptor channel. Moreover, our data indicate that activation of G-protein-coupled dopamine D1 receptors also induces a transporter-mediated increase in spontaneous GABA release, but through a different mechanism of action, i.e., through cAMP-dependent inhibition of Na(+)/K(+)-ATPase, inducing accumulation of intracellular sodium, reversal of the GABA carrier, and potentiation of NMDA-induced release. These receptor interactions may play a crucial role in the behavioral activating effects of psychostimulant drugs.

Heterogeneous expression of the SSX cancer/testis antigens in human melanoma lesions and cell lines.

The SSX genes, located on the X chromosome, encode a family of highly homologous nuclear proteins. The SSX1 and SSX2 genes were initially identified as fusion partners of the SYT gene in t(X;18)-positive synovial sarcomas. Recently, however, it was found that these two genes, as well as the highly homologous SSX4 and SSX5 genes, are aberrantly expressed in different types of cancers, including melanomas. Because normal SSX expression has been detected only in the testis and, at very low levels, the thyroid, these proteins are considered as new members of the still growing family of cancer/testis antigens. These antigens are presently considered as targets for the development of cancer immunotherapy protocols. In the present study, we developed a monoclonal antibody found to recognize SSX2, SSX3, and SSX4 proteins expressed in formaldehyde-fixed and paraffin-embedded tissues. This antibody was used to investigate SSX expression in normal testis and thyroid, benign melanocytic lesions, melanoma lesions, and melanoma cell lines. SSX nuclear expression in the testis was found to be restricted to spermatogenic cells, mainly spermatogonia. Of 18 melanoma cell lines analyzed, 9 showed SSX RNA and protein expression, although heterogeneously and at variable levels. Treatment of an SSX-negative cell line with 5-aza-2'-deoxycytidine, a demethylating agent, led to SSX RNA and protein expression, indicating a role for methylation in transcription regulation. Thirty-four of 101 primary and metastatic melanoma cases and 2 of 24 common nevocellular and atypical nevus cases showed SSX nuclear staining. Again, SSX expression was heterogeneous, ranging from widespread to scarce. Our findings stress the importance of assessing the a priori SSX expression status of melanoma cases that may be selected for immunotherapeutic trials.

Melanoma-inhibiting activity (MIA) mRNA is not exclusively transcribed in melanoma cells: low levels of MIA mRNA are present in various cell types and in peripheral blood.

The detection of minimal amounts of melanoma cells by tyrosinase reverse transcription polymerase chain reaction (RT-PCR) is seriously hampered by false negative reports in blood of melanoma patients with disseminated melanoma. Therefore, additional assays which make use of multiple melanoma markers are needed. It has been shown that introduction of multiple markers increases the sensitivity of detection. Melanoma inhibitory activity (MIA) is one such melanoma-specific candidate gene. To test the specificity of MIA PCR, we performed 30 and 60 cycles of PCR with two different sets of MIA specific primers on 19 melanoma and 16 non-melanoma cell lines. MIA mRNA was detected in 16 out of 19 melanoma cell lines and in seven out of 16 non-melanoma cell lines after 30 cycles of PCR. However, MIA mRNA could be detected in all cell lines after 60 cycles of PCR. Also, in 14 out of 14 blood samples of melanoma patients, five out of six blood samples of non-melanoma patients and in seven out of seven blood samples of healthy volunteers, MIA mRNA was detected after 60 cycles of PCR, whereas no MIA PCR product could be detected in any of the blood samples after 30 cycles of PCR. We conclude that low levels of MIA transcripts are present in various normal and neoplastic cell types. Therefore, MIA is not a suitable marker gene to facilitate the detection of minimal amounts of melanoma cells in blood or in target organs of the metastatic process.

Transcription of the MAGE-1 gene and the methylation status of its Ets binding promoter elements: a quantitative analysis in melanoma cell lines using a real-time polymerase chain reaction technique.

The human MAGE gene family comprises at least 12 highly homologous genes. This makes it very difficult to assess expression of a single member quantitatively by means of Northern blotting. In order to investigate expression of the MAGE-1 gene quantitatively we therefore used the recently developed real-time polymerase chain reaction (PCR), a novel fluorescence-based quantitative PCR technique. This powerful technique enables detection of expression levels which differ by as much as a factor of 10(5) in magnitude. MAGE-1 expression is known to correlate with demethylated status of the Ets binding sites of its promoter. In a panel of 19 melanoma and nine non-melanoma cell lines we were able to confirm the relationship between MAGE-1 expression and demethylation of the Ets binding promoter region. Five cell lines, however, showed only very slight expression, while the two essential Ets promoter elements were largely demethylated. Earlier studies have shown that treatment of MAGE-1-negative cell lines with the demethylating agent 5-aza-2'-deoxycytidine (DAC) is sufficient to induce MAGE-1 expression. We were able to induce clear MAGE-1 expression in two of the non-expressing cell lines by incubation with DAC, although this expression did not reach very high levels. Consistent with this low level of induction is the observation that the Ets binding sites of the MAGE-1 promoter were not completely demethylated in the DAC-treated cell populations. In conclusion, we show in this study that the real-time PCR technique is a very useful tool for the quantification of expression of highly homologous genes.

Reproducibility of detection of tyrosinase and MART-1 transcripts in the peripheral blood of melanoma patients: a quality control study using real-time quantitative RT-PCR.

In recent years, large discrepancies were described in the success rate of the tyrosinase reverse transcription polymerase chain reaction (RT-PCR) for detecting melanoma cells in the peripheral blood of melanoma patients. We present a quality control study in which we analysed the reproducibility of detection of tyrosinase and MART-1 transcripts in 106 blood samples from 68 melanoma patients (mainly stages III and IV). With this study, we aimed to improve insight in the reproducibility of a RT-PCR for the detection of (minimal) amounts of circulating melanoma cells. We performed two reverse transcriptions on each mRNA sample and performed tyrosinase and MART-1 nested PCRs in duplicate per cDNA sample. Thus, four tyrosinase and four MART-1 measurements were performed per blood sample. In our study, the majority of blood samples was negative for tyrosinase (80%) or MART-1 (66%). Only four samples were positive in all four determinations for tyrosinase and seven for MART-1. Variable results (1-3 times positive results) were obtained for tyrosinase and MART-1 in 16% and 27% respectively. MART-1 PCR had a better performance than tyrosinase PCR. Sensitivity increased when both markers were used. We reasoned that the low number of melanoma marker PCR-positive blood samples can be explained by differences in mRNA quality. By using real-time quantitative PCR, we found that this was not the case: amplification of porphobilinogen deaminase (PBGD), a low copy household gene, was not different in blood samples in which a melanoma marker was not detected from groups in which this marker was detected more or less consistently (1-4 times). When applying real-time quantitative PCR for tyrosinase and MART-1, we found that a low amount of SK-MEL-28 cell equivalents was present in the blood of melanoma patients, with a higher number of equivalents in the group with a consistently positive result. We conclude that low reproducibility of a repeated assay for the detection of circulating melanoma cells is not caused by differences in mRNA quality between the samples, but due to low numbers of amplifiable target mRNA molecules in the mRNA sample. Use of more than one marker and repetition of the assay will increase the probability of finding positive PCR results.

Opposing role of dopamine D1 and D2 receptors in modulation of rat nucleus accumbens noradrenaline release.

The role of dopamine receptors in the modulation of nucleus accumbens noradrenaline release was investigated in superfused rat brain slices. At concentrations of