Early intensification and autologous stem cell transplantation in patients with systemic AL amyloidosis: a single-centre experience.

Primary systemic amyloidosis (AL amyloidosis) continues to have a very poor prognosis. Most therapeutic strategies remain unsatisfactory. Conventional chemotherapy is known to offer at best only moderate efficacy. Several studies have yielded higher complete response rates after high-dose chemotherapy and autologous stem cell transplantation (ASCT) in addition to improving outcomes in a subgroup of patients. However, the superiority of an intensive approach in AL amyloidosis has not been confirmed in a randomised trial. The precise role of ASCT remains unclear. We report our experience in 16 patients diagnosed with AL amyloidosis and treated in a multidisciplinary approach with high-dose melphalan and ASCT. Median age was 59 (39-71) years. The kidneys were predominantly affected in 75% of cases; two or more organs were affected in 38%. Median time from diagnosis to transplantation was 2 (1-4) months. Three patients (19%) developed acute renal failure and required transient dialysis. Transplant-related mortality was 6% after 100 days. Haematological complete response (CR) was obtained in nine (56%) and organ response in six (38%) patients. Nine out of 12 patients (75%) with kidney involvement exhibited a sustained clinical benefit at 12 months. Half of all the patients (n = 8) were alive after a median follow-up of 33 months, including two in continuous CR. This suggests that high-dose chemotherapy and ASCT are still valid treatment options in AL amyloidosis and that a significant number of patients with renal involvement might benefit from this approach.

Peroxisome proliferator-activated receptor β/δ: a master regulator of metabolic pathways in skeletal muscle

Skeletal muscle is considered to be a major site of energy expenditure and thus is important in regulating events affecting metabolic disorders. Over the years, both in vitro and in vivo approaches have established the role of peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) in fatty acid metabolism and energy expenditure in skeletal muscles. Pharmacological activation of PPARβ/δ by specific ligands regulates the expression of genes involved in lipid use, triglyceride hydrolysis, fatty acid oxidation, energy expenditure, and lipid efflux in muscles, in turn resulting in decreased body fat mass and enhanced insulin sensitivity. Both the lipid-lowering and the anti-diabetic effects exerted by the induction of PPARβ/δ result in the amelioration of symptoms of metabolic disorders. This review summarizes the action of PPARβ/δ activation in energy metabolism in skeletal muscles and also highlights the unexplored pathways in which it might have potential effects in the context of muscular disorders. Numerous preclinical studies have identified PPARβ/δ as a probable potential target for therapeutic interventions. Although PPARβ/δ agonists have not yet reached the market, several are presently being investigated in clinical trials.

Differential effects of GABAB receptor subtypes, {gamma}-hydroxybutyric Acid, and Baclofen on EEG activity and sleep regulation.

The role of GABA(B) receptors in sleep is still poorly understood. GHB (γ-hydroxybutyric acid) targets these receptors and is the only drug approved to treat the sleep disorder narcolepsy. GABA(B) receptors are obligate dimers comprised of the GABA(B2) subunit and either one of the two GABA(B1) subunit isoforms, GABA(B1a) and GABA(B1b). To better understand the role of GABA(B) receptors in sleep regulation, we performed electroencephalogram (EEG) recordings in mice devoid of functional GABA(B) receptors (1(-/-) and 2(-/-)) or lacking one of the subunit 1 isoforms (1a(-/-) and 1b(-/-)). The distribution of sleep over the day was profoundly altered in 1(-/-) and 2(-/-) mice, suggesting a role for GABA(B) receptors in the circadian organization of sleep. Several other sleep and EEG phenotypes pointed to a more prominent role for GABA(B1a) compared with the GABA(B1b) isoform. Moreover, we found that GABA(B1a) protects against the spontaneous seizure activity observed in 1(-/-) and 2(-/-) mice. We also evaluated the effects of the GHB-prodrug GBL (γ-butyrolactone) and of baclofen (BAC), a high-affinity GABA(B) receptor agonist. Both drugs induced a state distinct from physiological sleep that was not observed in 1(-/-) and 2(-/-) mice. Subsequent sleep was not affected by GBL whereas BAC was followed by a delayed hypersomnia even in 1(-/-) and 2(-/-) mice. The differential effects of GBL and BAC might be attributed to differences in GABA(B)-receptor affinity. These results also indicate that all GBL effects are mediated through GABA(B) receptors, although these receptors do not seem to be involved in mediating the BAC-induced hypersomnia.

International Union of Pharmacology. LXI. Peroxisome proliferator-activated receptors.

The three peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors of the nuclear hormone receptor superfamily. They share a high degree of structural homology with all members of the superfamily, particularly in the DNA-binding domain and ligand- and cofactor-binding domain. Many cellular and systemic roles have been attributed to these receptors, reaching far beyond the stimulation of peroxisome proliferation in rodents after which they were initially named. PPARs exhibit broad, isotype-specific tissue expression patterns. PPARalpha is expressed at high levels in organs with significant catabolism of fatty acids. PPARbeta/delta has the broadest expression pattern, and the levels of expression in certain tissues depend on the extent of cell proliferation and differentiation. PPARgamma is expressed as two isoforms, of which PPARgamma2 is found at high levels in the adipose tissues, whereas PPARgamma1 has a broader expression pattern. Transcriptional regulation by PPARs requires heterodimerization with the retinoid X receptor (RXR). When activated by a ligand, the dimer modulates transcription via binding to a specific DNA sequence element called a peroxisome proliferator response element (PPRE) in the promoter region of target genes. A wide variety of natural or synthetic compounds was identified as PPAR ligands. Among the synthetic ligands, the lipid-lowering drugs, fibrates, and the insulin sensitizers, thiazolidinediones, are PPARalpha and PPARgamma agonists, respectively, which underscores the important role of PPARs as therapeutic targets. Transcriptional control by PPAR/RXR heterodimers also requires interaction with coregulator complexes. Thus, selective action of PPARs in vivo results from the interplay at a given time point between expression levels of each of the three PPAR and RXR isotypes, affinity for a specific promoter PPRE, and ligand and cofactor availabilities.

Design, synthesis and biological evaluation of a class of bioisosteric oximes of the novel dual peroxisome proliferator-activated receptor α/γ ligand LT175.

The effects resulting from the introduction of an oxime group in place of the distal aromatic ring of the diphenyl moiety of LT175, previously reported as a PPARα/γ dual agonist, have been investigated. This modification allowed the identification of new bioisosteric ligands with fairly good activity on PPARα and fine-tuned moderate activity on PPARγ. For the most interesting compound (S)-3, docking studies in PPARα and PPARγ provided a molecular explanation for its different behavior as full and partial agonist of the two receptor isotypes, respectively. A further investigation of this compound was carried out performing gene expression studies on HepaRG cells. The results obtained allowed to hypothesize a possible mechanism through which this ligand could be useful in the treatment of metabolic disorders. The higher induction of the expression of some genes, compared to selective agonists, seems to confirm the importance of a dual PPARα/γ activity which probably involves a synergistic effect on both receptor subtypes.

Neuroscience: Astrocytes as aide-mémoires.

Memory formation is known to occur at the level of synaptic contacts between neurons. It therefore comes as a surprise that another type of brain cell, the astrocyte, is also involved in establishing memory.

The direct peroxisome proliferator-activated receptor target fasting-induced adipose factor (FIAF/PGAR/ANGPTL4) is present in blood plasma as a truncated protein that is increased by fenofibrate treatment.

The fasting-induced adipose factor (FIAF, ANGPTL4, PGAR, HFARP) was previously identified as a novel adipocytokine that was up-regulated by fasting, by peroxisome proliferator-activated receptor agonists, and by hypoxia. To further characterize FIAF, we studied regulation of FIAF mRNA and protein in liver and adipose cell lines as well as in human and mouse plasma. Expression of FIAF mRNA was up-regulated by peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARbeta/delta agonists in rat and human hepatoma cell lines and by PPARgamma and PPARbeta/delta agonists in mouse and human adipocytes. Transactivation, chromatin immunoprecipitation, and gel shift experiments identified a functional PPAR response element within intron 3 of the FIAF gene. At the protein level, in human and mouse blood plasma, FIAF was found to be present both as the native protein and in a truncated form. Differentiation of mouse 3T3-L1 adipocytes was associated with the production of truncated FIAF, whereas in human white adipose tissue and SGBS adipocytes, only native FIAF could be detected. Interestingly, truncated FIAF was produced by human liver. Treatment with fenofibrate, a potent PPARalpha agonist, markedly increased plasma levels of truncated FIAF, but not native FIAF, in humans. Levels of both truncated and native FIAF showed marked interindividual variation but were not associated with body mass index and were not influenced by prolonged semistarvation. Together, these data suggest that FIAF, similar to other adipocytokines such as adiponectin, may partially exert its function via a truncated form.

Adenosine A1 receptor activation is arrhythmogenic in the developing heart through NADPH oxidase/ERK- and PLC/PKC-dependent mechanisms.

Whether adenosine, a crucial regulator of the developing cardiovascular system, can provoke arrhythmias in the embryonic/fetal heart remains controversial. Here, we aimed to establish a mechanistic basis of how an adenosinergic stimulation alters function of the developing heart. Spontaneously beating hearts or dissected atria and ventricle obtained from 4-day-old chick embryos were exposed to adenosine or specific agonists of the receptors A(1)AR (CCPA), A(2A)AR (CGS-21680) and A(3)AR (IB-MECA). Expression of the receptors was determined by quantitative PCR. The functional consequences of blockade of NADPH oxidase, extracellular signal-regulated kinase (ERK), phospholipase C (PLC), protein kinase C (PKC) and L-type calcium channel (LCC) in combination with adenosine or CCPA, were investigated in vitro by electrocardiography. Furthermore, the time-course of ERK phosphorylation was determined by western blotting. Expression of A(1)AR, A(2A)AR and A(2B)AR was higher in atria than in ventricle while A(3)AR was equally expressed. Adenosine (100μM) triggered transient atrial ectopy and second degree atrio-ventricular blocks (AVB) whereas CCPA induced mainly Mobitz type I AVB. Atrial rhythm and atrio-ventricular propagation fully recovered after 60min. These arrhythmias were prevented by the specific A(1)AR antagonist DPCPX. Adenosine and CCPA transiently increased ERK phosphorylation and induced arrhythmias in isolated atria but not in ventricle. By contrast, A(2A)AR and A(3)AR agonists had no effect. Interestingly, the proarrhythmic effect of A(1)AR stimulation was markedly reduced by inhibition of NADPH oxidase, ERK, PLC, PKC or LCC. Moreover, NADPH oxidase inhibition or antioxidant MPG prevented both A(1)AR-mediated arrhythmias and ERK phosphorylation. These results suggest that pacemaking and conduction disturbances are induced via A(1)AR through concomitant stimulation of NADPH oxidase and PLC, followed by downstream activation of ERK and PKC with LCC as possible target.

Effects of salbutamol on the contractile properties of human skeletal muscle before and after fatigue.

AIM: The study examined the effects of an oral acute administration of the beta2-agonist salbutamol (Sal) (6 mg) vs. placebo on muscle strength and fatigability in 12 non-asthmatic recreational male athletes in a randomized double-blind protocol. METHODS: Contractile properties of the right quadriceps muscle were measured during electrical stimulations, i.e. twitch, 1-s pulse trains at 20 (P(20) ) and 80 Hz (P(80) ) and during maximal voluntary isometric contraction (MVIC) before (PRE) and after (POST) a fatigue-producing protocol set by an electromyostimulation (30 contractions, frequency: 75 Hz, on-off ratio: 6.25-20s). In addition, the level of muscle voluntary activation was measured. RESULTS: In PRE and POST conditions, the peak torque (PT) of twitch, P(80) and MVIC were not modified by the treatment. The PT in POST P(20) was slightly, although not significantly, less affected by fatigue in Sal compared with placebo condition. Moreover, twitch half-relaxation time at PRE was smaller under Sal than under placebo (P < 0.05). No significant changes in the degree of voluntary activation were observed with Sal treatment in PRE or POST condition. CONCLUSION: Although these findings did not exclude completely an effect of Sal on peripheral factors of human skeletal muscle, oral acute administration of the beta2-agonist Sal seems to be without any relevant ergogenic effect on muscle contractility and fatigability in non-asthmatic recreational male athletes.

Ectodysplasin A (EDA) - EDA receptor signalling and its pharmacological modulation.

The TNF family ligand ectodysplasin A (EDA) regulates the induction, morphogenesis and/or maintenance of skin-derived structures such as teeth, hair, sweat glands and several other glands. Deficiencies in the EDA - EDA receptor (EDAR) signalling pathway cause hypohidrotic ectodermal dysplasia (HED). This syndrome is characterized by the absence or malformation of several skin-derived appendages resulting in hypotrychosis, hypodontia, heat-intolerance, dry skin and dry eyes, susceptibility to airways infections and crusting of various secretions. The EDA-EDAR system is an important effector of canonical Wnt signalling in developing skin appendages. It functions by stimulating NF-κB-mediated transcription of effectors or inhibitors of the Wnt, Sonic hedgehog (SHH), fibroblast growth factor (FGF) and transforming growth factor beta (TGFβ) pathways that regulate interactions within or between epithelial and mesenchymal cells and tissues. In animal models of Eda-deficiency, soluble EDAR agonists can precisely correct clinically relevant symptoms with low side effects even at high agonist doses, indicating that efficient negative feedback signals occur in treated tissues. Hijacking of the placental antibody transport system can help deliver active molecules to developing foetuses in a timely manner. EDAR agonists may serve to treat certain forms of ectodermal dysplasia.

Increased vulnerability to kainic acid-induced epileptic seizures in mice underexpressing the scaffold protein Islet-Brain 1/JIP-1.

Islet-Brain 1, also known as JNK-interacting protein-1 (IB1/JIP-1) is a scaffold protein mainly involved in the regulation of the pro-apoptotic signalling cascade mediated by c-Jun-N-terminal kinase (JNK). IB1/JIP-1 organizes JNK and upstream kinases in a complex that facilitates JNK activation. However, overexpression of IB1/JIP-1 in neurons in vitro has been reported to result in inhibition of JNK activation and protection against cellular stress and apoptosis. The occurrence and the functional significance of stress-induced modulations of IB1/JIP-1 levels in vivo are not known. We investigated the regulation of IB1/JIP-1 in mouse hippocampus after systemic administration of kainic acid (KA), in wild-type mice as well as in mice hemizygous for the gene MAPK8IP1, encoding for IB1/JIP-1. We show here that IB1/JIP-1 is upregulated transiently in the hippocampus of normal mice, reaching a peak 8 h after seizure induction. Heterozygous mutant mice underexpressing IB1/JIP-1 showed a higher vulnerability to the epileptogenic properties of KA, whereas hippocampal IB1/JIP-1 levels remained unchanged after seizure induction. Subsequently, an increasing activation of JNK in the 8 h following seizure induction was observed in IB1/JIP-1 haploinsufficient mice, which also underwent more severe excitotoxic lesions in hippocampal CA3, as assessed histologically 3 days after KA administration. Taken together, these data indicate that IB1/JIP-1 in hippocampus participates in the regulation of the neuronal response to excitotoxic stress in a level-dependent fashion.

Toll-like receptor 3 expressed by melanoma cells as a target for therapy?

PURPOSE: The immunomodulatory properties of Toll-like receptors (TLR) agonists have inspired their use as experimental adjuvants for vaccination of cancer patients. However, it is now well recognized that TLR expression is not restricted to immune cells but can also be found in many cell types, including those giving rise to tumors. It is therefore mandatory to explore the potential effects of TLR triggering directly on tumor cells. EXPERIMENTAL DESIGN: In the present work, we have investigated TLR3 protein expression in melanoma cell lines derived from patients, and analyzed the effects of TLR3 agonists on tumor cell survival. Moreover, we used RNA interference to stably knock down TLR3 expression and study the involvement of this receptor in dsRNA-induced effects on melanoma cells viability. RESULTS: Human melanoma cells can express functional TLR3 protein. Interestingly, the engagement of the receptor by TLR3 agonists can directly inhibit cell proliferation and induce tumor cell death when combined to treatment with either type I IFN or protein synthesis inhibitors. These effects were shown by RNA interference to be largely dependent on TLR3. Moreover, TLR3-mediated cell death involves the activation of caspases and engages both extrinsic and intrinsic apoptotic pathways. CONCLUSION: TLR3 protein can be expressed in human melanoma cells, where it can deliver proapoptotic and antiproliferative signaling. Altogether, these results suggest that TLR3 agonists represent very promising adjuvants for cancer vaccines not only based on their well-described immunostimulatory properties, but also due to their newly identified cytostatic and cytotoxic effects directly on tumor cells.

The CD8 beta polypeptide is required for the recognition of an altered peptide ligand as an agonist.

T cell activation is triggered by the specific recognition of cognate peptides presented by MHC molecules. Altered peptide ligands are analogs of cognate peptides which have a high affinity for MHC molecules. Some of them induce complete T cell responses, i.e. they act as agonists, whereas others behave as partial agonists or even as antagonists. Here, we analyzed both early (intracellular Ca2+ mobilization), and late (interleukin-2 production) signal transduction events induced by a cognate peptide or a corresponding altered peptide ligand using T cell hybridomas expressing or not the CD8 alpha and beta chains. With a video imaging system, we showed that the intracellular Ca2+ response to an altered peptide ligand induces the appearance of a characteristic sustained intracellular Ca2+ concentration gradient which can be detected shortly after T cell interaction with antigen-presenting cells. We also provide evidence that the same altered peptide ligand can be seen either as an agonist or a partial agonist, depending on the presence of CD8beta in the CD8 co-receptor dimers expressed at the T cell surface.

Regulation of human class I alcohol dehydrogenases by bile acids

Class I alcohol dehydrogenases (ADH1s) are the rate-limiting enzymes for ethanol and vitamin A (retinol) metabolism in the liver . Because previous studies have shown that human ADH1 enzymes may participate in bile acid metabolism, we investigated whether the bile acid-activated nuclear receptor farnesoid X receptor (FXR) regulates ADH1 genes. In human hepatocytes, both the endogenous FXR ligand chenodeoxycholic acid and synthetic FXR-specific agonist GW4064 increased ADH1 mRNA, protein, and activity. Moreover, overexpression of a constitutively active form of FXR induced ADH1A and ADH1B expression, whereas silencing of FXR abolished the effects of FXR agonists on ADH1 expression and activity. Transient transfection studies and electrophoretic mobility shift assays revealed functional FXR response elements in the ADH1A and ADH1B proximal promoters, thus indicating that both genes are direct targets of FXR. These findings provide the first evidence for direct connection of bile acid signaling and alcohol metabolism.