A randomized, intraindividual, non-inferiority, Phase III study comparing daylight photodynamic therapy with BF-200 ALA gel and MAL cream for the treatment of actinic keratosis.

BACKGROUND: The most effective treatment modality for actinic keratosis (AK) is photodynamic therapy (PDT). Major obstacles of PDT are the need of a special illumination device and pain accompanying the illumination. These issues may be overcome by replacing an artificial high-power light source with natural daylight for more extended illumination at lower light doses. OBJECTIVE: To determine whether BF-200 ALA (a nanoemulsion gel containing 7.8% 5-aminolaevulinic acid) is non-inferior to MAL (a cream containing 16% methyl-aminolaevulinate) in the treatment of mild-to-moderate AK with daylight PDT (dPDT). Non-inferiority of the primary efficacy variable (total lesion clearance rate per patient's side 12 weeks after PDT) is established if the mean response for BF-200 ALA is no worse than for MAL, within a statistical margin of Δ = -12.5%. METHODS: The study was performed as an intraindividual comparison with 52 patients in seven centres in Germany and Spain. Each patient received one dPDT. Results include clinical endpoints as well as 1-year follow-up results. RESULTS: Twelve weeks after a single dPDT, 79.8% of the AK lesions treated with BF-200 ALA gel and 76.5% of the lesions treated with MAL cream were completely cleared. The median of differences was 0.0 with a one-sided 97.5% CI of 0.0, establishing non-inferiority (P < 0.0001). Results for secondary efficacy parameters were in line with the primary outcome. Recurrence rates 1 year after the treatment were 19.9% for lesions treated with BF-200 ALA and 31.6% for lesions treated with MAL. Adverse reactions including pain were mostly mild and transient and identical to those previously described for dPDT. CONCLUSION: Daylight PDT of AK with BF-200 ALA is well-tolerated and non-inferior to MAL/dPDT. The study demonstrates a trend towards higher efficacies after 3 months and significantly lower recurrence rates after 1 year follow-up.

A randomized, multinational, noninferiority, phase III trial to evaluate the safety and efficacy of BF-200 aminolaevulinic acid gel vs. methyl aminolaevulinate cream in the treatment of nonaggressive basal cell carcinoma with photodynamic therapy.

BACKGROUND: Basal cell carcinoma (BCC) represents the most common nonmelanoma skin cancer worldwide, affecting mainly adult, fair-skinned individuals. The World Health Organization distinguishes aggressive and nonaggressive forms, of which prototypical variants of the latter are primary nodular and superficial BCC. OBJECTIVES: To demonstrate noninferiority of BF-200 ALA (a nanoemulsion gel containing 5-aminolaevulinic acid) compared with MAL (a cream containing methyl aminolaevulinate) in the treatment of nonaggressive BCC with photodynamic therapy (PDT). Noninferiority of the primary efficacy variable (overall patient complete response 12 weeks after last PDT) would be declared if the mean response for BF-200 ALA was no worse than that for MAL, within a statistical margin of Δ = -15%. METHODS: The study was a randomized, phase III trial performed in Germany and the U.K. with ongoing 5-year follow-up. Of 281 randomized patients, 138 were treated with BF-200 ALA and 143 with MAL. Patients received two PDT sessions 1 week apart. Remaining lesions 12 weeks after the second PDT were retreated. Illumination was performed with a red light source (635 nm, 37 J cm-2 ). The results shown include clinical end points and patients' reassessment 12 months after the last PDT. The study was registered with EudraCT (number 2013-003241-42). RESULTS: Of the BF-200 ALA-treated patients, 93·4% were complete responders compared with 91·8% in the MAL group. The difference of means was 1·6, with a one-sided 97·5% confidence interval of -6·5, establishing noninferiority (P < 0·0001). The results for secondary efficacy parameters were in line with the primary outcome. Recurrence rates 12 months after the last treatment were ≤ 10%. CONCLUSIONS: Treatment of nonaggressive BCC with BF-200 ALA-PDT is highly effective and well tolerated with proven noninferiority to MAL-PDT. It demonstrates low recurrence rates after 1 year of follow-up.

Patient-reported health outcomes in patients with non-melanoma skin cancer and actinic keratosis: results from a large-scale observational study analysing effects of diagnoses and disease progression.

BACKGROUND: Non-melanoma skin cancer (NMSC) and actinic keratosis (AK) are very common among fair-skinned individuals. A disease continuum from AK to squamous cell carcinoma (SCC) has been frequently postulated. AK and NMSC may influence quality of life (QL) of patients, and it can be suspected that disease progression entails a QL reduction. The purpose of this study was to document QL in patients with NMSC and AK using the health-outcome questionnaire EQ-5D-5L. METHODS: The study was designed as a non-interventional, prospective, cross-sectional study. Patients with AK, SCC, basal cell carcinoma (BCC) or multiple diagnoses were enrolled in this study in 29 dermatological centres across Germany. Patients were asked to complete the EQ-5D-5L (compromising EQ Index and EQ VAS), and the dermatologists provided diagnosis, disease history and treatment data. RESULTS: A total of 1184 patients were enrolled and diagnosed as follows: 73% AK, 49% BCC and 17% SCC. 66% had a single diagnosis, 28% two different diagnoses and 6% three different diagnoses. QL was strongly associated with patients' diagnosis. Patients with a single AK diagnosis had significantly higher mean EQ VAS (78) than patients with BCC (74), SCC (72), and BCC plus SCC (69), P < 0.050. When the effects of disease progression were calculated, patients with AK plus SCC reported significantly less mean EQ VAS (71) than patients with a single AK diagnosis (78), P < 0.011. CONCLUSIONS: While rarely being imminently life-threatening, NMSC and AK have an impact on QL as quantified by the EQ-5D-5L. This impact is associated with diagnosis (AK vs. NMSC) and clinical progression (AK vs. AK plus SCC). Both lead to a clear decline in QL. This shows that disease progression is perceived and judged as detrimental by patients and that AK and NMSC should be diligently treated to preserve and restore QL.

A randomized, double-blind, phase III, multicentre study to evaluate the safety and efficacy of BF-200 ALA (Ameluz(®) ) vs. placebo in the field-directed treatment of mild-to-moderate actinic keratosis with photodynamic therapy (PDT) when using the BF-RhodoLED(®) lamp.

BACKGROUND: Multiple actinic keratosis (AK) lesions may arise from the cancerization of large, sun-damaged skin areas. Although photodynamic therapy (PDT) is considered the most effective therapeutic option, the efficacy and safety of field treatment of multiple AK lesions with PDT has never before been tested in a pivotal trial. OBJECTIVES: To evaluate the efficacy, safety and cosmetic outcome of BF-200 ALA (a nanoemulsion formulation containing 10% aminolaevulinic acid hydrochloride) combined with the BF-RhodoLED(®) lamp for the field-directed treatment of mild-to-moderate AK with PDT. METHODS: The study was performed as a randomized, multicentre, double-blind, placebo-controlled, parallel-group, phase III trial with BF-200 ALA and placebo in seven centres in Germany. A total of 94 patients were enrolled in this study; 87 were randomized (55 patients received BF-200 ALA, 32 received placebo). Patients received one PDT. If residual lesions remained at 3 months after treatment, PDT was repeated. Illumination was performed with the PDT lamp BF-RhodoLED (635 nm ± 9 nm) until a total light dose of 37 J cm(-2) was achieved. RESULTS: BF-200 ALA was superior to placebo with respect to patient complete clearance rate (91% vs. 22%, P < 0·0001) and lesion complete clearance rate (94·3% vs. 32·9%, P < 0·0001) after a maximum of two PDTs. The confirmatory analysis of all key secondary variables supported this superiority" should not be skipped since this is an important result. Treatment-emergent adverse events (TEAEs) were experienced by 100% of the BF-200 ALA group and 69% of the placebo group. The most commonly reported TEAEs were TEAEs of the application site. The cosmetic outcome was improved in the BF-200 ALA group compared with placebo. CONCLUSIONS: Field-directed therapy with BF-200 ALA and BF-RhodoLED lamp is highly effective and well tolerated for multiple mild-to-moderate AK lesions, providing greatly improved skin quality.

Long-term (6 and 12 months) follow-up of two prospective, randomized, controlled phase III trials of photodynamic therapy with BF-200 ALA and methyl aminolaevulinate for the treatment of actinic keratosis.

BACKGROUND: Two phase III trials of photodynamic therapy (PDT) with BF-200 ALA, a recently approved nanoemulsion formulation of 5-aminolaevulinic acid (ALA) demonstrated high clearance rates in mild-to-moderate actinic keratosis (AK). The comparison to a registered methyl aminolaevulinate (MAL) cream demonstrated significantly superior total patient clearance rates. OBJECTIVES: To evaluate long-term efficacy and safety of PDT for AK 6 and 12 months after the last PDT with BF-200 ALA, MAL or placebo. METHODS: The follow-up phase (FUP) was performed with patients of two phase III studies. Both studies compared BF-200 ALA with placebo, one of the studies additionally with MAL. Overall recurrence rates and various subgroups (light source, lesion severity, lesion location, complete responders after first PDT) were assessed 6 and 12 months after the last PDT. RESULTS: Recurrence rates were similar for BF-200 ALA and MAL, with a tendency to lower recurrence rates for BF-200 ALA. The proportion of patients who were fully cleared during PDT and remained completely clear for at least 12 months after PDT were 47% for BF-200 ALA (both studies) and 36% for MAL treatment. The subgroup that was illuminated with narrow wavelength LED lamps reached 69% and 53% for BF-200 ALA (both studies, respectively) and 41% for MAL. No safety concerns were reported. CONCLUSIONS: The FUP data confirmed the high efficacy and safety of PDT with BF-200 ALA. The slightly lower recurrence rates after BF-200 ALA treatment compared with MAL treatment enhanced the better treatment outcome due to the significantly superior efficacy.

Photodynamic therapy with BF-200 ALA for the treatment of actinic keratosis: results of a multicentre, randomized, observer-blind phase III study in comparison with a registered methyl-5-aminolaevulinate cream and placebo.

BACKGROUND: Photodynamic therapy (PDT) with 5-aminolaevulinic acid (ALA) or its methylester [methyl-5-aminolaevulinate (MAL) or 5-amino-4-oxopentanoate] was recently ranked as first-line therapy for the treatment of actinic keratosis (AK) and is an accepted therapeutic option for the treatment of neoplastic skin diseases. BF-200 ALA (Biofrontera Bioscience GmbH, Leverkusen, Germany) is a gel formulation of ALA with nanoemulsion for the treatment of AK which overcomes previous problems of ALA instability and improves skin penetration. OBJECTIVES: To evaluate the efficacy and safety of PDT of AKs with BF-200 ALA in comparison with a registered MAL cream and with placebo. METHODS: The study was performed as a randomized, multicentre, observer-blind, placebo-controlled, interindividual trial with BF-200 ALA, a registered MAL cream and placebo in a ratio of 3:3:1. Six hundred patients, each with four to eight mild to moderate AK lesions on the face and/or the bald scalp, were enrolled in 26 study centres in Germany, Austria and Switzerland. Patients received one PDT. If residual lesions remained at 3months after treatment, PDT was repeated. RESULTS: PDT with BF-200 ALA was superior to placebo PDT with respect to patient complete clearance rate (78·2% vs. 17·1%; P<0·0001) and lesion complete clearance rate (90·4% vs. 37·1%) at 3months after the last PDT. Moreover, superiority was demonstrated over the MAL cream regarding the primary endpoint patient complete clearance (78·2% vs. 64·2%; P<0·05). Significant differences in the patient and lesion complete clearance rates and severity of treatment-related adverse events were observed for the narrow- and broad-spectrum light sources. CONCLUSIONS: BF-200 ALA is a very effective, well-tolerated new formulation for AK treatment with PDT and is superior to a registered MAL medication. Efficacies and adverse events vary greatly with the different light sources used.

Photodynamic therapy with BF-200 ALA for the treatment of actinic keratosis: results of a prospective, randomized, double-blind, placebo-controlled phase III study.

BACKGROUND: Photodynamic therapy (PDT) with 5-aminolaevulinic acid (ALA) provides a therapeutic option for the treatment of actinic keratosis (AK). Different strategies are applied to overcome the chemical instability of ALA in solution and to improve skin penetration. A new stable nanoemulsion-based ALA formulation, BF-200 ALA, is currently in clinical development for PDT of AK. OBJECTIVES: To evaluate the efficacy and safety of PDT of AK with BF-200 ALA. METHODS: The study was performed as a randomized, multicentre, double-blind, placebo-controlled, interindividual, two-armed trial with BF-200 ALA and placebo. A total of 122 patients with four to eight mild to moderate AK lesions on the face and/or the bald scalp were included in eight German study centres. The efficacy of BF-200 ALA after one and two PDT treatments was evaluated. BF-200 ALA was used in combination with two different light sources under illumination conditions defined by European competent authorities. RESULTS: PDT with BF-200 ALA was superior to placebo PDT with respect to patient complete clearance rate (per-protocol group: 64% vs. 11%; P < 0.0001) and lesion complete clearance rate (per-protocol group: 81% vs. 22%) after the last PDT treatment. Statistically significant differences in the patient and lesion complete clearance rates and adverse effect profiles were observed for the two light sources, Aktilite CL128 and PhotoDyn 750, at both time points of assessment. The patient and lesion complete clearance rates after illumination with the Aktilite CL128 were 96% and 99%, respectively. CONCLUSIONS: BF-200 ALA is a very effective new formulation for the treatment of AK with PDT. Marked differences between the efficacies and adverse effects were observed for the different light sources used. Thus, PDT efficacy is dependent both on the drug and on the characteristics of the light source and the illumination conditions used.

Expression and modulation of voltage-gated calcium channels after RNA injection in Xenopus oocytes.

Calcium ions flow into cells through several distinct classes of voltage-dependent calcium-selective channels. Such fluxes play important roles in electrical signaling at the cell membrane and in chemical signaling within cells. Further information about calcium channels was obtained by injecting RNA isolated from rat brain, heart and skeletal muscle into Xenopus oocytes. Macroscopic currents through voltage-operated calcium channels were resolved when the endogenous calcium-dependent chloride current was blocked by replacing external calcium with barium and chloride with methanesulfonate. The resulting barium current was insensitive to tetrodotoxin but was completely blocked by cadmium or cobalt. With both heart and brain RNA at least two distinct types of calcium ion conductance were found, distinguishable by their time course and inactivation properties. In oocytes injected with heart RNA, the slowly inactivating component was selectively blocked by the calcium-channel antagonist nifedipine. Barium ion currents induced by heart RNA were modulated by isoproterenol, cyclic adenosine monophosphate, and acetylcholine.

The influence of different illumination parameters on protoporphyrin IX induced cell death in squamous cell carcinoma cells.

BACKGROUND: Topical photodynamic therapy (PDT) is a highly effective therapy especially for extended cancerized fields of the skin. Whenever extended fields are treated pain management is advisable. Light source mediated pain management can be performed by reducing fluence rates, as long as this does not compromise efficacy. METHODS: Two squamous cell carcinoma cell lines (A431 and SCC-13) were subjected to in vitro PDT using two different ALA concentrations and synthesis intervals and protoporphyrin IX (PpIX) synthesis was assessed. Two total light doses (6 J/cm2 and 37 J/cm2) were applied at three different fluence rates and cell viability was measured using the MTS-test. RESULTS: Both cell lines synthetized PpIX at different kinetics. A431 cells produced a maximum 28.6 nmol/l PpIX, while SCC-13 reached only a production of 8.7 nmol/l. Illumination reduced cell viability depending on PpIX content and light dose. When a lower light dose (6 J/cm2) was applied, only the combination with the highest PpIX content was effective in A431 cells and no effect could be detected in SCC-13 cells. With a light dose of 37 J/cm2, lower PpIX amounts became effective in A431 and cell death could be induced in SCC-13 cells. Light fluence rate had no differential effect in this setup. CONCLUSIONS: In both, A431 and SCC-13 cells, total light dose is a key factor for photodynamic efficacy. Additionally, our results hint towards a threshold concentration of PpIX upon which a drastic loss of viability occurs. Light fluence rate in the analyzed range is not a limiting factor of photodynamic cytotoxicity. This may allow for the clinical implementation of low fluence rate protocols for pain management without compromising efficacy.

Characterization and comparison of receptors for 17 beta-estradiol and progesterone in human proliferative endometrium and endometrial carcinoma.

Sedimentation coefficients of cytoplasmic estradiol and progesterone receptors of human proliferative endometrium and endometrial carcinoma were determined by sucrose gradient centrifugation. In the absence of KCl, receptors from proliferative endometrium sedimented as single bands in the 8 S region and in the presence of 0.3M KCl in the 4 S region of the gradients. Receptors from endometrial carcinoma sedimented in several bands (between 3 and 9 S). When chromatographed on agarose gel comumns, the receptors (from both normal and neoplastic tissue) showed different molecular weights in the presence and absence of KCl (approximately 40,000 and 120,000, respectively). Elution profiles from agarose gel and ion exchange columns, as well as electrophoretic patterns from isoelectric focusing, revealed a similarity between biochemical properties of the receptors from endometrial carcinoma and proliferative endometrium. While the concentration of binding sites for estradiol and progesterone in normal endometrium depended on the day of the cycle, in endometrial carcinoma it depended on the degree of differentiation of the tumor. The binding of estradiol was highest at the beginning of the proliferative phase and declined continuously towards the 14th day of the cycle. In contrast, the concentration of progesterone binding sites was relatively low throughout the proliferative phase. In endometrial carcinoma low binding of estradiol was obtained in well differentiated tumors and high binding (as high as in proliferative endometrium) in undifferentiated tumors. For progesterone the contrary was the case. There was no difference in pH sensitivity between cytoplasmic receptors from normal and neoplastic tissue, optimal binding occurring at pH 7. Dissociation constants (Kd) for estradiol and progesterone depended on the degree of tumor differentiation. Kd values increased for E2 and decreased for P with increasing differentiation of the tumor. Competition studies with various unlabeled steroids revealed no significant difference between the specificity of the receptors from proliverative and neoplastic endometrium.

Progesterone metabolism in normal human endometrium during the menstrual cycle and in endometrial carcinoma.

Different subcellular fractions (purity checked by electron microscopy and respective marker enzymes) were incubated with 0.1 muCi 14C-progesterone (10 muM) in 0.15 M phosphate buffer at pH 7.4 and 37 C under air for varying periods of time in the presence of NAD(P)H (500 muM). By the preparation of chromic acid oxidation products and acetates, thin-layer chromatography, and crystallisation to constant specific activity, the following metabolites were identified: 20alpha-hydroxypregn-4-en-3-one, 20alpha-hydroxy-5alpha-pregnan-3-one, 20alpha-hydroxy-5beta-pregnan-3-one, 5alpha-pregnane-3,20-dione, and 5beta-pregnane-3,20-dione, indicating the presence of a 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) and 5alpha- and 5beta-reductases. Most of the 20alpha-HSD activity was located in mitochondria (associated mainly with outer membranes) and microsomes. Purified nuclei and cytosol contained 1/6 to 1/18 of the activity of mitochondria and microsomes, respectively. SUBFRACTIONS OF ENDOMETRIAL CELLS ONLY CONTAINED EITHER 5ALPHA- OR 5BETA-REDUCTASE ACTIVITY. 5alpha-reductase activity was mainly associated with microsomes, 5beta-reductase activity was found only in the cytosol. While in normal endometrium specific enzyme activities in subcellular fractions depended on the phase of the cycle, in endometrial carcinoma it depended on the degree of tumour differentiation. The highest values of 5alpha-reductase activity were found in the early proliferative phase. 20alpha-HSD activity was highest in the middle of the secretory phase. The specific activity of the 5alpha-reductase increased with decreasing differentiation of the tumour while the specific activity of the 20alpha-HSD decreased. Kinetic parameters (Km-values, coenzyme requirements and maximum velocities) were determined. The Km-value for progesterone of the 20alpha-HSD in proliferative endometrium was significantly higher than in secretory endometrium, while the Km-values of the 5alpha- and 5beta-reductases were considerably lower during the proliferative than secretory phase.

Expression and regulation of human and rat phosphodiesterase type IV isogenes.

Type IV phosphodiesterases (PDE IV) specifically hydrolyze cAMP and are inhibited by rolipram. RT-PCR was applied to analyze the expression patterns of mRNAs for four cloned human and rat phosphodiesterase type IV isogenes (PDE IV-A, -B, -C and -D). Although these patterns were mostly coincident for the human and rat PDE IV genes, some differences were found between the two species. PDE IV-A expression was detectable in human blood but not in rat blood, suggesting a species-specific difference in the expression of this PDE IV isogene. PDE IV-C was neither detected in human or rat blood nor in different cell populations of the human immune system. It is further demonstrated that the PDE IV isogene expression is differentially regulated by cAMP in different cell types.

Molecular cloning and functional expression in yeast of a human cAMP-specific phosphodiesterase subtype (PDE IV-C).

We have recently reported increased survival of dopaminergic substantia nigra neurons by inhibition of phosphodiesterase type IV enzymes. As a first step to unravel the involvement of PDE IV subtypes in this process, we isolated phosphodiesterase type IV cDNAs from human substantia nigra. One isolated partial cDNA clone was most homologous to the partially cloned rat and human PDE IV-C isogene. Distribution analysis revealed that the enzyme is expressed in various tissues but not in cells of the immune system. Isolation of the full-length human PDE IV-C isogene cDNA and expression in a PDE-deficient yeast strain resulted in functional complementation of the yeast heat shock response. Inhibition of the enzymatic activity by rolipram characterized this enzyme as a typical type IV phosphodiesterase.

Cloning and functional characterization of the human 5-HT2B serotonin receptor.

Recently, we have reported the cloning of the rat 5-HT2B receptor cDNA. This receptor is particularly interesting since it may be involved in diseases such as migraine. Here, we describe the isolation of a human 5-HT2B receptor clone from a cDNA library derived from SH-SY5Y cells. Although the receptor sequence was only 80% homologous to the rat sequence, the exon-intron distribution was conserved between the two species. In the human body, the receptor mRNA was detected in most peripheral organs. Only low expression levels were found in the brain. After expression in HEK 293 cells, activation of the receptor stimulated the production of phosphatidylinositol. The pharmacology of this functional response correlated well with that of the rodent receptor.

Cloning and characterization of MUPP1, a novel PDZ domain protein.

Using the yeast two-hybrid system we isolated a cDNA clone encoding a novel protein interacting with the C-terminal domain of the 5-HT2C receptor. The protein, named MUPP1 (multi-PDZ-domain protein), contains thirteen PDZ domains and no obvious catalytic domain; it is related to hINADL and a putative C. elegans polypeptide referred to as C52A11.4 containing six or ten PDZ domains, respectively. Domains highly similar to those of MUPP1 are arrayed in the same order in all three proteins. The MUPP1 gene is localized on human chromosome 9p24-p22. Transcripts encoding MUPP1 are abundant in the brain as well as in several peripheral organs.

Expression of serotonin receptor mRNAs in blood vessels.

Using RT-PCR we distinguished mRNAs for all known G-protein coupled serotonin receptors expressed in various rat and porcine blood vessels. Nearly all vessels expressed 5HT1D beta, 5-HT2A, 5-HT2B, 5-HT4, and 5-HT7 receptor mRNA to different extents. New splice variants of the porcine 5-HT4 receptor were observed. Similar PCR assays were performed with endothelial and smooth muscle cells from human pulmonary artery, aorta, and with endothelial cells from human coronary artery and umbilical vein. All endothelial cells expressed 5-HT1D beta, 5-HT2B, and 5-HT4 receptor mRNA, whereas in smooth muscle cells 5-HT1D beta, 5-HT2A, 5-HT7, and in some experiments 5-HT2B receptor mRNA were found. A model for the regulation of vascular tone by different 5-HT receptors is proposed.

Distribution and second messenger coupling of four somatostatin receptor subtypes expressed in brain.

The mRNA distribution in the brain and the coupling to cellular effector systems of four somatostatin receptors (SSTR1-4) was studied. All four SRIF receptor subtypes were expressed in cortex and hippocampus. In addition, SSTR1 mRNA was relatively abundant in the spinal cord whereas SSTR2 mRNA was also present in the striatum. The SSTR3 gene was predominantly expressed in the olfactory bulb and in the cerebellum. Conflicting results about the effector coupling of SSTR1-3 have been published previously. We have stably expressed human SSTR1-4 in HEK 293 human embryonal kidney cells. Agonist binding to the receptor subtypes, including the recently cloned SSTR4, inhibited the formation of forskolin-induced cAMP. Is is concluded that, in an appropriate cellular environment, all four receptor subtypes can functionally couple to the inhibition of adenylyl cyclase.

APP RNA splicing is not affected by differentiation of neurons and glia in culture.

Amyloid precursor protein (APP) gene expression was investigated in primary cultures of neurons, astrocytes, microglial cells and oligodendrocytes. Neurons from various rat brain regions, as well as oligodendrocytes, contained RNA encoding APP695, while astrocytes and microglial cells expressed high levels of RNAs for APP770 and APP751. It was studied whether the cell type-specific regulation of APP gene expression could be modified by induction of cellular differentiation in vitro. While neuronal differentiation of PC12 cells has been shown to correspond with an altered pattern of APP splicing, in the primary cultures neither the time in culture nor a treatment of the cells with appropriate differentiation factors affected this pattern.

The distribution and cellular localization of the serotonin 1C receptor mRNA in the rodent brain examined by in situ hybridization histochemistry. Comparison with receptor binding distribution.

The regional distribution and cellular localization of mRNA coding for the serotonin 1C receptor were investigated in tissue sections of mouse and rat brain by in situ hybridization histochemistry. Several 32P-labelled riboprobes derived from mouse genomic clones were used. The serotonin 1C receptor binding sites were visualized autoradiographically and quantified using [3H]mesulergine as ligand, in the presence of spiperone to block serotonin 1C receptors. Strong hybridization signal was observed in the choroid plexus of all brain ventricles. High levels of hybridization were also seen in the anterior olfactory nucleus, pyriform cortex, amygdala, some thalamic nuclei, especially the lateral habenula, the CA3 area of the hippocampal formation, the cingulate cortex, some components of the basal ganglia and associated areas, particularly the nucleus subthalamicus and the substantia nigra. The midbrain and brainstem showed moderate levels of hybridization. The distribution of the serotonin 1C receptor mRNA corresponded well to that of the serotonin 1C receptors. The highest levels of serotonin 1C receptor binding were observed in the choroid plexus. In addition, significant levels of the serotonin 1C receptor binding were seen in the anterior olfactory nucleus, pyriform cortex, nucleus accumbens, ventral aspects of the striatum, paratenial and paracentral thalamic nuclei, amygdaloid body and substantia nigra pars reticulata. The cingulate and retrosplenial cortices as well as the caudal aspects of the hippocampus (CA3) were also labelled. Binding in brainstem and medulla was low and homogeneously distributed. No significant binding was seen in the habenular and subthalamic nuclei. Similar findings were obtained in rat brain. These results demonstrate that, in addition to their enrichment in the choroid plexus, the serotonin 1C receptor mRNA and binding sites are heterogeneously distributed in the rodent brain and thus could be involved in the regulation of many different brain functions. The combination of in situ hybridization histochemistry with receptor autoradiography opens the possibility of examining the regulation of the serotonin 1C receptor synthesis after pharmacological or physiological alterations.

Cortical astrocytes activated by basic fibroblast growth factor secrete molecules that stimulate differentiation of mesencephalic dopaminergic neurons.

In reactive gliosis, astrocytes undergo morphological and biochemical changes which can be mimicked in vitro by treatment with bFGF (basic fibroblast growth factor) or cAMP. To investigate the influence of activated cortical astrocytes on central nervous system (CNSD) neurons, we studied the effect of the supernatant from bFGF-treated astrocytes on the development of dopaminergic neurons from rat mesencephalon. Conditioned medium of untreated astrocytes stimulated dopamine uptake of mesencephalic cultures. After activation of astrocytes with bFGF this effect was greatly enhanced. It was significantly more potent than stimulating effects of other neurotrophic factors. The supernatant of these astrocytes increased the biochemical differentiation but not the survival of dopaminergic neurons in our cell culture system. Trypsin digestion and gel chromatography revealed that the activity was due to one or several proteins with molecular mass above 5 kDa. We excluded the participation of several factors known to be produced by astrocytes or that are neurotrophic for substantia nigra cultures. In particular, we provide evidence that bFGF, BDNF, NT-3, Il-1, Il-6, S100 beta and alpha 2-macroglobulin were not involved in the effect of the conditioned medium. In vitro stimulation of astrocytes therefore triggers the expression of currently uncharacterized factors which influence the biochemical differentiation of mesencephalic dopaminergic neurons, the cells that degenerate in Parkinson's disease.