Randomized, double-blind, placebo-controlled study of safety and efficacy of miltefosine in antihistamine-resistant chronic spontaneous urticaria.

BACKGROUND: Chronic spontaneous urticaria (CSU), a mast cell-driven condition, is debilitating, common, and hard to treat. Miltefosine, a lipid raft modulator, can inhibit mast cell responses in vivo. OBJECTIVE: To study the safety and efficacy of systemic miltefosine treatment in CSU patients resistant to standard-dosed antihistamines. METHODS: In this investigator-initiated multicentre, randomized, double-blind, placebo-controlled study, CSU patients were treated for 4 weeks with daily doses of up to 150-mg miltefosine (n = 47) or placebo (n = 26). Disease activity was assessed using the urticaria activity score. Safety and tolerability of miltefosine were also assessed. RESULTS: After 4 weeks of treatment, Urticaria Activity Score (UAS7) levels were substantially more reduced in miltefosine-treated patients (-6.3 vs. -3.5 in placebo-treated patients; P = 0.05). Also, the number of weals, but not the intensity of pruritus, was significantly reduced in miltefosine-treated patients vs. placebo-treated patients (P = 0.02). In general, adverse events were frequent in both groups (miltefosine: 88%, placebo: 65% of patients) but mostly mild to moderate in severity. We did not observe any serious adverse events. CONCLUSIONS: The results of this study indicate that miltefosine is an effective and safe treatment option for CSU patients who do not respond to standard-dosed antihistamines.

Antisense oligomers for selective suppression of MCP-1 synthesis in human pulmonary endothelial cells.

Endothelial synthesis of the C-C chemokine monocyte chemotactic protein-1 (MCP-1) has been implicated in the regulation of monocyte recruitment for extravascular pools under both physiologic and inflammatory conditions. We designed and characterized five antisense phosphorothioate oligodeoxynucleotides (PS-ODN) targeting MCP-1 secretion by human pulmonary artery endothelial cells (HPAEC) and pulmonary microvascular endothelial cells (HMVEC-L). The most effective PS-ODN (MCP-1 AS 2) dose-dependently suppressed the secretion of MCP-1 but not the secretion of the C-X-C chemokine interleukin-8 (IL-8) in both HPAEC and HMVEC-L in the nanomolar concentration range. Mismatch controls bearing 2 or 4 bp substitutions showed markedly reduced inhibitory capacity. MCP-1 mRNA levels were not affected even at the highest PS-ODN doses employed (ribonuclease protection assay), suggesting a translational arrest of MCP-1 production. Accordingly, PS-ODN exhibited no nonspecific side effects on immediate-early gene regulation of the transcription factor nuclear factor-kappaB (NF-kappaB), as analyzed by gel shift assays. Antisense pretreatment of HPAEC reduced the monocyte chemotactic bioactivity liberated from tumor necrosis factor-alpha (TNF-alpha)-activated endothelial cells (EC) and reduced the TNF-alpha-induced transendothelial monocyte migration. We conclude that nanomolar concentrations of specific antisense oligodeoxynucleotides effectively inhibit human endothelial MCP-1 synthesis and may thus provide a rational approach to modulate monocyte recruitment under inflammatory conditions.

Cationic lipids employed for antisense oligodeoxynucleotide transport may inhibit vascular cell adhesion molecule-1 expression in human endothelial cells: a word of caution.

Antisense oligodeoxynucleotides (ODN) have become a powerful tool to achieve specific gene inhibition in various cell types, including endothelial cells. The low spontaneous cellular uptake of ODN, however, usually requires the employment of transmembrane carriers, such as the positively charged liposome formulation dioleyloxypropyltrimethyl ammonium chloride/dioleoylphosphatidylethanolamine (DOTMA/DOPE). In the present study, we observed that DOTMA/DOPE per se interferes with the inducible expression of vascular cell adhesion molecule-1 (VCAM-1) in human pulmonary artery endothelial cells (HPAEC). By RT-PCR analysis, a dose-dependent suppression of VCAM-1 but not intracellular adhesion molecule-1 (ICAM-1) mRNA levels in tumor necrosis factor-alpha (TNF-alpha)-challenged HPAEC pretreated with DOTMA/DOPE (5-20 microg/ml) was demonstrated. Correspondingly, a strong reduction of TNF-alpha-induced VCAM-1 but not ICAM-1 cell surface expression on HPAEC was observed. These DOTMA/DOPE-induced changes were not due to alterations in VCAM-1 mRNA stability, nor did DOTMA/DOPE inhibit TNF-alpha-induced NF-kappaB-like binding activity in nuclear extracts of HPAEC, as analyzed by electrophoretic mobility shift assay. In contrast, DOTMA/DOPE effected a dose-dependent increase in AP-1-like binding activity in nuclear extracts of HPAEC, as analyzed by Western blotting and EMSA. We conclude that positively charged liposome preparations may per se inhibit TNF-alpha-induced endothelial VCAM-1 expression, and this may be related to changes in AP-1 but not NF-kappaB-dependent transcriptional control. Notably, when used at concentrations below 5 microg/ml, DOTMA/DOPE may be employed for specific antisense-mediated downregulation of VCAM-1 in the absence of vehicle-related side effects on adhesion molecule transcription.

Changes in the expression of synapsin I and II messenger RNA during postnatal rat brain development.

Synapsin Ia, Ib, IIa, and IIb are neuronal phosphoproteins, which are supposed to play a role in the short-term regulation of neurotransmitter release. Besides a high degree of homology among the four synapsin subtypes, there are structural differences in the 3'end of their coding region. Here we present the first extensive study of the expression of their gene transcripts by using in situ hybridization and northern blot analysis. Our results show regionally and temporally distinct expression patterns of synapsin Ia, Ib, IIa, and IIb, which suggests different functional properties of the four synapsin subtypes. There was no specific messenger RNA (mRNA) expression of synapsin IIb in most brain regions apart from the cerebellum, suggesting a minor functional role of this synapsin subtype. Synapsin Ia, Ib, and IIa mRNA were expressed earlier in ontogenetically older brain regions such as the piriform cortex, the thalamus, and the hippocampus and later in ontogenetically younger areas such as the neocortex and the cerebellum. Owing to the distinct expression pattern of the synapsin subtypes, we suppose that the synapsins might be essential for the underlying molecular mechanism of pattern formation and plasticity in distinct brain regions during different states of rat brain development.

Differential expression of c-jun, junB and junD in rat hippocampal slices.

The temporal expression pattern of jun genes was studied in the hippocampal slice preparation. Slices were kept either in a physiological Ringer solution or a modified medium, substituting calcium (Ca2+) by magnesium (Mg2+). All three jun genes were expressed in a circumscribed, independent fashion with respect to distribution, intensity and time course. c-jun and junD were expressed strongly throughout the hippocampus with a shift from DG to CA1 to CA3. junB expression was confined to DG and CA1, but substitution of Ca2+ by Mg2+ led to profound changes: the signal rose earlier, was prolonged, strongly enhanced and more widespread. Our findings suggest, that changes in differential expression of jun gene transcripts are important for parallel processing of non-convergent stimuli.

The role of Jun transcription factor expression and phosphorylation in neuronal differentiation, neuronal cell death, and plastic adaptations in vivo.

1. To investigate the role of the Jun transcription factors in neuronal differentiation, programmed neuronal cell death, and neuronal plasticity, we used phosphorothioate oligodeoxynucleotides (S-ODN) to inhibit selectively the expression of c-Jun, JunB, and JunD. 2. We have shown previously that in contrast to c-Jun, the JunB and JunD transcription factors are negative regulators of cell growth in various cell lines. Here we confirm this finding in primary human fibroblasts. 3. c-Jun and JunB are counterplayers not only with respect to proliferation, but also in cell differentiation. Since JunB expression is essential for neuronal differentiation, we analyzed possible posttranslational modifications of JunB after induction of PC-12 cell differentiation by nerve growth factor (NGF). 4. JunB was strongly phosphorylated after induction of PC-12 cell differentiation with NGF but not after stimulation of cell proliferation with serum. Thus, while cell proliferation is associated with c-Jun phosphorylation, cell differentiation is correlated with JunB phosphorylation. This supports the finding that c-Jun and JunB play antagonistic roles in both proliferation and differentiation. 5. The JunB transcription factor together with the c-Fos transcription factor is also induced in vivo in the suprachiasmatic nucleus (SCN) of rat brain after a light stimulus that induces resetting of the circadian clock. 6. Using antisense oligonucleotides injected into the third ventricle, we selectively cosuppressed the two transcription factors in vivo as shown by immunohistochemistry. Expression of c-Jun, JunD, and FosB was not affected. Inhibition of JunB and c-Fos expression prevented the light-induced phase shift of the circadian rhythm. In contrast, rats injected with a randomized control oligonucleotide showed the same phase shift as untreated animals. 7. In primary rat hippocampal cultures, anti-c-jun S-ODN selectively inhibited neuronal cell death and promoted neuronal survival. This indicates a causal role of c-Jun in programmed neuronal cell death. 8. These findings demonstrate the essential role of inducible transcription factors in the reprogramming of cells to a different functional state. Jun transcription factors play an essential role not only in fundamental processes such as cell proliferation, differentiation, and programmed neuronal cell death, but also in such complex processes as plastic adaptations in the mature brain. The inhibition of neuronal cell death by anti-c-jun S-ODN shows the great therapeutic potential of selective antisense oligonucleotides.

Inhibition of p185c-erbB-2 proto-oncogene expression by antisense oligodeoxynucleotides down-regulates p185-associated tyrosine-kinase activity and strongly inhibits mammary tumor-cell proliferation.

The c-erbB-2 proto-oncogene codes for a 185-kd putative growth factor receptor that is highly homologous to but distinct from the epidermal growth factor (EGF) receptor. Amplification and overexpression of c-erbB-2 occurs in a number of human tumors, in some of which it is a negative prognostic factor. This study investigates the possibility of inhibiting tumor-cell proliferation by blocking c-erbB-2 expression in the human mammary carcinoma cell line SK-Br-3 using chemically modified antisense oligodeoxynucleotides. Expression of the p185c-erbB-2 protein product was selectively reduced within 48 hours and resulted in a growth arrest of SK-Br-3 cells. Biochemical studies of tyrosine-kinase and S6-kinase activities after antisense inhibition of c-erbB-2 show that p185c-erbB-2 activates the S6-kinase signalling pathway in a nonlinear, dose-dependent manner. This may be relevant for the design of therapeutic strategies involving the inhibition of c-erbB-2 (proto)- oncogene expression.

Changes of synapsin I messenger RNA expression during rat brain development.

Synapsin I is a synaptic phosphoprotein that is involved in the short-term regulation of neurotransmitter release. In this report we present the first extensive study of the developmental expression of its corresponding messenger ribonucleic acid (mRNA) by in situ hybridization and northern blot analysis in rat brain. Synapsin I mRNA showed pronounced differences in expression in different brain regions during postnatal development. The early expression of synapsin I mRNA in ontogenetically older regions such as the thalamus, the piriform cortex and the hippocampus coincides with the earlier maturation of these regions, in contrast to its later expression in ontogenetically younger areas such as the cerebellum and the neocortex. An intriguing expression pattern was found in the hippocampus. In all hippocampal subregions synapsin I mRNA expression increased from postnatal day (PND) 1 to 17. After PND 17, however, there was a marked dissociation between persisting high expression levels in CA3 and the dentate gyrus and a strong decline in synapsin I mRNA expression in CA1. The persistence of synapsin I in some adult rat brain regions indicates that it plays a part in synapse formation during plastic adaptation in neuronal connectivities.

Developmental expression of the transcription factor zif268 in rat brain.

Changes in the distribution pattern of mRNA encoding the zif268 transcription factor (also referred to as NGFI-A, Krox-24 or EGR-1) were investigated by in situ hybridization histochemistry during postnatal rat brain development. Marked changes in zif268 expression patterns were seen in particular in the cerebral cortex and the hippocampal formation during the first 3 wk. In the 1st postnatal week, zif268 mRNA levels were highest in the corpus striatum and the piriform cortex. In the neocortex, expression rose sharply in the sensorymotor area between postnatal days (PNDs) 10 and 12. In the frontal and occipital cortex, in contrast, an increase in zif268 mRNA levels was first seen on PND 14. After PND 17, levels decreased in the sensorymotor and the frontal cortex but remained high in the occipital and the piriform cortex. In the hippocampus, an initially uniform increase in expression during the 2nd week was followed by a marked dissociation in expression levels between CA1, with continuously high expression levels on the one hand, and CA3, CA4 and the dentate gyrus, with a strong decline of expression during the 3rd week, on the other hand. Our results indicate that zif268 expression displays a highly dynamic expression pattern during plastic adaptations of different cerebral subregions during postnatal development, suggesting a possible involvement in gene regulatory processes during these phases.

Opposite functions of jun-B and c-jun in growth regulation and neuronal differentiation.

Induction of the jun-B and/or c-jun transcription factors is part of the immediate early response to diverse stimuli that induce alterations in cellular programs. While c-jun is a protooncogene whose expression is required for induction of cell proliferation, jun-B has recently been found to be induced by stimuli inducing differentiation in various cell lines. Furthermore, its expression is largely restricted to differentiating cells during embryogenesis. To determine the functional significance of these findings, we used antisense phosphorothioate oligodeoxynucleotides to inhibit expression of the two genes in proliferating and neuronally differentiating cells. While selective inhibition of c-jun expression reduced proliferation rates, inhibition of jun-B protein synthesis markedly increased proliferation in 3T3 fibroblasts, human mammary carcinoma cells and PC-12 pheochromocytoma cells, suggesting jun-B involvement in negative growth control. Neuronal differentiation of PC-12 cells induced by nerve growth factor (NGF) was prevented by inhibition of jun-B protein synthesis. PC-12 cells not only failed to grow neurites but also remained in the proliferative state. Furthermore, in cultured primary neurons from rat hippocampus, inhibition of jun-B expression, again, markedly reduced morphological differentiation. Conversely, inhibition of c-jun protein synthesis enhanced morphological differentiation of both primary neurons and PC-12 tumor cells. Thus, jun-B expression is required for neuronal differentiation and its balance with c-jun activity is involved in regulating key steps in proliferation and differentiation processes.

Regional and temporal expression of sodium channel messenger RNAs in the rat brain during development.

The distribution of mRNA expression for three types of voltage gated neuronal sodium-channels was studied in the rat brain at different developmental stages (embryonal day E18, postnatal day P5 and adult). With the in-situ hybridization technique, using synthetic DNA-oligomer probes, pronounced regional and temporal variations in the expression levels of the different channel subtypes could be detected. In comparison with types I and III, sodium channel II mRNA was the most abundant subtype at all developmental stages. Maximal expression of sodium channel II mRNA was seen at P5 in virtually all parts of the grey matter, except for the cerebellum. In adult rat brain in contrast, sodium channel II mRNA levels were maximal in the granular layer of the cerebellum, whereas in all other regions expression had decreased to roughly 50% of postnatal levels. Na channel I expression was virtually absent at E18 and showed highest levels at P5, with maxima in the caudate nucleus and hippocampus. In the adult brain, expression of Na-channel I was nearly absent in the neocortex, but well detectable in the cerebellum and, at lower levels in the striatum and thalamus. Sodium channel III was mainly expressed at the embryonal stage and showed a decrease to very low levels with little regional preferences in the adult.

The topology of the thalamo-cortical projections in the marmoset monkey (Callithrix jacchus).

This paper addresses the question of a general topological principle of thalamo-cortical projections. In the lissencephalic primate brain of the common marmoset (Callithrix jacchus), large injections of horseradish peroxidase were made in various parts of the neocortex. These injections were placed in different animals and hemispheres along various caudo-rostral and medio-lateral gradients. Labelled cells in the thalamus were plotted and the labelling-zones resulting from several injections along a medio-lateral and two caudo-rostral cortical vectors were drawn into semi-schematic thalamic maps. These composite maps reveal a topological organization of the whole thalamo-cortical projection. The thalamic representation of the caudo-rostral and medio-lateral gradients indicate a rotation of the posterior relative to the anterior thalamus. An attempt is made to relate the organization of the thalamo-cortical projection to the development of the thalamus and the cortex. The cortex is divided into concentric zones around the sensory-motor and insular cortex. The thalamus is divided into corresponding projection zones. The topology of thalamo-cortical connections can then be regarded as a consequence of corresponding thalamic and cortical growth gradients. This is not only consistent with the general thalamo-cortical topology and the inversion of maps from thalamus to cortex, but also explains the continuity and overlap of thalamic projection zones in the pulvinar to widely separated cortical areas as the parietal, temporal and frontal association cortex.