Role of phosphorylcholine in Streptococcus pneumoniae and nontypeable Haemophilus influenzae adherence to epithelial cells.

OBJECTIVE: Phosphorylcholine (PC) is a structural component of Streptococcus pneumoniae (Spn) and nontypeable Haemophilus influenzae (NTHi), and is known to be associated with adherence through the platelet activating factor receptor (PAF-R). Furthermore, high PC expression is considered to be involved in Spn and NTHi virulence. In this study, we examined the influence of PC expression on the adherence of Spn and NTHi to epithelial cells in order to clarify the potential effectiveness of a vaccine targeting PC. METHODS: Twenty-seven strains of Spn and twenty-two strains of NTHi were used, cultured overnight, and PC expression was evaluated by fluorescence activated cell sorting; the strains were divided into two groups: PC low expression (PC-low) and PC high expression (PC-high) groups. Bacterial adherence was then examined using Detroit 562 cells and BALB/c mice. Bacterial invasion was then examined in Detroit 562 cells. RESULTS: The adherence of Spn and NTHi and invasion of NTHi in the PC-high group was significantly reduced by pretreatment with a monoclonal anti-PC antibody (TEPC-15), PAF-R antagonist (ABT-491), and PC-keyhole limpet hemocyanin (PC-KLH). However, such findings were not observed in the PC-low group. CONCLUSION: The present study suggests that PC is involved in the mucosal adhesion of Spn and NTHi, and the mucosal invasion of NTHi with PC-high strains, but not PC-low strains. These results suggest that a PC-targeting mucosal vaccine only affects PC-high Spn and NTHi strains and does not disturb commensal bacterial flora in the upper respiratory tract, which comprises nonpathogenic PC-low bacteria.

Biochemical and inhibition studies of glutamine synthetase from Leishmania donovani.

Leishmaniasis is a group of tropical diseases caused by protozoan parasites of the genus Leishmania. Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis, a fatal disease if left untreated. Chemotherapy for leishmaniasis is problematic as the available drugs are toxic, costly and shows drug resistance, hence, there is a necessity to look out for the novel drug targets, chemical entities and vaccine. Glutamine synthetase (GS) catalyzes the synthesis of glutamine from glutamate and ammonia. In the present study, we have identified and characterized GS from L. donovani. The nucleotide sequence encoding putative glutamine synthetase like sequence from L. donovani (LdGS, LDBPK_060370) was cloned. A 43.5 kDa protein with 6X-His tag at the C-terminal end was obtained by overexpression of LdGS in Escherichia coli BL21 (DE3) strain. Expression of native LdGS in promastigotes and recombinant L. donovani glutamine synthetase (rLdGS) was confirmed by western blot analysis. An increase in expression of GS was observed at different phases of growth of the parasite. Expression of LdGS in promastigote and amastigote was confirmed by western blot analysis. Immunofluorescence studies of both the promastigote and amastigote stages of the parasite revealed the presence of LdGS in cytoplasm. GS exists as a single copy gene in parasite genome. Kinetic analysis of GS enzyme revealed Km value of 26.3 ± 0.4 mM for l- glutamate and Vmax value of 2.15 ± 0.07 U mg-1. Present study confirms the presence of glutamine synthetase in L. donovani and provides comprehensive overview of LdGS for further validating it as a potential drug target.

Phosphorylcholine and SpaA, a choline-binding protein, are involved in the adherence of Erysipelothrix rhusiopathiae to porcine endothelial cells, but this adherence is not mediated by the PAF receptor.

A crucial event in the initiation of many bacterial infections is the adherence of the bacteria to host cells, and bacterial surface structures and their interactions with host cell receptors play an important role in this process. Erysipelothrix rhusiopathiae is the causative agent of swine erysipelas, which may cause acute septicemia or chronic endocarditis and polyarthritis. To study the pathogenic mechanism of the widespread vascular disease observed in the acute form of swine erysipelas, we investigated the role of phosphorylcholine (PCho), a component of the E. rhusiopathiae capsule, in bacterial adherence to porcine endothelial cells (PECs) in vitro. We found that adherence of E. rhusiopathiae strain Fujisawa to PECs was twice that of adherence to control COS-7 cells and that the adherence rates of PCho-defective mutants were approximately 30-50% lower than those of the Fujisawa strain. The adherence of the Fujisawa strain to COS-7 cells transfected with the porcine platelet-activating factor receptor (PAFR) gene, which encodes a G protein-coupled receptor that has been shown to directly bind to Streptococcus pneumoniae via PCho in the bacterial cell wall, was not enhanced. Treatment with a PAFR antagonist (WEB-2086) did not inhibit bacterial adherence to PECs. Incubation of the bacterial cells with an antibody against PCho or SpaA, a choline-binding protein anchored to PCho of the Fujisawa strain, reduced the adherence of the strain to PECs. This effect was not observed when PCho-defective mutants were used. These results suggest that E. rhusiopathiae adheres to PECs via PCho and SpaA and that the PCho-mediated adherence is independent of PAFR.

Ultraviolet B radiation of human skin generates platelet-activating factor receptor agonists.

Ultraviolet B radiation (UVB) is a potent stimulator of epidermal cytokine production. In addition to cytokines, such as tumor necrosis factor-alpha (TNF-alpha), UVB generates bioactive lipids including platelet-activating factor (PAF). Our previous in vitro studies in keratinocytes or epithelial cell lines have demonstrated that UVB-mediated production of PAF agonists is due primarily to the pro-oxidative effects of this stimulant, resulting in the nonenzymatic production of modified phosphocholines (oxidized glycerophosphocholines). The current studies use human skin to assess whether UVB irradiation generates PAF-receptor agonists, and the role of oxidative stress in their production. These studies demonstrate that UVB irradiation of human skin results in PAF agonists, which are blocked by the antioxidant vitamin C and the epidermal growth factor receptor inhibitor PD168393. Inasmuch as UVB-generated PAF agonists have been implicated in animal model systems as being involved in photobiologic processes including systemic immunosuppression and cytokine (TNF-alpha) production, these studies indicate that this novel activity could be involved in human disease.

Celecoxib antagonizes perifosine's anticancer activity involving a cyclooxygenase-2-dependent mechanism.

Perifosine is an orally bioavailable alkylphospholipid currently being tested in phase II clinical trials as a potential anticancer drug. In this study, we reveal a novel mechanism underlying the anticancer activity of perifosine that involves the induction of cyclooxygenase 2 (COX-2) in human cancer cells. Perifosine induced apoptosis and/or cell cycle arrest in several lung and head and neck cancer cell lines. However, the combination of perifosine with low concentrations of celecoxib rendered cells less sensitive to perifosine both in cell culture systems and in lung cancer xenograft models. Subsequently, we examined the effects of perifosine on COX-2 expression and activity in a set of lung and head and neck cancer cell lines, and found that perifosine rapidly and potently increased COX-2 levels and activity, the degrees of which correlated to the abilities of perifosine to inhibit the growth of cancer cells. We also detected increased COX-2 levels in lung cancer xenografts treated with perifosine. Moreover, blockage of COX-2 induction by both antisense and small interfering RNA approaches decreased cell sensitivity to perifosine. Collectively, these data indicate that the activation of COX-2 contributes to the anticancer activity of perifosine, including apoptosis induction and growth arrest. These data are clinically relevant as they suggest that the combination of perifosine and COX-2 inhibitors such as celecoxib, may produce a potential drug contradiction.

Lipids as targets for novel anti-inflammatory therapies.

Lipids serve important functions as membrane constituents and also as energy storing molecules. Besides these functions certain lipid species have now been recognized as signalling molecules that regulate a multitude of cellular responses including cell growth and death, and also inflammatory reactions. Bioactive lipids are generated by hydrolysis from membrane lipids mainly by phospholipases giving rise to fatty acids and lysophospholipids that either directly exert their function or are further converted to active mediators. This review will summarize the present knowledge about bioactive lipids that either promote or attenuate inflammatory reactions. These lipids include polyunsaturated fatty acids (PUFA), eicosanoids including the epoxyeicosatrienoic acids (EET), peroxisome proliferation activating receptor (PPAR) activators, cannabinoids and the sphingolipids ceramide, sphingosine 1-phosphate and sphingosylphosphorylcholine.

Effect of direct albumin binding to sphingosylphosphorylcholine in Jurkat T cells.

We investigated the effects of serum on lysophospholipid-induced cytotoxicity in Jurkat T cells. We found that sphingosylphosphorylcholine (SPC, also known as lysosphingomyelin) induced cytotoxicity and that albumin in serum could protect cells by binding directly to SPC. Furthermore, we also found that SPC induced ROS generation, increased [Ca(2+)](i), and decreased MMP. However, those effects were only observed at concentrations higher than 10 microM and were only induced in albumin-free media. Therefore, SPC may be trapped by albumin in plasma and unable to exert its effects under normal conditions, although at high concentrations, SPC could induce several responses such as ROS generation, increased [Ca(2+)](i), and decreased MMP in Jurkat T cells.

Blebbistatin inhibits sphingosylphosphorylcholine-induced contraction of collagen-gel fiber populated by vascular smooth-muscle cells.

We prepared a cell-populated collagen-gel fiber including GbaSM-4 cells derived from the basilar artery of guinea pigs. This fiber tended to be a differentiated contractile phenotype in electron-microscope observations. Sphingosylphosphorylcholine (SPC) can induce contraction of the fiber (EC50 = 0.70 +/- 0.05 microM), and blebbistatin can inhibit the SPC-induced contraction (IC50 = 22.8 +/- 1.26 microM). Phosphorylation of the 20 kD myosin light chain (MLC20) significantly increased in GbaSM-4 cells provided with 1 microM SPC (P<0.05), which was maintained in the presence of 1 to 100 microM blebbistatin. These results suggest that vascular smooth muscle can relax even if MLC20 is phosphorylated.

Searching new targets for anthelminthic strategies: Interference with glycosphingolipid biosynthesis and phosphorylcholine metabolism affects development of Caenorhabditis elegans.

Nematode infections are amongst the most abundant diseases of man and animals. They are characterised by a low mortality but high morbidity, thus reflecting the adaptation of these parasites to their hosts. Resistance as well as severe side-effects and efficacies restricted to distinct larval stages or parasites of the anthelmithics used at present require the urgent development of new and more nematode-specific drugs, targeting enzymes of parasite restricted biosynthetic routes. Caenorhabditis elegans has been found to be a good model system for parasitic nematodes, drug screening and developmental studies. Structural analyses have revealed nematode-specific glycosphingolipid structures of the arthro-series, carrying in part, phosphorylcholine substituents. These biomolecules appear to play important roles in nematode development, fertility and survival within the host and are, therefore, good target-candidates for the development of new anthelminthic strategies. Here we show that RNAi experiments targeting enzymes of glycosphingolipid biosynthesis or choline metabolism result, in part, in a drastic reduction of fertility. We further tested various chemical inhibitors of these pathways and found significant effects on the development of the worms, resulting in developmental arrest, sterility and, in part, lethality. Such inhibitors can, therefore, help to define new classes of anthelminthics.

Comparison of noradrenaline and lysosphingolipid-induced vasoconstriction in mouse and rat small mesenteric arteries.

1 We have compared vasoconstriction responses in isolated mesenteric small arteries from mice and rats as elicited by KCl, noradrenaline and the lysosphingolipids sphingosine-1-phosphate (S1P) and sphingosylphosphorylcholine (SPC). 2 Contractile responses to KCl and noradrenaline, but not those of S1P or SPC, were significantly related to vessel diameter in both species. 3 When comparing vessels of similar diameter, contractile responses for KCl and the three agonists were much smaller in mice than in rats, e.g. 8.3 +/- 0.4 vs. 14.7 +/- 0.7 mn for noradrenaline. 4 Based upon the antagonist rank order of potency of prazosin (pKB 8.80) > B8805-033 (pKB 7.89) > yohimbine (pKB 6.18) approximately BMY 7378 (pA2 6.03), noradrenaline responses in mice were mediated solely via alpha1A-adrenoceptors, similar to what repeatedly has been shown in rats. 5 The S1P3 receptor antagonist suramin (100 microM) significantly inhibited responses to S1P and SPC in rats but not in mice, and did not affect noradrenaline responses in either species. 6 We conclude that for any given diameter, mouse mesenteric arteries develop less contraction in response to various stimuli. Noradrenaline acts via alpha1A-adrenoceptors in both species. Responses to S1P and SPC differ between both species with regard to suramin-sensitivity indicating involvement of different receptor subtypes for lysosphingolipids in both species.

Perifosine, a novel alkylphospholipid, inhibits protein kinase B activation.

Perifosine is a novel p.o. bioavailable alkylphospholipid. Perifosine has displayed significant antiproliferative activity in vitro and in vivo in several human tumor model systems and has recently entered phase I clinical trials. Recent studies have identified that perifosine could cause cell cycle arrest with induction of p21(WAF1/CIP1) in a p53-independent fashion; however, the basis for that effect is not known. Structurally, perifosine resembles naturally occurring phospholipids. Therefore, we hypothesized that perifosine might perturb pathways related to phospholipids modulated by growth factor action. We demonstrate here that perifosine causes dose-dependent inhibition of protein kinase B/Akt phosphorylation and thus activation at concentrations causing growth inhibition of PC-3 prostate carcinoma cells. Only the myristoylated form of Akt (MYR-Akt), which bypasses the requirement for pleckstrin homology (PH) domain-mediated membrane recruitment, abrogated perifosine-mediated decrease of Akt phosphorylation and cell growth inhibition by perifosine. We demonstrate further that perifosine decreases the plasma membrane localization of Akt, and this is substantially relieved by MYR-Akt along with relief of downstream drug effect on induction of p21(WAF1/CIP1). Perifosine does not directly affect phosphoinositide 3-kinase (PI3K), phosphoinositide-dependent kinase 1, or Akt activity at concentrations inhibiting Akt phosphorylation and membrane localization. Our results demonstrate that Akt is an important cellular target of perifosine action. In addition, these studies show that the membrane translocation of certain PH domain-containing molecules can be greatly perturbed by the alkylphospholipid class of drugs and imply further that the PI3K/Akt pathway contributes to regulation of p21(WAF1/CIP1) expression.

Sphingosylphosphorylcholine-induced contraction of feline ileal smooth muscle cells is mediated by Galphai3 protein and MAPK.

We studied the mechanism of sphingosylphosphorylcholine (SPC)-induced contraction in feline ileal smooth muscle cells. Western blotting revealed that G protein subtypes of Galpha(i1), Galpha(i3) and Galpha(o) existed in feline ileum. Galpha(i3) antibody penetration into permeabilized cells decreased SPC-induced contraction. In addition, incubation of [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) with membrane fraction increased its binding to Galpha(i3) subtype after SPC treatment, suggesting that the signalling pathways invoked by SPC were mediated by Galpha(i3) protein. MAPK kinase (MEK) inhibitor PD98059 blocked the contraction significantly, but p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 did not. Chelerythrine and neomycin also inhibited the contraction. However, cotreatment of PD98059 and chelerythrine showed no significant difference. Phosphorylation of p44/42 MAPK was increased by SPC treatment, which was reversed by pretreatment of inhibitors of signalling molecules that decreased SPC-induced contraction previously. The same result was obtained in the assay of MAPK activity.

The inhibitory effect of C-reactive protein on bacterial phosphorylcholine platelet-activating factor receptor-mediated adherence is blocked by surfactant.

Numerous major bacterial pathogens in the human respiratory tract, including Streptococcus pneumoniae and Haemophilus influenzae, express cell-surface phosphorylcholine (ChoP), a ligand for the receptor for platelet-activating factor (rPAF). ChoP is also bound by C-reactive protein (CRP), which, in the presence of complement, may be bactericidal. This study found that CRP can block the attachment of bacteria expressing cell-surface ChoP to host cells. Concentrations of CRP equivalent to those on the mucosal surface of the human airway blocked most adherence of both S. pneumoniae and H. influenzae to human pharyngeal epithelial cells. ChoP-mediated adherence was also reduced in the presence of an rPAF antagonist. The antiadhesive effects of the rPAF antagonist and CRP were not additive, suggesting that CRP activity is specific to the area of adherence mediated by the receptor. The binding of CRP to ChoP and the effect of CRP on adherence were inhibited by human surfactant (primarily ChoP). The antiadhesive effect of CRP may be diminished in the terminal airway, where surfactant is abundant.

Sphingosylphosphorylcholine induces endothelial cell migration and morphogenesis.

Sphingosylphosphorylcholine (SPC) is one of the biologically active phospholipids that may act as extracellular messengers. Particularly important is the role of these lipids in the angiogenic response, a complex process involving endothelial cell migration, proliferation, and morphologic differentiation. Here we demonstrate that SPC and its hydrolytic product, sphingosine, induce chemotactic migration of human and bovine endothelial cells. The response is approximately equal to that elicited by vascular endothelial cell growth factor. The effect of SPC and sphingosine was associated with a rapid down-regulation of Edg1, a sphingosine 1-phosphate (SPP)-specific receptor involved in endothelial cell chemotaxis. Both SPC and sphingosine induced differentiation of endothelial cells into capillary-like structures in vitro. Thus, SPC and sphingosine join SPP among the biologically active lipids with angiogenic potential. Since neuronal abnormalities accompany pathological accumulation of SPC in brain tissue, it is possible that SPC is a modulator of angiogenesis in neural tissue upon its release from brain cells following trauma or neoplastic growth.

Activation of type D phospholipase by serum stimulation and ras-induced transformation in NIH3T3 cells.

Mitogenic stimulation of NIH3T3 fibroblasts with growth factors or ras oncogenes is associated with an increase in the levels of phosphorylcholine and diacylglycerol. Both metabolites could be generated as a result of direct activation of a phosphatidylcholine-specific phospholipase C (PC-PLC) or by a more complex pathway, involving activation of phospholipase D followed by choline kinase and phosphatidic acid-hydrolase. We show evidence indicating that the generation of phosphorylcholine and diacylglycerol follow independent mechanisms in both serum-treated and in ras-transformed NIH3T3 cells. No significant activation of a PC-PLC enzyme was observed. Instead, activation of a phosphatidylcholine-specific phospholipase D (PC-PLD) was detected. Moreover, while a fivefold constitutive activation of the endogenous PLD activity and a twofold increase on the levels of phosphatidic acid were observed in ras-transformed cells, very small alterations on these parameters were detected at late times after serum stimulation of quiescent cells. Thus, cell proliferation induced by ras oncogenes in fibroblasts cells may be functionally linked to activation of a PC-PLD enzyme. The differences found in the activation of this enzyme between ras-transformed and normal cells may constitute an important difference in mitogenic signalling between normal and transformed cells.

The synergistic and antagonistic effects of cytotoxic and biological agents on the in vitro antitumour effects of suramin.

Suramin has shown antitumour activity in vitro and in vivo. At plasma levels higher than 200 microM there is, however, excessive toxicity. We have, therefore, attempted to improve the antitumour effects of suramin in vitro by combining it with several other antitumour agents. The MCF-7 mammary carcinoma and PC3 prostate cancer cell lines were exposed continuously to suramin and the other agents for 6 days. The sulphorhodamine B (SRB) assay was used for the assessment of growth inhibition. The dose-response interactions were evaluated using the median-effect analysis with the Chou and Talalay computer programme. In the MCF-7 cell line, the combination of suramin plus doxorubicin (DXR), cisplatin (CDDP), 5-fluorouracil (5-FU) or tumour necrosis factor (TNF) resulted in synergistic growth inhibition, whilst its combination with miltefosine (HPC) was antagonistic. In the PC-3 cell line, suramin plus CDDP or TNF was synergistic, whilst its combination with DXR, 5-FU and HPC was antagonistic. All tested combinations with interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma) and with the combination of both IFN-alpha+IFN-gamma were not synergistic. The synergistic effect of suramin with DXR was schedule dependent. Pretreatment (addition of DXR on day 1 and suramin on days 2-5) was additive at the IC50 level, in both cell lines. Addition of DXR at day 5 was more effective than simultaneous exposure. We found a synergistic effect for the combination of suramin with CDDP and TNF in both cell lines. In addition the combination with DXR and 5-FU was synergistic in MCF-7. Sequential administration of DXR-suramin or suramin-DXR increased the growth inhibition.

Selection of the expressed B cell repertoire by infusion of normal immunoglobulin G in a patient with autoimmune thyroiditis.

In the present study we have analyzed the changes in the expressed antibody repertoire and in temporal fluctuations of antibody levels in serum that followed infusion of normal IgG (IVIg) in a patient with autoimmune thyroiditis. Administration of IVIg resulted in the stimulation of IgM production, in alterations of expressed antibody activity in serum that could not merely be accounted for by the passive transfer of antibody specificities contained in IVIg, in transient down-regulation of B cells clones expressing a specific disease-related idiotype and in the increase in serum in recipient's autoantibodies specifically reactive with F(ab')2 fragments of IVIg. In addition, infusion of IVIg shifted the pattern of spontaneous fluctuations of autoantibody activities in the patient's serum from a pattern indicative of disconnected events in the immune network to a pattern similar to that which is consistently observed in healthy controls. These results suggest that normal IgG may modulate autoreactivity by selecting expressed antibody repertoire through V region-dependent interactions with antibodies.

Induction of alkaline phosphatase activity in HeLa cells by choline chloride.

Choline chloride produced a dose-dependent induction of alkaline phosphatase activity in HeLa cells. At the highest concentration tested, 40 mM, there was a 5- to 7-fold increase in alkaline phosphatase activity, a significantly greater induction than that produced by equiosmolar additions of either NaCl or sucrose. Enzyme activity was higher than control values by 24 hr after the addition of the salt, although the largest increases in activity occurred between 36 and 72 hr. The induction of alkaline phosphatase activity by choline chloride could be inhibited in a dose-dependent manner by the simultaneous addition of either caffeine or theophylline. At comparable concentrations of inhibitor, the magnitude of the inhibition of the induction produced by choline chloride was greater than that observed when the xanthines were used to inhibit the induction by either 5-iodo-2'-deoxyuridine or NaCl. Choline chloride, like NaCl, produced a proportionately greater increase in the heat-stable rather than the heat-labile form of alkaline phosphatase activity.