Successful treatment of SAPHO syndrome with apremilast.

SAPHO (synovitis, acne, pustulosis, hyperostosis and osteitis) syndrome is a rare disease with inflammatory osteoarticular and skin involvement. The pathogenesis of SAPHO syndrome remains unclear, but evidence suggests it may be an autoinflammatory disease triggered upon exposure to infectious agents in genetically predisposed individuals. Induction of the interleukin (IL)-23/T helper 17 axis in addition to neutrophil activation seem to play a key role, and therapies targeting these immunological pathways, including tumour necrosis factor (TNF) inhibitors, ustekinumab, secukinumab and the IL-1 inhibitor anakinra, are potential treatment options that need further investigation. Here we report a case of a 24-year-old woman with SAPHO syndrome who presented at our clinic with palmoplantar pustulosis and sternoclavicular joint involvement. Previous treatments with topical steroids and keratolytics combined with nonsteroidal anti-inflammatory drugs, intravenous methylprednisolone, methotrexate and sulfasalazine had all failed to improve symptoms. Therapy with etanercept was not tolerated, and because of a previous demyelinating peripheral neuropathy, further treatment with TNF inhibitors was avoided. We initiated ustekinumab 45 mg, which improved skin manifestations but not joint pain. Dose escalation to 90 mg initially improved joint pain, but the dose had to be reduced to 45 mg again because of increased infections. During subsequent 45-mg ustekinumab treatment, joint pain exacerbated so we switched to adalimumab which caused an exacerbation of the disease, so we switched to secukinumab, which improved skin and joint symptoms significantly but was associated with a pustular hypersensitivity reaction. Finally, we began treatment with apremilast, a pan-cytokine approach, resulting in stabilization of the skin and joint symptoms without side-effects. To our knowledge, this is the first case report of apremilast as a treatment for SAPHO syndrome.

Hydroxytyrosol modulates the levels of microRNA-9 and its target sirtuin-1 thereby counteracting oxidative stress-induced chondrocyte death.

OBJECTIVE: Nutraceutical compounds, such as hydroxytyrosol (HT), have been found to exert protective effects in osteoarthritis (OA) by affecting a variety of key molecular and cellular processes in chondrocytes. However, to our knowledge, no relationship has been reported between nutraceuticals and microRNA (miR) network in OA models. Here, we identified a miR that is implicated in HT-mediated chondroprotection following oxidative stress condition by targeting sirtuin-1 (SIRT-1). METHODS: Human primary and C-28/I2 chondrocytes were pre-treated with 100 µM HT 30 min before 100 µM H2O2 addition. In silico analyses were exploited to select putative candidate miRs able to target SIRT-1 mRNA. Luciferase-based gene reporter assay was employed to demonstrate the direct link between miR-9 and its putative mRNA target. Transient transfection approach was performed to examine the effects of miR-9 levels on caspase activity, cell viability and expression of OA-related genes. RESULTS: MiR-9 was identified and confirmed as a post-transcriptional regulator of SIRT-1. MiR-9 and SIRT-1 levels showed opposite changes in chondrocytes following H2O2 and HT treatment. Moreover mir-9 silencing inhibited cell death induced by H2O2 partly through down-regulation of SIRT-1, whereas miR-9 overexpression markedly reduced the protective effect of HT. The manipulation of miR-9 levels also resulted in the modulation of OA-related gene expression, including MMP-13, VEGF and RUNX-2. CONCLUSIONS: These results show that miR-9 is a critical mediator of the deleterious and OA-related effects of oxidative stress in chondrocytes and that modulation of miR expression may be a crucial mechanism underlying the protective action of HT.

What to Do, and What Not to Do, When Diagnosing and Treating Breakthrough Cancer Pain (BTcP): Expert Opinion.

Clinical management of breakthrough cancer pain (BTcP) is still not satisfactory despite the availability of effective pharmacological agents. This is in part linked to the lack of clarity regarding certain essential aspects of BTcP, including terminology, definition, epidemiology and assessment. Other barriers to effective management include a widespread prejudice among doctors and patients concerning the use of opioids, and inadequate assessment of pain severity, resulting in the prescription of ineffective drugs or doses. This review presents an overview of the appropriate and inappropriate actions to take in the diagnosis and treatment of BTcP, as determined by a panel of experts in the field. The ultimate aim is to provide a practical contribution to the unresolved issues in the management of BTcP. Five 'things to do' and five 'things not to do' in the diagnosis and treatment of BTcP are proposed, and evidence supporting said recommendations are described. It is the duty of all healthcare workers involved in managing cancer patients to be mindful of the possibility of BTcP occurrence and not to underestimate its severity. It is vital that all the necessary steps are carried out to establish an accurate and timely diagnosis, principally by establishing effective communication with the patient, the main information source. It is crucial that BTcP is treated with an effective pharmacological regimen and drug(s), dose and administration route prescribed are designed to suit the particular type of pain and importantly the individual needs of the patient.

Stem cell-mediated muscle regeneration and repair in aging and neuromuscular diseases.

One of the most exciting aspirations of current medical science is the regeneration of damaged body parts. The capacity of adult tissues to regenerate in response to injury stimuli represents an important homeostatic process that until recently was thought to be limited in mammals to tissues with high turnover such as blood and skin. However, it is now generally accepted that each tissue type, even those considered post-mitotic, such as nerve or muscle, contains a reserve of undifferentiated progenitor cells, loosely termed stem cells, participating in tissue regeneration and repair. Skeletal muscle regeneration is a coordinate process in which several factors are sequentially activated to maintain and preserve muscle structure and function upon injury stimuli. In this review, we will discuss the role of stem cells in muscle regeneration and repair and the critical role of specific factors, such as IGF-1, vasopressin and TNF-alpha, in the modulation of the myogenic program and in the regulation of muscle regeneration and homeostasis.

Hypertrophy and transcriptional regulation induced in myogenic cell line L6-C5 by an increase of extracellular calcium.

Calcium plays a pivotal role in the establishment of the differentiated phenotype in myogenic cells but the involved molecular mechanisms are still matter of debate. Here we studied the effects of exposing L6-C5 myogenic cells to high extracellular Ca2+ concentration ([Ca2+]o), which induces an increase of intracellular calcium ([Ca2+]i) without involving Ca2+ release from the intracellular stores but exclusively due to plasma membrane influx (Naro et al., 2003). Exposure of L6-C5 cells to [Ca2+]o up to 20 mM for 30 min, before shifting them into a differentiative medium, induced the appearance of multinucleated, myosin-positive myotubes, much larger than in control cells with an increased protein/DNA ratio. These large myotubes showed nuclear accumulation of the hypertrophy marker GATA-2. The hypertrophic growth of these cells was blocked by cyclosporin A (CsA), FK506, or overexpression of a calcineurin-dominant negative protein, suggesting the involvement in this process of the Ca2+ responsive phosphatase calcineurin. Furthermore, transient exposure of L6-C5 cells to high [Ca2+]o increased the expression of luciferase reporter driven by myoglobin (Mb) and beta-MHC promoters but not IIB-MHC and MCK promoters. Luciferase transcription driven by CK promoter was, instead, enhanced by mobilizing Ca2+ from the intracellular stores. These data indicate that a transient increase of [Ca2+]i due to plasma-membrane influx is sufficient to induce a hypertrophic phenotype and an increased expression of slow-fiber genes but not fast-fiber genes.

Surgical results and functional outcome after total anorectal reconstruction by double graciloplasty supported by external-source electrostimulation and/or implantable pulse generators: an 8-year experience.

BACKGROUND AND AIMS: Surgical and functional results after abdominoperineal resection and total anorectal reconstruction by electrostimulated gracilis muscle transposition are still poorly documented. This study prospectively evaluated surgical and functional outcome over time in our patients. PATIENTS AND METHODS: Twenty-three patients underwent abdominoperineal resection, coloperineal pullthrough, double graciloplasty, and loop abdominal stoma. Temporary external-source intermittent electrostimulation, biofeedback training, and selective delayed stimulator implantation to improve unsatisfactory results were carried out in the first 13 patients (1st series); thereafter (2nd series) the stimulator was implanted during graciloplasty. Surgical and oncological results were followed up in all patients. Functional results were evaluated in 16 patients who underwent abdominal stoma takedown, eight in each of the two series, by anomanometry (up to 1 year) and our own 0-20 scoring system (up to 8 years from initial surgery). RESULTS: The rate of major and minor postoperative complications was 21.7% and 65%, respectively. Continuous electrostimulation proved effective on resting anal pressure. Early clinical assessments showed satisfactory functional results (considered as having a score < or =8) in all first-group patients, including five who had stimulator support, and in one-half of second-group patients. After impairment (at least 2 points) at 1 year in five patients, four of whom were from the first group, all functional results improved and became satisfactory from 5 years on (1st series) and from 4 years on (2nd series). CONCLUSION: Despite marked morbidity the high rate of good results, which improved over time, suggests that total anorectal reconstruction is worth being performed as part of abdominoperineal resection in well-selected patients with a strong motivation to avoid a permanent colostomy.

A patient-rated, surgeon-corrected scale for functional assessment after total anorectal reconstruction. An adaptation of the Working Party on Anal Sphincter Replacement scoring system.

BACKGROUND AND AIMS: To test current systems evaluating for fecal continence after total anorectal reconstruction (TAR) we adapted the incontinence plus evacuation scoring system proposed by the Working Party on Anal Sphincter Replacement (WPASR). PATIENTS AND METHODS: We examined 51 monthly diaries recorded by 14 patients after TAR or at yearly checks (up to 5 years). A form detailing all items and frequencies of the WPASR system was given to 12 patients who assigned a rating to each item in a frequency cell. The mean values of cells were rounded off, and a 0-20 scoring scale was obtained. We corrected the scores using previously defined criteria aimed at complying with an objective rating of severity while preserving the overall patient rating. Diaries were reevaluated by both patient-rated and surgeon-corrected scales, and the correlation was calculated to each other and to Jorge and Wexner's and the Williams et al. systems; correlations between incontinence and evacuation scores were also calculated. RESULTS: The surgeon-corrected system tended to have a lower mean score than the patient-rated one and was strongly correlated with it ( r=0.984), a significantly higher mean score than the Jorge and Wexner scale ( r=0.893), and a significantly lower mean score than the Williams et al. classification (quadrupled scores; r=0.857). No correlations between incontinence and evacuation were found. CONCLUSIONS: Although not validated, the patient-rated, surgeon-corrected adjustment of the WPASR system proved in our patients a reliable instrument for functional assessment. Its consensus administration to any given patient samples requires further research.

Surface remodeling associated with vasopressin-induced membrane traffic in L6 myogenic cells.

The plasma membrane is dynamically remodeled as a function of the cell cycle, motility and membrane traffic. We have previously shown that arg8-vasopressin (AVP) stimulation of L6 myoblasts induces the activation of phosholipase D during the first minutes of stimulation, and the differentiation of 1,6 myoblasts as a long term effect. We now report that AVP also induces two types of morphological responses in L6 cells within a few minutes of stimulation: exocytosis, apparent as uncoated pits, and the generation of membrane projections and reffles. Thus, such an experimental model is suitable for the study of hormone-induced morphological surface modifications and their regulatory mechanisms. In L6 cells, AVP-induced projection generation depends on the integrity of microfilaments, intermediate filaments, and microtubules. Moreover, projection generation and exocytosis appear to be independently regulated phenomena: in fact, inhibition of the de novo synthesis of phosphatidylcholine inhibits membrane traffic but fails to block projection appearance. Conversely, the latter phenomenon, unlike exocytosis, is mediated by PI3-kinase signaling. Thus, AVP induces two early, independently regulated morphological modifications in L6 cells: exocytosis, involved in plasma membrane phospholipid turnover, and membrane projections, likely involved in cell migration.

Involvement of type 4 cAMP-phosphodiesterase in the myogenic differentiation of L6 cells.

Myogenic cell differentiation is induced by Arg(8)-vasopressin, whereas high cAMP levels and protein kinase A (PKA) activity inhibit myogenesis. We investigated the role of type 4 phosphodiesterase (PDE4) during L6-C5 myoblast differentiation. Selective PDE4 inhibition resulted in suppression of differentiation induced by vasopressin. PDE4 inhibition prevented vasopressin-induced nuclear translocation of the muscle-specific transcription factor myogenin without affecting its overall expression level. The effects of PDE4 inhibition could be attributed to an increase of cAMP levels and PKA activity. RNase protection, reverse transcriptase PCR, immunoprecipitation, Western blot, and enzyme activity assays demonstrated that the PDE4D3 isoform is the major PDE4 expressed in L6-C5 myoblasts and myotubes, accounting for 75% of total cAMP-hydrolyzing activity. Vasopressin cell stimulation caused a biphasic increase of PDE4 activity, which peaked at 2 and 15 min and remained elevated for 48 h. In the continuous presence of vasopressin, cAMP levels and PKA activity were lowered. PDE4D3 overexpression increased spontaneous and vasopressin-dependent differentiation of L6-C5 cells. These results show that PDE4D3 plays a key role in the control of cAMP levels and differentiation of L6-C5 cells. Through the modulation of PDE4 activity, vasopressin inhibits the cAMP signal transduction pathway, which regulates myogenesis possibly by controlling the subcellular localization of myogenin.

Vasopressin and insulin-like growth factors synergistically induce myogenesis in serum-free medium.

Terminal differentiation of myogenic cells has long been known to be positively regulated by insulin-like growth factors (IGFs). Arg8-vasopressin (AVP) has been recently reported to potently induce myogenic differentiation. In the present study, the effects and the mechanisms of action of AVP and IGFs on myogenic cells have been investigated under conditions allowing growth and differentiation of myogenic cells in a simple serum-free medium. Under these conditions, L6 and L5 myogenic cells slowly proliferate and do not undergo differentiation (less than 1% fusion up to 7 days). AVP rapidly (2-3 days) and dose-dependently induces the formation of multinucleated myotubes. Creatine kinase activity and myosin accumulation are strongly up-regulated by AVP. Insulin or IGF-I or IGF-II, at concentrations that cause extensive differentiation in serum-containing medium, induces a modest degree of differentiation in serum-free medium. The simultaneous presence of AVP and of one of the IGFs in the synthetic medium induces maximal differentiation of L6, L5, and satellite cells. The expression of both myogenin and Myf-5 is dramatically stimulated by AVP. Our results indicate that AVP induces a significant level of myogenic differentiation in the absence of other factors. Furthermore, they suggest that to express their full myogenic potential, IGFs require the presence of other factors normally present in serum and fully mimicked by AVP. These studies support the conclusion that terminal myogenic differentiation may depend on the presence of differentiation factors rather than the absence of growth factors.

Characterization of the retinoid binding properties of the major fusion products present in acute promyelocytic leukemia cells.

The bcr1- and bcr3- promyelocytic leukemia/retinoic acid receptor alpha (PML/RAR alpha) are the two major fusion proteins expressed in acute promyelocytic leukemia (APL) patients. These proteins, which are present in different lengths of PML (amino acids 1-552 and 1-394, respectively), contain most of the functional domains of PML and RAR alpha, bind all-trans-retinoic acid (t-RA), and act as t-RA-dependent transcription factors. T-RA is an effective inducer of clinical remission only in patients carrying the t(15;17) and expressing the PML/RAR alpha products. However, in APL patients achieving complete remission with t-RA therapy the bcr3-PML/RAR alpha product has been found associated with a poorer prognosis than bcr1-PML/RAR alpha. In the present study we have investigated the structural and functional properties of the bcr3-PML/RAR alpha in comparison to the previously characterized bcr1-PML/RAR alpha. In particular, we have measured the binding properties of the two endogenous ligands t-RA and 9-cis-RA to both of these isoforms. T-RA binding analysis of nuclear and cytosolic extracts prepared from bcr3-PML/RAR alpha APL patients and from bcr3-PML/RAR alpha COS-1 transfected cells indicates that this protein is present only as high-molecular-weight nuclear complexes. Using saturation binding assays and Scatchard analyses we found that t-RA binds with slightly less affinity to the bcr3-PML/RAR alpha receptor than to bcr1-PML/RAR alpha or RAR alpha (Kd = 0.4 nmol/L, 0.13 nmol/L or 0.09 nmol/L, respectively). Moreover, two different high-affinity 9-cis-RA binding sites (Kd = 0.45 and 0.075 nmol/L) were detectable in the bcr3-PML/RAR alpha product but not in the bcr1-PML/RAR alpha product (Kd = 0.77 nmol/L). By competition binding experiments we showed that 9-cis-RA binds with higher specificity to the bcr3-PML/RAR alpha isoform than to the bcr1-PML/RAR alpha or RAR alpha. Consistent with these data, the binding of 9-cis-RA to the bcr3-PML/RAR alpha product resulted in increased transcriptional activation of the RA-responsive element (RARE) TRE, but not of the betaRARE, in transiently transfected COS-1 cells. These results provide evidence indicating that preferential retinoid binding to the different PML/RAR alpha products can be measured.

Correlation between changes in host behaviour and octopamine levels in the tobacco hornworm Manduca sexta parasitized by the gregarious braconid parasitoid wasp Cotesia congregata

The parasitoid wasp Cotesia congregata lays its eggs within the body of its host, the larval form of the tobacco hornworm Manduca sexta. Host behaviour appeared normal until approximately 8 h prior to the emergence of the parasitoids from their host at which time M. sexta feeding and locomotion declined irreversibly. This change in host behaviour may be to the advantage of the wasp since unparasitized M. sexta presented with wasp pupae ate them. Despite the decline in feeding and locomotion, hosts with emerged parasitoids had normal reflexes and showed no other signs of debilitation. Concomitant with the change in host behaviour, octopamine concentration measured using high-performance liquid chromatography with electrochemical detection (HPLC-ED) increased from 22.2±2.1 pg µl-1 to 143.7±7.8 pg µl-1 in the haemolymph of the host. In unparasitized M. sexta, however, increased octopamine levels were correlated with increased activity. We discuss possible explanations for the co-occurrence of high haemolymph octopamine levels and low behavioural arousal in parasitized M. sexta.

Retinoid-induced differentiation of acute promyelocytic leukemia involves PML-RARalpha-mediated increase of type II transglutaminase.

All-trans retinoic acid (t-RA) administration leads to complete remission in acute promyelocytic leukemia (APL) patients by inducing growth arrest and differentiation of the leukemic clone. In the present study, we show that t-RA treatment dramatically induced type II transglutaminase (type II TGase) expression in cells carrying the t(15;17) translocation and expressing the PML-RARalpha product such as the APL-derived NB4 cell line and fresh leukemic cells from APL patients. This induction correlated with t-RA-induced growth arrest, granulocytic differentiation, and upregulation of the leukocyte adherence receptor beta subunit (CD18) gene expression. The increase in type II TGase was not abolished by cycloheximide treatment, suggesting that synthesis of a protein intermediate was not required for the induction. t-RA did not significantly alter the rate of growth arrest and did not stimulate differentiation and type II TGase activity in NB4.306 cells, a t-RA-resistant subclone of the NB4 cell line, or in leukemic cells derived from two patients morphologically defined as APL but lacking the t(15;17). However, in NB4.306 cells, t-RA treatment was able to increase CD18 mRNA expression in a manner similar to NB4 cells. The molecular mechanisms involved in the induction of these genes were investigated. In NB4 cells, using novel receptor-selective ligands such as 9-cis-RA, TTNPB, AM580, and SR11217, we found that RAR- and RARalpha-selective retinoids were able to induce growth arrest, granulocytic differentiation, and type II TGase, whereas the RXR-selective retinoid SR11217 was inactive. Moreover, an RAR alpha-antagonist completely inhibited the expression of type II TGase and CD18 induced by these selective retinoids in NB4 cells. In NB4.306 cells, an RARalpha-dependent signaling pathway was found involved in the modulation of CD18 expression. In addition, expression of the PML-RARalpha gene in myeloid U937 precursor cells resulted in the ability of these cells to induce type II TGase in response to t-RA. On the basis of these results we hypothesize a specific involvement of a signaling pathway involving PML-RAR alpha for the induction of growth arrest, granulocytic differentiation, and type II TGase by retinoids in APL cells.

Rapid activation and down-regulation of protein kinase C alpha in 12-O-Tetradecanoylphorbol-13-acetate-induced differentiation of human rhabdomyosarcoma cells.

Human rhabdomyosarcoma RD cells express the myogenic regulatory factors MyoD and myogenin but differentiate spontaneously very poorly. Prolonged treatment of RD cells with the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA) induces growth arrest and myogenic differentiation as shown by the accumulation of alpha-actin and myosin light and heavy chains, without affecting the expression of MyoD and myogenin. In this study, we show that short-term phorbol ester treatment of the cultures is sufficient to trigger myogenic differentiation but not growth arrest. Furthermore, PKC inhibitors, such as staurosporine or calphostin C, prevent TPA-induced differentiation but not cell growth arrest. These data suggest that the two events are mediated by different pathways; a possible interpretation is that the activation of one or more PKC isoforms mediates the induction of differentiation, whereas the down-regulation of the same or different isoforms mediates the growth arrest. To address the mechanism whereby TPA affects cell growth and differentiation in RD cells, we first analyzed PKC isoenzyme distribution. We found that RD cells express the alpha, beta 1, gamma, and sigma PKC isoenzymes. Only the alpha isoform is exclusively found in the soluble fraction, but it translocates to the membrane fraction within 5 min of TPA treatment and is completely down-regulated after 6 h. The other isoenzymes are found associated to both the soluble and the particulate fractions and are down-regulated after long-term TPA treatment. By immunofluorescence analysis, we show that the PKC alpha down-regulation is specific for those cells that respond to TPA by activating the muscle phenotype. We propose that TPA-induced differentiation in RD cells is mediated by the transient activation of PKC alpha, which activates some of the intracellular events that are necessary for MyoD and myogenin transacting activity and for the induction of terminal differentiation of RD cells. By contrast, the constitutively active beta 1 and sigma are responsible for the maintenance of cell growth, and their down-regulation is responsible for long-term TPA-induced cell growth arrest.

Arginine-vasopressin induces differentiation of skeletal myogenic cells and up-regulation of myogenin and Myf-5.

The neurohypophyseal nonapeptide arginine8-vasopressin (AVP) induces phosphoinositide turnover and calcium and pH changes in skeletal myogenic cells in culture. In order to investigate the effect of AVP on skeletal myogenesis, we examined the effect of this hormone on proliferating mononucleated L6 myoblast cultures. Addition of AVP to the medium resulted in the formation of much larger myotubes than those formed in its absence and in a significant increase (2.2-fold) of the percentage of fusion within 3-4 days of treatment. The effect of AVP was dose dependent, in the 10 nM to 1 microM range, and was observed also in primary cultures of mouse satellite cells. The rate of growth of L6 cells was not affected by AVP treatment. The induction of morphological differentiation by AVP correlated with an increased expression of muscle-specific gene products, such as myosin, and an increased number of acetylcholine receptor sites. The accumulation of mRNA transcripts of the acetylcholine receptor subunits was also enhanced by AVP. The mechanism involved in the myogenic action of AVP was investigated. Using AVP-related peptides and antagonists, we found that a specific chemical structure is required and that V1 receptors probably mediate the effect on myogenesis. Expression of muscle-specific transcription factor genes Myf-5 and myogenin and their products are strongly upregulated by AVP. Our findings support the hypothesis that AVP may represent a novel physiological modulator of skeletal muscle differentiation through its action on muscle regulatory genes.

12-O-tetradecanoylphorbol-13-acetate-induced differentiation of a human rhabdomyosarcoma cell line.

The effect of 12-O-tetradecanoyl phorbol-13-acetate (TPA) on proliferation and differentiation of the human embryonal rhabdomyosarcoma cell line RD was investigated. The proliferation of RD cells is drastically and reversibly inhibited by 100 nM TPA. The effect is evident after 24 h of treatment and is maximal after 50-70 h. The reduction of proliferation in treated cells is followed by increased expression of differentiative characters such as a large increase in muscle myosin expression and in the binding of 125I-alpha-bungarotoxin. Moreover TPA induces the appearance of myotube-like structures, which contain bundles of thick and thin myofilaments along with Z bodies. The described effects are not observed if the TPA-containing medium is replaced daily, thus suggesting that these effects might be related to substances secreted by treated cells. The phosphorylation of three proteins is significantly stimulated by TPA within minutes of its administration to RD cells. Although with a different pattern, the stimulation of protein phosphorylation is still clearly detectable after 6 days of incubation with TPA. These results on human rhabdomyosarcoma cells are, to our knowledge, the first evidence for a growth-inhibiting and a differentiative effect of TPA on a solid tumor of mesodermal origin.