Duane R. Schultz

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.

Lupus nephritis: A clinical review for practicing nephrologists

The renal manifestations in systemic lupus erythematosus (SLE) are protean and difficult to categorize into clinical syndromes and histologic classes. Lupus nephritis is frequently unrecognized until full-blown nephritic and/or nephrotic syndrome with renal failure emerge. Epidemiologically, approximately one third of SLE patients from unselected populations have renal involvement early during the disease. Most renal abnormalities emerge within the first few years of SLE diagnosis. Currently, most nephrologists agree that an early renal biopsy is worthwhile in those SLE patients with abnormal urinalysis and/or reduced renal function. First, it provides a histologic categorization of the glomerulonephritis as well as an assessment of the degree of activity and chronicity. Second, it provides vital prognostic information. Third, it is beneficial in planning a more rational therapy with or without potentially toxic immunosuppressive agents. Over the last 3 decades, many controlled clinical trials for treatment of lupus nephritis have been completed with a few therapeutic immunosuppressive regimens. Among those agents used. cyclophosphamide and azathioprine provide a reduction of morbidity in those patients afflicted with proliferative forms of lupus glomerulonephritis. A new immunosuppressive agent, mycophenolate mofetil, is being studied for treatment of proliferative forms of lupus glomerulonephritis in a controlled clinical trial at our institution. Immunosuppressive agents and the availability of dialysis and transplantation have improved the survival of patients with lupus nephritis, in particular those with proliferative forms.

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.

Antiphospholipid antibodies: Basic immunology and assays

Great progress has been made within the past 10 years in characterizing, assaying, and describing mechanism(s) of action in vitro of antiphospholipid antibodies (a-PL Abs); three prominent members are reagin, anticardiolipin antibodies (a-CL Abs), and the lupus anticoagulants (LAC). The major focus of this review is on basic and current biochemical and immunologic research. First, the biochemistry, structural composition, and sources of anionic and dipolar ionic (zwitterionic) phospholipids are discussed together with several serum antibodies directed to these phospholipids. Cardiolipin, the most acidic phospholipid (net negative charge of 2 at pH 7.0) has been historically important as an antigen for testing reagin in syphilis serology, and currently is part of the antigenic composition used in the Venereal Disease Research Laboratory (VDRL) tests. In this connection, the chronic biological false-positive test for syphilis and the LAC are discussed in association with autoimmune disorders such as systemic lupus erythematosus. Second, a naturally occurring plasma anticoagulant in vitro and a critical cofactor for binding of purified autoimmune a-CL Abs to cardiolipin is considered, the beta 2-glycoprotein I (beta 2-gpI). This single-chain plasma polypeptide is highly glycosylated, has 326 amino acids, a molecular weight of 50 kD, and is characterized by repeating amino acid motifs or domains that structurally resemble multiple loops. The highly cationic C-terminal fifth domain binds to anionic phospholipids. The beta 2-gpI is a member of the short consensus repeat superfamily of proteins, and is compared with other proteins with similar domains. Third, experiments are detailed for defining LAC and distinguishing it from other a-CL Abs. Cofactors are also associated with LAC and include beta 2-gpI, prothrombin, protein C, protein S, tissue factor, and factor XI. Thus, LAC antibodies are heterogeneous, and no individual assay can detect all LACs. Because patients with syphilis and other infectious diseases have no cofactor associated with a-CL Abs, their plasma LACs are negative. The a-CL Abs found in infection are not associated with the clinical features of the antiphospholipid syndrome. LAC assays are important because of the pathogenetic association with clinical observations of venous and arterial thrombosis, thrombocytopenia, and recurrent fetal loss. Finally, reports leading to development of currently used direct solid-phase enzyme-linked immunosorbent assays (ELISA) for testing a-PL Abs are outlined; these developments have greatly increased understanding of the basic immunology of target antigens and their respective antibodies. Of significance, a-CL Abs cross-react with other anionic phospholipids. Additionally, the results of these assays led to the realization that high levels of circulating a-PL Abs over long periods are associated with a number of clinical problems now known collectively as the antiphospholipid syndrome.

Antineutrophil cytoplasmic antibodies : major autoantigens, pathophysiology, and disease associations

Antineutrophil cytoplasmic antibodies (ANCA) are important serological markers for the primary systemic vasculitides, including microscopic polyarteritis and necrotizing crescentic glomerulonephritis. Numerous reports have established the clinical utility of ANCA titer in monitoring disease activity, relapses, and response to treatment. ANCA, detected by indirect immunofluorescence (IIF) assays using patients serum and ethanol-fixed human neutrophils, produce two common fluorescent staining patterns: cytoplasmic (C-ANCA), involving a 29-kD neutral serine protease termed proteinase 3 (PR3), and perinuclear (P-ANCA), the result mainly of myeloperoxidase (MPO), but occasionally by other components of the azurophilic granules including lysozyme, elastase, cathepsins, and lactoferrin. Some sera contain granulocyte-specific antinuclear antibodies (GS-ANA), which require formaldehyde fixation of neutrophils to cross link cytoplasmic antigens for distinguishing between ANCA and the GS-ANA by IIF. Positive IIF is confirmed by Western blot analysis or specific enzyme-linked immunosorbent assay for PR3, MPO, and other neutrophil granule antigens. The C-ANCA pattern is highly specific for Wegeners granulomatosis, a disease characterized by granulomatous inflammation, necrotizing and crescentic glomerulonephritis, and vasculitis; P-ANCA is found in sera of individuals with vasculitis, glomerulonephritis, and several other diseases. ANCA are predominantly immunoglobulin (Ig)G isotype, but may be IgM and IgA. Various pathophysiologic mechanisms have been proposed involving ANCA-mediated neutrophil activation in a hypothetical model of vasculitic diseases: positive signals via the FcgammaRII (CD32) receptor after IgG-ANCA binding to membrane-associated PR3, relevant cytokines, production of adhesion molecules on both activated neutrophils and endothelial cells, and the release of neutrophil reactive oxygen species and degranulation causing endothelial cell damage. Interference of C-ANCA with PR3 proteolysis and PR3 inhibition physiologically by the alpha1-proteinase inhibitor may have a pathogenic role. No convincing data have been reported for the existence of autoreactive T lymphocytes reactive to any degree with the neutrophil azurophilic enzymes. Studies of various drug- and infectious agent-related diseases and ANCA may contribute to understanding the mechanism(s) involved in some vasculitides.

Systemic lupus erythematosus in two adults with human immunodeficiency virus infection

T WO PATIENTS with strong clinical and serological evidence of systemic lupus erythematosus (SLE) and renal disease are presented to illustrate the difficulties of correctly diagnosing the underlying nephropathy in the presence of human immunodeficiency virus (HIV) infection. Although the first patient demonstrated typical changes consistent with SLE nephritis, the renal pathological condition in the second patient was that of collapsing glomerulosclerosis.

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.