Metabolism of platelet-activating factor in rat epididymal spermatozoa

A role for platelet-activating factor (PAF) in sperm function has been proposed. In the present investigation the metabolism of PAF was examined in sperm and epididymal tissue. The major regulatory enzymes for the synthesis of PAF via the de novo pathway have been established in spermatozoa; these include acetyltransferase and cholinephosphotransferase specific for PAF biosynthesis. De novo acetyltransferase activity for PAF biosynthesis in spermatozoa was significantly higher than that of the acetyltransferase of the remodeling pathway. A metabolic pathway was described for the catabolism of PAF in sperm, involving PAF-acetylhydrolase (-AH), lysophospholipase D, and a phosphohydrolase. An isozyme of PAF-AH similar to that reported in sera was also demonstrated in epididymal fluid and tissue. This isozyme was distinctly different from that found in the spermatozoa. The partial inactivation of PAF-AH by the vaginal pH, and/or its detachment from sperm during migration to the site of fertilization, may allow increased motility and migration to the site of fertilization. It is suggested that a decapacitation factor previously described may be related to PAF-AH.     

The role of recombinant platelet-activating factor acetylhydrolase in a neonatal rat model of necrotizing enterocolitis

Previous studies have shown that the endogenous inflammatory mediator platelet-activating factor (PAF) plays an important role in the pathophysiology of neonatal necrotizing enterocolitis (NEC). This study was designed to investigate the role of the PAF-degrading enzyme acetylhydrolase (PAF-AH) in a neonatal rat model of NEC. To study the absorption, localization, and activity of human recombinant PAF-AH (rPAF-AH), newborn rats were treated with enteral rPAF-AH, and plasma and intestines were sampled at 8 and 24 h for determination of PAF-AH enzyme activity and rPAF-AH concentration using a specific enzyme-linked immunoassay. To study the effect of rPAF-AH on neonatal NEC, rats were treated with rPAF-AH via the enteral route every 3 h, and then subjected to formula feeding and asphyxia per an established neonatal rat protocol for NEC. Pretreatment with enteral rPAF-AH significantly reduced the incidence of NEC compared with controls (6/26 versus 19/26, p < 0.001). We found that enteral rPAF-AH administration resulted in significant intestinal PAF-AH activity but no circulating PAF-AH activity despite immunohistochemical localization of the administered rPAF-AH to the intestinal epithelial cells. These findings suggest that rPAF-AH is functional and stable in the gut of neonatal rats. We conclude that enteral administration of rPAF-AH remains locally active and reduces the incidence of NEC in our experimental animal model.     

A role for CD4 in peripheral T cell differentiation

A rapid method for the simultaneous determination of cholesterol and its oxidation products as well as alpha-to-copherol and tocopherolquinone in brain subcellular fractions is described. The samples are saponified and extracted with hexane. It is not necessary to remove cholesterol in the sample before analyzing for oxysterols. The hexane extract can be used for the assay of cholesterol compounds by capillary gas chromatography and tocopherol compounds by liquid chromatography using a procedure reported previously. Oxidation of synaptosomes by a mixture of Fe2+ plus ascorbate resulted in the production of 7-keto-, 7 alpha-hydroxy-, 7 beta-hydroxy-, and 5 alpha, 6 alpha-epoxycholesterols. The identities of these products were confirmed with gas chromatography/mass spectrometry. Cholesterol oxidase treatment did not result in the formation of any of the above compounds. Thus the types and amounts of the products of oxidation of cholesterol were dependent upon the oxidizing agent. Extraction of the oxysterols under milder conditions without saponification using sodium dodecyl sulfate cannot be used since such treatment results in low recovery of oxysterols. Oxidation of synaptosomes by low concentrations of ferrous iron and ascorbate resulted in (i) low levels of oxidation of cholesterol which could be followed by estimating the production of oxysterols and (ii) oxidation of a substantial percentage of alpha-tocopherol. The proposed procedure will be useful in monitoring the oxidation of small quantities of membrane cholesterol in vitro.     

Effect of endotoxin and platelet-activating factor on rat vascular permeability: role of vasoactive mediators

The aim of the study was to correlate the DNA Index (DI) and S-phase fraction (SPF) values determined by multiparametric flow cytometry in breast cancer (mainly T1 and T2 stages) with several clinico-pathologic variables and other biological parameters. For this purpose, a total of 136 breast cancers were submitted to flow cytometry and to several types of immunohistochemical analyses. Among clinico-pathologic data we considered pT, pN and grade and among immunohistochemical markers, hormonal receptors, P53, c-erbB-2 and MIB-1. We found that DNA aneuploidy was strongly correlated with high tumoral grade, absence of hormonal receptors, high proliferation, as shown by high MIB-1 (> or = 36%) and SPF values (> or = 13.3%), and P53 positivity. Grouping the tumours according to their DI values, we observed a relative significantly higher correlation of the near-triploid range carcinomas (DI 1.40-1.60) with immunohistochemical expression of P53 (p = 0.0001). Near-triploid DI was also associated with a high proliferative activity, expressed both by SPF (p = 0.0001) and MIB-1 reactivity (p = 0.0001), high tumoral grade (p = 0.0001) and presence of axillary metastases (p = 0.03). These data suggest that DNA near-triploid tumours in breast cancer may have a more aggressive behaviour in comparison with other DI classes.     

The role of oxygen free radicals in isoniazid-induced hepatotoxicity

This report describes the glycosaminoglycans, collagen and elastin--composition of leiomyosarcoma. Studies were performed on leiomyosarcoma removed during surgery. The material was taken from 7 patients. Rearrangement of extracellular matrix in leiomyosarcoma has been found. There was an increase of total collagen content in comparison to control uterus. In both tissues type I collagen was found to be the predominant one. Both tissues, normal and neoplastic contain all known glycosaminoglycans types. Among them heparan sulphate was found to be the most abundant. A slight decrease in the content of this glycosaminoglycan was observed in leiomyosarcoma. A possible role of these alterations in tumour biology is discussed.     

Potential angiogenic role of platelet-activating factor in human breast cancer

This study investigated the presence of platelet-activating factor (PAF) in the lipid extracts of 18 primary breast carcinomas and 20 control breast tissues. The amount of PAF detected in breast carcinomas was significantly higher than in controls. The mass spectrometric analysis of PAF-bioactive lipid extract from breast carcinomas showed the presence of several molecular species of PAF, including C16-alkylPAF, C18-lysophosphatidylcholine (LPC), C16-LPC, lyso-PAF, and C16-acylPAF. The amount of bioactive PAF extracted from breast specimens significantly correlated with tumor vascularization revealed by the number of CD34-and CD31-positive cells. As C16-alkylPAF was previously shown to induce angiogenesis in vivo, we evaluated whether the thin layer chromatography-purified lipid extracts of breast specimens elicited neoangiogenesis in a murine model of subcutaneous Matrigel injection. The lipid extracts from specimens of breast carcinoma containing high levels of PAF bioactivity, but not from breast carcinomas containing low levels of PAF bioactivity or from normal breast tissue, induced a significant angiogenic response. This angiogenic response was significantly inhibited by the PAF receptor antagonist WEB 2170. T47D and MCF7 breast cancer cell lines, but not an immortalized nontumor breast cell line (MCF10), released PAF in the culture medium. A significant in vivo neoangiogenic response, inhibited by WEB 2170, was elicited by T47D and MCF7 but not by MCF10 culture medium. These results indicate that an increased concentration of PAF is present in tumors with high microvessel density and that PAF may account for the neoangiogenic activity induced in mice by the lipid extracts obtained from breast cancer. A contribution of PAF in the neovascularization of human breast cancer is suggested.     

Activities of enzymes involved in the metabolism of platelet-activating factor in neural cell cultures during proliferation and differentiation

Platelet-Activating Factor (PAF) is a potent lipid mediator involved in physiological and pathological events in the nervous tissue where it can be synthesized by two distinct pathways. The last reaction of the de novo pathway utilizes CDPcholine and alkylacetylglycerol and is catalyzed by a specific phosphocholinetransferase (PAF-PCT) whereas the remodelling pathway ends with the reaction catalyzed by lyso-PAF acetyltransferase (lyso-PAF AcT) utilizing lyso-PAF, a product of phospholipase A2 activity, and acetyl-CoA. The levels of PAF in the nervous tissue are also regulated by PAF acetylhydrolase that inactivates this mediator. We have studied the activities of these enzymes during cell proliferation and differentiation in two experimental models: 1) neuronal and glial primary cell cultures from chick embryo and 2) LA-N-1 neuroblastoma cells induced to differentiate by retinoic acid (RA). In undifferentiated neuronal cells from 8-days chick embryos the activity of PAF-PCT was much higher than that of lyso-PAF AcT but it decreased during the period of cellular proliferation up to the arrest of mitosis (day 1-3). During this period no significant changes of lyso-PAF AcT activity was observed. Both enzyme activities increased during the period of neuronal maturation and the formation of cellular contacts and synaptic-like junctions. The activity of PAF acetylhydrolase was unchanged during the development of the neuronal cultures. PAF-PCT activity did not change during the development of chick embryo glial cultures but lyso-PAF AcT activity increased up to the 12th day. RA treatment of LA-N-1 cell culture in proliferation decreased PAF-PCT activity and had no significant effect on lyso-PAF AcT and PAF acetylhydrolase indicating that the synthesis of PAF by the enzyme catalyzing the last step of the de novo pathway is inhibited when the LA-N-1 cells are induced to differentiate. These data suggest that: 1) in chick embryo primary cultures, both pathways are potentially able to contribute to PAF synthesis during development of neuronal cells particularly when they form synaptic-like junctions whereas, during development of glial cells, only the remodelling pathway might be particularly active on synthesizing PAF; 2) in LA-N-1 neuroblastoma cells PAF-synthesizing enzymes coexist and, when cells start to differentiate the contribution of the de novo pathway to PAF biosynthesis might be reduced.     

Leukemia inhibitory factor inhibits osteogenic differentiation in rat calvaria cell cultures

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine with both anabolic and catabolic effects on bone tissue. To investigate the effect of LIF on bone formation in the absence of a resorption cycle, we used fetal rat calvaria cell cultures and quantified bone nodule production, which provides a colony assay to analyze the effects of factors on osteoprogenitor differentiation and bone formation. In these cultures, dexamethasone (Dex) stimulates bone nodule formation. In dose-response experiments, LIF inhibited bone nodule formation by cells cultured with (+Dex; ID50 = 250 U/ml) or without (-Dex; ID50 = 30 U/ml) 10(-8) M Dex. Residual nodules were small and poorly mineralized. Continuous exposure to LIF (500 U/ml) up to day 25 did not affect either the growth rate or saturation density of the cultures, but decreased alkaline phosphatase activity and bone nodule production, with greater inhibition in -Dex cultures. Exposure to LIF (500 U/ml) for 3 days early during nodule formation (about day 10) reduced bone nodule numbers to the same extent as continuous treatment in -Dex cultures and significantly, but less markedly, in +Dex cultures; earlier and later pulses had no effect. Northern blot analysis of expression of messenger RNAs of bone related proteins in cultures pulsed (-Dex) at various stages of development showed marked inhibition of alkaline phosphatase, bone sialoprotein, and osteocalcin; slight inhibition of type I collagen; early stimulation of osteopontin; and no effect on Secreted Protein, Acidic and Rich in Cysteine/osteonectin. These results suggest that LIF is an inhibitor of bone nodule formation in these cultures, acting at a stage when late osteoprogenitors and/or early osteoblasts are present, and that Dex may modulate the effects of LIF by shifting effective doses to higher concentrations.     

Synergistic effects of colony-stimulating factor 1 and leukemia inhibitory factor in inducing early myeloid cell differentiation

Cells of the M1D+ murine myeloid leukemic cell line differentiate into macrophages in response to either leukemia inhibitory factor (LIF) or interleukin 6. Previously, it was shown that LIF treatment of M1D+ cells leads to an increased expression of colony-stimulating factor (CSF) receptor mRNA encoded by c-fms. CSF-1, a macrophage growth factor, induces the survival, growth, and differentiation of mononuclear phagocytes but has not been implicated in the regulation of early myeloid cell differentiation. Here we show that low-dose LIF treatment of M1D+ cells results in CSF-1 secretion and CSF-1 receptor up-regulation. CSF-1, when applied alone, induces some M1D+ adherence and the up-regulation of lysozyme M, a macrophage-specific marker. Finally, we show that when applied together, LIF and CSF-1 act synergistically to induce macrophage morphology, phagocytosis, and the expression of the macrophage-specific markers CD11b/Mac-1 alpha chain, lysozyme M, FcgammaRII, and JE/MCP.1. These results indicate that instead of being part of exclusive pathways, as thought until this work, LIF and CSF-1 can function synergistically to further stimulate the early stages of myeloid differentiation.     

Involvement of platelet-activating factor in cell death induced under ischemia/postischemia-like conditions in an immortalized hippocampal cell line

The involvement of platelet-activating factor (PAF) in cell damage induced by ischemia/postischemia-like conditions was studied in a hippocampus-derived cell line, HN33.11. Cells exposed to N2-saturated glucose-free HEPES-buffered saline (ischemia) for 5 h followed by 18 h of incubation in serum-free control medium (postischemia reincubation) remained 67.4 +/- 2.4% viable in comparison with sham-treated cells. Analysis of DNA fragmentation in combination with Hoechst 33258 staining indicates that apoptosis is the dominant mode of cell death in the present model. PAF level during 10 h of ischemia was unchanged. However, an increase in PAF accumulation was found early during the reincubation period that followed 5 h of ischemia. Peak PAF concentrations were noted at 2 h after initiation of reincubation and rapidly declined to control level after 7 h of reincubation. Consistent with a role of PAF in mediating cell death under ischemia/postischemia reincubation in this model, the PAF antagonist BN 50739 exerted a dose-dependent protective effect. Maximal protection (85.7 +/- 5.4%) of the cells from ischemia/reincubation-induced cell damage was achieved at 0.1 microM BN 50739. The PAF antagonist lacked any protective effect against ischemia-induced cell death. On the other hand, the addition of the stable PAF analogue 1-O-hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (MC-PAF) at the onset of ischemia potentiated ischemia/reincubation-induced apoptosis--an effect that was blocked by BN 50739. Pretreatment of HN33.11 cells with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester (BAPTA-AM) also provided a protective effect against ischemia/reincubation-induced cell damage. BAPTA-AM increased cell viability by 50%. Pretreatment with BAPTA-AM also decreased ischemia/reincubation-induced PAF accumulation in HN33.11 cells. The results suggest that PAF, acting via a PAF receptor, is at least in part mediating apoptosis under ischemia/postischemia-like conditions in HN33.11 cells.     

The role of neutrophils and platelet-activating factor in mediating experimental pancreatitis

BACKGROUND & AIMS: Pancreatitis is characterized by inflammation and death of acinar cells. Death can occur by either necrosis or apoptosis. The initial injury may cause expression of cytokines that mediate activation and infiltration of neutrophils. The aim of this study was to assess the effect of neutrophils and platelet-activating factor (PAF) in cell death responses. METHODS: The effects of neutrophil depletion with antineutrophil serum (ANS) and a PAF antagonist (BN52021) were measured in the cerulein model of pancreatitis. Rats received a 6-hour intravenous infusion of cerulein either alone or after treatment with ANS, BN52021, or both. RESULTS: Cerulein-induced pancreatitis was characterized by neutrophilic infiltration, vacuolization of acinar cells, and foci of necrosis. Treatment with ANS and BN52021 prevented the inflammatory response caused by cerulein and decreased the cell damage. Treatment with ANS increased apoptosis in cerulein-infused animals. When BN52021 was added, apoptosis was abolished. The measurement of PAF in pancreatic tissue showed a ninefold increase with cerulein treatment alone and a 14-fold increase in cerulein-infused, neutrophil-depleted animals. CONCLUSIONS: The results indicate that cerulein stimulates pancreatic production of PAF. PAF mediates both apoptosis and neutrophil chemotaxis in the pancreas. Neutrophils in turn may convert acinar cells undergoing apoptosis into necrotic cells.     

Characterization of platelet-activating factor synthesis in glomerular endothelial cell lines

Platelet-activating factor synthesis in two transformed lines of glomerular endothelial cells was characterized and contrasted with platelet-activating factor production in macrovascular-derived endothelial cells as well as with glomerular cells of mesenchymal origin. Platelet-activating factor synthesis was assessed in intact cells and in cell-free preparations. Glomerular endothelial cells constitutively synthesize bio-active alkyl-PAF, and this basal activity can be chronically augmented by various inflammatory and thrombotic agents. In contrast, thrombin-mediated platelet-activating factor formation in bovine pulmonary aortic endothelial cells as well as in glomerular mesangial cells is acute and transient. The potential role of anti-inflammatory prostanoids to function as negative feedback modulators of thrombin- or endothelin-mediated platelet-activating factor synthesis was also investigated, as the synthesis of platelet-activating factor is often associated with the formation of these prostanoids. Indomethacin augmented receptor-mediated platelet-activating factor synthesis while prostanoids of the E and I series reduced agonist-stimulated PAF synthesis. In summary, the unique capacity of glomerular endothelial cells to respond to inflammatory stimuli with sustained platelet-activating factor synthesis is a clear indication of this cell's pivotal role in augmenting the inflammatory response in the limited environment of the glomerulus.     

Predominant expression of platelet-activating factor receptor in the rat brain microglia

Cellular localization of platelet-activating factor (PAF) receptor in the rat brain was determined by (1) in situ hybridization, (2) Northern blot analysis in primary cell cultures of neurons, microglia, astrocytes, and fibroblasts, and (3) Ca2+ imaging in hippocampal culture. In situ hybridization revealed that the PAF receptor mRNA is expressed intensely in microglia and moderately in neurons. Northern blot analysis revealed that PAF receptor mRNA is the most abundant in microglia. In primary hippocampal cultures, PAF elevated intracellular Ca2+ concentration in microglia and also in neurons, but to a lesser extent. These results suggest predominant presence of PAF receptor in microglia. Cultured microglia also expressed cPLA2 mRNA the most intensely. PAF-activated microglia released arachidonic acid in a Ca(2+)-dependent manner and without conversion to its derivatives. We propose that microglia as well as neurons contribute to PAF-related events in the CNS by releasing arachidonic acid.     

A possible role for the high mobility group box transcription factor Tcf-4 in vertebrate gut epithelial cell differentiation

Antinuclear Abs are the hallmark of the autoimmune disease systemic lupus erythematosus (SLE). The ability of self reactive autoantibodies to bind to DNA and nucleosomes is partly conferred by an increased number of arginine and asparagine residues in the heavy chain third complementarity determining region. This increased content of cationic residues is primarily the result of unusual VH-D-JH rearrangements, which include D-D fusions and D gene inversions. While self Ag-driven clonal expansion is a major contributor to the production of antinuclear Abs in lupus, we explore in this study the hypothesis that newly emerging B cells from autoimmune mice display more frequently these unusual VH-D-JH rearrangements. To this end, libraries of PCR-generated VH-D-JH junctions from MRL and C3H newborn livers were analyzed. When compared with the C3H controls, D and JH gene usage in MRL junctions suggests a greater frequency of secondary D-JH rearrangements in this strain. Furthermore, B cells from the autoimmune-prone MRL mice have significantly increased numbers of atypical VH-D-JH rearrangements (D-D fusions and D inversions). Therefore, B cells from MRL mice manifest intrinsic defects that could confer an increased propensity to produce unusual VH-D-JH rearrangements early in ontogeny.     

The role of active oxygen radicals in blood system reactions in inflammation

On the model of carrageenan-induced acute aseptic peritonitis in rats with using of alpha-tocopherol it is shown that the active oxygen metabolites increase granulomonocytopoiesis, leukocytosis and stimulation of blood neutrophils, accumulation and activation of neutrophils of an inflammatory focus, decrease accumulation and stimulation of monocytes of an inflammatory focus and thus they are pro-inflammatory modulators of the blood system's reactions.     

A role for phosphatidylinositol 3-kinase in generating T cell help for B cell growth and differentiation

Evidence supporting the importance of the 3-phosphoinositide signaling pathway in lymphocyte activation is rapidly accumulating. In our study, we assessed the effects of two PI 3-kinase inhibitors, wortmannin and LY294002, on T cells as a means to analyze the role of the PI 3-kinase-signaling pathway in the generation of T cell help for B cell growth and differentiation. For these studies, B cells were cocultured with CD3-activated mitomycin C-treated T cells to induce B cell responsiveness. Of interest, wortmannin or LY294002 pretreatment of the T cell population significantly inhibited T cell-dependent induction of B cell proliferation and differentiation. The failure of wortmannin-treated CD3-activated mitomycin C-treated T cells to provide help in driving the differentiation of B cells to Ig-secreting cells could not be corrected by the addition of exogenous IL-2. Further studies designed to elucidate the mechanism by which wortmannin-treated T cells failed to provide B cell help indicated that wortmannin and LY294002 significantly inhibited the induction of CD40 ligand and, to a lesser extent, intercellular adhesion molecule-1 expression. These results suggest that the PI 3-kinase-signaling pathway, or other wortmannin- and LY294002-sensitive pathways, may be important for the induction of expression of crucial interaction molecules, such as CD40 ligand, on T cells and thus indicates that D-3 phosphoinositides play a pivotal role in regulating T cell-dependent B cell activation.