Patricia I. Arnold

Properties of four acute phase proteins : C-reactive protein, serum amyloid A protein, α1-acid glycoprotein, and fibrinogen

Four plasma proteins, referred to as positive acute phase proteins because of increases in concentration following inflammatory stimuli, are reviewed: C-reactive protein (CRP), serum amyloid A protein (SAA), alpha 1-acid glycoprotein (AAG), and fibrinogen. The CRP and SAA may increase in concentration as much as 1000-fold, the AAG and fibrinogen approximately twofold to fourfold. All are synthesized mainly in the liver, but each may be produced in a number of extrahepatic sites. The role of cytokines in induction of the acute phase proteins is discussed, particularly the multiple functional capabilities of interleukin-6 (IL-6). Other cytokines that regulate acute phase gene expression and protein synthesis include IL-1, tumor necrosis factor alpha, interferon gamma, as well as other stimulatory factors and cofactors. The physicochemical characteristics of each protein are reviewed together with the molecular biology. For each protein, the known biological effects are detailed. The following functions for CRP have been described: reaction with cell surface receptors resulting in opsonization, enhanced phagocytosis, and passive protection; activation of the classical complement pathway; scavenger for chromatin fragments; inhibition of growth and/or metastases of tumor cells; modulation of polymorphonuclear function; and a few additional diverse activities. The role of plasma SAA is described as a precursor of protein AA in secondary amyloidosis; other functions are speculative. AAG may play an immunoregulatory role as well as a role in binding a number of diverse drugs. In addition to clot formation, new data are described for binding of fibrinogen and fibrin to complement receptor type 3. Finally, the concentration of each protein is discussed in a wide variety of noninfectious and infectious disease states, particularly in connective tissue diseases. The quantification of the proteins during the course of various acute and chronic inflammatory disorders is useful in diagnosis, therapy, and in some cases, prognosis.

An immunological study of predation on hatchery-reared, juvenile red drum (Sciaenops ocellatus, Linnaeus): description of an ELISA and predator-prey studies in nature

This report is a continuation of an on-going study to develop immunological methods for eventual use in determining the predation mortality of newly released, hatchery-reared red drum (Sciaenops ocellatus, Linnaeus). Using a specific goat antiserum produced to a purified 80 kDa red drum glycoprotein, we detected the glycoprotein routinely in soluble extracts of red drum by Western blots. To supplement the immunoblotting, we proceeded to develop a highly sensitive and specific ELISA (enzyme-linked immunosorbent assay). The major problem in developing the ELISA was defining conditions to eliminate a natural inhibitor in soluble extracts of red drum that prevented the 80 kDa protein from binding to microtiter plates. The technical difficulties for a successful ELISA were resolved by adjusting extracts to pH 4.7 and 0.3 M NaCl, based on conditions developed for purification of the 80 kDa protein on a cationic-exchange gel by fast protein liquid chromatography (FPLC). The defined parameters eliminated the inhibition and resulted in optimal binding of the glycoprotein to the polymer surface of plates for ELISA. Approximately 10 h after the release of tens of thousands of red drum fingerlings at two sites in Biscayne Bay, FL, USA, a center-bag haul seine was used to sample the fish and capture predators. Two species, Sphyraena barracuda (Walbaum), great barracuda, and Strongylura notata (Poey), redfin needlefish, were the major predators. ELISA and Western blots were used to identify visually difficult or unidentifiable Sciaenops ocellatus in gut contents of the predators. Based on nine samples from seven Strongylura notata, and 10 samples from eight Sphyraena barracuda, 100% of the samples were identified as red drum in the needlefish and 50% in the great barracuda. These studies confirm the feasibility of using immunological methods to identify otherwise unidentifiable prey in gut contents of predators in nature.

Heat shock (stress) proteins and autoimmunity in rheumatic diseases

The rheumatic diseases (RDs) are characterized by acute and chronic inflammation, and autoimmunity plays a major role in their pathogenesis. RDs are for the most part of unknown etiology, but recent evidence indicates that heat shock or stress proteins (HSPs) may have an important role in the etiology/pathogenesis of RDs. HSPs are produced by prokaryotic and eukaryotic cells and are grouped according to molecular weight. Phylogenetically, HSPs are very old and are remarkably conserved molecules in evolution from bacteria to humans. HSPs are induced by a variety of cellular stresses in addition to heat; cognates are expressed constitutively and are essential in a number of normal functions. Some HSPs serve as molecular chaperones, the latter defined as proteins that mediate folding of other polypeptides and either promote their assembly into oligomeric structures or disassemble the final product. Conservation of structure and function of many HSPs may provide a link between immunity to infection and the autoimmune features of RDs. Evidence is reviewed from clinical and laboratory observations that diverse microbial agents, including viruses, bacteria, and parasites, may have putative roles in the development and pathogenesis of some RDs. HSPs also are discussed in relation to the major histocompatibility complex, HLA antigens, and disease associations and how they may alter the balance between tolerance and autoimmunity. Studies are reviewed that are supportive or nonsupportive of the concept of microbial infection associated with autoimmunity; individuals first react to microbial immunizations or infections with enhanced cellular/humoral responses to the agents HSPs. With the enhanced immune response, cross-reactivity may occur with an HSP of the stressed host because of structural similarities to the microbial HSP. If all of these events occur, the hosts homologous HSP or stressed cells now become true autoantigen(s). This sequence has implications for the etiology of immune-mediated RDs, the concept of epitope sharing, and the accompanying autoimmunity. A recurring theme emphasized in some reports to understand better the role of HSPs in autoimmunity is the need to select patients with early-onset disease. A minor subpopulation of T lymphocytes express a CD3-associated T-cell receptor (TCR) heterodimer composed of gamma and delta polypeptide chains. The gamma delta + T cells have several unique features. When analyzed by the polymerase chain reaction, lymphocytes with TCR-gamma delta appear to reflect the polyclonal expansion of preexisting gamma delta clones. They are found in peripheral lymphoid tissue in very low percentage (< 5%) but may represent the majority of T cells within epithelial tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Complement activation in septic shock patients.

To evaluate the status of the complement system and to determine the effects of corticosteroids on complement component levels in septic shock, C3, C4, and Factor B were measured in 42 patients with severe late septic shock. Serum levels of C4 and Factor B correlated with C3 levels (r = 0.48 and 0.64, respectively; p < .01) in patients in shock for more than 4 h, but only Factor B correlated with C3 (r = 0.85; p < .01) in patients in shock for 4 h or less. C3 and Factor B levels were significantly (p < .05) lower in patients who died (12,174 ± 1,524 CH50 U/ml and 14 ± 1 mg/dl, respectively) than in patients who survived (18,418 ± 2,833 CH50 U/ml and 21 ±2 mg/dl, respectively). Corticosteroids did not alter complement component levels.The alternative pathway appears to be activated early in septic shock, whereas the classical pathway is activated later. C3 and Factor B levels may predict survival of patients in septic shock. In this study, corticosteroids did not change the complement component levels of patients in late severe septic shock.

Agonist-induced capping of adhesion proteins and microparticle shedding in cultures of human renal microvascular endothelial cells.

Capping and release of membranous, small (< 1.5 microm) endothelial microparticles were quantified by immunofluorescence microscopy and flow cytometry after treatment of cultures of human renal microvascular endothelial cells with agonists tumor necrosis factor-alpha (TNF-alpha) or mitomycin C. For constitutive marker CD31, both agonist-treated attached, monolayer, and detached, free endothelial cells formed caps and released microparticles. TNF-alpha and mitomycin C induced dissimilar appearing CD31-containing caps after 3 h, followed by endothelial microparticle release after 6 h. The degree of capping correlated with increasing counts of released microparticles. For lymphokine-inducible CD54, TNF-alpha also induced CD54-containing caps and microparticle release, but mitomycin C failed to induce the expression of either entity. Neither capping nor microparticle release caused by TNF-alpha was part of an apoptotic pathway that involved caspase 3. Mitomycin C treatment of endothelial cells caused capping and microparticle release with a time course similar to TNF-alpha induction for 15 to 24 h, but assays for caspase 3 were positive, confirming the apoptotic action of mitomycin C. Membrane capping and microparticle release from endothelial cells are a convenient experimental model for studying protein movement, release of microparticles, and their possible biological significance.

Isolation and partial characterization of a polysaccharide in ant venom (Pseudomyrmex sp.) that activates the classical complement pathway

Abstract A heterogeneous polysaccharide (PS) was isolated from the venom of the tropical ant Pseudomyrmex sp. which activates the classical complement (C) pathway. Six sugars were identified in the PS by gas chromatography, and the molar ratios were determined. The sugars are mannose, N -acetylglucosamine, galactose, fucose, N -acetylgalactosamine, and glucose. A small PS, estimated to have a mol. wt of 3000 daltons on Sephadex G-25, was separated from a larger species, and may be a cleavage product of the larger PS. Both species were potent activators of the classical C pathway. The large and small PS have a strong net negative charge because of hexuronic acid(s), and both bind mainly to β-lipoprotein in serum to form a visible precipitate. Precipitation is prevented by ethylenediaminetetraacetate. partially prevented by 300 m M NaCl, but not prevented by heating the serum at 56° C for 30 min. It is not certain if binding to β-lipoprotein is an absolute necessity for C1 activation and the resulting C4 and C2 consumption. The PS caused C4 and C2 consumption in a serum deficient in apolipoprotein B, but no visible precipitation was observed. In studies of the mechanism of C1 activation. 125 I-C1 q could not be shown to bind to the PS or to PS-β-lipoprotein complexes by a method used to show antigen-antibody complexes in serum. However, C4 and C2 were not consumed in a serum deficient in C1 q . Supplementing this serum with a physiological amount of highly purified C1 q resulted in C4 and C2 consumption after addition of the PS, demonstrating that C1 q was necessary for the reaction.

Acquired HDL Deficiency Associated With Apolipoprotein A-I Reactive Monoclonal Immunoglobulins

To the Editor: Immunoglobulins (Igs) directed at components of lipoproteins to cause altered lipoprotein metabolism have been described in patients with multiple myeloma,1,2⇓ xanthomatosis,3 benign gammopathies,4 rheumatoid arthritis,5 systemic lupus erythematosus (SLE), and primary antiphospholipid syndrome (APS).6,7⇓ In most cases, hyperlipidemia results from Igs reactive with apolipoprotein (apo) B present on very low–density lipoproteins and LDLs. Less common have been associations of Igs with hypolipidemia or reactive with HDLs. Here we describe two patients who developed low HDL-cholesterol (HDL-C) levels (<5th percentile) associated with benign gammopathy and the presence of serum Igs reactive with apo A-I. Patient 1 (P1) was a morbidly obese (body mass index, 48 kg/m2), 45-year-old woman with mild normocytic, normochromic anemia (hematocrit, 33 g/L; hemoglobin, 11.6 g/L) and low HDL-C (0.22 mmol/L [8 mg/dL]). Serum Ig levels were elevated. Antinuclear antibodies were detected but were nonreactive with dsDNA, Sm, RNP, SSA, SSB, SCL-70, and histone. Anticardiolipin and anti–β2-glycoprotein I antibodies were negative. Immunofixation electrophoresis (IFE) identified monoclonal IgGκ and IgGλ bands. No Igs or light chains were detected in her urine. Serum lipid levels showed that HDL-C was in the low-normal to normal range between 1992 and 1996 (1.0 to 1.4 mmol/L) but at the time of presentation …

Functional Properties of Heterogeneous Human Asialo-C4 and Its Isotypes C4A and C4B

The fourth component of human complement (C4) is encoded at two separate but closely linked loci within the MHC on the short arm of chromosome 6. Thus, there are two types of C4 protein in most individual and pooled normal human sera (NHS): C4A and C4B. Incubation of individual sera, pooled NHS, or purified heterogeneous C4 (C4A/C4B) with bacterial sialidase at 37 degrees C increased C-mediated hemolysis of antibody-sensitized sheep erythrocytes 1.54- to 1.93-fold. Comparative studies of Tmax of human C2, using asialo-C4 or buffer-treated C4 on EAC1gp and extrapolation to time 0 indicated a z value 4-fold higher with asialo-C4. This indicated that more hemolytically active C42 complexes are available with sialidase-treated C4 compared to untreated C4. There was no appreciable difference in the % 125I-C4 bound to EAC1gp (sialidase- or buffer-treated). Sera from two different blood donors with C4A3 phenotype (C4BQ0), two different donors with C4B1 phenotype (C4AQ0), and serum from an individual heterozygous deficient at both C4A3 and C4B1 regions (A3, AQ0; B1, BQ0) were investigated. The C4 allotypes, purified from these sera, were treated with sialidase; the C4A3 was enhanced in hemolytic assays by sialidase-treatment (1.52- to 2.3-fold), whereas the C4B1 allotype was not enhanced. Fluorometric determinations revealed that approximately the same percentage of sialic acid was released from sialidase-treated C4A3 and C4B1. Therefore, the increase in hemolytic titer observed after treatment of NHS or purified heterogeneous C4 with sialidase is a property of C4A3 but not a property of C4B1.