Circulating tumour cells--a bona fide cause of metastatic cancer.

Circulating tumour cells (CTCs) are emerging as important prognostic markers and have potential clinical utility as tumour biomarkers for targeted cancer therapy. Although CTCs were proposed more than 100 years ago as potential precursors that may form metastatic lesions, formal evidence that CTCs are indeed capable of initiating metastases is limited. Moreover, the process of CTCs shedding into the circulation, relocating to distant organ sites and initiating metastatic foci is complex and intrinsically inefficient. To partially explain the metastatic process, the concepts of CTCs as metastatic precursors or pre-metastatic conditioners have been proposed; however, it is questionable as to whether these are both variable pathways to metastasis or just markers of metastatic burden. This review explores the evidence for CTCs in the initiation and progression of metastatic cancer and the data supporting these different concepts in an attempt to better understand the role of CTCs in metastasis. A greater understanding of the metastatic potential of CTCs will open new avenues for therapeutic interventions in the future.

Overlapping gene expression profiles in rheumatoid fibroblast-like synoviocytes induced by the proinflammatory cytokines interleukin-1 beta and tumor necrosis factor.

OBJECTIVE AND DESIGN: The development of therapies directed against TNF alpha and IL-1 beta has underscored the importance of these cytokines in rheumatoid arthritis (RA). In this study, oligonucleotide microarrays were used to identify novel transcriptional events mediated by TNF alpha and IL-1 beta. METHODS: In this study we have used Affymetrix U95A GeneChips representing 12,600 full-length human genes to identify transcriptional events mediated by these cytokines. Fibroblast-like synoviocytes were cultured from rheumatoid synovium from RA patients and stimulated with TNF alpha and IL-1 beta. Gene transcript levels were determined using Affymetrix U95A GeneChips representing 12,600 full-length human genes. RESULTS: A large number of differentially regulated genes were identified (1.7% of array-displayed genes for TNF alpha and 2.4% for IL-1 beta), and the validity of the array protocol was subsequently confirmed using real-time PCR. The majority of the differentially expressed genes were regulated by both TNF alpha and IL-1 beta, reflecting the distal signaling pathways shared by these cytokines. A large number of novel TNF alpha and IL-1 beta-regulated genes were identified. CONCLUSIONS: A panel of novel TNF alpha- and IL-1 beta-regulated genes was identified, and these are promising candidates for further study in relation to RA and other inflammatory diseases.

A novel approach to the design of inhibitors of human secreted phospholipase A2 based on native peptide inhibition.

Human Type IIA secreted phospholipase A(2) (sPLA(2)-IIA) is an important modulator of cytokine-dependent inflammatory responses and a member of a growing superfamily of structurally related phospholipases. We have previously shown that sPLA(2)-IIA is inhibited by a pentapeptide sequence comprising residues 70-74 of the native sPLA(2)-IIA protein and that peptides derived from the equivalent region of different sPLA(2)-IIA species specifically inhibit the enzyme from which they are derived. We have now used an analogue screen of the human pentapeptide (70)FLSYK(74) in which side-chain residues were substituted, together with molecular docking approaches that modeled low-energy conformations of (70)FLSYK(74) bound to human sPLA(2)-IIA, to generate inhibitors with improved potency. Importantly, the modeling studies showed a close association between the NH(2) and COOH termini of the peptide, predicting significant enhancement of the potency of inhibition by cyclization. Cyclic compounds were synthesized and indeed showed 5-50-fold increased potency over the linear peptide in an Escherichia coli membrane assay. Furthermore, the potency of inhibition correlated with steady-state binding of the cyclic peptides to sPLA(2)-IIA as determined by surface plasmon resonance studies. Two potential peptide interaction sites were identified on sPLA(2)-IIA from the modeling studies, one in the NH(2)-terminal helix and the other in the beta-wing region, and in vitro association assays support the potential for interaction of the peptides with these sites. The inhibitors were effective at nanomolar concentrations in blocking sPLA(2)-IIA-mediated amplification of cytokine-induced prostaglandin synthesis in human rheumatoid synoviocytes in culture. These studies provide an example where native peptide sequences can be used for the development of potent and selective inhibitors of enzyme function.

Crystallization and preliminary X-ray diffraction studies of a new crystal form of human secretory type IIA phospholipase A2.

Human synovial type IIA phospholipase A(2) (sPLA(2)-IIA) has been implicated in the pathogenesis of a number of inflammatory diseases and is a target for the development of therapeutically useful inhibitors. Biochemical evidence suggests a novel mechanism of inhibition for a series of peptide inhibitors originally derived from the primary sequence of the protein. On co-incubation with one of these inhibitors, single crystals of a hitherto unreported crystallographic form of sPLA2-IIA suitable for diffraction analysis were obtained. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 140.8, b = 38.9, c = 109.1 A, beta = 125.1 degrees, and diffraction at 2.4 A resolution has been observed.

Type IIA secretory phospholipase A2 up-regulates cyclooxygenase-2 and amplifies cytokine-mediated prostaglandin production in human rheumatoid synoviocytes.

Human type IIA secretory phospholipase A2 (sPLA2-IIA) is induced in association with several immune-mediated inflammatory conditions. We have evaluated the effect of sPLA2-IIA on PG production in primary synovial fibroblasts from patients with rheumatoid arthritis (RA). At concentrations found in the synovial fluid of RA patients, exogenously added sPLA2-IIA dose-dependently amplified TNF-alpha-stimulated PGE2 production by cultured synovial fibroblasts. Enhancement of TNF-alpha-stimulated PGE2 production in synovial cells was accompanied by increased expression of cyclooxygenase (COX)-2 and cytosolic phospholipase A2 (cPLA2)-alpha. Blockade of COX-2 enzyme activity with the selective inhibitor NS-398 prevented both TNF-alpha-stimulated and sPLA2-IIA-amplified PGE2 production without affecting COX-2 protein induction. However, both sPLA2-IIA-amplified PGE2 production and enhanced COX-2 expression were blocked by the sPLA2 inhibitor LY311727. Colocalization studies using triple-labeling immunofluorescence microscopy showed that sPLA2-IIA and cPLA2-alpha are coexpressed with COX-2 in discrete populations of CD14-positive synovial macrophages and synovial tissue fibroblasts from RA patients. Based on these findings, we propose a model whereby the enhanced expression of sPLA2-IIA by RA synovial cells up-regulates TNF-alpha-mediated PG production via superinduction of COX-2. Therefore, sPLA2-IIA may be a critical modulator of cytokine-mediated synovial inflammation in RA.

Functional coupling and differential regulation of the phospholipase A2-cyclooxygenase pathways in inflammation.

Prostaglandins generated by the phospholipase A2 (PLA2)/cyclooxygenase (COX) pathway are well known to mediate diverse intracellular and extracellular effects that regulate mammalian development, vascular function, renal physiology, parturition, and immune responses to infection or wounding. In immune-mediated diseases and in certain cancers, this pathway is aberrantly up-regulated and excessive prostaglandin production contributes to the pathology. It is now known that there are two isoforms of COX and multiple secreted and intracellular PLA2 enzymes. The use of isoform-specific inhibitors, coupled with antisense and in vivo gene deletion experiments, has identified independent pathways of arachidonic acid metabolism, which are differentially regulated at the levels of gene expression, protein phosphorylation, and cellular localization. There is cross-talk between the pathways at the level of PLA2 and substrate supply to the two isoforms of COX is apparently compartmentalized. Knockout studies have shown that the two COX isoforms play independent roles in immediate and delayed agonist-induced prostaglandin synthesis. Cytosolic PLA2-alpha is essential for both responses. Inducible secreted forms of PLA2 are, as yet, not essential for either response with the exception of the in vitro murine mast cell immediate response and instances of murine macrophage prostaglandin synthesis. These enzymes amplify the delayed response and are likely to modulate the severity of immune-mediated diseases.

Increased expression of human type IIa secretory phospholipase A2 antigen in arthritic synovium.

OBJECTIVE: To determine the localisation and level of expression of human type IIa secretory phospholipase A2 (sPLA2) in the synovium of rheumatoid arthritis (RA), osteoarthritis (OA), and non-arthritic (NA) patients and to examine the relation between sPLA2 and histological features of inflammation. METHODS: Immunoperoxidase staining using the anti-sPLA2 monoclonal antibody 9C1 was performed on frozen sections of knee synovium of 10 RA, 10 OA, and 10 NA patients. sPLA2 positive cells were scored on a scale of 0-3 in 10 fields of a representative tissue section from each case. Double labelling immunofluorescence confocal microscopy with antibodies to CD14 or CD45 and 9C1 was used to determine cell type specificity. Inflammation was assessed by semiquantitative scoring of lining layer thickness and mononuclear cell infiltrates (MC) and a cumulative inflammation score, generated by summing the two parameters. Scores in each group were compared using non-parametric statistical analysis. RESULTS: sPLA2 was localised to endothelium (EC), vascular smooth muscle (VSM), and mast cells (M) in all tissue sections. In RA and OA sections, staining was seen in both macrophage-like and fibroblast-like cells in the synovial lining layer (LL) and subsynovial lining layer (SLL). Perineural cells stained positively. Subintimal lymphoid aggregates (LA) were negative in all sections. The RA group showed significantly greater staining in extravascular synovial tissue (median 3.6, range 1.5-6.0) than the OA (median 1.95, range 0-5.3) or NA (median 0, range 0-5.9) groups (p < 0.05). LL staining was significantly higher in RA than both OA and NA sections (p < 0.05). The OA group showed a trend to higher staining scores than the NA group that did not reach significance. There was a significant correlation between the sPLA2 staining score and inflammation score within the RA patient group (p < 0.05). CONCLUSIONS: The synovium is a site of increased expression of sPLA2 antigen in both RA and OA relative to NA. Its presence in both fibroblast and macrophage-like cells in the LL and SLL of synovial tissue in RA and OA, but not NA, indicates that the enzyme is specifically induced in these regions in both conditions with expression in the LL being particularly characteristic of RA. The widespread expression of sPLA2 in synovium suggests it is likely to play a significant part in synovial pathology.

Contribution of type II phospholipase A2 to in vitro phospholipase A2 enzymatic activity in human term placenta.

Although phospholipase A2 (PLA2) enzymatic activities have been implicated in the regulation of phospholipid metabolism and eicosanoid formation in human gestational tissues, the role and contribution made by individual PLA2 isozymes has not been established. The aim of this study, therefore, was to determine the contribution made by Type II PLA2 to PLA2 enzymatic activity present in human term placenta. The experimental paradigm used to establish the contribution made by Type II PLA2 to total in vitro PLA2 enzymatic activity present in placental extracts was to remove Type II PLA2 by immunoaffinity extraction and then to quantify residual PLA2 enzymatic activity. Before immunoaffinity extraction, Type II PLA2 immunoactivity and total PLA2 enzymatic activity present in placental extracts averaged 28.0 +/- 10.0 ng/mg protein and 1040 +/- 367 pmol/h per mg protein (n = 3) respectively. After solid-phase immunoaffinity batch extraction of placental extracts, immunoreactive Type II PLA2 was not detectable by ELISA, and PLA2 enzymatic activity was decreased by 82 +/- 1% (P < 0.001). Residual (i.e. non-Type II) PLA2 enzymatic activity was further characterised by Western blot analysis and enzyme activity assay. The data obtained are consistent with a contribution by both cytosolic PLA2 and other secretory PLA2 isozymes (i.e. non-Type II) to residual PLA2 enzymatic activity. The results obtained in this study support the conclusion that Type II PLA2 is quantitatively the primary PLA2 isozyme that contributes to in vitro PLA2 enzymatic activity present in extracts of human term placenta, accounting for at least 80% of total activity. These data further support the involvement of this extracellularly active isozyme in the regulation of placental phospholipid metabolism and eicosanoid formation during late gestation.

Secretory phospholipase A2 and lipoprotein lipase enhance 15-lipoxygenase-induced enzymic and nonenzymic lipid peroxidation in low-density lipoproteins.

The oxidation of low-density lipoprotein (LDL) is thought to contribute to atherogenesis. 15-Lipoxygenase (15LO) induces LDL oxidation, and phospholipase A2 enhances this process [Sparrow, C. P. , Parthasarathy, S., and Steinberg, D. (1988) J. LipidRes. 29, 745-753]. As the underlying mechanism of the enhancing effect has not been investigated previously, we here show that in the presence of soybean 15LO (SLO) or human 15LO (rhLO), the addition of lipoprotein lipase, porcine pancreatic, or human type IIa secretory phospholipase A2 (sPLA2) greatly enhanced the accumulation of hydro(pero)xides of all major classes of LDL's lipids. Hydroperoxides of free fatty acids accumulated exclusively as enzymic products with kinetics reflecting both the formation of free fatty acids and the initial 'build-up' of alpha-tocopheroxyl radical. In contrast, hydroperoxides of cholesteryl esters and phosphatidylcholine accumulated linearly over comparatively longer periods of time and, in the case of rhLO, well beyond inactivation of the oxygenase. With SLO, formation of oxidized esterified lipids occurred nonenzymically, independent of the presence of lipase and despite the oxygenase remaining active until the end of the incubation. Enhancement of rhLO-induced LDL lipid peroxidation by sPLA2 was eliminated by a neutralizing anti-sPLA2 antibody, indicating that lipolytic activity was required for this effect. LDL depleted of alpha-tocopherol was resistant to oxidation by 15LO alone, whereas lipase overcame this resistance, demonstrating that lipases enhance 15LO-induced enzymic and nonenzymic peroxidation of LDL lipids. This is likely due to provision of free fatty acid substrate, resulting in an enhanced rate of free radical formation which itself causes nonenzymic peroxidation of esterified lipids. As lipases and 15LO are present in atherosclerotic lesions, our findings could be of pathophysiological significance.

Release of Type II phospholipase A2 immunoreactivity and phospholipase A2 enzymatic activity from human placenta.

The aim of this study was to determine whether Type II phospholipase A2 (PLA2) is released from late pregnant human placental tissue. Placental explants were incubated in vitro and the release of immunoreactive (ir) Type II PLA2 and PLA2 enzymatic activity into the medium was determined. Both irType II PLA2 and PL2 enzymatic activity accumulated in the incubation medium in a time-dependent manner (P < 0.0001). This release was not associated with a loss of cell membrane integrity, as indicated by measurement of the intracellular enzyme, lactate dehydrogenase, in the incubation medium. The concentration of irType II PLA2 and PLA2 enzyme activity present in incubation medium were significantly correlated (P < 0.01). Consistent with the hypothesis that Type II PLA2 may be store in secretory granules within human placental tissue, incubation in the presence of a membrane depolarising concentration of KCI (60 mM) caused the release of irType II PLA2 2.0-fold (P < 0.001). PLA2 enzyme activity released into the incubation medium displays biochemical characteristics consistent with those previously reported for secretory PLA2 isozymes, that is, a requirement for millimolar concentrations of calcium for optimal enzyme activity, inhibited by reducing agents, such as dithiothreitol and insensitive to heat inactivation. The data obtained in this study establish that irType PLA2 is released from term placenta, when incubated in vitro. The release of this extracellularly-active PLA2 isozyme may contribute to gestational and labour-associated increases in glycerophospholipid metabolism and prostaglandin formation.

Native peptide inhibition. Specific inhibition of type II phospholipases A2 by synthetic peptides derived from the primary sequence.

The binding of low molecular weight type II phospholipase A2 (EC) to membrane surfaces and hydrolysis of phospholipid are thought to involve the formation of a hydrophobic channel into which a single substrate molecule diffuses before cleavage. The floor and right side of the channel are provided by hydrophobic residues 2, 5, and 9 of an amphipathic amino-terminal helix. The channel is postulated to form via a conformational change in this helix and inward movement of a hydrophobic flap (residue 69 side chain). We show that the amino-terminal tryptic peptide of human type II phospholipase A2 forms a noncovalent complex with the tryptic peptide from residues 70-74 of the enzyme. Further, the 70-74-peptide sequence (FLSYK) dose-dependently inhibits phospholipid hydrolysis in a mixed micelle assay. This native peptide inhibition also occurred with type II enzymes from Crotalus durissus and Crotalus atrox, which have different amino acid sequences at the amino terminus as well as different 70-74 regions of the molecules. Despite significant conservation of tertiary structure among the enzymes, inhibition by each peptide is specific to the enzyme from which the peptide sequence is derived. We propose that these native peptides inhibit enzyme activity via a sequence-specific, noncovalent interaction with the amino-terminal residues of the enzyme, thereby preventing the conformational change on binding to the micelle interface. These experiments demonstrate a new method for specific inhibition of phospholipase A2 which, in principle, would be applicable to other biologically active polypeptides and proteins.

Localization of type II phospholipase A2 messenger RNA and immunoactivity in human placenta and fetal membranes.

Although Type II phospholipase A2 (PLA2) immunoactivity has been identified in homogenates of human placenta and fetal membranes, there is a paucity of information concerning the sites of synthesis of this secreted PLA2 isozyme. The aim of this study, therefore, was to establish the cellular localization of Type II PLA2 messenger RNA (mRNA) in human term placental and fetal membranes by in situ hybridization. In addition, the co-localization of immunoreactive Type II PLA2 in gestational tissues was determined, and the effect of labour status and pre-eclampsia on immunolabelling intensity were established. Type II PLA2 mRNA was identified in all tissue sections examined and was localized principally in placental villous vasculature and mesenchymal elements of placenta, chorion and amnion. Within the vasculature, Type II PLA2 mRNA was associated with smooth muscle cells. Immunoreactive Type II PLA2 was identified in the fibroblast and spongy layers of the amnion, the fibroblast and reticular layer of the chorion, and in the mesenchymal core and trophoblasts of placental villi. Immunolabelling staining intensity was greater in placenta and chorion than that observed in amnion, however, staining intensity was unaffected by labour status. Pre-eclampsia was associated with increased immunolabelling for Type II PLA2 in placenta but not fetal membranes. The data obtained clearly established that Type II PLA2 is synthesized by multiple cell types within human gestational tissues and co-localization of Type II PLA2 mRNA and immunoreactive protein has been established. The role of Type II PLA2 in gestational tissue phospholipid metabolism and, in particular, in vascular and mesenchymal elements, has yet to be established. Possible roles for this isozyme may include, the provision of substrate for eicosanoid synthesis, maintaining cell membrane phospholipid asymmetry and prevention of clot formation within the placental vascular bed.

Circulating group II phospholipase A2 activity and antilipocortin antibodies in systemic lupus erythematosus. Correlative study with disease activity.

OBJECTIVE: Lipocortin (LC) and phospholipase A2 (PLA2) are involved in phospholipid metabolism, and on the cellular level LC seems to be an antagonist of PLA2. Since anti-LC1 autoantibodies were found in systemic lupus erythematosus (SLE), we undertook a study of the relationship between PLA2, anti-LC1, and disease activity in a large group of patients with SLE. METHODS: Sera from 81 patients with SLE were tested for the activity of extracellular PLA2 and the presence and level of antilipocortin 1 [anti-LC1 (IgM) and anti-LC1 (IgG)] antibodies. Both were compared to SLE activity. RESULTS: Mean PLA2 activity was 4.6-fold higher in patients with SLE than in healthy controls (707 +/- 219 vs 154 +/- 6 u/ml, p < 0.01). PLA2 activity correlated significantly with PLA2 immunoreactivity as estimated by an ELISA method using monoclonal antibodies against "synovial type" PLA2 (n = 21, r = 0.984, p < 0.001). Anti-LC1 IgM and IgG antibody levels were significantly higher in SLE than in healthy individuals [anti-LC1 (IgM) 54.5 +/- 4.6 vs 22.6 +/- 2.3 EU/ml, p < 0.001 and anti-LC1 (IgG) 54.3 +/- 3.4 vs 22.9 +/- 2.3 EU/ml, p < 0.001]. There was no correlation between PLA2 activity and anti-LC1 antibody titers. Elevated levels of PLA2 [> normal mean + 2 SD (i.e., > 300 u/ml)] were found in 41/81 patients with SLE. Anti-LC1 antibody titers were high (> 64 EU/ml) in 23/41 patients; 14/40 patients with SLE with normal PLA2 (< 300 u/ml) also had higher titers of anti-LC1 antibodies. PLA2 activity was significantly associated with the presence of synovitis, being markedly increased in 11/12 patients. Mean PLA2 in this group of patients (1593 +/- 957 u/ml) was significantly higher (p < 0.001) than that (553 +/- 188 u/ml) in the group of 69 patients with SLE without synovitis. CONCLUSIONS: There was no correlation of PLA2 activity with the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) or the Lupus Activity Criteria Count (LACC). Circulating PLA2 activity in SLE correlated only with active synovitis. There was no correlation of anti-LC1 titers with duration of the disease, age, steroid dosage, SLEDAI, or LACC or any individual clinical or laboratory variable included in the assessment of SLEDAI and LACC.

Type II phospholipase A2 in human gestational tissues: extractable immuno- and enzymatic activity in fetal membranes.

In this study, we have established the presence of immunoreactive (ir) Type II PLA2 in human amnion and choriodecidua obtained from women at term prior to the onset of labour. The content of irType II PLA2 present in 1 M NaCl extracts of choriodecidua and amnion averaged 3.5 +/- 3.1 and 10.6 +/- 5.2 ng/mg tissue protein (n = 3), respectively. PLA2 enzymatic activity present in the same tissues averaged 1.3 +/- 0.2 and 1.9 +/- 0.7 nmol phosphatidylethanolamine (PE) hydrolysed/mg tissue protein per h (n = 3), respectively. To allow intra-patient comparison of the relative distribution in gestational tissues, irType II PLA2 and PLA2 enzymatic activity was also determined in placenta obtained from the same group of women, and averaged 26.0 +/- 7.0 ng/mg tissue protein and 3.5 +/- 1.0 nmol PE hydrolysed/mg protein per h (n = 3), respectively. As has been previously reported for human placenta, the recovery of Type II PLA2 and PLA2 enzymatic activity from amnion and choriodecidua was increased between 16- and 25-fold when tissues were homogenized in high-ionic strength media (i.e., 10% (w/v) ammonium sulphate or 1 M NaCl) compared with that recovered when tissues were homogenized in low-ionic strength media (i.e., 0.32 M sucrose-20 mM Hepes). The data obtained represent the first quantitative estimates of immunoreactive Type II PLA2 in human amnion and choriodecidua, and support the conclusion that previous analyses of the PLA2 enzymatic activity present in gestational tissues have essentially excluded the contribution made by this PLA2 isozyme to net enzymatic activity. We suggest that this isozyme represents a major component of the PLA2 enzymatic activity present in human gestational tissues at term and that it contributes significantly to the phospholipid metabolism and arachidonic acid release which occurs during late pregnancy and at the time of labour.

Type II phospholipase A2 in human gestational tissues: subcellular distribution of placental immuno- and catalytic activity.

The aims of this study were to determine the subcellular distribution of Type II phospholipase A2 immunoactivity (irPLA2) and in vitro net PLA2 catalytic activity in human term placenta and to establish the efficacy of previously utilised homogenisation procedures with respect to the quantitative recovery of Type II PLA2 immunoreactive and in vitro net PLA2 catalytic activity. Type II PLA2 immunoactivity and PLA2 catalytic activity recovered in 900 x g supernates prepared from placental tissue (n = 3) homogenised in low ionic strength media (sucrose 0.32 M Hepes 20 mM; phosphate-buffered saline or phosphate-buffered saline containing 3 mM EGTA) was less than 10% of that recovered following homogenisation in high ionic strength medium (ammonium sulphate 10%, w/v). The subcellular distribution of Type II PLA2 immunoactivity and PLA2 catalytic activity was established by the differential centrifugation (10,000, 20,000 and 100,000 x g) of placental homogenates (n = 3). Although Type II PLA2 immunoactivity was equally distributed throughout the particulate subcellular fractions examined, PLA2 catalytic activity increased by comparison in 100,000 x g particulate material. This apparent dissociation between irType II PLA2 and catalytic activity may indicate the presence of other types of PLA2 in this fraction. The data obtained in this study indicate that previous studies which have utilised low ionic strength extractions of human gestational tissue to characterise PLA2 catalytic activity and subcellular distribution have largely excluded the contribution made by Type II PLA2. Consequently, much of the available published data on the role of PLA2 in human parturition is inadequate. A reappraisal of this enzyme's contribution to the biochemical events associated with human pregnancy and labour is required.

Measurement of human phospholipase A2 in arthritis plasma using a newly developed sandwich ELISA.

A sandwich enzyme-linked immunosorbent assay (ELISA) for human non-pancreatic phospholipase A2 (PLA2) was developed using monoclonal antibodies raised against purified recombinant PLA2. This assay was shown to be specific for human non-pancreatic PLA2, showing no cross-reactivity with human pancreatic PLA2 or with snake venom PLA2 (Crotalus durissus). The immunoassay showed no cross-reactivity with plasma components, and was reproducible and quantitative between 39 pmol and 2.7 nmol PLA2/1. The levels of non-pancreatic PLA2 in the plasma of patients with arthritis was measured using this immunoassay. There were significantly higher levels of PLA2 in patients with rheumatoid arthritis than in those with osteoarthritis or healthy controls. Plasma PLA2 was highest in those patients with active rheumatoid arthritis.

Isolation and characterization of Rhizobium (IC3342) genes that determine leaf curl induction in pigeon pea.

Nodulation by the Rhizobium strain IC3342 causes a leaf curl syndrome in certain tropical legumes such as pigeon pea (Cajanus cajan) (N.M. Upadhyaya, J.V.D.K. Kumar Rao, D.S. Letham, and P.J. Dart, Physiological and Molecular Plant Pathology 39:357-373, 1991). Transposon (Tn5) mutagenesis of this leaf curl-inducing (Curl+) Rhizobium strain yielded two Curl- Fix- and three Curl- Fix+ mutants. Plasmid visualization and subsequent Southern blot hybridization analyses with Tn5, nif and nod gene probes showed that the Tn5 element had inserted into the symbiotic (Sym) plasmid in three of the mutants. Restriction endonuclease analyses indicated that none of the Tn5 insertions were closely linked. Tn5-containing EcoRI fragments were cloned from each mutant and used as probes to isolate the corresponding wild-type DNA fragments from a cosmid (pLAFR3) genomic library. Fix+ and/or Curl+ phenotypes were restored in each mutant by the introduction of cosmids containing the corresponding wild-type DNA. A closely related but Curl- Rhizobium strain ANU240 was shown, by Southern hybridization, to contain conserved DNA sequences of all but one of the identified genetic regions of the Curl+ Rhizobium strain IC3342. Cosmids containing the genetic region unique to the strain IC3342, designated lcr1, conferred a Curl+ phenotype on the strain ANU240. DNA sequence analysis of the cloned lcr1 region revealed five open reading frames (ORFs). The ORF2 showed homology with the Escherichia coli regulatory gene ompR, and ORF4 showed homology with E. coli and Rhizobium meliloti regulatory genes fnr and fixK, respectively.

Elevated maternal plasma immunoreactive phospholipase A2 in human preterm and term labour.

Peripheral plasma concentrations of immunoreactive phospholipase A2 (irPLA2) (Type II, non-pancreatic) were determined in 110 women during pregnancy. The concentration of irPLA2 did not significantly change during pregnancy (5.7 +/- 0.6 ng/ml, n = 72) until the onset of labour. When compared with non-labouring women, irPLA2 concentrations were significantly elevated in association with both preterm labour (13.3 +/- 2.4 ng/ml, n = 15, p less than 0.02) and labour at term (10.4 +/- 1.7, n = 23, p less than 0.02). These data suggest that maternal plasma irPLA2 may be reflective of the mechanism(s) underlying the labour-associated increase in human gestational tissue eicosanoid formation.

Circulating phospholipase A2 activity associated with sepsis and septic shock is indistinguishable from that associated with rheumatoid arthritis.

Elevation of circulating phospholipase A2 (PLA2) activity is associated with sepsis and septic shock. Elevated levels of PLA2 activity also are seen in association with chronic inflammatory disorders such as rheumatoid arthritis. The relationship between these phospholipases is unclear. We have developed a highly specific enzyme-linked immunosorbent assay (ELISA) capable of measuring human synovial PLA2 in plasma, using monoclonal antibodies raised to recombinant synovial PLA2. This ELISA has been used to quantitate circulating PLA2 levels in patients clinically diagnosed with sepsis. These elevated levels positively correlated with the elevation seen in plasma PLA2 enzyme activity. The antibodies also have been used to purify immunoreactive PLA2 from plasma of patients with sepsis, thus enabling characterization of the purified protein by amino-terminal sequence analysis. We conclude from this study that the increase in PLA2 activity seen in association with sepsis and septic shock results from a dramatic elevation in levels of a circulating PLA2 enzyme. This inflammatory PLA2 is indistinguishable, both immunologically and chemically, from that associated with rheumatoid arthritis. Therapeutic agents directed towards inhibition of this inflammatory PLA2 enzyme may have utility in the treatment of both chronic and acute inflammatory disease.