Lack of effect of opioid peptides,morphine and naloxone on superoxide formation in human neutrophils and HL-60 leukemic cells

Summary There are controversial reports in the literature concerning the effects of opioids on superoxide (O ₂ ^– ) formation in phagocytes, these agents being either inhibitory or stimulatory. We re-examined this issue and compared the effects of the Chemotactic peptide, N-formyl-l,-methionyl-l-leucyl-l-phenylalanine (fMet-Leu-Phe), phorbol myristate acetate (PMA), ATP, platelet activating factor (PAF), cytochalasin B (CB) and prostaglandin E₁ (PGE₁) with those of various opioids on O ₂ ^– formation in human neutrophils and HL-60 leukemic cells under defined experimental conditions. In the presence of CB, fMet-Leu-Phe and PAF concentration-dependently activated O ₂ ^– formation in neutrophils with EC₅₀ values of 20 nM and 100 nM, respectively. In the absence of CB, fMet-Leu-Phe and PAF were much less effective. PAF synergistically enhanced O ₂ ^– formation induced by fMet-Leu-Phe. ATP at a concentration of 100 M and the opioids, methionine enkephalin, -endorphin, dynorphin, [d-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin, [d-Ala²-d-Leu⁵]-enkephalin and morphine at concentrations between 10 pM to 1 M did not activate O ₂ ^– formation. ATP but not \-endorphin potentiated fMet-Leu-Phe-induced O ₂ ^– formation. O ₂ ^– formation induced by a maximally stimulatory concentration of PMA (100 ng/ml) was enhanced by fMet-Leu-Phe but was unaffected by methionine enkephalin or PGE₁. PMA at a non-stimulatory concentration (2 ng/ml) potentiated the effect of fMet-Leu-Phe but did not induce responsiveness to PAF, ATP or -endorphin. PGE₁ strongly inhibited fMet-Leu-Phe-induced O ₂ ^– formation, whereas morphine, methionine enkephalin and the opioid antagonist, naloxone, were without effect. In HL-60 cells differentiated with dibutyryl cAMP, fMet-Leu-Phe, PAF and ATP but not -endorphin activated O ₂ ^– formation. Our results show that O ₂ ^– formation is differentially regulated by various classes of intercellular signal molecules and that opioids do not play a role in the regulation of O ₂ ^– formation. The precise definition of the experimental conditions and control experiments with established modulators of O ₂ ^– formation are essential to evaluate the role of opioids in the regulation of this effector system.Send offprint requests to R. Seifert at the above address     

Accumulation of p53 in response to adenovirus early region 1A sensitizes human cells to tumor necrosis factor alpha-induced apoptosis

Many tumor cells are resistant to tumor necrosis factor alpha (TNFalpha)-induced apoptosis. Adenovirus early region 1A (AdE1A) sensitizes the otherwise resistant cells to TNFalpha. AdE1A also stabilizes the p53 protein. The present study demonstrates a correlation between AdE1A-induced sensitization and stabilization of p53 in TNFalpha-induced apoptosis since the N-terminal and CR2 regions, the binding sites for CBP/p300, Rb and 26S proteasome regulatory components, are required for both these actions of AdE1A. TNFalpha does not induce apoptosis and AdE1A fails to sensitize TNFalpha cytotoxicity in p53-negative cells. However, introduction of exogenous p53 overcomes the cellular resistance to TNFalpha toxicity and enhances AdE1A sensitization, demonstrating that AdE1A sensitizes TNFalpha-induced apoptosis by its stabilization of p53. A proteasome inhibitor, lactacystin, enhances TNFalpha cytotoxicity in p53-positive and -negative cells, suggesting that accumulation of cellular proteins other than p53 might also regulate the cellular response to TNFalpha signaling.     

Cationic-amphiphilic arpromidine-derived guanidines and a histamine trifluoromethyl-toluidide derivative may activate pertussis toxin-sensitive G-proteins by a receptor-independent mechanism

Formyl peptides activate superoxide anion (O₂ ^–) formation in human neutrophils and in HL-60 cells via pertussis toxin (PTX)-sensitive guanine nucleotide-binding proteins (G-proteins), and histamine (HA) mediates inhibition of O₂ ^– formation via H₂-receptors. We have studied the effects of lipophilic arpromidine-derived guanidines, which are potent, full H₂-receptor agonists in the guinea pig atrium, on O₂ ^– formation and on activation of G-proteins in HL-60 membranes and on purified G-proteins. We have also studied the effects of a HA trifluoromethyl-toluidide derivative (HTMT), a cationic-amphiphilic HA derivative which activates O₂ ^– formation in HL-60 cells through a mechanism which is independent of known HA receptor subtypes, on G-protein activation. Guanidines, at concentrations, up to 30 mol/l inhibited and, at concentrations above 30 mol/l, enhanced formyl peptide-induce O₂ ^– formation in neutrophils. In HL-60 cells, guanidines per se activated O₂ ^– formation. The stimulatory effects of guanidines on O₂ ^– formation were not inhibited by H₁- or H₂-receptor antagonists. In HL-60 membranes, guanidines and HTMT, activated high-affinity GTPase in a PTX-sensitive manner. These substances also increased GTP hydrolysis effected by transducin and G_i/G_o-proteins. Our data suggest that lipophilic guanidines and HTMT may act as receptor-independent activators of PTX-sensitive G-proteins, resulting in stimulation of O ₂ ^– formation.     

Elevated levels of the antimicrobial peptide LL‐37 in hidradenitis suppurativa are associated with a Th1/Th17 immune response

Hidradenitis suppurativa (HS) is an inflammatory skin disease with poorly understood immunopathogenic mechanisms. LL‐37 is an antimicrobial peptide, which is transcribed from the CAMP (cathelicidin antimicrobial peptide) gene. Previous reports showed upregulated levels of CAMP and LL‐37 in HS lesions, and therefore, the aim of this study was to compare levels of LL‐37 in HS to other inflammatory skin diseases and to establish immunomodulatory functions of LL‐37 in HS. We confirm an upregulation of the LL‐37 peptide in lesional HS skin with comparable levels as in psoriasis patients and are able to positively correlate the presence of LL‐37 in HS with the presence of T cells, macrophages, neutrophils, IFN‐γ, IL‐17, IL‐23, TNF‐α, IL‐32 and IL‐1β. Mechanistically, LL‐37 boosts the proliferation of unspecifically activated CD4⁺ T cells via an increased calcium signalling independent of antigen‐presenting cells. Targeting LL‐37 may therefore represent a new therapeutic option for the treatment of this recalcitrant disease, but it has to be kept in mind that LL‐37 also has an antimicrobial function.     

Phage Display-directed Discovery of LEDGF/p75 Binding Cyclic Peptide Inhibitors of HIV Replication

The interaction between the human immunodeficiency virus (HIV) integrase (IN) and its cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is crucial for HIV replication. While recently discovered LEDGINs inhibit HIV-1 replication by occupying the LEDGF/p75 pocket in IN, it remained to be demonstrated whether LEDGF/p75 by itself can be targeted. By phage display we identified cyclic peptides (CPs) as the first LEDGF/p75 ligands that inhibit the LEDGF/p75–IN interaction. The CPs inhibit HIV replication in different cell lines without overt toxicity. In accord with the role of LEDGF/p75 in HIV integration and its inhibition by LEDGINs, CP64, and CP65 block HIV replication primarily by inhibiting the integration step. The CPs retained activity against HIV strains resistant to raltegravir or LEDGINs. Saturation transfer difference (STD) NMR showed residues in CP64 that strongly interact with LEDGF/p75 but not with HIV IN. Mutational analysis identified tryptophan as an important residue responsible for the activity of the peptides. Serial passaging of virus in the presence of CPs did not yield resistant strains. Our work provides proof-of-concept for direct targeting of LEDGF/p75 as novel therapeutic strategy and the CPs thereby serve as scaffold for future development of new HIV therapeutics.     

Triple‐echo steady‐state T2 relaxometry of the human brain at high to ultra‐high fields

Quantitative MRI techniques, such as T₂ relaxometry, have demonstrated the potential to detect changes in the tissue microstructure of the human brain with higher specificity to the underlying pathology than in conventional morphological imaging. At high to ultra‐high field strengths, quantitative MR‐based tissue characterization benefits from the higher signal‐to‐noise ratio traded for either improved resolution or reduced scan time, but is impaired by severe static (B₀) and transmit (B₁) field heterogeneities. The objective of this study was to derive a robust relaxometry technique for fast T₂ mapping of the human brain at high to ultra‐high fields, which is highly insensitive to B₀ and B₁ field variations. The proposed method relies on a recently presented three‐dimensional (3D) triple‐echo steady‐state (TESS) imaging approach that has proven to be suitable for fast intrinsically B₁‐insensitive T₂ relaxometry of rigid targets. In this work, 3D TESS imaging is adapted for rapid high‐ to ultra‐high‐field two‐dimensional (2D) acquisitions. The achieved short scan times of 2D TESS measurements reduce motion sensitivity and make TESS‐based T₂ quantification feasible in the brain. After validation in vitro and in vivo at 3 T, T₂ maps of the human brain were obtained at 7 and 9.4 T. Excellent agreement between TESS‐based T₂ measurements and reference single‐echo spin‐echo data was found in vitro and in vivo at 3 T, and T₂ relaxometry based on TESS imaging was proven to be feasible and reliable in the human brain at 7 and 9.4 T. Although prominent B₀ and B₁ field variations occur at ultra‐high fields, the T₂ maps obtained show no B₀‐ or B₁‐related degradations. In conclusion, as a result of the observed robustness, TESS T₂ may emerge as a valuable measure for the early diagnosis and progression monitoring of brain diseases in high‐resolution 2D acquisitions at high to ultra‐high fields. Copyright © 2014 John Wiley & Sons, Ltd.     

Stenting for restenotic lesions with the BARD XT stent

BACKGROUND: Conventional PTCA for the treatment of restenotic lesions is associated with a high rate of recurrence (30-50%). Primary stenting decreases the restenosis rate at long-term follow-up. METHODS: One-hundred consecutive patients with restenosis received a Bard XT stent. Follow-up angiography was performed after 6 months. Angiograms were compared by means of computed quantitative analysis. RESULTS: The mean pretreatment reference diameter was 2.88 +/- 0.51 mm. The mean minimal luminal diameter (MLD) increased from 1.09 +/- 0.57 mm to 2.70 +/- 0.44 mm. The percent diameter stenosis decreased from 66 +/- 13% to 15 +/- 10%. The procedural success rate was 99%. At 6 month follow-up repeat angiography was performed in 86 patients. The mean MLD was 1.74 +/- 0.67 mm with a mean diameter stenosis of 41 +/- 20%. Residual anginal complaints were reported in 29% of patients. In-stent restenosis (defined as diameter stenosis of more than 50%) occurred in 18% of the patients. CONCLUSION: Placement of the Bard XT stent in restenotic lesions is feasible, has an excellent short term outcome and yields a favorable result at 6 month follow-up angiography.     

Cutting balloon for in-stent restenosis: acute and long-term results

INTRODUCTION: Conventional percutaneous coronary intervention for the treatment of in-stent restenosis (ISR) has shown a high rate of ISR (30-55%). Considering the need for both extrusion of hyperplastic intima and additional stent expansion, a cutting balloon might be more effective for the treatment of ISR. METHODS: We prospectively assessed the immediate and 8-month outcome of balloon angioplasty using the Barath Cutting Balloon in 100 consecutive patients (mean age: 60.5 +/- 10.8 years, 71% male). RESULTS: In 73 lesions (73%), a good result was reached with the cutting balloon only. In 21 lesions (21%) postdilatation and in 6 lesions (6%) predilatation with a conventional balloon was necessary. The mean inflation pressure was 8.7 +/- 2.0 (range: 6.0-18.0) atm. Before the procedure the mean minimal luminal diameter (MLD) was 0.95 +/- 0.45 mm. Quantitative coronary analysis showed a mean diameter stenosis of 65%+/- 16%. Immediately after the procedure the mean MLD was 2.42 +/- 0.54 mm with a mean diameter stenosis of 19%+/- 13%. Two patients died during the follow-up period (1 stroke, 1 nonvascular). At 8-month follow-up 26 patients (26%) reported to have anginal complaints CCS class II-IV of whom 16 (16%) needed target lesion revascularization. CONCLUSION: Treatment of ISR using the Barath Cutting Balloon can be performed safely with good immediate results and a relatively low need for repeated target lesion revascularization at 8-month follow-up.     

Insulin-like Growth Factor and its Therapeutic Potential for Diabetes Complications - Mechanisms and Metabolic Links: A Review

BACKGROUNDThe insulin-like growth factor (IGF) system is an important system in normal physiological functioning of the body. In diabetes mellitus, alterations of IGF-binding protein (IGFBP) levels have been described, mainly in vascular complications.AIMThe aim of this review was to explore the role of the IGF system in reducing diabetes complications and its role as potential therapeutic target.RESULTSIGF-1 plays a role in neuronal growth and developmental processes. Low concentrations of IGF-1 have been associated with neuropathy and other diabetes complications. Moreover, impaired IGF synthesis and function may result in cellular senescence and impaired vascular endothelial proliferation, adhesion, and integration. Of note, high IGF-1 bioavailability may prevent or delay the inception of diabetes-associated complications in diabetes patients. The mechanism of normal functioning IGF-1 is induced by increasing nitric oxide synthesis and potassium ion channel opening in cardiovascular physiology, which improves impaired small blood vessel function and reduces the occurrence of diabetes complications associated with reduced concentrations of IGF-1.CONCLUSIONSIGF may be considered an alternative therapy for diabetes and diabetes-associated complications. Therefore, future studies should focus on the mechanism of action and therapeutic potential of IGFs in reducing the risk of development and progression of the disease in different clinical settings.