Pentraxin binding to isolated rat liver nuclei.

The interaction of human C-reactive protein (CRP) and serum amyloid P-component (SAP) with isolated rat liver nuclei was studied to identify nuclear ligands for each pentraxin using the iodinatable heterobifunctional thiol-cleavable cross-linking reagent sulphosuccinimidyl-2-(p-azidosalicylamido)-1,3'-dithiopropio nate (SASD). Nuclei (100 micrograms of DNA) bound 21 pmol of 125I-labelled CRP Ca(2+)-dependently at saturation with half-saturation occurring at 200 pmol of 125I-CRP. By contrast, only 2.7 pmol of 125I-labelled SAP was bound at saturation, with half-saturation at 50 pmol. The binding of pentraxins to nuclei is, in addition to putative chromatin binding, due to nuclear-envelope binding, where 3.2 pmol 125I-labelled CRP binds Ca2+ dependently to nuclear envelopes (25 micrograms) at saturation, but only 0.62 pmol SAP is required to saturate. Specific photocross-linking of 125I-2-(p-azidosalicylamido)-1,3'-dithiopropionate (125I-ASD)-CRP and 125I-ASD-SAP to nuclei revealed transfer of 125I-photoreactive azides to nuclear-envelope proteins of 43, 46, 52 and 70 kDa. In addition, SAP binding to histones H2A, H2B, H3 and H4 was detected, whereas CRP bound only to H4. Neither pentraxin cross-linked to histone H1.

Neurotensin, vaso-active intestinal polypeptide and gastrin levels in plasma and portal venous blood in experimental mesenteric ischaemia.

The effect of mesenteric ischaemia on the levels of neurotensin, vaso-active intestinal polypeptide and gastrin in portal venous blood and in the peripheral circulation was studied in two groups of 7 and 6 baboons (Papio ursinus). In peripheral blood a decreasing trend in levels of neurotensin was observed, while vaso-active intestinal polypeptide and gastrin levels were unchanged. There was a similar trend in neurotensin levels in portal venous blood, together with an increasing trend in levels of vaso-active intestinal polypeptide. Gastrin levels were unchanged. Further investigation of these apparent trends in a large number of animals is warranted.

Peptides generated from C-reactive protein by a neutrophil membrane protease. Amino acid sequence and effects of peptides on neutrophil oxidative metabolism and chemotaxis.

We have recently provided evidence that C-reactive protein (CRP) could act as an up-regulatable substrate for membrane-associated neutrophil serine protease(s). The resultant degradation of CRP yielded small soluble bioactive peptides that inhibit many of the proinflammatory functions of activated neutrophils and could oppose the tissue destructive potential of these cells. We report on the reverse phase HPLC separation of the small TCA-soluble peptides obtained when CRP is degraded with nonstimulated or PMA-stimulated neutrophils and purified neutrophil membranes. The amino acid sequence of seven peptides isolated from the CRP digest has been ascertained and synthetic peptides homologous to these sequences have been synthesized. Three of the synthetic peptides corresponding to residues 201-206 (CRP-III), 83-90 (CRP-IV), and 77-82 (CRP-V) of the intact protein were identified to significantly inhibit superoxide production from activated neutrophils at 50 microM whereas CRP-III and CRP-V in addition inhibited neutrophil chemotaxis at this concentration. These peptides act additively and their action likely involves the signal transduction pathways for neutrophil activation.

C-reactive protein and serum amyloid A protein in pregnancy and labour.

Serum levels of C-reactive protein (CRP) and amyloid A protein (SAA) were measured prospectively using immunoradiometric assays in normal pregnant women, newborn infants and women with prelabour rupture of membranes (PROM), focusing on the peripartum period. CRP levels in 50 healthy women at 38 weeks gestation did not differ significantly from previously established normal values. CRP levels in 67 healthy women sampled serially in labour from admission to 96 h postpartum confirm the physiological occurrence of a major acute phase response. The serial CRP levels of 16 women with PROM did not differ significantly from the wide range of CRP levels found in the normal postpartum period. This complicates the use of CRP as an early predictor of clinical chorio-amnionitis. Serial SAA levels in 17 women at 38 weeks gestation, immediately postpartum and 24 h postpartum showed a parallel rise to CRP in the peripartum period. Significant differences between maternal and neonatal CRP and SAA levels were demonstrated, implying a lack of transplacental transfer during labour.

Generation of biologically active C-reactive protein peptides by a neutral protease on the membrane of phorbol myristate acetate-stimulated neutrophils.

The association of human C-reactive protein (CRP) with nonstimulated and PMA-stimulated human neutrophils and the concomitant degradation of CRP (monitored by TCA-soluble peptides and SDS-PAGE analysis) has been studied. Maximum association of 125I-labeled CRP with neutrophils and 125I-labeled CRP degradation during association with these cells was achieved by stimulating the neutrophils with PMA at 10 ng/ml; a concentration in which azurophil granule release was not significant. For PMA-stimulated neutrophils, the association of 125I-labeled CRP was 1.8 times higher and PMA-stimulated neutrophil-mediated degradation of the ligand was three times faster than that for nonstimulated cells. The neutrophil-associated 125I-labeled CRP in the absence and presence of PMA proved on SDS-PAGE analysis to be approximately 50% degraded. There was a positive correlation between the extent of CRP degradation and the association of 125I-labeled CRP with neutrophils. In addition to generation of neutrophil associated CRP intermediates, small soluble CRP peptides were generated during association of CRP with neutrophils. These peptides inhibited superoxide production from opsonized zymosan-activated neutrophils by approximately 40% at 10 micrograms/ml. 125I-labeled CRP degradation mediated by nonstimulated neutrophils, and neutrophil-conditioned medium (from both non-stimulated and PMA-stimulated cells) was inhibitable by alpha 1-antitrypsin and approximately seven times less at 1 h than that occurring during 125I-labeled CRP-association with PMA-stimulated neutrophils. The degradation of 125I-labeled CRP mediated by PMA-stimulated neutrophils was not fully inhibitable by alpha 1-antitrypsin. The data point to the involvement of a membrane-associated serine protease, which is maximally activated by PMA, in the degradation of 125I-labeled CRP during association with neutrophils. Our results indicate that at an inflammatory site CRP-derived peptides can be produced that inhibit the action of activated neutrophils.

Human serum amyloid A protein. The assignment of the six major isoforms to three published gene sequences and evidence for two genetic loci.

Serum amyloid A protein (apo-SAA) is an acute-phase reactant and an apolipoprotein of high density lipoproteins (HDL). Six major isoforms of apo-SAA occur in humans (pI 6.0, 6.4, 7.0, 7.4, 7.5, 8.0). In this report we have rationalized the phenotypic expression of apo-SAA isoforms with published apo-SAA structures predicted from apo-SAA cDNA's pA1 and pSAA82 and the genomic DNA SAAg9. The six apo-SAA isoforms fall into three pairs, pI 6.0/6.4, 7.0/7.5, and 7.4/8.0, which are products of cDNA pA1, cDNA pSAA82, and genomic DNA SAAg9, respectively. The second of each isoform pair (i.e. pI 6.4, 7.5, and 8.0) is the "primary" product: a 104-residue peptide with the NH2-terminal sequence Arg-Ser-Phe-Phe. Each primary product is processed either to a major 103-residue peptide with the NH2-terminal sequence Ser-Phe-Phe or processed to a minor 102-residue product which results from the loss of both an Arg and a Ser residue from the NH2 termini. These "secondary" products have the lower pI values of 6.0, 7.0, and 7.4, respectively. The isoelectric points of the SAAg9 products were confirmed by expression of SAAg9 in transfected mouse L-cells. Both the pI 8.0 and 7.4 isoforms were present in cellular extracts, suggesting that post-translational modification of apo-SAA may occur intracellularly. However, the greater relative abundance of the pI 7.4 isoform extracellularly suggests that the major conversion may occur after secretion. Whereas the gene corresponding to the pA1 cDNA sequence does not show allelic variation, the segregation characteristics of the pI 7.0/7.5 and 7.4/8.0 isoform pairs amongst individuals suggests that these isoforms are the products of genes (with sequences corresponding to pSAA82 and SAAg9, respectively) which are allelic variants at a single locus distinct from that for the pI 6.0/6.4 isoform pair.

Human serum amyloid A protein. Behaviour in aqueous and urea-containing solutions and antibody production.

Human serum amyloid A protein (apo-SAA) can be prepared by gel filtration of delipidated acute-phase high-density lipoprotein in the presence of urea. The resultant apo-SAA is soluble (greater than 90% solubility) in a wide range of buffer solutions, with all of the six major isoforms of apo-SAA being equally soluble. In urea-containing solutions the isoforms behave qualitatively differently in various urea concentrations, probably reflecting subtle primary-structure variations. The higher-pI isoforms are only completely unfolded at greater than 7 M-urea. By immunizing with apo-SAA adsorbed to acid-treated bacteria (Salmonella minnesota R595), high-titre antibodies can easily be elicited in rabbits.

Characterization of protein kinase C and its isoforms in human T lymphocytes.

Protein kinase C (PKC) regulates numerous T cell functions and is present in abundance in normal human T cells and certain T cell lines. Although crude Triton X-100 soluble material obtained from T cell pellets contains minimal PKC activity, DEAE chromatography revealed that 12 to 37% of cellular PKC was membrane associated, probably due to removal of an inhibitor through column chromatography. As in other tissues, PKC from lymphoid tissue was phospholipid and Ca2+ dependent and diolein reduced the Ca2+ requirements for enzyme activity. Hydroxylapatite chromatography revealed that T cells possess two major peaks of PKC activity. Although, the enzyme in these peaks had similar m.w. and identical iso-electric mobility, the proteins differed with respect to their autophosphorylation sites and immunoreactivity toward an isoform specific antibody. Furthermore, differences in their activities in the presence of phospholipid, diolein, and limiting amounts of Ca2+ imply that these isoforms may be differentially activated. We discuss optimal conditions for activation of PKC and its isoforms for study of T lymphocyte cellular function.

Phosphorylation of human serum amyloid A protein by protein kinase C.

Monokine-induced hepatic secretion of serum amyloid A protein (apo-SAA), an acute-phase reactant, is followed by rapid association with high-density lipoprotein (HDL) in plasma. Plasma clearance of apo-SAA is more rapid than any of the other HDL apolipoproteins. It has been shown that, of the acute-phase HDL3 apolipoproteins, apo-SAA preferentially associates with neutrophil membranes. HDL apolipoproteins have been shown to activate protein kinase C in endothelial cells. We therefore investigated potential phosphorylation of HDL3 apolipoproteins by protein kinase C. Apo-SAA was the only apolipoprotein phosphorylated (Km = 12 mM). Phosphorylation of the apo-SAA-containing HDL3 particle was selective for the more basic isoforms of apo-SAA (pI 7.0, 7.4, 7.5 and 8.0), with more acidic isoforms being phosphorylated when delipidated acute-phase apolipoproteins were used as substrate. However, phosphorylation was not in itself responsible for the establishment of the apo-SAA isoforms.

Circulating acute phase reactive proteins as indicators of infection in poorly controlled diabetes mellitus.

Serum levels of six acute phase proteins (APP)--C-reactive protein (CRP), serum amyloid A (SAA), alpha 1-antitrypsin, haptoglobin and complement fractions C3 and C4--were serially studied in 24 patients with poorly controlled diabetes mellitus, ten of whom had unequivocal evidence of an underlying infection. In diabetic patients without infection, no change in APP levels was noted suggesting that hyperglycaemia per se does not quantitatively influence the acute phase response. No correlation between the presence of infection, and fever, leukocytosis, a raised erythrocyte sedimentation rate, or serum levels of alpha 1-antitrypsin, haptoglobin or complement was apparent in these patients. However, serum CRP and SAA were initially increased 10-100 times above normal in diabetic patients with an underlying infection (P less than 0.01); during the following week circulating levels of CRP and SAA decreased steadily in response to the infection being brought under control. We conclude that serial measurement of CRP and/or SAA is a sensitive, albeit non-specific, parameter to detect and monitor the activity of infection in patients with diabetes.

Identification of three isoform patterns of human serum amyloid A protein.

Three patterns of human apo-SAA (serum amyloid A protein) isoforms have been identified by electrofocusing. In pattern 1, six major apo-SAA isoforms of pI 6.0, 6.4, 7.0, 7.4, 7.5 and 8.0 were found. In pattern 2, the apo-SAA isoforms of pI 7.4 and 8.0 were not detected, whereas in pattern 3 the pI-7.0 and -7.5 isoforms were lacking. Six patients displayed apo-SAA isoform pattern 1, 11 displayed pattern 2 and one displayed pattern 3.

Antibodies reactive with streptococcal peptidoglycan-polysaccharide complexes in rheumatic fever, subacute bacterial endocarditis and tuberculosis.

Serum antibodies reactive with streptococcal peptidoglycan-polysaccharide complexes (PG-PS) have been estimated by enzyme-linked immunosorbent assay in patients with acute rheumatic fever (ARF), tuberculosis (TB) and subacute bacterial endocarditis (SBE) compared with normal age- and sex-matched controls. IgG2 and IgG3 were the predominant subclasses of antibodies in TB and SBE, whereas in ARF IgG1, IgG2 and IgG3 antibodies were detected. IgG4 antibodies were detected only infrequently. Antibodies of the IgG subclasses were detected in a greater proportion of sera when the PG-PS antigen was further purified by extraction with sodium dodecyl sulphate.

Serum amyloid A-containing human high density lipoprotein 3. Density, size, and apolipoprotein composition.

Serum amyloid A protein (apo-SAA), an acute phase reactant, is an apolipoprotein of high density lipoproteins (HDL), in particular the denser subpopulation HDL3. The structure of HDL3 isolated from humans affected by a variety of severe disease states was investigated with respect to density, size, and apolipoprotein composition, using density gradient ultracentrifugation, gradient gel electrophoresis, gel filtration, and solid phase immunoadsorption. Apo-SAA was present in HDL particles in increasing amounts as particle density increased. Apo-SAA-containing HDL3 had bigger radii than normal HDL3 of comparable density. Purified apo-SAA associated readily with normal HDL3 in vitro, giving rise to particles containing up to 80% of their apoproteins as apo-SAA. The addition of apo-SAA resulted in a displacement of apo-A-I and an increase in particle size. Acute phase HDL3 represented a mixture of particles, polydisperse with respect to apolipoprotein content; for example, some particles were isolated that contained apo-A-I, apo-A-II, and apo-SAA, whereas others contained apo-A-I and apo-SAA but no apo-A-II. We conclude that apo-SAA probably associates in the circulation of acute phase patients with existing HDL particles, causing the remodeling of the HDL shell to yield particles of bigger size and higher density that are relatively depleted of apo-A-I.

C-reactive protein levels in ectopic pregnancy, pelvic infection and carcinoma of the cervix.

The value of C-reactive protein (CRP) levels in the differential diagnosis of pelvic infection and ectopic pregnancy, in the staging of carcinoma of the cervix, and after necrotizing irradiation for tumour was assessed. CRP was measured using a sensitive magnetizable solid-phase immunoradiometric assay. There was an obvious difference in CRP levels between patients with ectopic pregnancies and acute pelvic infections, but CRP levels failed to differentiate between stages IIB and IIIB carcinoma of the cervix, the majority of patients not having a significant acute-phase response. During radiotherapy there was wide variation and substantial individual differences in CRP levels which could have been caused by undiagnosed infective complications.

CRP and neutrophils: functional effects and complex uptake.

The uptake of C-reactive protein (CRP)-pneumococcal C-polysaccharide (CPS) complexes by neutrophils was studied. A specific CRP dependent mechanism of uptake was demonstrated. This promoted CPS (complexed to CRP) clearance which was further enhanced by additional complement activation. Physiological concentrations of low density lipoprotein inhibited entry of complexed CPS into neutrophils but had no effect on entry of CRP alone. Pure human CRP was shown to have no effect on neutrophil chemotaxis and oxidative metabolism.

Standardisation of the quantitation of serum amyloid A protein (SAA) in human serum.

An adequate method for standardising the quantitation of serum amyloid A protein (SAA) in human serum was developed. Acute phase high density lipoprotein3 (HDL3) was used as a standard. The concentration of the SAA in the standard was determined by the use of purified SAA. After protein determination, various concentrations of purified SAA were run on SDS-polyacrylamide gel together with the HDL3 standard containing an unknown amount of SAA amongst the apolipoproteins. From the standard curve obtained by pyridine extraction (Coomassie blue colour yield at A605 nm) the concentration of SAA in the HDL3 standard was determined. An established immunoradiometric assay (IRMA) for SAA was standardised with the HDL3. SAA concentrations in normal and acute phase sera were determined.

Cerebrospinal fluid C-reactive protein in infective meningitis in childhood.

The value of cerebrospinal fluid C-reactive protein (CSF CRP) determination as a diagnostic aid in infective meningitis has been investigated in four groups of children. In a "no meningitis" group of 10 children, a median CSF CRP value of 0.08 micrograms/ml was obtained (range 0 to 0.31 micrograms/ml); in a viral meningitis group of 21 children a median value of 0.01 micrograms/ml (range 0 to 3.06 micrograms/ml); in a bacterial meningitis group of 27 children a median value of 9.6 micrograms/ml (range 0 to 31.5 micrograms/ml); and in a tuberculous meningitis group of 18 children a median value of 0.29 micrograms/ml (range 0 to 4.9 micrograms/ml). CSF CRP values in the bacterial meningitis group differed significantly from those of each of the other groups (P less than 0.01), but considerable overlap between the groups detracted from the diagnostic value of the test. In six patients with bacterial meningitis with ambiguous conventional CSF chemistry results, normal CSF CRP values were found. Simultaneous serum CRP was determined in nine patients with tuberculous meningitis and 11 with bacterial meningitis, and the CRP response in both the serum and CSF appears subdued in tuberculous meningitis in comparison with bacterial meningitis. CSF CRP and total protein values were determined intermittently during a 24-hour period in ventricular CSF from two children with tuberculous meningitis who underwent temporary direct ventricular drainage. A considerable and apparently parallel diurnal variation in both values was seen. CSF CRP values have limited application in the etiologic diagnosis of meningitis.

Acute phase response in bronchiectasis and bronchus carcinoma.

Measurement of the acute phase response in patients suffering from bronchiectasis, emphysema, bronchus carcinoma and various benign space-occupying lesions was undertaken, using sensitive immunoradiometric assays for C-reactive protein (CRP) and serum amyloid-A protein (SAA). In some patients with bronchiectasis, clinically judged to be in remission, the results show a major ongoing acute phase response. Such a response could predispose these patients to the development of reactive secondary amyloidosis. In bronchus carcinoma the application of these measurements to judge the extent of tumor growth is limited as infection-complicating obstruction is a more potent initiator of the acute phase response than the neoplastic process per se.

C reactive protein concentrations during long distance running.

Long distance runners competing in events ranging from 15 to 88 km showed a distance related acute phase response as indicated by significantly raised serum C reactive protein concentrations. In trained athletes only a small rise in C reactive protein concentrations was seen after races of less than 21 km. After an 88 km ultramarathon concentrations comparable to those found in patients with small myocardial infarctions were detected. Indomethacin did not affect the increases in C reactive protein after the ultramarathon. This study has established serial C reactive protein concentrations for given race distances. These data may help in diagnosing myocardial infarction during long distance running. The acute phase response should be measured in untrained people running shorter distances to provide comparative data for the physically untrained population.

Value of C reactive protein measurement in tuberculous, bacterial, and viral meningitis.

The value of C reactive protein measurement in the differential diagnosis of meningitis was assessed in a population where tuberculous meningitis is prevalent. C reactive protein was measured serially with a sensitive radioimmunoassay in sera from 31 children with bacterial meningitis, 15 with tuberculous meningitis (6 with miliary tuberculosis), and 28 with viral meningitis. Concentrations of C reactive protein in patients with tuberculous meningitis lay between those of patients with bacterial and viral meningitis--a finding which detracts from the virtually absolute discrimination C reactive protein measurement allows between bacterial and viral meningitis. In all but two of the patients with tuberculous meningitis, C reactive protein concentrations fell rapidly after treatment began and became normal after 10 days. This fall did not, however, exclude the development of hydrocephalus as a complication. Measurement of C reactive protein remains a useful additional parameter in the diagnosis and management of the various types of meningitis.