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Biomedical subjects

W Pruzanski

Publications and source records attributed to W Pruzanski.

At least 19 recordsLinked to original sources
PubMed1995

A natural disruption of the secretory group II phospholipase A2 gene in inbred mouse strains.

The synovial fluid or group II secretory phospholipase A2 (sPLA2) has been implicated as an important agent involved in a number of inflammatory processes. In an attempt to determine the role of sPLA2 in inflammation, we set out to generate sPLA2-deficient mice. During this investigation, we observed that in a number of inbred mouse strains, the sPLA2 gene was already disrupted by a frameshift mutation in exon 3. This mutation, a T insertion at position 166 from the ATG of the cDNA, terminates out of frame in exon 4, resulting in the disruption of the calcium binding domain in exon 3 and loss of both activity domains coded by exons 4 and 5. The mouse strains C57BL/6, 129/Sv, and B10.RIII were found to be homozygous for the defective sPLA2 gene, whereas outbred CD-1:SW mice had variable genotype at this locus. BALB/c, C3H/HE, DBA/1, DBA/2, NZB/BIN, and MRL lpr/lpr mice had a normal sPLA2 genotype. The sPLA2 mRNA was expressed at very high levels in the BALB/c mouse small intestine, whereas in the small intestine of the sPLA2 mutant mouse strains, sPLA2 mRNA was undetectable. In addition, PLA2 activity in acid extracts of the small intestine were approximately 40 times higher in BALB/c than in the mutant mice. Transcription of the mutant sPLA2 gene resulted in multiple transcripts due to exon skipping. None of the resulting mutant mRNAs encoded an active product. The identification of this mutation should not only help define the physiological role of sPLA2 but also has important implications in mouse inflammatory models developed by targeted mutagenesis.

Amino Acid Sequence↗
PubMed1995

Serum amyloid A protein enhances the activity of secretory non-pancreatic phospholipase A2.

The acute-phase proteins serum amyloid A protein (SAA) and secretory phospholipase A2 (sPLA2) are simultaneously expressed during inflammatory conditions. SAA associates with high-density lipoprotein (HDL) altering its physicochemical composition. We found that purified acute-phase SAA, but not the constitutive form, markedly enhances the lipolytic activity of sPLA2 in a dose-related manner with phosphatidylcholine/lysophosphatidylcholine or phosphatidylethanolamine/lysophosphatidylethanolamine liposomal substrates. Normal HDL was found to reduce activity of sPLA2 in a dose-dependent manner, but when acute-phase HDL containing 27% SAA was tested, it enhanced sPLA2 activity. Immunopurified monospecific antibodies against SAA completely abolished the enhancing activity of SAA and acute-phase HDL. Given the central role of HDL in lipoprotein metabolism, the interaction between HDL, SAA and sPLA2 may account for changes detected in lipoprotein metabolism during the acute phase.

Acute-Phase Proteins↗
PubMed1995

Inhibition of human group II phospholipase A2 by C-reactive protein in vitro.

Endotoxin or cytokine-induced expression of a secretory non-pancreatic phospholipase A2 (sPLA2) has been implicated in the pathogenesis of multisystem organ dysfunction or failure in patients with septic shock. Circulating sPLA2 levels increase as much as 1000-fold during the course of septic shock. However, the mode of regulation of the activity of this lipolytic enzyme is unknown, since circulating inhibitors have not been identified. We investigated the potential inhibitory activity of the acute phase reactant, C-reactive protein (CRP), a phospholipid-binding protein whose expression increases as much as 1000-fold during severe infections. Serum CRP and sPLA2 profiles were highly concordant in patients with septic shock. In studies in vitro, human CRP inhibited hydrolysis of PC-lyso-PC (2:1) multilamellar liposomes by human recombinant sPLA2 in a dose-dependent manner, with an apparent IC50 of 50 micrograms/ml CRP and maximal inhibition at 100 micrograms/ml. Inhibition of sPLA2 activity by CRP was substrate concentration-dependent, and increasing substrate concentrations reversed the inhibitory effect of CRP using the PC-lyso-PC system. Preincubation of CRP with phosphorylcholine led to a concentration-dependent loss of CRP-induced inhibition of substrate hydrolysis. These observations are consistent with a substrate-depletion model of inhibition of sPLA2 activity by CRP.

C-Reactive Protein↗
PubMed1994

Impact of intravenous infusions of low and high doses of gamma globulins (IVIG) on phagocytic functions in adults with primary humoral immunodeficiency.

Twelve adult patients with primary humoral immunodeficiency were treated for at least six months with IVIG 200 mg/kg/mo and then crossed over to a high dose of 600 mg/kg/mo. Polymorphonuclear and mononuclear cells of these patients were tested after the third infusion in the low-dose cycle and then after the third infusion in the high-dose cycle, each time a day before, four days after, and 14 days after intravenous infusion. Each time, patients' cells and normal cells were tested using normal sera and patients' sera. IVIG infusions led to a significant increase in the level of circulating IgG, which was much more prominent in the high-dose group. Phagocytosis, phagocytic index, intracellular bactericidal activity and chemotaxis of polymorphonuclear cells (PMNs) were at least as active as in healthy controls. Actually in both cycles patients' PMN's had slightly higher phagocytic activity than normal cells. Patients' serum in the high dose cycle supported chemotaxis better than normal serum. Efficient phagocytic activity was maintained throughout the cycle; however, it was more active (P < 0.0125) in the midcycle in the high-dose cycle. Superoxide generation was normal in all conditions. Monocytic function was also normal in all conditions tested. It may be concluded that as far as cellular phagocytic functions are concerned, the high dose of IVIG does not protect the host more efficiently than the low dose.

Adult↗
PubMed1994

Phospholipase A2 in juvenile rheumatoid arthritis: correlation to disease type and activity.

OBJECTIVE: Secretory nonpancreatic phospholipase A2 (snpPLA2) is a proinflammatory enzyme and its activity in serum correlates with disease activity in adults with rheumatoid arthritis. Juvenile rheumatoid arthritis (JRA) may be stratified into 3 clinical types with differing degrees of disease activity. Since in JRA there are no reliable indices of disease activity, our objective was to find whether the level of circulating snpPLA2 correlates with the severity of inflammation and with JRA activity. METHODS: PLA2 enzymatic activity was assayed using E. coli membranes labelled with (14C)-oleic acid. SnpPLA2 immunoreactivity was tested by ELISA technique using monoclonal antibodies against recombinant human (rh) snpPLA2. SnpPLA2 activity was determined in sera of 127 children including 25 with systemic (S-JRA), 50 with polyarticular (Po-JRA) and 52 with pauciarticular (Pa-JRA) types of JRA. Twenty-five patients with active disease, were subsequently restudied in an inactive phase. RESULTS: Markedly increased snpPLA2 (> mean + 2 SD of normal mean, i.e., > 575 U/ml) was found during the active disease in 100% S-JRA, 57% Po-JRA and 25% Pa-JRA patients. The differences in the mean and median PLA2 activity among these 3 subtypes of JRA were highly significant (p < 0.001) with the highest levels found in S-JRA and the lowest in Pa-JRA. Presence of rheumatoid factor and/or of antinuclear antibody had no relation to the level of snpPLA2. SnpPLA2 activity became markedly lower when active inflammation became quiescent. In the whole group, snpPLA2 activity correlated highly with the Lansbury index, number of involved joints and number of effusions. A significant positive correlation was also found between snpPLA2 and erythrocyte sedimentation rate (ESR) and neutrophil count, while a significant negative correlation was noted with the level of albumin and hemoglobin. With the exception of snpPLA2, other laboratory variables did not correlate with the number of effusions or the number of active joints. However a negative correlation was noted between both hemoglobin and albumin, and Lansbury index. CONCLUSION: Circulating snpPLA2 significantly correlates with JRA activity and may serve as an index of activity in JRA especially in patients with systemic type of disease.

Adolescent↗
PubMed1992

Inhibition of enzymatic activity of phospholipases A2 by minocycline and doxycycline.

Extracellular phospholipases A2 play an important role in articular and extra-articular inflammatory processes. Secretory non-pancreatic phospholipase A2 (PLA2) has been implicated in the pathogenesis of articular inflammation in rheumatoid arthritis, whereas pancreatic PLA2 contributes to the tissue damage associated with acute pancreatitis. Since in experimental models lipophilic tetracyclines such as minocycline and doxycycline are antiinflammatory, we examined their effects on PLA2 activity using two assay systems in vitro. We found that minocycline and to a lesser degree doxycycline were markedly inhibitory to both pancreatic and non-pancreatic PLA2. Using [14C]oleic acid labeled Escherichia coli membrane phospholipids as substrate, the IC50 values for minocycline and doxycycline were 3.6 x 10(-5) M (18 micrograms/mL) and 0.98 x 10(-4) M (47 micrograms/mL), respectively. In a scooting mode assay using the synthetic phospholipid 1-palmitoyl-2-(10-pyrenedecanoyl)-3-L-phosphatidylmethanol as substrate, IC50 values for minocycline were 5 microM (2.47 micrograms/mL) for non-pancreatic PLA2 and 8 microM (3.95 micrograms/mL) for pancreatic PLA2. Addition of excess calcium up to 50 mM did not reverse the inhibitory activity of tetracyclines. We conclude that lipophilic tetracyclines inhibit PLA2, probably by interaction with the substrate, and may be a useful adjunct in the therapy of inflammatory conditions in which PLA2 is implicated pathogenetically.

Animals↗
PubMed1992

Enzymatic activity and immunoreactivity of extracellular phospholipase A2 in inflammatory synovial fluids.

Synovial fluid PLA2 concentration was measured by an ELISA technique using monoclonal antibodies raised against human recombinant "synovial-type" group II phospholipase A2. This ELISA was specific for synovial-type PLA2 and did not detect pancreatic (group I) PLA2. In all synovial fluids examined, including rheumatoid, osteoarthritic, psoriatic, and gouty fluids, synovial fluid PLA2 enzyme activity significantly correlated with PLA2 immunoreactivity (P < 0.001). Within the limits of the ELISA technique, there was no evidence for the presence of specific or nonspecific modulation of PLA2 activity by either putative PLA2 activating or inhibitory proteins.

Arthritis↗
PubMed1992

Hyperphospholipasemia A2 in human volunteers challenged with intravenous endotoxin.

Phospholipase A2 (PLA2) activity was measured in the serum of 23 individuals infused intravenously with endotoxin (EN) at a dose of 4 ng/kg body weight. A marked increase in PLA2 was noted 3 h after EN challenge (mean 828 +/- 513 units/ml), reached its maximum at 24 h after the challenge (mean 2667 +/- 2442 units/ml), and was still evident at 48 h (mean 763 +/- 366 units/ml). In contrast, TNF levels were maximal (mean 712 +/- 375 pg/ml) 90 min after the EN challenge and subsided to very low values (5 +/- 5 pg/ml) 5 h after the challenge. There was a positive correlation between the maximum response of TNF and that of PLA2 (r = 0.82, P < 0.01). Administration of ibuprofen or pentoxifylline did not alter the PLA2 response. EN challenge did not affect serum pancreatic PLA2 concentration or that of the lysosomal cationic enzyme, lysozyme. Neutralizing antibody against human recombinant (synovial type) PLA2 completely abolished PLA2 activity in the sera tested. We conclude that EN infusions cause marked intravascular release of nonpancreatic secretory PLA2 and that the magnitude of this response seems to be related to the prior generation of TNF.

Endotoxins↗
PubMed1992

Serum phospholipase A2 enzyme activity and immunoreactivity in a prospective analysis of patients with septic shock.

Massive elevations of serum phospholipase A2 activity have been documented in patients with septic shock. Serum PLA2 activity correlated to the degree and duration of circulatory collapse, while purified native PLA2 reproduced hypotension in experimental animals. In a prospective study of patients with septic shock, we have determined the relationship of PLA2 enzyme activity to PLA2 immunoreactivity using radiolabelled E. coli phospholipid substrate and an ELISA specific for group II human nonpancreatic PLA2. In all patients, there was a clear concordance of the two assays. Maximal PLA2 concentration was increased a mean of 554-fold over normal levels. We found no evidence to support the presence of activating or inhibitory proteins. These data confirm that the observed increase in serum PLA2 activity in septic shock is due to intravascular release of group II nonpancreatic PLA2.

Antibodies, Monoclonal↗
PubMed1992

Induction of circulating phospholipase A2 activity by intravenous infusion of endotoxin in patients with neoplasia.

The purpose of this study was to evaluate the impact of repeated intravenous infusions of endotoxin (EN) in patients with cancer on the systemic release of extracellular proinflammatory phospholipase A2 (PLA2) and its relationship to the release of tumor necrosis factor (TNF) and interleukin-6 (IL-6). Six patients received 15 infusion of EN isolated from Salmonella abortus equi at a dose of 4 ng/kg. Marked increase in the activity of circulating PLA2 was noted within 3 h after the first EN infusion and reached a maximal level of 20.4-fold greater than baseline 24 h after infusion. In five patients challenged with EN 2 weeks later, PLA2 reached peak levels 15.5-fold greater than baseline. In two patients who received three sequential daily infusions, the incremental increase in PLA2 activity after the second and third challenge reached maximum levels 6 h after EN infusion. PLA2 response followed those of TNF and IL-6 but was quantitatively different. Whereas maximal levels of TNF and IL-6 declined substantially after repeat EN challenges, no such decline occurred in PLA2 activity. Since, in the clinical setting of gram-negative sepsis, there is recurrent increase in circulating EN, our study approximates this clinical situation and shows that extracellular release of PLA2 follows temporally that of proximal cytokines such as TNF and IL-6. These cytokines may be related to PLA2 release and sustained high activity in the systemic circulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1992

Induction of circulating group II phospholipase A2 expression in adults with malaria.

High levels of interleukin 1 and tumor necrosis factor are found in both cases of malaria and cases of septic shock. Since both interleukin 1 and tumor necrosis factor induce expression of the proinflammatory enzyme phospholipase A2 (PLA2), we examined serum PLA2 levels in 14 adults with malaria. Mean serum PLA2 activity was elevated 40-fold above normal (P less than 0.001). Serum PLA2 activity correlated with PLA2 immunoreactivity (r = 0.987; P less than 0.001) by an enzyme-linked immunosorbent assay specific for human group II PLA2, showing that serum PLA2 in cases of malaria is host derived. This article describes the novel finding of elevated PLA2 levels in cases of malaria, further strengthening the notion that mediators of the host response in cases of malaria are similar to those in cases of septic shock.

Adult↗
PubMed1991

Variable region gene analysis of pathologic human autoantibodies to the related i and I red blood cell antigens.

To investigate the molecular basis of the autoimmune response to the related i and I carbohydrate antigens, we studied cold agglutinins (CA) from B-cell clones and from the peripheral circulation of patients with lymphoproliferative syndromes. Sequence analyses of expressed variable region genes indicate that both anti-i and anti-I specificities from B-cell clones from two patients are encoded by the VH4.21 or a very closely related VH4 heavy chain gene, whereas the expressed light chain genes differed. The anti-i-secreting B-cells express unmutated germline-encoded VH4.21 and VKI gene sequences. The VH region gene encoding anti-I has the closest homology (97%) to the VH4.21 germline gene and differs at the protein level by only three amino acids. In contrast, while the VL region gene encoding anti-I is most homologous (96%) to the VKIII, kv328 germline gene, there are seven amino acid differences due to nonrandom replacement mutations, which suggests a role for antigen-mediated selection in the anti-I response of this individual. These studies were extended by a structural survey of 20 additional serum CA using antipeptide antibodies specific for determinants in VH and VL regions. All anti-I and anti-i CA were shown to express VH4 heavy chains, and 14 of 17 CA expressed a previously described VH4 second hypervariable region determinant, termed VH4-HV2a. We also found that 13 of 14 anti-I CA used VKIII light chains, while the anti-i CA used light chains from at least three VL families. Taken together, the data show that anti-i and anti-I CA probably both derive from the VH4.21 gene (or a closely related gene). Furthermore, the restricted VH and different VL gene use in anti-i and anti-I CA may reflect the close structural relationship of the i and I antigens.

Autoantibodies↗
PubMed1991

Interleukin-1 alpha stimulates the release of prostaglandin E2 and phospholipase A2 from fetal rat calvarial cells in vitro: relationship to bone nodule formation.

We have shown previously that interleukin-1 (IL-1) has biphasic effects on the formation of bone nodules in long-term cultures of fetal rat calvarial (RC) cells (Ellies and Aubin, Cytokine 2:430-437, 1990). To determine the role of arachidonic acid metabolism in this process, we have examined the release of prostaglandin E2 (PGE2) and phospholipase A2 (PLA2) from RC cells under conditions that allowed concomitant analysis of the formation of bone nodules. Recombinant human IL-1 alpha (rhIL-1 alpha) stimulated PGE2 and PLA2 release in a time- and dose-dependent manner. PGE2 release was highest in preconfluent cultures (days 1-6) and was stimulated up to 8.5-fold in response to 50 U/ml of rhIL-1 alpha. In contrast, extracellular PLA2 activity was maximal in postconfluent cultures, with 50 U/ml of rhIL-1 alpha causing a 20-fold increase by day 15. PLA2 release by RC cells was not significantly affected by PGE2, the glucocorticoid dexamethasone, or the cyclooxygenase inhibitor indomethacin. Indomethacin partially blocked the inhibition of bone nodule formation caused by rhIL-1 alpha, and exogenous PGE2 reversed this effect. Addition of group I PLA2 from Naja naja venom to RC cells had no effect on bone nodule development; however, group II PLA2 from Crotalus adamanteus venom inhibited the formation of bone nodules in a dose range similar to that induced by rhIL-1 alpha. These results indicate that PGE2 release does not have a direct temporal correlation with increases in PLA2 activity. In addition, the data show that only part of the inhibition of bone formation seen with rhIL-1 alpha is mediated by PGE2 and suggest that extracellular PLA2 also accounts for part of the inhibition.

Animals↗
PubMed1991

Comparison of group I and II soluble phospholipases A2 activities on phagocytic functions of human polymorphonuclear and mononuclear phagocytes.

Soluble phospholipase A2 (PLA2) purified from rheumatoid synovial fluid (group II) and repurified Naja naja venom PLA2 (group I) were compared for their influence on phagocytic activity of human polymorphonuclear (PMN) and mononuclear (MO) phagocytes. Group II PLA2 reduced chemotaxis, adhesiveness, and intracellular bactericidal activity (ICBA) and induced release of muramidase from PMNs. Group I PLA2 suppressed chemotaxis, and enhanced ICBA but had no influence on other phagocytic functions. Group II PLA2 purified from synovial fluid or from placenta caused marked spontaneous superoxide generation followed by inhibition of phagocytosis-induced burst of energy. Group I Naja naja and porcine pancreatic PLA2 had no effect on superoxide generation. Group II but not group I PLA2 reduced markedly ICBA of monocytes. It may be concluded that human group II soluble PLA2, in concentrations comparable to those present in inflamed joints or in sera of patients with active arthritis or septic shock, causes spontaneous formation of the oxygen radical superoxide and release of lysosomal enzymes, and suppresses conventional phagocytic activities of PMNs and monocytes. Marked differences between group I and group II PLA2s may mean that these enzymes exert different influences on cell membrane.

Arthritis, Rheumatoid↗
PubMed1991

Enzymatic activity and distribution of phospholipase A2 in human cartilage.

Extracellular phospholipase A2 (PLA2) with proinflammatory activity has recently been discovered in synovial fluids in inflammatory arthritides. In the search for the sources of synovial fluid PLA2, human synovium and articular cartilage were found to contain large quantities of the enzyme. In rheumatoid arthritis (RA), PLA2 activity in synovium, superficial and deep layers of articular cartilage was 20 +/- 14 (SEM), 168 +/- 62 and 533 +/- 176 nmol/min/mg protein respectively. Corresponding values in osteoarthritis (OA) were 49 +/- 11, 569 +/- 109 and 1709 +/- 243 nmol/min/mg protein, all significantly higher (p less than .01) than in RA. Nasal septal cartilage contained much less PLA2, 19 +/- 5.6. PLA2 in human articular and nasal cartilage has sn-2 specificity, a neutral pH optimum and absolute calcium dependence. High PLA2 concentration in articular cartilage may imply that, at least in part, cartilage is the source of PLA2 in the joint space. Since RA cartilage and synovium have less PLA2 activity than the corresponding OA tissues, additional sources of PLA2 in RA synovial fluids are implicated.

Arthritis, Rheumatoid↗
PubMed1991

Extracellular phospholipase A2 secretion is a common effector pathway of interleukin-1 and tumour necrosis factor action.

Inflammatory processes are characterized by increased levels of extracellular phospholipase A2 (PLA2) and cytokines such as interleukin 1 (IL-1) and tumour necrosis factor (TNF). IL-1, TNF and PLA2 share a number of proinflammatory, arthritogenic effects. The sequential induction, first of the cytokines followed by PLA2, suggests that these cytokines may regulate synthesis and secretion of PLA2. To test this postulate, foetal rat calvarial bone-forming cells (FRCC) were treated with recombinant human IL-1 and TNF and extracellular PLA2 release was quantitated. Both IL-1 and TNF induced the de novo synthesis of PLA2 in a concentration-dependent manner. Continuous exposure of FRCC in primary culture to IL-1 (50 units/ml) over 15 days resulted in as much as 100-fold increase in PLA2 secretion. IL-1 (50 units/ml) added to post-confluent cultures for a 48-h pulse increased PLA2 activity 9.4-fold. The combination of IL-1 (50 units/ml) and TNF (500 units/ml) was synergistic with an observed increase in extracellular PLA2 secretion of 146-fold following a 48-h pulse. Interleukin-6, alone or in combination with IL-1 or TNF, did not further enhance PLA2 synthesis of secretion. Cytokine-induced synthesis of PLA2 was inhibited 80% by 10 microM cycloheximide but not by dexamethasone over the range of 10(-6) to 10(-8) M. FRCC-derived PLA2 was neutral-active with a pH optimum of 6-7.5 and was calcium-dependent with optimal activity in the presence of 2-7 mM calcium. It had absolute 2-acyl specificity using micellar phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗