Stefano Coaccioli

Constitutively activated Notch signaling is involved in survival and apoptosis resistance of B-CLL cells

Notch signaling is involved in tumorigenesis, but its role in B-chronic lymphocytic leukemia (B-CLL) pathogenesis is not completely defined. This study examined the expression and activation of Notch receptors in B-CLL cells and the role of Notch signaling in sustaining the survival of these cells. Our results show that B-CLL cells but not normal B cells constitutively express Notch1 and Notch2 proteins as well as their ligands Jagged1 and Jagged2. Notch signaling is constitutively activated in B-CLL cells, and its activation is further increased in B-CLL cells, which resist spontaneous apoptosis after 24-hour ex vivo culture. Notch stimulation by a soluble Jagged1 ligand increases B-CLL cell survival and is accompanied by increased nuclear factor-kappa B (NF-kappaB) activity and cellular inhibitor of apoptosis protein 2 (c-IAP2) and X-linked inhibitor of apoptosis protein (XIAP) expression. In contrast, Notch-signaling inhibition by the gamma-secretase inhibitor I (GSI; z-Leu-Leu-Nle-CHO) and the specific Notch2 down-regulation by small-interfering RNA accelerate spontaneous B-CLL cell apoptosis. Apoptotic activity of GSI is accompanied by reduction of NF-kappaB activity and c-IAP2 and XIAP expression. Overall, our findings show that Notch signaling plays a critical role in B-CLL cell survival and apoptosis resistance and suggest that it could be a novel potential therapeutic target.

Group B Streptococcus Induces Macrophage Apoptosis by Calpain Activation

Group B Streptococcus (GBS) has developed several strategies to evade immune defenses. We show that GBS induces macrophage (Mφ) membrane permeability defects and apoptosis, prevented by inhibition of calcium influx but not caspases. We analyze the molecular mechanisms of GBS-induced murine Mφ apoptosis. GBS causes a massive intracellular calcium increase, strictly correlated to membrane permeability defects and apoptosis onset. Calcium increase was associated with activation of calcium-dependent protease calpain, demonstrated by casein zymography, α-spectrin cleavage to a calpain-specific fragment, fluorogenic calpain-substrate cleavage, and inhibition of these proteolyses by calpain inhibitors targeting the calcium-binding, 3-(4-Iodophenyl)-2-mercapto-(Z)-2-propenoic acid, or active site (four different inhibitors), by calpain small-interfering-RNA (siRNA) and EGTA. GBS-induced Mφ apoptosis was inhibited by all micro- and m-calpain inhibitors used and m-calpain siRNA, but not 3-(5-Fluoro-3-indolyl)-2-mercapto-(Z)-2-propenoic acid (micro-calpain inhibitor) and micro-calpain siRNA indicating that m-calpain plays a central role in apoptosis. Calpain activation is followed by Bax and Bid cleavage, cytochrome c, apoptosis-inducing factor, and endonuclease G release from mitochondria. In GBS-induced apoptosis, cytochrome c did not induce caspase-3 and -7 activation because they and APAF-1 were degraded by calpains. Therefore, apoptosis-inducing factor and endonuclease G seem the main mediators of the calpain-dependent but caspase-independent pathway of GBS-induced apoptosis. Proapoptotic mediator degradations do not occur with nonhemolytic GBS, not inducing Mφ apoptosis. Apoptosis was reduced by Bax siRNA and Bid siRNA suggesting Bax and Bid degradation is apoptosis correlated. This signaling pathway, different from that of most pathogens, could represent a GBS strategy to evade immune defenses.

Modulation of Pro- and Antiapoptotic Molecules in Double-Positive (CD4+CD8+) Thymocytes following Dexamethasone Treatment

Glucocorticoids play a role in regulation of T lymphocytes homeostasis and development. In particular, glucocorticoid treatment induces massive apoptosis of CD4+CD8+ double-positive (DP) thymocytes. This effect is due to many mechanisms, mainly driven by modulation of gene transcription. To find out which genes are modulated, we analyzed DP thymocytes treated for 3 h with dexamethasone (a synthetic glucocorticoid) by global gene expression profiling. Results indicate modulation of 163 genes, also confirmed by either RNase protection assay or real-time polymerase chain reaction. In particular, dexamethasone caused down-regulation of genes promoting DP thymocyte survival (e.g., Notch1, suppressor of cytokine signaling 1, and inhibitor of DNA binding 3) or modulation of genes activating cell death through the ceramide pathway (UDP-glucose ceramide glucosyltransferase, sphingosine 1-phosphate phosphatase, dihydroceramide desaturase, isoform 1, and G protein-coupled receptor 65) or through the mitochondrial machinery. Among the latter, there are Bcl-2 family members (Bim, Bfl-1, Bcl-xL, and Bcl-xβ), genes involved in the control of redox status (thioredoxin reductase, thioredoxin reductase inhibitor, and NADP+-dependent isocitrate dehydrogenase) and genes belonging to Tis11 family that are involved in mRNA stability. Our study suggests that dexamethasone treatment of DP thymocytes modulates several genes belonging to apoptosis-related systems that can contribute to their apoptosis.

Fibromyalgia: nosography and therapeutic perspectives.

▪ Abstract:  Fibromyalgia (FM) is an important cause of morbidity and health expenditure. Severe widespread extra‐articular chronic pain, along with nonrestorative sleep, dominates the clinical syndrome. The pathogenesis of FM remains unclear. While dysfunction in serotoninergic neurotransmission is believed to play an important role, several neurologic and immuno‐endocrine mechanisms may also be relevant. A theory is advanced based on an inherited imbalance in neuro‐vegetative systems resulting from increased sympathetic tone because of a metabolic deficiency in the serotoninergic system that, when exposed to a precipitating event, leads to the development of the clinical manifestations of FM. The importance of both nonpharmacological treatments and multimodal medication management is stressed. ▪

Mitochondrial dysfunction and effect of antiglycolytic bromopyruvic acid in GL15 glioblastoma cells

Most cancer cells, including GL15 glioblastoma cells, rely on glycolysis for energy supply. The effect of antiglycolytic bromopyruvate on respiratory parameters and viability of GL15 cells was investigated. Bromopyruvate caused Δψm and MTT collapse, ATP decrease, and cell viability loss without involving apoptotic or necrotic pathways. The autophagy marker LC3-II was increased. Δψm decrease was accompanied by reactive oxygen species (ROS) increase and cytochrome c (cyt c) disappearance, suggesting a link between free radical generation and intramitochondrial cyt c degradation. Indeed, the free radical inducer menadione caused a decrease in cyt c that was reversed by N-acetylcysteine. Cyt c is tightly bound to the inner mitochondrial membrane in GL15 cells, which may confer protein peroxidase activity, resulting in auto-oxidation and protein targeting to degradation in the presence of ROS. This process is directed towards impairment of the apoptotic cyt c cascade, although cells are committed to die.

Improved Cancer Pain Treatment Using Combined Fentanyl‐TTS and Tramadol

▪ Abstract:  The aim of the study was to facilitate dose escalation of strong opioids. In this randomized open‐label study the influence of tramadol on dose adjustment of transdermal fentanyl in advanced cancer pain control was prospectively evaluated. Seventy patients affected by intractable cancer disease with visual analog scale (VAS) score >3 were enrolled. Thirty‐five patients were treated conventionally with increasing transdermal fentanyl dosage as required (group F) and 35 patients received oral tramadol added to their transdermal fentanyl before each increment of the transdermal opioid dosage (group T). Pain control was equally satisfactory in the two groups. VAS scores at baseline (T: 4.36 ± 1.53; F: 4.51 ± 1.36; n.s.) and at the end of the study (T: 1.8 ± 1.6; F: 1.6 ± 1.5; n.s.) did not differ. However, in the tramadol group this level of pain control was achieved with much slower dose escalation of fentanyl. The mean application time of the fentanyl‐Transdermal Therapeutic System patch for each dosage (25, 50, 75 μg/hour) was significantly greater in patients receiving tramadol. No patient in group T escalated to the 100 μg/hour patch, while in 12 patients of group F the 100 μg/hour patch was applied after a 75 μg/hour patch mean application period of 18.6 ± 4.7 days. The number of fentanyl‐TTS dosage changes was significantly lower in group T (1.2 ± 0.4 vs. 2.3 ± 0.5; P < 0.05). The mean total duration of treatment in group T, was 37.1 ± 11.6 days. The amount of fentanyl used at study end was 56.6 ± 11.2 μg/hour plus 141.1 ± 151.9 mg tramadol per day (median: 200 mg/day) in group T patients compared with 84.1 ± 12.2 μg/hour in group F patients (P < 0.05). The combination of a strong opioid with a weak opioid to treat severe cancer pain allowed a more gradual increase of analgesic delivery than was possible using fentanyl‐TTS alone, minimizing periods of under‐ and overdosing. In addition, it considerably slowed the pace of fentanyl dose escalation. In conclusion, this TTS fentanyl‐tramadol analgesic protocol provides a useful alternative to the usual treatment of cancer pain with fentanyl‐TTS alone, especially in case of quick progression of disease and pain. ▪

Omeprazole induces apoptosis in normal human polymorphonuclear leucocytes.

We investigated in vitro apoptosis in human polymorphonuclear neutrophils (PMN) induced by omeprazole. This drug, both in the native (OM) and acidified (OM-HCl) form, is a potent inducer of PMN apoptosis. The effect is time- and dose-dependent. OM-HCl is more efficient than OM in inducing PMN apoptosis. In fact, after 24 h incubation in vitro at 1×10 −4M OM-HCl induces apoptosis in 70% of the cell population compared to 37% induced by OM. Apoptosis induced by both forms of the drug is caspase dependent being significantly reduced by pretreating cells with the caspase 3 inhibitor (DEVDH-CHO). However, some differences in the apoptosis mechanisms between the two forms of the drug seem to exist because PMN treatment with the specific caspase 8 inhibitor (Z-IETD-FMK) only blocks OM-HCl mediated apoptosis. We observed cleavage of caspase 8 only in the cells incubated with OM-HCl while the executioner caspase 3 was activated with both forms of the drug. Furthermore, pretreatment with GM-CSF, a known activator of intracellular survival pathways in PMN, partially protected cells from OM-HCl induced apoptosis but did not contrast the apoptotic effect of OM. Cysteine cathepsin proteases also seem involved in the apoptotic mechanism of both drug forms since the specific inhibitor E64d gave a significant protection. To verify if OM-HCl induced apoptosis was dependent on the sulfenamide bound with the cell sulfhydryl groups we used molecules with thiol groups such as β-mercaptoethanol (β-ME) and reduced glutathione (GSH). Reactions of OM-HCl with cellular sulfhydryl groups are strongly involved in both the triggering and evolving phase of the apoptotic mechanism since significant protection from apoptosis was obtained when PMN were pretreated for 1h with β-ME (lipid-permeable) or GSH (lipid-impermeable). These results show that OM and OM-HCl induce apoptosis in human PMN and suggest that the second binds the sulfhydryl groups, present on the cell membrane, to then penetrate the cell thus causing a further significant increase in apoptosis. OM-induced PMN apoptosis during the treatment of gastric inflammatory disease could be an advantage for the resolution of the phlogosis state. However, this aspect should be further elucidated to assess the optimal therapeutical regimen for gastric diseases which are related to infective agents.

Strong analgesics: Working towards an optimal balance between efficacy and side effects

Inadequately treated chronic pain remains a major cause of suffering. A recent survey found that moderate to severe chronic pain affects 19% of adult Europeans (Breivik et al., 2006). Other large scale surveys in Australia (Blyth et al., 2001), Denmark (Sjogren et al., 2009) and Norway (Rustoen et al., 2004) have given similar results. These also represent a substantial socioeconomic burden (Dunn and Croft, 2004). Moreover, these surveys may underestimate the true incidence, because many patients paradoxically report their analgesia to be satisfactory despite being in pain. One study found that over 75% of patients declared themselves satisfied or very satisfied with their overall pain management, despite almost 50% reporting recent moderate to severe pain (Dawson et al., 2002). The cornerstone of treatment for moderate to severe pain is classical opioid therapy; the WHO Pain Analgesic Ladder recommends weak opioids for Step 2 and strong opioids for Step 3 in the management of chronic pain (WHO, 1996), even if this recommendation has been criticized (Marinangeli et al., 2004). However, the prescription of opioids is still limited by medical, ethical, cultural and legal issues (Bhamb et al., 2006; Jacobsen et al., 2007). One major factor is opiophobia; physicians’ and patients’ concerns about the appropriate use of opioids, and their fears and misunderstandings in connection with these agents. Patients may be reluctant to take opioids because of a perceived association with drug abuse and criminality (Fields, 2007) or with terminal illness. Classic opioid therapy also has shortcomings; specifically, in providing sustained analgesic efficacy over long periods, reliable efficacy in difficult-to-treat pain syndromes, and acceptable levels of tolerability. Balancing adequate pain relief with minimal side effects is difficult, and sometimes impossible. Long term opioid efficacy may be compromised by the development of tolerance and/or hyperalgesia. The current clinical strategy for managing these phenomena is opioid rotation, utilising the incomplete cross-tolerance between opioids in order to recover efficacy (DuPen et al., 2007). However, the evidence for this strategy is largely anecdotal or based on observational and uncontrolled studies, and there are no predictive criteria for the best drug option. A recent approach has been to combine drugs with different modes of action, such as morphine with N-methyl-D-aspartate (NMDA) antagonists, anticonvulsants or antidepressants. A few clinical studies have been undertaken with morphine plus the NMDA antagonist dextromethorphan. The combination has so far failed to demonstrate either enhanced opioid analgesia or reduced tolerance compared to morphine alone, and – in addition – discontinuation rates have been higher with the combination (Galer et al., 2005). Difficult-to-treat pain syndromes are essentially those where the pain is neuropathic in origin or has a neuropathic component. Examples include diabetic peripheral neuropathy, post-herpetic neuralgia and mixed cancer pain. Many different drugs are currently used to treat these syndromes and there is no gold standard for treatment, demonstrated by a review of 105 high-quality, randomised, placebo-controlled clinical trials (Finnerup et al., 2005). Experienced pain therapists use a combination of analgesics (often an opioid and a non-opioid) with different modes of action. Animal studies have suggested that combining morphine with an alpha2 delta ligand such as gabapentin (Matthews and Dickenson, 2002), a tricyclic antidepressant such as amitriptyline (Luccarini et al., 2004), or an NMDA antagonist such as ketamine (Pelissier et al., 2003) all offer some benefit. However, some unresolved issues remain with this approach (such as the safety of polypharmacy and its effect on compliance), while clinical trials suggest that any improvement in efficacy may be outweighed by the increased incidence of side effects (Hanna et al., 2008; Gilron et al., 2005). Tolerability problems are common with opioids, particularly nausea and vomiting during the first one or two weeks, and constipation throughout long term treatment. Anti-emetics and laxatives may be used both for prophylaxis and treatment of these adverse events, but the current pharmacological approach is often unsatisfactory and/or the regimen is too complex, so patients discontinue treatment because of additional and intolerable side effects. One recent alternative is to combine a classical opioid with a systemic mu opioid receptor antagonist (Muller-Lissner et al., 2007), but further studies are needed. Peripheral selective and non-selective mu opioid receptor antagonists have been developed. These act on peripheral receptors, including those located within the GI tract, and do not cross the blood–brain barrier. Some clinical trials has shown that subcutaneous methylnaltrexone rapidly induces laxation in patients with advanced illness and opioid-induced consti-

Degenerative Joint Diseases and Neuroinflammation.

Rheumatic and joint diseases, as exemplified by osteoarthritis and rheumatoid arthritis, are among the most widespread painful and disabling pathologies across the globe. Given the continuing rise in life expectancy, their prevalence is destined to grow. Osteoarthritis, a degenerative joint disease, is, in particular, on its way to becoming the fourth leading cause of disability worldwide by 2020, with the rising incidence of obesity in addition to age being important factors. It is estimated that 25% of osteoarthritic individuals are unable to perform daily activities. Accompanying osteoarthritis is rheumatoid arthritis, which is a chronic systemic disease that often causes pain and deformity. At least 50% of those affected are unable to remain gainfully employed within 10 years of disease onset. A growing body of evidence now points to inflammation, locally and more systemically, as a promoter of damage to joints and bones, as well as joint‐related functional deficits. The pathogenesis underlying joint diseases remains unclear; however, it is currently believed that cross‐talk between cartilage and subchondral bone—and loss of balance between these two structures in joint diseases—is a critical element. This view is amplified by the presence of mast cells, whose dysregulation is associated with alterations of junction structures (cartilage, bone, synovia, matrix, nerve endings, and blood vessels). In addition, persistent activation of mast cells facilitates the development of spinal neuroinflammation mediated through their interaction with microglia. Unfortunately, current treatment strategies for rheumatic and articular disease are symptomatic and do little to limit disease progression. Research now should be directed at therapeutic modalities that target osteoarticular structural elements and thereby delaying disease progression and joint replacement.

Chronic Pain and Neurodegenerative Processes in Elderly People

There is an increasing interest in the relationship between anatomo-physiological modifications of the nervous system and chronic pain, especially in the elderly. Apparently, contradictory data have been reported, as in the high incidence of chronic pain in the older population despite a significant elevation in the pain perception threshold in the same age group. Those data, also confirmed by the fMRI, might be related to the progressive hypofunctionality of Ad fibers. Nevertheless, the tolerance to pain seems unaltered or even reduced with age. Data indicate a hyposensitivity to low intensity stimuli and a hypersensitivity to high intensity stimuli. In other words, it seems that the elderly perceive pain later, but once perceived the pain becomes rapidly intolerable. This reduced early sensitivity might in part be responsible for the high incidence of accidents and thus contributes to the high prevalence of chronic pain reported in the elderly. Additionally, there are other important modifications of the pain perception system in this age group. For example, there is a clear reduction in the descending inhibitory capacity with an associated increase in central sensitization. These epiphenomena can be postulated to be the consequence of yet other anatomo-physiological modifications coincident with aging. There are many experimental data clearly showing the importance of cells in the nervous system other than neurons, such as mast cells and microglia. In older people, the production of factors essential for normal function of the stromal cells, also present in the nervous system, is compromised. However, the production of mast cell precursors is not affected. Moreover, mast cells in many tissues of the elderly are hypersensitive to inflammatory mediators and show an increased capacity for degranulation. In other tissues, the mast cell density is significantly increased with age. In the endoneural compartment, such an increase might also be caused by progressive neuronal damage. The increased endoneural number of mast cells and their progressive hyper-reactivity with age play a major role in the determination of the altered functionality of the pain receptors and the pain primary fibers. Moreover, hyper-reactive endoneural mast cells seem to be responsible for the progressive deterioration of the Ad fibers via the uncontrolled release of enzymes. Like the mast cells, the microglia (which are arguably the most important immune cells of the central nervous system [CNS]) are functionally very much modified with age. In normal conditions, microglia are present in the CNS in a physiological branched form. When stimulated, they become immediately activated or proinflammatory. This modification is responsible for the rapid reduction of the neuroinflammation and a rapid restoration of the neurohomeostasis. In elderly people, microglia are present in a primed phenotype, which is responsible for a more intense and long-lasting response. This abnormally exuberant reaction seems to be a main cause for persistent neuroinflammation and irreversible neuronal damage. The primed microglia present at spinal cord level and in the thalamus, because of an excessive response to peripheral painful stimuli, seem to facilitate the initiation of chronic and neuropathic pain. Moreover, direct action on secondand third-order neurons in the pain pathway seems to be responsible for the neuronal hyperexcitability (central pain). Proinflammatory cytokines over-released by activatedmicroglia may also damage the integrity of the white substance and the ultrastructure of the myelin sheath. In fact, a reduction in myelin proteins has been related to the increased glial activation. These data implicate deep modifications in Ad fibers with age as major contributors to chronic pain states, as opposed to alterations in the integrity of C fibers. The altered reactivity of mast cells and microglia in the older people is increasingly emerging as a cofactor, if DOI: 10.1111/papr.12254