Tat-induced platelet-activating factor synthesis contributes to the angiogenic effect of HIV-1 Tat

This study shows that human umbilical cord vein-derived endothelial cells (HUVEC) stimulated with HIV-1 Tat synthesized platelet-activating factor (PAF), a phospholipid mediator of inflammation that possesses angiogenic properties. The synthesis of PAF by HUVEC stimulated with Tat was dose and time dependent. Moreover, in vitro experiments were performed to evaluate whether migration of HUVEC induced by Tat was dependent on the synthesis of PAF. It was found that the cell motility induced by Tat was inhibited by WEB 2170, a specific PAF receptor antagonist. In vivo, the neoangiogenesis induced by Tat was also inhibited by WEB 2170 in a murine model, in which matrigel subcutaneously injected was used as substratum for angiogenesis. These results suggest that the synthesis of PAF by endothelial cells mediates, at least in part, the angiogenic activity of Tat by promoting the endothelial cell migration.     

Synthesis of platelet-activating factor by human monocytes stimulated by platelet-activating factor

The capacity of platelet-activating factor (PAF) to stimulate its own synthesis by human monocytes was examined. Adherent human monocytes of greater than 85% purity were incubated with 100 fM to 10 nM of PAF in the presence of 20 microCi of [3H]acetic acid to radiolabel newly synthesized PAF. After incubation for 15 minutes to 15 hours, PAF was purified by high-performance liquid chromatography, and newly synthesized PAF was quantified by its radioactivity. PAF stimulated its own synthesis in a dose-related manner with a maximal twofold to threefold increase in synthesis at 10 pM to 100 pM of PAF. Maximal PAF synthesis occurred after incubation for 6 to 8 hours. There was a good correlation (r = 0.95) between PAF quantified by [3H]acetic acid incorporation and by rabbit platelet aggregation bioassay, indicating that the radioactive material is PAF. The protein synthesis inhibitor, cycloheximide, did not inhibit delayed PAF synthesis, indicating that delayed PAF synthesis does not require protein synthesis. PAF is metabolized rapidly in vivo. The capacity of PAF to stimulate its own synthesis would result in a prolonged effective half-life in vivo. This prolonged half-life could contribute to the capacity of PAF to induce prolonged inflammatory reactions in vivo.     

Protein kinase C regulates the synthesis of platelet-activating factor by human monocytes.

Human peripheral blood monocytes synthesize the potent lipid autacoid platelet-activating factor (PAF) following appropriate stimulation. We examined the role of protein kinase C (PKC) in regulating the synthesis of PAF by stimulated monocytes. 4 beta-phorbol 12-myristate 13-acetate (PMA) and 1,2-dioctanoyl-sn-glycerol, which directly activate PKC, stimulated the synthesis of PAF. Sphingosine, a long-chain amine that inhibits PKC, blocked both the binding of phorbol esters to monocytes and the synthesis of PAF in response to PMA (half-maximal inhibition at 5 to 10 microM and complete inhibition at 10 to 30 microM sphingosine). Thus, the activation of PKC was necessary and sufficient for PAF synthesis in response to phorbol ester. Sphingosine also blocked PAF synthesis in response to the calcium ionophore A23187 and opsonized zymosan particles by specific inhibition of PKC. Two other PKC inhibitors, stearylamine and staurosporine, also blocked PAF synthesis following A23187 or opsonized zymosan stimulation. These experiments demonstrated that PKC activation was required for PAF synthesis in response to the calcium signal generated by A23187 or a receptor-mediated agonist, opsonized zymosan. The synthesis of PAF and leukotriene B4 were temporally coupled following cell stimulation. Further, production of these two lipid mediators, and the release of arachidonic acid, were inhibited in parallel by sphingosine. Thus, PKC regulate the synthesis of both PAF and leukotriene B4 at a common step, probably phospholipase A2.     

Regulation of platelet-activating factor synthesis in human monocytes by dipalmitoyl phosphatidylcholine

Platelet-activating factor (PAF) has a major role in inflammatory responses within the lung. This study investigates the effect of pulmonary surfactant on the synthesis of PAF in human monocytic cells. The pulmonary surfactant preparation Curosurf significantly inhibited lipopolysaccharide (LPS)-stimulated PAF biosynthesis (P<0.01) in a human monocytic cell line, Mono mac-6 (MM6), as determined by (3)H PAF scintillation-proximity assay. The inhibitory properties of surfactant were determined to be associated, at least in part, with the 1,2-dipalmitoyl phosphatidylcholine (DPPC) component of surfactant. DPPC alone also inhibited LPS-stimulated PAF biosynthesis in human peripheral blood monocytes. DPPC treatment did not affect LPS-stimulated phospholipase A(2) activity in MM6 cell lysates. However, DPPC significantly inhibited LPS-stimulated coenzyme A (CoA)-independent transacylase and acetyl CoA:lyso-PAF acetyltransferase activity. DPPC treatment of MM6 cells decreased plasma membrane fluidity as demonstrated by electron paramagnetic resonance spectroscopy coupled with spin labeling. Taken together, these findings indicate that pulmonary surfactant, particularly the DPPC component, can inhibit LPS-stimulated PAF production via perturbation of the cell membrane, which inhibits the activity of specific membrane-associated enzymes involved in PAF biosynthesis.     

Signaling pathways triggered by HIV-1 Tat in human monocytes to induce TNF-alpha

In this study we investigated the signaling pathways triggered by Tat in human monocyte to induce TNF-alpha. In monocytes, the calcium, the PKA, and the PKC pathways are highly implicated in the expression of cytokine genes. Thus, these three major signaling pathways were investigated. Our data show that (i) PKC and calcium pathways are required for TNF-alpha production, whereas the PKA pathway seems to be not involved; (ii) downstream from PKC, activation of NFkappaB is essential while ERK1/2 MAP kinases, even though activated by Tat, are not directly involved in the pathway signaling leading to TNF-alpha production.     

Histamine potentiates human platelet aggregation induced by platelet-activating factor.

Using human platelet-rich plasma (PRP) we found that histamine concentration-dependently potentiated platelet aggregation induced by platelet-activating factor (PAF). When added separately, neither histamine nor chloropyramine, a competitive antagonist of H1 receptors, influenced platelet aggregation, however BN 52021, a specific PAF receptor antagonist, concentration-dependently inhibited the response. Chloropyramine reduced the potentiating effect of histamine on PAF-induced platelet aggregation, and when it was coadministered with BN 52021, inducing 63% inhibition of PAF-induced aggregation, the histamine-potentiated PAF response was completely abolished. This observation draws attention to the importance of cooperation between histamine and PAF in mediation of platelet aggregation in human PRP, and points to the powerful antiaggregatory effect of BN 52021 combined with an antagonist of H1 receptors.     

Rickettsial stimulation of endothelial platelet-activating factor synthesis.

Endothelial cells (EC) synthesize platelet-activating factor (PAF) when activated by agents such as ATP or thrombin, and PAF production occurs as a consequence of endothelial phospholipase A activity. Because interactions between Rickettsia prowazekii and a variety of host cells result in the expression of phospholipase A activity, we assessed the relative abilities of uninfected and rickettsia-infected EC to synthesize PAF. Endothelial cells were infected with rickettsiae and examined at 24-h intervals for rickettsial multiplication, EC viability, and PAF synthesis. By 24 h postinfection, 80% of the EC were infected with an average of 10.6 rickettsiae per cell; by 72 h, the rickettsiae were too numerous to count and the numbers of viable EC began to decrease. Both rickettsia-infected and sham-treated EC synthesized PAF when stimulated with either thrombin or ATP, but rickettsia-infected EC synthesized about three times as much PAF in response to cell activation as did their uninfected counterparts. Additionally, unlike their uninfected counterparts, rickettsia-infected EC synthesized significant amounts of PAF in the absence of cell activation; rickettsia-infected EC synthesized as much PAF in the absence of activation as did uninfected EC in response to ATP. In each case, essentially all of the newly synthesized PAF remained with the cell pellet. Finally, EC incubated with high numbers of rickettsiae (1,000 rickettsiae per EC) for 30 min synthesized more PAF when activated with ATP than did their sham-treated activated counterparts.     

Interaction of platelet-activating factor with interferon-gamma in the stimulation of interleukin-1 production by human monocytes

Platelet-activating factor (PAF) produces both early and late inflammatory responses in vivo. The late and persistent responses to PAF may result from the production of cytokines, such as interleukin-1 (IL-1). The effects of PAF on IL-1 release by human monocytes were studied with PAF alone and in combination with interferon-gamma. These studies involved the use of D10.G4.1 cells in a proliferation assay to determine the actual concentration of active IL-1 in monocyte supernatant and pellet fractions. These studies confirmed that PAF stimulates IL-1 release at two different ranges of PAF concentrations, 100 fM to 1 pM and 100 pM to 1 nM. PAF inhibited IL-1 release at 1 or 10 pM. PAF effects on IL-1 release were specific to the form of PAF that is biologically active in most system; (S)-PAF and lyso-PAF had no effect. When monocytes were incubated with both PAF and interferon-gamma, IL-1 release was greatly stimulated at the same two ranges of PAF concentrations, 100 fM to 1 pM and 100 pM to 1 nM of PAF. PAF and interferon-gamma interacted synergistically to enhance significantly the release of IL-1.     

Safety and immunogenicity of HIV-1 Tat toxoid in immunocompromised HIV-1-infected patients

OBJECTIVES: To antagonize the deleterious effects of the HIV-1 toxin extracellular Tat on uninfected immune cells, we developed a new strategy of anti-HIV-1 vaccine using an inactivated but immunogenic Tat (Tat toxoid). Tat toxoid has been assayed for safety and immunogenicity in seropositive patients. METHOD: The phase I vaccine clinical trial testing Tat toxoid preparation in Seppic Isa 51 oil adjuvant was performed on 14 HIV-1-infected asymptomatic although biologically immunocompromised individuals (500-200 CD4+ cells/mm3). RESULTS: Following as many as 8 injections, no clinical defects were observed. All patients exhibited an antibody (Ab) response to Tat, and some had cell-mediated immunity (CMI) as evaluated by skin test in vivo and T-cell proliferation in vitro. CONCLUSION: These results provide initial evidence of safety and potency of Tat toxoid vaccination in HIV-1-infected individuals.     

HIV-1 Tat基因的融合构建及在大肠杆菌中的高效表达

目的：在E．coli BL21(DE3)中高效表达Tat蛋白。方法：用PCR方法构建HIV-1 Tat基因全序列，同时将Tat基因进行定点突变(AAG28替换为CAG，AAG50替换为CAG)，以消除天然．Tat蛋白的转录活性。将突变的Tat基因与伴侣10(chap10)基因连接后，共同亚克隆人表达载体pET28a中，并在Ecoli中表达，表达产物用Western blot进行鉴定。结果：分别通过3轮PCR，成功地构建了Tat基因全序列。构建的重组质粒pET28a-chap10-Tat在Ecoli BL21(DE3)中得到高效表达。Western blot分析表明，在相对分子质量(Mr)为24000处有1条特异性的带。结论：chap10基因与HIV-1 Tat全基因的融合构建，使Tat蛋白在大肠杆菌中得到高效表达，为其在爱滋病发病中作用研究奠定了基础。     

Characterization of cutaneous vascular permeability induced by platelet-activating factor in guinea pigs and rats and its inhibition by a platelet-activating factor receptor antagonist

Mechanisms of platelet-activating factor (PAF)-induced increases of cutaneous vascular permeability in guinea pigs and in rats were further explored. PAF so far is the most potent vasoactive mediator, being more than 1000-fold more potent than histamine and bradykinin in both species. In guinea pigs, there is a time delay of 5 to 10 minutes before PAF action, whereas, in the rat, the increased vasopermeability occurs immediately following the intradermal PAF injection. Relative vasoactive potencies of PAF and several structure-related analogues in both species correlate very well with their relative inhibition of the binding of 3H-PAF to specific receptor sites on isolated rabbit platelet plasma membranes and their aggregatory abilities of rabbit platelets. Furthermore, the PAF-induced cutaneous vascular permeability is inhibitable by a competitive specific PAF receptor antagonist, kadsurenone, suggesting that binding of PAF to its specific receptor site is the first step to initiate its action of increased cutaneous vascular permeability. Several pure cyclooxygenase inhibitors, including indomethacin, diflunisal, and flurbiprofen, and the dual cyclooxygenase/lipoxygenase inhibitor, BW755C, but not the histamine antagonists, inhibit the PAF-induced vasopermeability in guinea pigs. The inhibition by indomethacin or BW755C can be fully reversed by coinjection intradermally with PAF and prostaglandin E1 but not leukotriene B4. Also, prostaglandin E1 but not leukotriene B4 enhances the guinea pig in vivo response to PAF in this model. However, in rats, none of the cyclooxygenase inhibitors, histamine antagonists, or BW755C inhibit the PAF effect of cutaneous phenomena even though prostaglandin E1 also enhances the PAF potency of the increased cutaneous vascular permeability. Kadsurenone, a competitive specific receptor antagonist, inhibits both histamine- and bradykinin-induced rat cutaneous vascular permeability which suggests that PAF may be involved in the vasopermeability induced by histamine and bradykinin.