曲美他嗪抑制自噬减少中性粒细胞胞外核酸网形成

目的研究曲美他嗪(trimetazidine TMZ)对氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)体外诱导中性粒细胞胞外核酸网(neutrophil extracellular traps,NETs)形成的影响及其与自噬的关系。方法密度梯度离心法提取小鼠骨髓中性粒细胞,ox-LDL体外干预建立NETs诱导模型;采用TMZ、LY294002、Rapamycin干预ox-LDL对NETs的诱导;免疫荧光法观察NETs标志物MPO-DNA的释放;PicoGreen试剂盒定量检测cfDNA/nets含量;Western blot检测髓过氧化物酶(MPO)、自噬相关蛋白Beclin-1、LC3b的表达。结果 ox-LDL以浓度-时间依赖的方式刺激中性粒细胞释放MPO-DNA复合物,使上清cfDNA/nets含量明显增高;细胞自噬相关蛋白LC3b、Beclin-1及MPO的蛋白水平亦随ox-LDL浓度增加上调; TMZ预处理能抑制NETs释放,降低LC3b、Beclin-1及MPO的蛋白表达,该结果可被PI3K通路阻断剂LY294002模拟,...     

SB-656933, a novel CXCR2 selective antagonist, inhibits ex vivo neutrophil activation and ozone-induced airway inflammation in humans

AIMS

To determine the safety and tolerability of a novel selective CXCR2 antagonist and assess its pharmacodynamic effects using measures of neutrophil activation and function, including CD11b expression in whole blood and ozone-induced airway inflammation in healthy subjects.

METHODS

Flow cytometric determination of ex vivo CXCL1-induced CD11b expression on peripheral blood neutrophils was performed following single dose oral administration of SB-656933 (dose range 2–1100 mg). A subsequent randomized study (placebo, 50 mg and 150 mg) was performed to explore the dose–response for ozone-induced airway inflammation, as measured by sputum biomarkers.

RESULTS

Oral administration of SB-656933 resulted in significant inhibition of CXCL1-induced CD11b expression on peripheral blood neutrophils at single doses greater than or equal to 50 mg. Maximum inhibition (70%) relative to placebo was observed following administration of SB-656933 400 mg (95% CI 60%, 77%). This was sustained up to a dose of 1100 mg. Single doses of SB-656933 reduced ozone-induced airway inflammation in a dose-dependent manner. Relative to placebo, there were 55% (95% CI 20%, 75%) and 74% (95% CI 55%, 85%) fewer neutrophils in the sputum of subjects after a single dose of 50 mg or 150 mg, respectively. There was a corresponding reduction in myeloperoxidase concentrations in the sputum supernatant of 32.8% (95% CI 9.2, 50.3) and 50.5% (95% CI 33.3, 63.3). SB-656933 was safe and well-tolerated at all doses.

CONCLUSIONS

SB-656933 is a CXCR2 antagonist that demonstrates dose-dependent effects on neutrophil activation and recruitment within a well-tolerated dose range. These data suggest that SB-656933 may be an effective agent in neutrophil-predominant diseases.     

Self-assembling,pH-responsive nanoflowers for inhibiting PAD4 and neutrophil extracellular trap formation and improving the tumor immune microenvironment

Self-assembling carrier-free nanodrugs are attractive agents because they accumulate at tumor by an enhanced permeability and retention (EPR) effect without introduction of inactive substances, and some nanodrugs can alter the immune environment. We synthesized a peptidyl arginine deiminase 4 (PAD4) molecular inhibitor, ZD-E-1M. It could self-assembled into nanodrug ZD-E-1. Using confocal laser scanning microscopy, we observed its cellular colocalization, PAD4 activity and neutrophil extracellular traps (NETs) formation. The populations of immune cells and expression of immune-related proteins were determined by single-cell mass cytometry. ZD-E-1 formed nanoflowers in an acidic environment, whereas it formed nanospheres at pH 7.4. Accumulation of ZD-E-1 at tumor was pH-responsive because of its pH-dependent differences in the size and shape. It could enter the nucleus and bind to PAD4 to prolong the intracellular retention time. In mice, ZD-E-1 inhibited tumor growth and metastasis by inhibiting PAD4 activity and NETs formation. Besides, ZD-E-1 could regulate the ratio of immune cells in LLC tumor-bearing mice. Immunosuppressive proteins like LAG3 were suppressed, while IFN-γ and TNF-α as stimulators of tumor immune response were upregulated. Overall, ZD-E-1 is a self-assembling carrier-free nanodrug that responds to pH, inhibits PAD4 activity, blocks neutrophil extracellular traps formation, and improves the tumor immune microenvironment.     

ELR+ chemokine‐mediated neutrophil recruitment is involved in 2,4,6‐trinitrochlorobenzene‐induced contact hypersensitivity

Contact dermatitis is a form of delayed‐type hypersensitivity characterized by localized thickening, papules, redness and vesicles of the skin. A model of contact dermatitis involving repeated challenge of a hapten is adapted to assess dermatitis as characterized by skin thickening. Recently, it was reported that neutrophils have crucial roles in contact hypersensitivity. We thus examined the involvement of CXC chemokines bearing the glutamic acid–leucine–arginine (ELR) motif (“ELR⁺ chemokines”) and neutrophils in the ear swelling induced by 2,4,6‐trinitrochlorobenzene (TNCB) challenges in the present study. Mice were sensitized by application of TNCB on their abdominal skin. They were then challenged thrice with TNCB to the ear. The CXCR2 antagonist SB225002 (9 mg/kg, i.p.) was administered before each TNCB challenge. Gene expressions and protein levels of the ELR⁺ chemokines CXCL1, 2 and 5 was increased markedly in mouse ear after the final TNCB challenge. In addition, we indicated that gene expression of CXCL1 was enhanced in the epidermis and dermis upon TNCB challenge. Expression of the CXCL2 gene was enhanced in the epidermis, and that of the CXCL5 gene was enhanced in the dermis. The swelling induced by TNCB challenges was significantly attenuated by SB225002. Furthermore, the increases in myeloperoxidase activity, and expression of myeloperoxidase and neutrophil elastase induced by TNCB challenge in mouse ear were inhibited by SB225002. These data suggest that ear swelling resulting from TNCB challenges might be concerned by upregulated ELR⁺ chemokine‐induced neutrophil recruitment.     

Clinical relevance of deferasirox trough levels in β‐thalassemia patients

We evaluated the role of deferasirox therapeutic drug monitoring in order to avoid toxicity or treatment failure. Plasma concentrations, measured between two consecutive liver iron determinations, were determined at the end of dosing interval. Fifty‐four β‐thalassemic adult patients were enrolled: 50% were males; median age was 32.3 years (IQR 19.1‐41.7 years) and median body mass index was 22.25 kg/m² (IQR 20.24‐23.75 kg/m²). The mean deferasirox dose was 28.6 ± 6.3 mg/kg/d and mean plasma concentration was 17.3 ± 16.8 μg/mL. Drug levels showed lower results in males. Deferasirox concentration was significantly correlated with serum creatinine levels (P = .01) and serum ferritin (P < .0001). The assessment of deferasirox therapeutic drug monitoring could help clinicians to predict patient responses and to optimize the therapy.     

Effect of the anti-anginal agent, perhexiline, on neutrophil, valvular and vascular superoxide formation

The prophylactic anti-anginal agent, perhexiline, may also be effective in acute coronary syndromes and advanced aortic valvular stenosis, conditions associated with enhanced inflammation. Its potential effects on superoxide formation via NADPH oxidase were measured by lucigenin-mediated chemiluminescence. Perhexiline inhibited superoxide formation in intact neutrophils stimulated with formyl Met Leu Phe (fMLP) 4 muM or with phorbol myristate acetate (PMA) 162 nM - IC50 2.3 microM (1.5-3.6), n=4. Sub-unit assembly of NADPH oxidase by PMA was unaffected by pretreatment with perhexiline 2 microM, a concentration which reduced superoxide formation by 44+/-5% (n=4) in intact neutrophils. Perhexiline inhibited preassembled neutrophil NADPH oxidase and that in membranes of pig valve interstitial cells, human umbilical vein endothelial cells (HUVECs) and cardiac fibroblasts, but not that in rat aorta (rings or membrane preparations). These data imply that perhexiline inhibits the phagocytic NADPH oxidase directly, and that pig aortic valvular interstitial cells possess a similar enzyme, a conclusion supported by immunohistochemical localisation of the gp91phox subunit in these cells. However further study is required to clarify the effect of perhexiline on different NADPH oxidase isoforms particularly in the vasculature.     

中性粒细胞CD64指数、中性粒细胞与淋巴细胞比值、白介素-6对呼吸机相关性肺炎的早期诊断价值

目的 观察中性粒细胞CD64指数、中性粒细胞与淋巴细胞比值(NLR)、白介素-6(IL-6)在呼吸机相关性肺炎(VAP)病人外周血中的水平及探讨三者诊断VAP的价值.方法 前瞻性选取2014年5月至2019年4月哈尔滨医科大学附属第一医院收治的行机械通气重症监护室病人149例,依据7 d内是否发生VAP分为非VAP组8...     

The intravenous administration of tumor necrosis factor alpha,interleukin 8 and macrophage-derived neutrophil chemotactic factor inhibits neutrophil migration by stimulating nitric oxide production

1. The i.v. administration of tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8) and the recently described macrophage-derived neutrophil chemotactic factor (MNCF) inhibits the recruitment of neutrophils to the inflammatory site.
2. Pretreatment of mice with the NO synthase antagonist, N^G-monomethyl-L-arginine (L-NMMA, 15–60 mg kg⁻¹), but not the inactive enantiomer D-NMMA (30 mg kg⁻¹), prevented in a dose-dependent manner the TNF-α, IL-8 and MNCF-mediated inhibition of neutrophil migration into thioglycollate-challenged peritoneal cavities.
3. Treatment of the neutrophils with TNFα (10⁻⁷ M), IL-8 (10⁻⁷ M) or MNCF blocked their migration towards FMLP in the chemotaxis assay. The pretreatment of the neutrophils with L-NMMA (50–200 μM) prevented in a dose-dependent manner the inhibition of FMLP-induced chemotaxis by IL-8, but did not alter the inhibition caused by TNF-α or MNCF. Different concentrations of the NO donors, S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholino-sydnonimine (SIN-1), did not alter this chemotaxis.
4. Preincubating the neutrophils with L-NMMA (200 μM) significantly increased the TNF-α (10⁻⁷ M) and MNCF-mediated neutrophil adhesion to unstimulated endothelial cells, but had no effect on IL-8 (10⁻⁷ M)-mediated adhesion.
5. Although NO donors did not directly affect the mechanisms of neutrophil motility, NO is involved in the in vitro inhibitory action of IL-8 on chemotaxis. The TNF-α and MNCF-mediated inhibition of neutrophil migration seems to be indirect, by affecting the mechanisms of adhesion. It was concluded that TNF-α-, IL-8- and MNCF-mediated inhibition of neutrophil migration is associated with the stimulation of NO production.

     

Synthesis and evaluation of a novel samarium‐153 bifunctional chelating agent for radioimmunotargeting applications

A new bifunctional chelating agent (BCA), 3‐(4‐isothiocyanatobenzyl)triethylenetetraaminehexaacetic acid ( 9 ), has been synthesized in fast and easy conditions. An improved synthesis of its position isomer 1‐(4‐isothiocyanatobenzyl)triethylenetetraaminehexaacetic acid ( 19 ) is also described. Stability in serum media of the two corresponding aminobenzyl derivatives‐samarium‐153 complexes, respectively, 3‐(4‐aminobenzyl)triethylenetetraaminehexaacetic acid—samarium‐153 and 1‐(4‐aminobenzyl)triethylenetetraaminehexaacetic acid—samarium‐153, have been evaluated. The 3‐(4‐aminobenzyl)triethylenetetraaminehexaacetic acid complex revealed excellent stability in serum media, and therefore 3‐(4‐isothiocyanatobenzyl)triethylenetetraaminehexaacetic acid ( 9 ) appears useful for future in vivo radioimmunotherapy investigations. Copyright © 2005 John Wiley & Sons, Ltd.     

Inhibition of ex vivo neutrophil activation by oral LY293111, a novel leukotriene B4 receptor antagonist

1 The effects of orally administered LY293111 on ex vivo neutrophil Mac-1 upregulation were determined in a total of 24 healthy male subjects within three study periods.
2 In the first period, eight volunteers received 60  mg LY293111 or placebo three times daily in 22 total doses over 8 days followed by a 1 week follow-up. The average ex vivo Mac-1 response of the LY293111 group was 56% of the pre-dose control (95% confidence interval (CI) 44.3 to 67.9%; P <0.01). The inhibitory effect was maximum at the end of dosing and had disappeared by day 14.
3 In the second period, eight subjects received 120  mg LY293111 or placebo three times daily in 22 total doses over 8 days followed by a 1 week follow-up. The average response of the LY293111 group was 70% of the pre-dose control (95% CI 59.7 to 81.0%; P <0.01). The inhibitory effect was maximum the day following the initial dose and continued throughout the dosing period.
4 In the third period, eight subjects received 200  mg LY293111 or placebo twice daily in 15 total doses over 8 days followed by a 1 week follow-up. Mac-1 upregulation was 64% of pre-dose levels (95% CI 53.8 to 75.1%; P <0.01) over the course of the study period. The inhibition had disappeared 2 days following the final dose. Alternate neutrophil stimulation by fMLP was not inhibited.
5 No statistically significant inhibition was observed for placebo-treated subjects.
6 No statistically significant differences were apparent between the active dose regimens.
7 The results indicate that orally administered LY293111 is pharmacologically active in humans. Results from this study may be useful in determining dose selection for efficacy trials.     

The anti‐Trichomonas vaginalis phloroglucinol derivative isoaustrobrasilol B modulates extracellular nucleotide hydrolysis

Trichomonas vaginalis causes trichomoniasis, a neglected sexually transmitted disease. Due to severe health consequences and treatment failure, new therapeutic alternatives are crucial. Phloroglucinols from southern Brazilian Hypericum species demonstrated anti‐T. vaginalis and anti‐Leishmania amazonensis activities. The modulation of biochemical pathways involved in the control of inflammatory response by ectonucleotidases, NTPDase, and ecto‐5′‐nucleotidase represents new targets for combating protozoa. This study investigated the activity of phloroglucinol derivatives of Hypericum species from southern Brazil against T. vaginalis as well as its ability on modulating parasite ectonucleotidases and, consequently, immune parameters through ATP and adenosine effects. Phloroglucinol derivatives screening revealed activity for isoaustrobrasilol B (IC₅₀ 38 μm ) with no hemolytic activity. Although the most active compound induced cytotoxicity against a mammalian cell lineage, the in vivo model evidenced absence of toxicity. Isoaustrobrasilol B significantly inhibited NTPDase and ecto‐5′‐nucleotidase activities, and the immune modulation attributed to extracellular nucleotide accumulation was evaluated. The production of ROS and IL‐6 by T. vaginalis‐stimulated neutrophils was not affected by the treatment. Conversely, IL‐8 levels were significantly enhanced. The associative mechanism of trophozoites death and ectonucleotidases modulation by isoaustrobrasilol B may increase the susceptibility of T. vaginalis to host innate immune cell like neutrophils consequently, contributing to parasite clearance.     

Peptides derived from histidine and methionine‐rich regions of copper transporter 1 exhibit anti‐angiogenic property by chelating extracellular Cu

Angiogenesis is a process of synthesis of new blood vessels from preexisting vasculature. Copper (Cu) as a micronutrient is important to many proteins for their physiological roles. Cu is transported by ceruloplasmin from liver to other parts of the body. Copper transporter 1 (CTR1) is a transmembrane protein which participate in Cu transport across the cell. It is also known to be involved in angiogenesis. In this study, we have designed three peptides from copper‐binding regions of CTR1 which are rich in histidine and methionine. These peptides were screened for their inhibitory effect on angiogenesis in the HUVEC model. Mass spectroscopy studies revealed that all the three peptides derived from CTR 1 (Pep 1, 2, and 3) bound to Cu. The intracellular Cu levels estimated by atomic absorption spectroscopy showed decreased levels of copper in peptide‐treated cells as compared to control. These peptides inhibited proliferation, migration, and tube formation in HUVEC by sequestering copper, preventing its entry into the cell and thereby inhibiting angiogenesis.     

A novel adenosine analog, AMP579, inhibits neutrophil activation, adherence and neutrophil-mediated injury to coronary vascular endothelium

We hypothesized that 1S-[1a,2b,3b, 4a(S*)]-4-[7-[[1-[(3-chloro-2-thienyl)methylpropyl]propyl-amino]-3H-i midazo[4,5-b] pyridyl-3-yl]-N-ethyl-2,3-dihydroxycyclopentane carboxamide (AMP579), a new adenosine analog, inhibits superoxide anion (O(2)(-)) generation and degranulation from canine neutrophils, neutrophil adherence and neutrophil-induced dysfunction to canine coronary artery endothelium by adenosine receptor-mediated mechanisms. AMP579 inhibited O(2)(-) generation (nM/20x10(6) neutrophils) from platelet activating factor (PAF)-activated neutrophil in concentration-dependent manner (4.1+/-0.8 at 10 microM vs. 16.7+/-2.1 in PAF group, P<0.05). This inhibitory effect was blocked by the adenosine A(2A) receptor-selective antagonist, 4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3, 5]triazin-5-ylamino]ethyl)phenol (ZM241385, 17.7+/-2.8, P<0.05), but not by either the adenosine A(1) receptor-selective antagonist, 8-Cyclopentyl-1.3-dipropylxanthine (DPCPX) or the adenosine A(3) receptor-selective antagonist, 9-Chloro-2-(2-furanyl)-5-[(phenylacetyl)amino][1,2,4]-triazolo[1, 5-c]quinazoline (MRS1220). AMP579 inhibited neutrophil degranulation dose-dependently by 38+/-2% at 10 microM (P<0.05). The inhibitory effect of AMP579 was not altered by either DPCPX or MRS1220, but was partially reversed by ZM241385 (69+/-8%, P<0.05 vs. AMP579 10 microM). A total of 10 microM AMP579 reduced neutrophil adherence to thrombin-stimulated endothelium (neutrophils/mm(2)) from 269+/-16 to 44+/-4 (P<0.05); this was reversed by ZM241385, but not by DPCPX or MRS1220. After coincubation of unstimulated neutrophil with thrombin-stimulated endothelium, concentration-relaxation responses to the endothelium receptor-dependent vasodilator, acetylcholine, were reduced (maximum 57+/-5% vs. 120+/-5% in controls, P<0.05). This endothelial dysfunction was attenuated by AMP579 (116+/-7%, P<0. 05). We conclude that AMP579 inhibits neutrophil activation and neutrophil-mediated coronary endothelial dysfunction, primarily by an adenosine A(2A) receptor mechanism.     

An anticancer agent icaritin induces sustained activation of the extracellular signal-regulated kinase (ERK) pathway and inhibits growth of breast cancer cells

Icaritin, a prenylflavonoid derivative from Epimedium Genus, regulates many cellular processes. However, the function and the underlying mechanisms of icaritin in breast cancer cell growth have not been well established. Here, we report that icaritin strongly inhibited the growth of breast cancer MDA-MB-453 and MCF7 cells. At concentrations of 2-3 μM, icaritin induced cell cycle arrest at the G(2)/M phase accompanied by a down-regulation of the expression levels of the G(2)/M regulatory proteins such as cyclinB, cdc2 and cdc25C. Icaritin at concentrations of 4-5 μM, however, induced apoptotic cell death characterized by the accumulation of the annexin V- and propidium iodide-positive cells, cleavage of poly ADP-ribose polymerase (PARP) and down-regulation of the Bcl-2 expression. In addition, icaritin also induced a sustained phosphorylation of extracellular signal-regulated kinase (ERK) in these breast cancer cells. U0126, a specific ERK activation inhibitor, abrogated icaritin-induced G2/M cell cycle arrest and cell apoptosis. Icaritin more potently inhibited growth of the breast cancer stem/progenitor cells compared to anti-estrogen tamoxifen. Our results indicate that icaritin is a potent growth inhibitor for breast cancer cells and provide a rationale for preclinical and clinical evaluations of icaritin for breast cancer therapy.     

B‐Type natriuretic peptide suppression of neutrophil superoxide generation: mechanistic studies in normal subjects

Many acute cardiovascular disease states are associated with neutrophil infiltration of myocardium and subsequent release of superoxide (O₂^?) and myeloperoxidase (MPO), which contribute to inflammatory reactions. B‐Type natriuretic peptide (BNP) is known to exert anti‐inflammatory and antifibrotic effects, but it is not known whether these may include interactions with neutrophils. In neutrophils isolated from 20 healthy subjects, we assessed the effect of BNP on the ‘neutrophil burst’ (O₂^? production and MPO release) stimulated by phorbol myristate acetate (PMA) and N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP), respectively. Effects of BNP on cGMP accumulation, and the effects of the cell‐permeable cGMP analogue 8‐(4‐chlorophenylthio) guanosine‐cGMP (8‐p‐CPT‐cGMP) and protein kinase G (PKG) inhibition with KT5823 on the neutrophil–BNP interaction were also evaluated. B‐Type natriuretic peptide suppressed O₂^? release from neutrophils by 23 ± 6% (P < 0.001) and 24 ± 8% (P < 0.05) following PMA and fMLP stimulation, respectively. Although BNP did not significantly increase cGMP formation, 8‐p‐CPT‐cGMP suppressed both PMA‐ and fMLP‐induced neutrophil O₂^? release by 16% and 28%, respectively (P < 0.05). The PKG inhibitor KT5823 attenuated the effects of BNP on both fMLP‐ and PMA‐associated O₂^? production. Neither BNP nor 8‐p‐CPT‐cGMP significantly affected MPO release from neutrophils. Suppression of O₂^? release from neutrophils by BNP may contribute to its anti‐inflammatory and antifibrotic actions.     

子痫前期中性粒细胞胞外诱捕网的形成与血管内皮损伤的相关性研究

目的 研究子痫前期(PE)病人血清中中性粒细胞胞外诱捕网(NETs)来源,并分析与血管内皮损伤的相关性。方法选取2020年9月至2021年9月潍坊市人民医院收治的30例PE病人作为PE组,30例正常分娩的健康产妇作为Nor-P组,同期于该院进行孕前检查的健康未孕妇女30例作为对照组。比较三组的血白细胞及中性粒细胞数量、髓过氧化物酶、NETs、内皮损伤指标含量的差异。采用Pearson检验评估NETs与内皮损伤的相关性。结果 PE组白细胞数量(12.01±0.88)×10⁹/L、中性粒细胞数量(10.22±0.99)×10⁹/L及髓过氧化物酶(15.39±0.84)μg/L、NETs(124.68±3.63)ng/L、血管性血友病因子(vWF)(122.79±4.05)μg/L水平高于对照组和Nor-P组(P<0.05)。Pearson相关性分析发现,PE病人血清NETs水平与中性粒细胞数量呈正相关(r=0.63,P<0.05),同时NETs水平与vWF水平呈正相关(r=0.83,P<0.05)。结论 PE病人血清中的NETs...     

Ginsenoside-Rp1 inhibits platelet activation and thrombus formation via impaired glycoprotein VI signalling pathway, tyrosine phosphorylation and MAPK activation

BACKGROUND AND PURPOSE

Ginsenosides are the main constituents for the pharmacological effects of Panax ginseng. Such effects of ginsenosides including cardioprotective and anti-platelet activities have shown stability and bioavailability limitations. However, information on the anti-platelet activity of ginsenoside-Rp1 (G-Rp1), a stable derivative of ginsenoside-Rg3, is scarce. We examined the ability of G-Rp1 to modulate agonist-induced platelet activation.

EXPERIMENTAL APPROACH

G-Rp1 in vitro and ex vivo effects on agonist-induced platelet-aggregation, granule-secretion, [Ca²⁺]_i mobilization, integrin-α_IIbβ₃ activation were examined. Vasodilator-stimulated phosphoprotein (VASP) and MAPK expressions and levels of tyrosine phosphorylation of the glycoprotein VI (GPVI) signalling pathway components were also studied. G-Rp1 effects on arteriovenous shunt thrombus formation in rats or tail bleeding time and ex vivo coagulation time in mice were determined.

KEY RESULT

G-Rp1 markedly inhibited platelet aggregation induced by collagen, thrombin or ADP. While G-Rp1 elevated cAMP levels, it dose-dependently suppressed collagen-induced ATP-release, thromboxane secretion, p-selectin expression, [Ca²⁺]_i mobilization and α_IIbβ₃ activation and attenuated p38^MAPK and ERK2 activation. Furthermore, G-Rp1 inhibited tyrosine phosphorylation of multiple components (Fyn, Lyn, Syk, LAT, PI3K and PLCγ2) of the GPVI signalling pathway. G-Rp1 inhibited in vivo thrombus formation and ex vivo platelet aggregation and ATP secretion without affecting tail bleeding time and coagulation time, respectively.

CONCLUSION AND IMPLICATIONS

G-Rp1 inhibits collagen-induced platelet activation and thrombus formation through modulation of early GPVI signalling events, and this effect involves VASP stimulation, and ERK2 and p38^-MAPK inhibition. These data suggest that G-Rp1 may have therapeutic potential for the treatment of cardiovascular diseases involving aberrant platelet activation.     

Preparation and biological evaluation of 99mTc‐stannous fluoride colloid‐labelled‐leucocytes in rats 99mTc‐stannous fluoride‐labelled‐leucocytes in rats

High splenic activity is always visible in ^99mTc‐stannous fluoride (SnF₂)‐labelled‐leucocytes scans. In an attempt to reduce this activity, this study investigated the effect of pre‐injected SnF₂ colloid on the distribution of ^99mTc‐SnF₂ colloid, ^99mTc‐SnF₂‐labelled‐leucocytes, and opsonized ^99mTc‐SnF₂ colloid in rats. The radiopharmaceuticals ^99mTc‐SnF₂ colloid and ^99mTc‐SnF₂‐leucocytes were each found to exhibit identical biodistributions in separate experiments. SnF₂ colloid pre‐injection (26 μg) resulted in reduced splenic uptake of ^99mTc‐SnF₂ colloid (38%) and ^99mTc‐SnF₂‐labelled‐leucocytes (30%), but not for opsonized ^99mTc‐SnF₂ colloid. This indicates that the level of opsonization of radiocolloid is rate limiting rather than the phagocytic capacity of liver and spleen macrophages. There is a low level of ^99mTc‐SnF₂‐labelled‐leucocytes dominated by unopsonized radiocolloid in the ex vivo whole blood dose. Following administration of this dose, free radiocolloid is present in vivo that predominantly localizes in the liver and spleen. This uptake can be challenged with non‐radioactive stannous fluoride colloid pre‐injection, where splenic activity can be significantly reduced by up to 52%. Copyright © 2003 John Wiley & Sons, Ltd.     

KF4939, a new anti-platelet agent, inhibits activation of phospholipase C and A2 in rabbit platelets

Effect of KF4939 on activation of phospholipase C and A2 was examined by using rabbit platelets prelabelled with [14C]-arachidonic acid. The results showed that KF4939 inhibited the activation of the two phospholipases, separately. The inhibition of phospholipase C activation is thought to be a possible mechanism of KF4939 to inhibit aggregation and secretion which are independent of the synthesis of cyclooxygenase products such as thromboxane A2.     

Effects of tenoxicam on superoxide anion formation, β-glucuronidase release and fMLP binding in human neutrophils: Comparison with other NSAIDs

Non-steroidal anti-inflammatory drugs (NSAIDs) are considered to exert their activity by interfering with the generation of arachidonate metabolites in various cells, mainly in neutrophils and monocytes. The inhibition of cellular cyclooxygenase enzyme, however, does not always correlate with the in vivo activity of these drugs. Recent evidence indicates that several NSAIDs may interfere with the stimulus-response coupling of inflammatory cells. In this study, the effects of tenoxicam, an oxicam derivative with a thienothiazine structure, on neutrophil activation were evaluated by the assessment of the following parameters: (1) superoxide anion generation by neutrophils and whole blood stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP), the calcium ionophore A23187 and serum treated zymosan (STZ); (2) beta-glucuronidase release from neutrophils stimulated with fMLP, A23187 and STZ; (3) binding of [3H]fMLP to intact neutrophils. The results were compared to those obtained using piroxicam and diclofenac. Tenoxicam, added in vitro to whole blood, at concentrations ranging between 10(-5) and 3 x 10(-4) M, significantly inhibited the generation of superoxide anion induced by fMLP, A23187 and STZ. The activity of tenoxicam on whole blood was similar to that of piroxicam, whereas diclofenac had only minimal effects on this experimental system. In isolated cells tenoxicam inhibited the generation of superoxide anion induced by A23187 and STZ. In addition, at the 3 x 10(-4) M concentration, tenoxicam and diclofenac similarly inhibited O2- generation by neutrophils stimulated with fMLP, whereas piroxicam only minimally affected this parameter. Tenoxicam also slightly, but not significantly, inhibited beta-glucuronidase release by isolated neutrophils induced by all the agonists used. Specific binding of [3H]fMLP to neutrophils was inhibited by the three NSAIDs tested in a dose-dependent fashion and tenoxicam was the most potent. The affinities (Kd) of tenoxicam, piroxicam and diclofenac were 1.11, 1.80 and 2.70 x 10(-5) M, respectively. The mechanism of inhibition of [3H]fMLP binding by tenoxicam was non-competitive. It is concluded that tenoxicam, at concentrations achievable in plasma at steady state, effectively inhibits some of the processes involved in neutrophil activation, which bear some relevance in the inflammatory disease.