Draft Genome Sequence of Clostridium difficile Belonging to Ribotype 018 and Sequence Type 17.

Clostridium difficile, belonging to ribotype 018 (RT018), is one of the most prevalent genotypes circulating in hospital settings in Italy. Here, we report the draft genome of C. difficile CD8-15 belonging to RT018, isolated from a patient with fatal C. difficile-associated infection.

Long-term clinical outcomes in critical limb ischemia--A retrospective study of 181 patients.

OBJECTIVE: Critical limb ischemia (CLI) is the most severe manifestation of the peripheral arterial disease. To date, several prognostic factors have been identified but the data of long-term follow-up in real life setting are scarce. The aim of our study is to describe a large group of CLI patients and identify possible prognostic factors, in a long-term follow-up. PATIENTS AND METHODS: Case-control, retrospective study. 181 consecutive CLI patients with a minimum follow-up of 5 years were included in the study. RESULTS: Overall mortality was 15%, 24%, and 43% at 1, 2, and 5 years, respectively. Among known risk factors, only arterial hypertension was significantly correlated with survival rate; no differences were found between diabetics and non-diabetics. Patients treated with intravenous iloprost (46%), compared to untreated patients, showed a better (p < 0.0001) long-term outcome in terms of major amputation (6% vs. 21%), subsequent vascular surgery (4% vs. 32%) and survival rates (69% vs. 47%), at 5-year follow-up. Major amputations were significantly correlated with lower median forefoot transcutaneous values of O2 (0/3 mmHg, p < 0.001) and higher median values of CO2 (83/53 mmHg, p < 0.0001) in supine/dependent position, respectively. CONCLUSIONS: Our results confirm the poor prognosis of CLI patients in a very long-term follow-up and the severe metabolic damage caused by ischemia. A favourable role of iloprost was observed, in agreement with previous evidence in the literature.

Gender-related differential effect of tachykinin NK2 receptor-mediated visceral hyperalgesia in guinea pig colon.

BACKGROUND AND PURPOSE: The tachykinin NK2 receptor antagonist ibodutant is under Phase III clinical investigation to treat female patients with irritable bowel syndrome. The aim of this study was to investigate the NK2 receptor-related gender specificity in a model of colitis. EXPERIMENTAL APPROACH: Colitis was induced by rectal instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS, 0.5 mL, 30 mg·mL(-1) in 30% ethanol) in female and male guinea pigs. Electromyographic recording of the responses to colorectal distension (CRD) was made 3 days later. Ibodutant (0.33 , 0.65, 1.9 and 6.5 mg·kg(-1) ) was given s.c., 30 min before CRD. Release of neurokinin A and substance P from isolated mucosal and smooth muscle tissues following treatment with KCl (80 mM) or capsaicin (10 µM) was measured by EIA. Plasma pharmacokinetics of ibodutant following a single s.c. administration (0.73 or 2.1 mg·kg(-1) ) were measured over 24 h. KEY RESULTS: Ibodutant did not affect abdominal contractions in control animals. After TNBS-induced colitis, ibodutant prevented the increased visceral hypersensitivity to CRD in females, at lower doses than in males. Ibodutant pharmacokinetics did not differ between females and males. Tachykinins release was greater in smooth muscle than in mucosal samples. Capsaicin-stimulated release of tachykinins from inflamed mucosal samples from females was significantly lower than in males. CONCLUSIONS AND IMPLICATIONS: Ibodutant prevented abdominal nociception in a model of visceral hypersensitivity in guinea pigs with a greater efficacy in females than in males. Our results highlight a gender-related difference in colonic visceral hypersensitivity and mucosal nerve activation.

Long-term survival of patients with critical limb ischemia treated with iloprost: response rate and predictive criteria. A retrospective analysis of 102 patients.

OBJECTIVE: Critical limb ischemia (CLI) patients have poor long-term prognosis. We showed that iloprost improves outcomes (major amputation and survival) up a 5-year follow-up, but it is not known if in this length of time the survival curves, of clinical responders and non-responders, differ. PATIENTS AND METHODS: A retrospective study enrolling 102 consecutive patients between 2004-2008, with clinical and instrumental (ultrasound, angiography, transcutaneous tensiometry of oxygen TcpO2 and carbon dioxide TcpCO2 in the affected and contralateral limbs) diagnosis of critical ischemia. All patients received the best medical therapy. Iloprost was administered (0.5-2 ng/kg/min 6 hours/day for 2-4 weeks) in all patients initially considered unsuitable for revascularization, repeating it regularly in time every six-twelve months in the case of positive response. The minimum expected follow-up was 4 years. RESULTS: 71.5% of patients were treated with iloprost and the responder rate was 71.2%. Most of the patients were regularly retreated with repeated cycles. Initial median supine TcpCO2 in symptomatic limb was higher in untreated patients than those treated (58 vs. 49 mmHg; p < 0.05) and in non-responders compared to responders (60 vs. 49 mmHg; p < 0.05). TcpCO2 directly and significantly correlated with the highest risk of mortality and seems to represent a new accurate prognostic criterion of unfavourable short and long-term response to prostanoid. In iloprost group, major amputations were significantly reduced. Revascularization was significantly higher in non-responders (57.1% vs. 11.5%; p < 0.05). There was a significantly higher prevalence of subsequent myocardial infarction in the non-iloprost group (27.6% vs. 9.6%; p < 0.05). The survival rate of non-responders was higher than untreated up until the second year (76.2% vs. 62%; p < 0.05). At 4 years we found higher survival in patients treated with iloprost (64.3% vs. 41% in untreated; p < 0.05) and in responders (75% vs. 38.1% in non-responders; p < 0.05). CONCLUSIONS: Our results confirm the favourable role of iloprost on the long-term outcome in patients with CLI. In particular, the maximum benefit is obtained in responder patients treated with multiple cycles of infusion.

Synovial fluid levels of bradykinin correlate with biochemical markers for cartilage degradation and inflammation in knee osteoarthritis.

OBJECTIVE: To determine the content of bradykinin (BK) and markers of cartilage degradation and inflammation in the synovial fluid (SF) of patients with knee osteoarthritis (OA), and to evaluate correlations with biomarkers or clinical parameters. METHODS: SFs were obtained from 30 patients with knee OA. Levels of basal and generated BK, cartilage oligomeric matrix protein (COMP), interleukin (IL) 1, IL-6, IL-8 and matrix metalloprotease (MMP) 1, MMP-3, MMP-13 and sulfated glycosaminoglycans (GAGs) were measured by enzyme-linked immunosorbent assay (ELISA) or colorimetric assays. RESULTS: The mean concentration of basal BK (in the presence of peptidase and protease inhibitors to avoid degradation and de novo formation of BK) was 422 pg/ml (95% confidence interval, CI, 281-563) whereas that of in vitro generated BK (in the presence of peptidase inhibitors SFs were incubated 60 min at 37°C to measure the potential capability to generate BK) was 3427 pg/ml (2591-4264). The content of MMP-13, IL-1α, and IL-1ß was under assay sensitivity. Basal BK levels positively correlated (Spearman's rank correlation) with GAGs (40 µg/ml, 26-54, r = 0.4834, P = 0.0308) and IL-6 (553 pg/ml, 171-935, r = 0.3946, P = 0.0377) similarly to the generated BK (GAGs, r = 0.4563, P = 0.0431; IL-6, r = 0.5605, P = 0.0019). Statistical analysis of basal BK and biomarkers was significant (P = 0.0483). When applying a stepwise logistic regression analysis considering biomarkers together with clinical parameters, results indicated that K/L radiographic OA grade and COMP improved the model (P = 0.0032). CONCLUSION: The presence of BK in the knee OA SF and its correlations with cartilage degradation and inflammation markers of OA support its participation in OA pathology.

Blockade of nociceptive sensory afferent activity of the rat knee joint by the bradykinin B2 receptor antagonist fasitibant.

OBJECTIVE: The aim of this study was to determine in intact and inflamed knee joints of the rat, the effect of the bradykinin (BK) B2 receptor antagonist fasitibant (MEN16132) on nociceptor mechanosensitivity and hyperalgesia. METHODS: Joint afferent sensory fibers of the medial articular nerve of anesthetized animals were electrophysiologically recorded, measuring nerve impulse activity evoked by passive innocuous and noxious movements of the joint, in intact and kaolin and carrageenan-injected joints. Knee joints of rats were also acutely inflamed by intra-articular injection of carrageenan alone. Long term duration of fasitibant antinociceptive effects were behaviorally evaluated using the incapacitance test. RESULTS: BK (100 µM) injected into the saphenous artery, induced excitation and sensitization of multi- and single unit recordings. Fasitibant (300 µM) injected prior to BK, reduced its excitatory effects as well as the overall increase of movement-evoked activity resulting from repeated injections of BK. Fasitibant did not affect movement-evoked activity of sensory fibers of intact, non-inflamed knee joints. Intra-articular fasitibant (100 µg/knee) significantly reduced the carrageenan-induced inflammatory hyperalgesia measured with the incapacitance test up to four days after treatment. This antinociceptive effect was not obtained with systemic endovenous injection of the drug. CONCLUSIONS: Fasitibant prevents B2 receptor-mediated activation and sensitization of peripheral joint afferents and the ensuing inflammatory hyperalgesia, and may be a useful, novel drug for arthritis pain treatment.

Thermal and electrochemical decomposition of lithium peroxide in non-catalyzed carbon cathodes for Li-air batteries.

The decomposition of lithium peroxide during the charging process of lithium-air batteries is investigated. A novel preparation method for electrodes in the discharged state, i.e., prefilled with Li2O2 using polyethylene oxide as a binder, is presented. The composition and reactivity of Li2O2-prefilled electrodes are examined by thermal analysis coupled with on-line mass spectrometry. Voltage profiles and gas evolution during the charging process of Li2O2-prefilled electrodes in battery cells are correlated with the thermal decomposition process of Li2O2 and its impact on other electrode compounds. It is found that both thermal Li2O2 decomposition and the electrochemical decomposition of Li2O2 during charging enhance the oxidation of the electrolyte, the binder, and/or carbon, which is suggested to be due to the formation of "nascent" oxygen during Li2O2 decomposition into O2 and Li2O (thermally) or into O2 and lithium ions (electrochemically).

Fasitibant prevents the bradykinin and interleukin 1ß synergism on prostaglandin E2 release and cyclooxygenase 2 expression in human fibroblast-like synoviocytes.

This study investigates the effect of the selective and potent B(2) receptor antagonist fasitibant (MEN16132) on the proinflammatory effect of bradykinin (BK) and its interaction with interleukin 1ß (IL-1ß) in human synoviocytes. PGE(2) content was detected in the surnatants and COX-2 and COX-1 gene and protein expression determined in the cells. Radioligand binding ([(3) H]BK) and BK-induced inositolphosphate experiments were performed. Incubation of synoviocytes with BK induced a sustained production of PGE(2) and transient COX-2 gene expression that were prevented by pretreatment with fasitibant (1 µM, 30 min preincubation). IL-1ß increased PGE(2) release and COX-2 expression more than BK alone. The combined treatment of cells with BK and IL-1ß induced an even increase of released PGE(2) and COX-2 gene and protein expression indicating a synergistic rather than an additive effect, not related to an increase of B(2) receptors density or its coupling. These potentiating effects of BK on PGE(2) production and increased COX-2 expression produced by IL-1ß were B(2)-receptor-mediated as fasitibant could prevent them. None of the treatments induced changes in the COX-1 expression. The synergistic PGE(2) production was abolished by the specific NF-kappaB inhibitor (BAY-117085), whereas specific inhibitors for the p38 (SB203580), JNK (SP600125), and ERK1/2 (PD98059) mitogen-activated protein kinases could prevent the prostanoid release. BK can potentiate the COX-2 gene expression and consequent prostanoid production induced by IL-1ß. The prevention of this synergism by fasitibant indicates BK B(2) receptor blockade as an alternative symptomatic therapy for osteoarthritis.

Comparison of the molecular interactions of two antagonists, MEN16132 or icatibant, at the human kinin B2 receptor.

BACKGROUND AND PURPOSE: Icatibant is a well-known kinin B2 receptor antagonist currently used for angiooedema attacks. MEN16132 is a non-peptide B2 receptor antagonist, more potent and long lasting than icatibant in different models. Here we studied the reasons for these differences between the two antagonists. EXPERIMENTAL APPROACH: Rate of reversibility (over about 3 h) of the functional receptor blockade exerted by the antagonists was compared (inositol phosphates accumulation assay) in CHO cells expressing the human B2 receptor and in human synovial cells. Antagonist pretreated cells were washed with medium and the time taken to restore bradykinin (BK) response measured. Antagonist affinity was measured by radioligand binding to wild type and mutated B2 receptors. KEY RESULTS: Recovery of BK-induced responses was slower in cells pretreated with MEN16132 than in those treated with icatibant. The affinity of icatibant (for the [³H]-BK or the B2 receptor antagonist [³H]-MEN11270 binding site) was compared to that of MEN16132 using a panel of point-mutated receptors with mutations located at the transmembrane regions of the B2 receptor, previously shown to decrease MEN16132 high affinity interaction. No consistent decrease of icatibant affinity was observed. From the different affinity of MEN16132 derivatives at wild type and W86A (transmembrane 2 region) receptors, and by evaluating its antagonist profile at the D266A/D284A double mutant receptor, a model of the MEN16132-B2 receptor complex is proposed. CONCLUSIONS AND IMPLICATIONS: MEN16132 dissociated from the B2 receptor compartment more slowly than icatibant and interacted at a deeper level in transmembrane regions of the receptor.

Bradykinin and B2 receptor antagonism in rat and human articular chondrocytes.

BACKGROUND AND PURPOSE: In osteoarthritis (OA), bradykinin (BK) is known to contribute to pain and synovitis, but not to cartilage degradation. Here, we investigated effects of BK and its antagonists on chondrocytes, cells involved in cartilage homeostasis. EXPERIMENTAL APPROACH: BK receptor density and affinities of BK, its analogues and antagonists were measured in cultured human and rat chondrocytes by radioligand binding. Effects of BK were assessed by accumulation of inositol phosphates (IP) and release of interleukin (IL)-6 and IL-8. KEY RESULTS: Density of [³H]-BK binding sites was higher (13-30-fold) and BK evoked a greater (48-fold) IP production, in human than in rat chondrocytes. The BK B2 receptor antagonists MEN16132 and icatibant displayed similar binding affinity. MEN16132 was 40-fold more potent than icatibant in the IP assay. In human chondrocytes, BK increased release (over 24 h) of IL-6 and IL-8, effects blocked by MEN16132 but not by the B1 receptor antagonist Lys-[Leu8][desArg9]BK. BK-induced release of IL-6, but not of IL-8, was partially inhibited by indomethacin (10 µM) and nordihydroguaiaretic acid (10 µM). Antagonists for the prostanoid EP receptors (AH6809 10 µM; L-798,196, 200 nM; L-161,982, 1 µM) were ineffective. Dexamethasone (100 nM) partially inhibited release of both IL-6 and IL-8. Inhibitors of intracellular downstream signalling pathways (SB203580 10 µM; PD98059, 30 µM; SP600125, 30 µM; BAY-117085, 5 µM) indicated the involvement of p38 MAPK and the activation of NF-κB. CONCLUSION AND IMPLICATIONS: BK mediated inflammatory changes and cartilage degradation and B2 receptor blockade would, therefore, be a potential treatment for OA.

Novel effects mediated by bradykinin and pharmacological characterization of bradykinin B2 receptor antagonism in human synovial fibroblasts.

BACKGROUND AND PURPOSE: Bradykinin (BK) and B2 receptors have been implicated in the pathophysiology of osteoarthritis (OA), and synovitis is one of its hallmarks. Here, the selective B2 receptor antagonists MEN16132 and icatibant have been pharmacologically characterized in human synovial cells. EXPERIMENTAL APPROACH: Radioligand and functional studies (inositol phosphate (IP) accumulation, interleukin (IL)-6 and IL-8 release) were performed in cultured synoviocytes. KEY RESULTS: [3H]-BK saturation studies indicated receptor density (Bmax) and K(d) values of 121,550 sites per cell and 1.14 nM respectively. In synoviocytes, MEN16132 (pK(I) 8.9) was threefold more potent than icatibant (pK(I) 8.4). Both antagonists showed competitive antagonism in the BK-induced IP assay (control EC50 0.45 nM), with pK(B) values of 9.9 (MEN16132) and 8.1 (icatibant). 24h incubation with BK induced IL-6 (EC50 216 nM) and IL-8 (EC50 53 nM) release. Both MEN16132 (IL-6: pIC50 8.1; IL-8: pIC50 8.4) and icatibant (IL-6: pIC50 6.6; IL-8: pIC50 6.7) completely prevented this BK-induced release. Indomethacin did not affect the basal or the IL-6/IL-8 release induced by BK, whereas nordihydroguaiaretic acid decreased the basal release, although BK still increased IL-6 and IL-8 production. BK-induced IL-8 release was attenuated by inhibitors of phospholipase C (U73122), p38 (SB203580), JNK (SP600125), ERK 1/2 (PD98059) MAPKs, phosphoinositide 3-kinase (LY294002), NF-kappaB (BAY-117085) and by the glucocorticoid dexamethasone. CONCLUSIONS AND IMPLICATIONS: Bradykinin via B2 receptors can participate in inflammatory events in synovitis. MEN16132 is a highly potent B2 receptor antagonist capable of blocking pro-inflammatory responses to BK evoked in human synoviocytes.

Knee osteoarthritis: a role for bradykinin?

Osteoarthritis (OA) is a painful and degenerating progressive disease of the joints which affects millions of patients worldwide. The cause of OA is largely unknown. Among the potential therapies for the symptomatic treatment of OA, the intra-articular administration of a specific bradykinin (BK) B2 receptor antagonist has been reported to produce a long lasting analgesic effect in patients affected by knee OA. BK is a vasodilator and inflammatory nonapeptide which is generated in OA synovium. It contributes to the initiation and maintenance of inflammation, to exciting and sensitizing sensory nerve fibres, thus producing pain, and to activating synoviocytes and chondrocytes which are the main cells involved in the homeostasis of synovial fluid and cartilage, respectively. Moreover, BK synergistically potentiates the effects produced by pro-inflammatory cytokines. Biochemical and preclinical evidence supporting the therapeutic relevance of B2 receptor blockade in OA pathophysiology are reviewed in this publication.

Bradykinin analogs containing the 4-amino-2-benzazepin-3-one scaffold at the C-terminus.

High affinity peptide ligands for the bradykinin (BK) B(2) subtype receptor have been shown to adopt a beta-turn conformation of the C-terminal tetrapeptide (H-Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)-OH). We investigated the replacement of the Pro(7)-Phe(8) dipeptide moiety in BK or the D-Tic(7)-Oic(8) subunit in HOE140 (H-D-Arg(0)-Arg(1)-Pro(2)-Hyp(3)-Gly(4)-Thi(5)-Ser(6)-D-Tic(7)-Oic(8)-Arg(9)-OH) by 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one templates (Aba). Binding studies to the human B(2) receptor showed a correlation between the affinities of the BK analogs and the propensity of the templates to adopt a beta-turn conformation. The L-spiro-Aba-Gly containing HOE140 analog BK10 has the best affinity, which correlates with the known turn-inducing property of this template. All the compounds did not modify basal inositolphosphate (IP) output in B(2)-expressing CHO cells up to 10 microM concentration. The antagonist properties were confirmed by the guinea pig ileum smooth muscle contractility assay. The new amino-benzazepinone (Aba) substituted BK analogs were found to be surmountable antagonists.

Comparative antagonist pharmacology at the native mouse bradykinin B2 receptor: radioligand binding and smooth muscle contractility studies.

BACKGROUND AND PURPOSE: The aim was to characterize the recently discovered non-peptide antagonist MEN16132 at the mouse B2 receptor, relative to other antagonists. EXPERIMENTAL APPROACH: [3H]-BK binding experiments used mouse lung and ileum tissue membranes and antagonist potency was measured in the isolated ileum contractility assay. KEY RESULTS: Two BK binding sites resulted from saturation and homologous competition experiments. A role for the B1 receptor was excluded because of the poor affinity of B1 receptor ligands (pIC50<5). MEN16132, and the other reference antagonists, inhibited only one portion of BK specific binding, and the rank order of potency was (pIC50): Icatibant (lung 10.7; ileum 10.2)=MEN11270 (lung 10.4; ileum 9.9)=MEN16132 (lung 10.5; ileum 9.9).>LF16-0687 (lung 8.9; ileum 8.8)>FR173657 (lung 8.6; ileum 8.2). BK homologous curves performed with lung membranes after treatment with the antagonist MEN16132 or Icatibant (10 nM) displayed only the low affinity site. The functional antagonism by MEN16132 (pA2 9.4) and Icatibant (pA2 9.1), towards BK (control EC50 6.1 nM) induced ileum contractions, was concentration-dependent and surmountable, but the Schild plot slope was less than unity. CONCLUSIONS AND IMPLICATIONS: In mouse tissue, radiolabelled BK recognizes two binding sites and B2 receptor antagonists can compete only for the higher affinity one. The pharmacological profile of the novel non-peptide antagonist MEN16132 indicates that it exhibits subnanomolar affinity and potency for the mouse B2 receptor and is suitable for further characterization in in vivo pathophysiological models.

Characterization of kinin receptors in human cultured detrusor smooth muscle cells.

BACKGROUND AND PURPOSE: Kinins have an important role in inflammatory cystitis and in animal pathophysiological models, by acting on epithelium, fibroblasts, sensory innervation and smooth muscle. The aim of this study was to characterize the receptors responsible for direct motor responses induced by kinins on human detrusor. EXPERIMENTAL APPROACH: Human detrusor cells from biopsies were isolated and maintained in culture. B(1) and B(2) kinin receptors were characterized by means of radioligand and functional experiments (PI accumulation and PGE(2) release). KEY RESULTS: [(3)H]-[desArg(9)]-Lys-BK and [(3)H]-BK saturation studies indicated receptor density (B(max)) and K (d) values of 19 or 113 fmol mg(-1), and 0.16 or 0.11 nM for the B(1) or B(2) receptors, respectively. Inhibition binding studies indicated the selectivity of the B(1) receptor antagonist [desArg(9)Leu(8)]-Lys-BK and of the B(2) receptor antagonists Icatibant and MEN16132. [DesArg(9)]-Lys-BK and BK induced PI accumulation with an EC(50) of 1.6 and 1.4 nM and different maximal responses (E(max) of [desArg(9)]-Lys-BK was 10% of BK). BK also induced prostaglandin E(2) release (EC(50) 2.3 nM), whereas no response was detected with the B(1) receptor agonist. The incubation of detrusor smooth muscle cells with interleukin 1beta (IL-1beta) or tumour necrosis factor-alpha (TNF-alpha) (10 ng ml(-1)) induced a time-dependent increase in radioligand-specific binding, which was greater for the B(1) than for the B(2) receptor. CONCLUSIONS AND IMPLICATIONS: Human detrusor smooth muscle cells in culture retain kinin receptors, and represent a suitable model to investigate the mechanisms and changes that occur under chronic inflammatory conditions.

Extracorporeal blood oxygenation and ozonation (EBOO): a controlled trial in patients with peripheral artery disease.

BACKGROUND: Since 1990 our group has been using extracorporeal circulation to ozonate blood by an original method, known as extracorporeal blood oxygenation and ozonation (EBOO), with the aim of amplifying the results observed with ozone autohemotherapy. OBJECTIVE: To verify the hypothesis that EBOO improves the skin lesions typical of peripheral artery disease (PAD) patients. METHODS: Twenty-eight patients with PAD were randomized to receive EBOO or intravenous prostacyclin in a controlled clinical trial. The primary efficacy parameters were regression of skin lesions and pain,and improvement in quality of life and vascularisation. RESULTS: Patients treated with EBOO showed highly significant regression of skin lesions with respect to patients treated with prostacyclin. Other parameters that were significantly different in the two groups of patients were pain,pruritus, heavy legs and well-being. No significant differences in vascularisation of the lower limbs before and after treatment were found in either group. No side effects or complications were recorded during the 210 EBOO treatments. CONCLUSION: EBOO was much more effective than prostacyclin for treating skin lesions in PAD patients and also had a positive effect on patient general condition without any apparent change in arterial circulation. This suggests other mechanisms of action of EBOO.

Iloprost treatment reduces TNF-alpha production and TNF-RII expression in critical limb ischemia patients without affecting IL6.

Iloprost, a stable prostacyclin analogue, regulates expression of genes that are involved in inflammation and in cell growth and inhibits the in vitro production of cytokines. We evaluated the effect of an in vivo weekly iloprost treatment on TNF-alpha and IL6 monocyte production (evaluated by ELISA), on monocyte apoptosis (Annexin V/uptake of propidium iodide by flow cytometry) and on peripheral blood mononuclear cell (PBMC) TNF-alpha receptors (TNF-RI and TNF-RII) mRNA expression (RT-PCR) in 14 atherosclerotic critical limb ischemia patients. PBMC were stimulated with LPS for 24h. TNF-alpha production was significantly reduced by iloprost whereas IL6 production was not affected. Iloprost did not accelerate monocyte apoptosis. TNF-RI mRNA expression was not modified by iloprost, whereas TNF-RII mRNA expression was significantly reduced. Our data show that iloprost may have anti-inflammatory effects in addition to the well-known vasodilatatory and anti-aggregant ones.

Short-term and long-term effects of one-week treatment with intravenous iloprost in critical limb ischemia patients (Leriche-Fontaine stage III and IV).

AIM: Iloprost, usually administered through intravenous infusion for 6 hours per day for at least 21 days, is the main medical treatment for critical limb ischemia in patients unsuitable for surgical or endovascular approach. We evaluated the tolerance and the short-term and long-term effects of a single 1-week treatment in critical limb ischemia patients. METHODS: Twenty-nine patients in Leriche-Fontaine III and IV stage were treated with iloprost infusions for 16 hours per day for 7 days, achieving a maximal dose of 1.5 ng/kg/min. Tolerance and clinical assessment after treatment discontinuation and after 1 and 6 months were recorded; clinical evaluation (rest pain, trophic lesions), ankle/brachial pressure index (ABPI) and treadmill exercise test were performed before, immediately after treatment and after 1 and 6 months. RESULTS: No discontinuation of treatment occurred because of intolerance to iloprost. At the end of the treatment 69% of patients were responders, 55.2% at 1 month, 37.9% after 6 months. ABPI and treadmill maximum walking distance were improved by the treatment at every timepoint. After 6 months 10.3% mortality and 3.4% major amputation rates were recorded. There was a higher percentage of non-responders amongst women vs men, in diabetic patients vs non diabetic and in stage IV patients vs stage III. CONCLUSIONS: One-week treatment with iloprost is safe and effective in both Leriche-Fontaine stage III and IV patients. Clinical effects are persistent over time, often lasting up to the 6th month, similarly to the commonly used 28-day treatment, with clear implications in terms of patient's compliance and medical cost containment.

In vitro and in vivo activity of analogues of the kinin B2 receptor antagonist MEN1 1270.

In this study, we describe the in vitro and in vivo activities of a series of cyclic peptide analogues of the selective kinin B2 receptor antagonist MEN11270 on Chinese hamster ovary cells expressing the human B2 receptor (hB2R), the human isolated umbilical vein (hUV), the isolated guinea pig ileum (gpI), and bradykinin (BK) induced bronchoconstriction (BC) and hypotension in anaesthetized guinea pigs. Substitutions in the backbone of MEN1 1270 (H-DArg-Arg-Pro-Hyp-Gly-Thi-c(Dab-DTic-Oic-Arg)c(7gamma-10alpha)) aimed to increase the potency in inhibiting bronchospasm versus hypotension following the topical (intratracheal (i.t.)) or systemic (intravenous (i.v.)) application of these antagonists. A series of analogues were left unprotected from N-terminal cleavage by aminopeptidases (MEN12739, MEN13052, MEN13346, and MEN13371): these compounds maintained sizeable affinities for the hB2R (pKi = 9.4, 9.6, 9.7, and 8.6, respectively) and antagonist activities toward BK in the hUV (pA2 = 7.9, 8.3, 8.2, and 7.5) and gpI assays (pK(B) = 7.4, 7.8, 7.9, and 7.9), but the inhibition of BK-induced BC and hypotension in vivo was negligible following either i.v. or i.t. administration. Two analogues (MEN12388 and MEN13405) could be potential substrates of angiotensin-converting enzyme: these have good activity in the hB2R (pKi = 9.5 and 8.9, respectively), hUV (pA2 = 8.2 for MEN12388), and gpI assays (pK(B) = 8.4 and 8.0) but an in vivo activity 10- to 30-fold lower than the parent compound MEN1 1270 (pKi = 9.4, pA2 = 8.1, pKB = 8.3) when given by either the i.v. or the i.t. route. Other analogues were functionalized with a quaternary ammonium Lys derivative (MEN13031, MEN12374, and the previously mentioned MEN13052) or with an ethyl group on Arg (MEN13655 and the previously mentioned MEN13346 and MEN13405) in order to hinder or facilitate local absorption. MEN13346 and MEN13031 (pKi = 9.7and 9.5, pA2 = 8.2 and 7.9, pKB = 7.9 and 8.5, respectively) were 10- to 30-fold less active in vivo than MEN1 1270, without improving the discrimination between BK-induced BC and hypotension after either systemic or topical administration. It is concluded that the decreased in vivo activities of cyclic analogues of MEN11270 on BK-induced BC and hypotension following either their intratracheal or their intravenous routes of administration might be due in large part to metabolic degradation.