BRONJ in patients with rheumatoid arthritis: a multicenter case series.

OBJECTIVE: Osteonecrosis of the jaw (ONJ) is a potentially severe adverse effect of various medications (bisphosphonates, anti-resorptive, and anti-angiogenic drugs). ONJ pathogenesis is still unclear although some risk factors have been recognized. Of these, rheumatoid arthritis (RA) has been hypothesized as a potential risk factor for developing ONJ. This observational study will describe a multicenter case series of patients affected with RA and ONJ, and it will attempt to evaluate the association between features of ONJ and pharmacological, systemic, and site variables. METHODS: Demographic, pharmacological, and clinical data from 18 RA patients with ONJ were collected and registered from three Italian centers (i.e., Palermo, Verona, and Padua) from 2004 to 2013. RESULTS: Sixteen (88.9%) patients were in therapy for RA: 9 of 18 (50.0%) with systemic steroids, 3 of 18 (16.7%) with methotrexate, and 4 of 18 (22.2%) with both medications. Two patients were not receiving treatment for RA. All patients took NBPs for secondary osteoporosis (average NBP duration of 69 months, range: 20-130): Fifteen (83.3%) patients were treated with single NBPs, while three (16.7%) with different molecules; one patient was also treated with denosumab. Mandible was affected more frequently (66.7%) than maxilla (33.3%); one patient presented multiple ONJ events. CONCLUSIONS: This is the first multicenter case series in the international literature regarding our topic. Focusing on our data, it could be hypothesized that patients with RA may be more susceptible to ONJ than the majority of osteometabolic patients. In our opinion, it could be important to monitor also denosumab or other biological drug side effects.

Prevalence survey of healthcare-associated infections and antimicrobial use at the University Hospital "Paolo Giaccone", Palermo, Italy.

INTRODUCTION: Healthcare-associated infections (HAIs) and antimicrobial resistance are well known major public health threats. The first goal of our study was to describe the prevalence of HAI, while the second goal was to describe the antibiotic consumption at our University Hospital, "P. Giaccone" in Palermo, Italy. METHODS: A standardized methodology for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use in European acute care hospital developed by the European Centre for Disease Prevention and Control (ECDC) was piloted across Europe. The teaching Hospital "P. Giaccone" in Palermo, Italy, participated in the study. RESULTS: Out of 328 surveyed patients, 12 (3.6%) had an HAI and 159 (48.5%) were receiving at least one antimicrobial agent. Prevalence results were highest in intensive care units, with 17.6% patients with HAI. Bloodstream infections represented the most common type (50%) of HAI. Surgical prophylaxis was the indication for antimicrobial prescribing in 59 (37.1%) out of 159 patients and exceeded 24 hours in 54 (91.5%) cases. DISCUSSION: The results suggest that in our hospital there was a frequent and inappropriate use of antimicrobials, especially in the setting of surgical prophylaxis.

Chemotherapy-induced cardiotoxicity: role of the conventional echocardiography and the tissue Doppler.

AIM: The cardiotoxicity of anticancer drugs is an emerging problem and only an identification of the early signs of cardiotoxicity by conventional echocardiography and not (tissue Doppler imaging, TDI), will limit and contain the long-term cardiotoxicity effects. The aim of this study was to identify, through conventional echocardiography and TDI, parameters to use as early "signs" of cardiotoxicity. METHODS: A prospective study was performed using patients with breast cancer (72 women, median age 57 ± 12) treated with anticancer drugs (adjuvant chemotherapy). All patients underwent a careful cardiological evaluation before starting treatment (T0) and during follow-up at 3 months (T1), 6 months (T2) and 1 year (T3). Electrocardiography and echocardiography were performed in all patients in these times. Echocardiography evaluation considered the following parameters: systolic and diastolic diameters and volumes, LVEF, MAPSE, TAPSE, E/A TDI (Em, Am, Sm, IVCT, IVRT, ET, TEI index). On the basis of chemotherapy treatment, patients were divided into 5 groups: A=FEC (fluorouracil, epirubicin, cyclophosphamide), B=FEC+trastuzumab, C=trastuzumab, D=FEC+taxotere, E=FEC+taxolo+trastuzumab. RESULTS: A significant reduction in the echo parameters of TDI was observed. TDI appears to offer important advantages over traditional techniques in revealing the presence of early signs of cardiotoxicity. CONCLUSION: The TDI should be utilized to complement conventional echocardiography in the assessment of cardiotoxicity.

Regional changes in the metabolite profile after long-term hypoxia-ischemia in brains of young and aged rats: a quantitative proton MRS study.

Quantitative proton magnetic resonance spectroscopy (MRS) was used to determine region-specific metabolic changes in young and aged animals subjected to a long-term hypoxic-ischemic injury. Focal ischemia, which was studied as an experimental stroke model, was induced in 3- and 24-month-old rats by unilateral common carotid artery occlusion associated with 24 h of hypoxia. Eight metabolites were quantified from extracts in three different brain regions (hippocampus, frontoparietal and occipital cortices) from both the ipsilateral and contralateral sides. Our findings showed significant differences in lactate and myo-inositol concentration values in the hippocampus of the aged rats as compared to the same area of the young adult group under normoxic conditions. After hypoxia-ischemia (HI), the most relevant changes in metabolite concentrations were found in the hippocampal region of both young and aged groups as compared to their age-matched controls. Of the three brain areas under investigation, the hippocampus proved to be particularly susceptible to the prolonged hypoxia-ischemia perturbation. The effects were more evident in the aged animals.

Sex hormones and risk of liver tumor.

The liver is morphologically and functionally modulated by sex hormones. Long-term use of oral contraceptives (OCs) and anabolic androgenic steroids (AASs) can induce both benign (hemangioma, adenoma, and focal nodular hyperplasia [FNH]) and malignant (hepatocellular carcinoma [HCC]) hepatocellular tumors. Hepatic adenomas (HAs) are rare, benign neoplasms usually occurring in young women, the development and the complications of which have been related to the strength of OCs and the duration of their use. HA incidence has fallen since the introduction of pills containing smaller amounts of estrogens. FNH is a benign lesion, most commonly seen in young women, which is thought to represent a local hyperplastic response of hepatocytes to a vascular abnormality. Because of the female predominance and the young age at onset, a role of female hormones has been suggested. Furthermore, a large proportion of women with FNH (50-75%) are OC users. Liver hemangiomas (LHs) are the most common benign liver tumors and are seen more commonly in young adult females. The female predilection and clinical observations of LH growth under conditions of estrogenic exposure suggest a possible role for estrogen in the pathogenesis of LHs. HCC has become one of the most widespread tumors in the world in recent years, representing the sixth leading cancer and the third most common cause of death from cancer. Apart from liver cirrhosis, numerous other factors responsible for its onset have been proposed: hepatitis infections from virus B (HBV) and C (HCV), alcohol, smoking, and aflatoxin. However, regardless of etiology, chronic liver diseases progress at unequal rates in the two sexes, with the major sequelae, such as cirrhosis and HCC, being more frequent in men than in women. These epidemiological data have prompted researchers to investigate the relationship between sex hormones and liver tumors. The human liver expresses estrogen and androgen receptors and experimentally both androgens and estrogens have been implicated in stimulating hepatocyte proliferation and may act as liver tumor inducers or promoters.

Circulating IL-6 and sIL-6R in patients with hepatocellular carcinoma.

Interleukin-6 plays a central role in regulating the immune system, hematopoiesis, and acute phase reaction. It interacts with a receptor complex consisting of a specific ligand-binding protein (IL-6R, gp80) and a signal transduction protein (gp130). In this report, serum levels of IL-6 and a soluble form of the interleukin-6 receptor (sIL-6R) were evaluated in patients with hepatocellular carcinoma. The correlation between IL-6 and sIL-6R values, the stage of hepatocellular carcinoma, and main liver function tests was also studied.

Expression of HIP/PAP mRNA in human hepatoma cell lines.

The present study attempts to shed more light on the role of hepatocarcinoma-intestine-pancreas/pancreatic associated protein (HIP/PAP) in hepatoma cells. We initially examined, by reverse transcription-polymerase chain reaction (RT-PCR), the HIP/PAP transcripts present in human hepatoma cell lines of different origins and with different grades of differentiation and genetic profiles. We also used DNA sequencing analysis to investigate the structure of the HIP/PAP gene. Further investigation is necessary to define the role of HIP/PAP during the development of human hepatocellular carcinoma and to ascertain whether the use of different transcripts is helpful in regulating HIP/PAP expression in transformed liver cells.

Heterocycle-containing retinoids. Discovery of a novel isoxazole arotinoid possessing potent apoptotic activity in multidrug and drug-induced apoptosis-resistant cells.

In a search for retinoic acid (RA) receptor ligands endowed with potent apoptotic activity, a series of novel arotinoids were prepared. Because the stereochemistry of the C9-alkenyl portion of natural 9-cis-RA and the olefinic moiety of the previously synthesized isoxazole retinoid 4 seems to have particular importance for their apoptotic activity, novel retinoid analogues with a restricted or, vice versa, a larger flexibility in this region were designed and prepared. The new compounds were evaluated in vitro for their ability to activate natural retinoid receptors and for their differentiation-inducing activity. Cytotoxic and apoptotic activities were, in addition, evaluated. In general, these analogues showed low cytotoxicity, with the restricted structures being slightly more active than the more flexible ones. As an exception, however, the isoxazole retinoid 15b proved to be particularly able to induce apoptosis at concentrations <5 microM, showing a higher activity than the classical retinoids such as all-trans-RA, 13-cis-RA, and 9-cis-RA and the previously described synthetic retinoid 4. 15b also exhibited a good affinity for the retinoid receptors. Interestingly, another important property of 15b was its ability to induce apoptosis in the HL60R multidrug-resistant (MDR) cell line, at the same concentration as is effective in HL60. Therefore, 15b represents a new retinoid possessing high apoptotic activity in an MDR cell line. The ability of 15b to act on K562 and HL60R cells suggests that this compound may have important implications in the treatment of different leukemias, and its structure could offer an interesting model for the design of new compounds endowed with apoptotic activity on MDR- and retinoid-resistant malignancies.

Downregulation of wild-type beta-catenin expression by interleukin 6 in human hepatocarcinoma HepG2 cells: a possible role in the growth-regulatory effects of the cytokine?

We investigated the antitumour effects of interleukin 6 (IL-6) on hepatocarcinoma HepG2 cells, endowed with high levels of a mutated, non-degradable, beta-catenin. IL-6 produced minimal growth-inhibitory effects and no apoptosis or gross changes in cell adhesion. Interestingly, however, it caused a consistent decrease in the cytoplasmic levels of wild-type, but not of mutated, beta-catenin protein. There was no effect on E-cadherin or gamma-catenin and a reduction in alpha-catenin occurred only at high concentrations. IL-4, a non-related cytokine, did not modify the content of beta-catenin. IL-6 did not influence beta-catenin mRNA levels. LiCl, a potent inhibitor of Glycogen Synthase Kinase 3beta (GSK3beta) activity, abrogated the IL-6-induced inhibition of wild-type beta-catenin. This indicates that IL-6 can affect wild-type beta-catenin through a post-transcriptional mechanism, probably involving degradation of the protein. This effect might be related to the growth-regulatory activities of IL-6 in other situations, but can not counteract the oncogenic expression of mutated beta-catenin in HepG2 cells or possibly in other tumour cells with similar gene mutations.

Calmodulin distribution and the actomyosin cytoskeleton in Toxoplasma gondii.

The gliding motility of the protozoan parasite Toxoplasma gondii and its invasion of cells are powered by an actin-myosin motor. We have studied the spatial distribution and relationship between these two cytoskeleton proteins and calmodulin (CaM), the Ca(2+)-dependent protein involved in invasion by T. gondii. A 3D reconstruction using labeling and tomographic studies showed that actin was present as a V-like structure in the conoidal part of the parasite. The myosin distribution overlapped that of actin, and CaM was concentrated at the center of the apical pole. We demonstrated that the actomyosin network, CaM, and myosin light-chain kinases are confined to the apical pole of the T. gondii tachyzoite. MLCK could act as an intermediate molecule between CaM and the cytoskeleton proteins. We have developed a model of the organization of the actomyosin-CaM complex and the steps of a signaling pathway for parasite motility.

Programmed cell death (PCD) associated with the stilbene motif of arotinoids: discovery of novel apoptosis inducer agents possessing activity on multidrug resistant tumor cells.

Considering that the stereochemistry of the C9-C10 alkenyl portion of natural 9-cis-RA, as the one of the olefinic moiety of the previously described isoxazole retinoid 4, seems of particular importance for their apoptotic activity, we prepared a novel class of TTNPB analogues bearing both the cis or trans configuration of the alkenyl portion. The compounds were evaluated in vitro for their cytotoxic and apoptotic activities. We discovered that the cis-TTNPB 9c possesses apoptotic activity comparable with that of the retinoid 4. Moreover, the amino arotinoid 16c showed potent apoptotic activity in HL60 promyelocytic leukemia cells. Interestingly, 16c proved to be a particularly potent apoptosis-inducing agent active in multidrug resistant (MDR) cell lines. Therefore, to the best of our knowledge, 16c may represent the first known aminoarotinoid endowed with potent apoptotic activity in MDR cells. Taken together, these results seem to point out that the cis-stilbene motif of arotinoids may be at least an important feature in conferring cytotoxic and apoptotic activity to this class of compounds.

Induction of apoptosis by arachidonic acid in human retinoblastoma Y79 cells: involvement of oxidative stress.

Arachidonic acid administration caused apoptosis in Y79 cells, as shown by typical morphological changes, phosphatidylserine externalization, chromatin condensation, processing and activation of caspase-3 and cleavage of the endogenous caspase substrate poly-(ADP-ribose)-polymerase. Arachidonic acid also caused lamin B cleavage, suggesting caspase-6 activation. Arachidonic acid treatment was accompanied by increased formation of the lipid peroxidation end products malondialdehyde and 4-hydroxy-2-nonenal, lowering in reduced glutathione content and in mitochondrial membrane potential. Inhibiting glutathione synthesis sensitized Y79 cells to apoptosis-inducing stimuli, whilst replenishing reduced glutathione attenuated arachidonic acid toxicity. Similar findings were obtained using hydroperoxyeicosatetranoic acids (oxygenated metabolites of arachidonic acid which deplete the reduced glutathione pool) and nordihydroguaretic acid, a general inhibitor of lipooxygenase pathway. which may also trigger rapid depletion of reduced glutathione. Melittin, which is known to activate phospholipase A2, also potently induced apoptosis. Arachidonic acid toxicity was inversely related to cell density. This could depend on an increased production of molecules with antiapoptotic effect; insulin-like growth factors could most likely be one of these molecules. These results propose a role for oxidative stress in the cytotoxicity induced by arachidonic acid in Y79 cells and suggest that these cells could be protected from such toxicity as long as sufficient levels of reduced glutathione and survival factors are present.

Bovine seminal RNase induces apoptosis in normal proliferating lymphocytes.

Bovine seminal ribonuclease is a member of the RISBAses (ribonucleases with special biological actions) family. It exerts specific anti-tumor, embryotoxic, aspermatogenic and immunosuppressive activities. The cytotoxic effect of bovine seminal ribonuclease on tumor cells is accompanied by the induction of apoptosis. We provide ultrastructural and flow cytometry evidence of apoptotic death following bovine seminal ribonuclease treatment, in normal cells and phytohemagglutinin-stimulated lymphocytes. Transmission and scanning electron microscopy, which were fully supported by flow cytometry data, showed typical features of apoptosis, such as decreased cell size, chromatin condensation, fragmentation in micronuclei, and the presence of apoptotic bodies.

Structure-activity relationship studies of novel heteroretinoids: induction of apoptosis in the HL-60 cell line by a novel isoxazole-containing heteroretinoid.

In a search for retinoic acid receptor (RAR and RXR)-selective ligands, a series of isoxazole retinoids was synthesized and evaluated in vitro in transcriptional activation and competition binding assays for RARs and RXRs. In addition, these compounds were evaluated for their differentiating, cytotoxic, and apoptotic activities. In general, these derivatives showed scarcely any binding affinity and were not active in the transcriptional assay. However, among these isoxazole derivatives, the cis-isomer 14b was identified as a potent inducer of apoptosis, and its activity was found to be 6.5 and 4 times superior than that of 13-cis- and 9-cis-retinoic acids, respectively. On the other hand, compound 13b, which has the trans stereochemistry at the double bond, was found not to be active in the apoptotic assay, but it was endowed with appreciable differentiating activity. Therefore, it seems that the different stereochemistry of the double bond may be associated with a different biological activity: potent apoptotic activity for the cis-isomer but differentiating activity for the trans structure. This biological behavior was found, at least in part, for the 9-cis- and 13-cis-retinoic acids with respect to the all-trans-retinoic acid. Thus, structure 14b could offer an interesting model for the design of new compounds endowed with apoptotic activity.

Blocking signaling through the Gp130 receptor chain by interleukin-6 and oncostatin M inhibits PC-3 cell growth and sensitizes the tumor cells to etoposide and cisplatin-mediated cytotoxicity.

BACKGROUND: The mechanisms of drug resistance associated with advanced, hormone-independent prostate carcinoma are poorly understood. The human prostate carcinoma PC-3 cell line, derived from a metastatic tumor and lacking androgen receptors, represents a useful model to investigate drug resistance. METHODS: The effects of oncostatin M (OM), antiinterleukin-6 (IL-6) treatment, or interference with the gp130-mediated signaling on etoposide- or cisplatin-mediated cytotoxicity were investigated. RESULTS: Both endogenous and exogenous IL-6 and exogenous OM up-regulated cell growth and enhanced resistance of PC-3 tumor cells to both etoposide and cisplatin. The influence of IL-6 is controlled by treating PC-3 tumor cells with anti-IL-6 neutralizing antibody and, more efficiently, by a mutated IL-6, Sant7. Sant7 has a high affinity binding to the IL-6 receptor-alpha (IL-6Ralpha) subunit, but does not bind to the signaling subunit gp130; therefore, it behaves as a receptor antagonist. Both IL-6- and OM-mediated effects are inhibited by the treatment of PC-3 with an antisense oligodeoxynucleotide against gp130, the protein kinase inhibitor genistein (GNS), or the monoterpene perillic acid (PA), a posttranslational inhibitor of p21ras isoprenylation. CONCLUSIONS: These results demonstrate the protective roles in drug sensitivity of IL-6 and OM through signaling of the common chain gp130 and, most likely, a downstream ras-dependent pathway in PC-3 tumor cells. These findings suggest the potential clinical application of anticytokine therapy or interference with gp130 signaling in the treatment of drug resistant prostate carcinoma.

Dexamethasone-induced cytotoxic activity and drug resistance effects in androgen-independent prostate tumor PC-3 cells are mediated by lipocortin 1.

We have examined the effects that dexamethasone (DEX), alone or in combination with doxorubicin (DOX), cisplatin (CDDP), or etoposide (VP-16), exerts on the growth of the androgen-independent prostate cancer PC-3 cells. DEX exhibited only a limited cytotoxicity (growth inhibition of about 28% or 20% after 24 or 72 h of exposure, respectively, in the range of DEX 10-100 nM) and did not induce apoptosis in the cells. This cytotoxicity of DEX was mimicked by an active peptide (peptide Ac2-26) drawn from the human lipocortin 1 N-terminus region and abrogated by an antibody to human lipocortin 1. Two inhibitors of arachidonic acid metabolism, tenidap and indomethacin, also caused cytotoxicity. The cytotoxic effects of DEX in combination with DOX, CDDP, or VP-16 were antagonistic when the steroid was administered 3 h before or simultaneously with the drugs. Other schedule-dependency experiments further clarified that, at least in the case of the combination with DOX, it is the steroid that desensitizes the cells to the drug. When peptide Ac2-26, tenidap, or indomethacin were tested in combination with DOX, antagonism was also observed. DEX treatment neither modified the ability of the cells to accumulate DOX nor changed their weak expression of P-glycoprotein. PC-3 cells also produce IL-6, which autocrinally stimulates their growth, and whose gene expression may be reduced by glucocorticoids. In the present experiments DEX only slightly decreased the production and secretion of IL-6 by the cells. The present findings suggest that the slight cytotoxic activity and the drug resistance effects of DEX on PC-3 cells are mediated by induction of lipocortin 1 and inhibition of arachidonic acid metabolism, with no relationship to downregulation of IL-6 levels. These findings indicate also that the combination of DEX with conventional chemotherapeutic agents may result in antagonistic antitumor effects.

The apoptotic signaling of TNF-alpha in multidrug resistant Friend leukemia cells.

Drug resistance, especially in its multiple forms (multidrug resistance, MDR), is a major and difficult problem to resolve in cancer therapy. Certain cytokines might be capable of bypassing this process and here we report on the in vitro effects of Tumor Necrosis Factor alpha, (TNF) on a MDR variant (FLC/DOX) of Friend leukemia. Drug resistance of FLC/DOX is associated with at least two mechanisms, i.e. overexpression of P-glycoprotein and increase in glutathione-related detoxifying activities. Nevertheless, TNF exerts more cytotoxicity in FLC/DOX than in its parental, drug-sensitive, counterpart and this effect is related to the induction of apoptosis. In contrast, Doxorubicin (DOX) never induces apoptosis in FLC/DOX, even when applied at high, fully cytotoxic, concentrations. We have tried to elucidate TNF signaling in FLC/DOX. The results have indicated that in this cell line TNF-triggered apoptosis exhibits some distinct features. It occurs mostly through type I (p55) TNF receptors, probably involves a calphostin-C sensitive protein kinase C activity and requires synthesis of proteins (it is inhibited by actinomycin D or cycloheximide) and of inducible nitric oxide (NO) synthase (it is inhibited by NG-methyl-L-arginine or aminoguanidine). Further, it is not influenced by agents which increase or decrease cell sulfhydryl groups, such as N-acetylcysteine or buthionine sulfoximine, respectively. These steps appeared to be either not or dissimilarly involved in the resistance to DOX of the same cells. In particular, DOX activity was stimulated by calphostin C and buthionine sulfoximine, and reduced by N-acetyl-cysteine. These findings illustrate that TNF may activate fresh cytotoxic pathways in tumor cells which are multidrug resistant, also owing to multifactorial causes.

Selective induction of apoptosis in multidrug resistant HL60R cells by the thiazolobenzoimidazole derivative 1-(2,6-difluorophenyl)-1H,3H-thiazolo [3,4-a] benzimidazole (TBZ).

We investigated the antitumour effects of 1-(2,6-difluorophenyl)-1H,3H-thiazolo [3,4-a]benzimidazole (TBZ) a new anti-HIV-1 agent, on human promyelocytic HL60 leukaemia, both a parental and a multidrug resistant form (HL60R). HL60R overexpresses P-glycoprotein and, like HL60, lacks p53 protein expression. HL60 and HL60R show similar levels of Bcl-2 protein. In contrast to the conventional chemotherapeutic agents daunorubicin, etoposide and mitoxantrone, TBZ caused equal or even greater cytotoxicity in HL60R than in HL60, and this result was associated with a more marked induction of apoptosis in the drug resistant cells. The antitumour activity of TBZ occurred in the range of concentrations higher than those required to exert antiviral activity. TBZ seems to act in the presence of P-glycoprotein and Bcl-2 and in the absence of p53 and is able to circumvent the mechanisms of drug resistance and anti-apoptosis present in HL60R cells.

Characterization of Seven New Hydrocarbon Compounds Present in the Aroma of Virgin Olive Oils.

Seven isomeric hydrocarbons were detected in the volatile fraction of three samples of virgin olive oil. Chemical ionization mass spectrometry was used to assign the molecular formulas. Their structures were confirmed by comparison of retention time and mass spectral data with those of a synthetic sample obtained by pentene radical coupling. A final characterization of each chromatographic peak was done by means of a chiral capillary column to distinguish the optically active compounds from the isomers without chiral centers. For the quantitation the recovery factor in the oily matrix during the extraction of the volatile fraction was obtained using a related chemical, the commercially available beta-citronellene. On the basis of the previous literature and the present experiments a tentative rationalization of the involved biochemical pathway is proposed.