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PubMed1981

Screening for deficiencies in the classical and alternative pathways of complement by hemolysis in gel.

Two assays based on hemolysis in gel were assessed for screening complement (C) component deficiencies. In one assay sensitized sheep erythrocyte (EA) were incorporated in agarose gel containing Ca2+ and Mg2+, in the other guinea pig erythrocytes (GpE) were used in the presence of Mg2+and EGTA. With few exceptions, fresh samples from healthy individuals produced homogeneous areas of complete hemolysis in both assays. Clearly aberrant patterns were observed in approximately 4% of healthy blood donors. Sera from patients having complete deficiencies of Clq, C2 or C4 produced clear lysis of GpE only, whereas in sera lacking C3 or C8 lysis was grossly impaired in both assays. Properdin deficient serum produced very slight lysis of GpE but normal lysis of EA. Reconstitution of these C-deficient sera gave normal lysis. Together, the two assays supplement immunochemical C3 and C4 determinations for screening out C disorders.

Animals↗
PubMed1982

Immunohistological studies on valvular vegetations in nonbacterial thrombotic endocarditis (NBTE).

Thrombi and thrombus formation in nonbacterial thrombotic endocarditis (NBTE) were studied using light microscopy and immunohistology. Samples from vegetations on cardiac valves were taken at autopsy from five patients with NBTE and adenocarcinoma as an underlying disease. Morphological studies disclosed proliferative changes underneath the thrombi. In immunofluorescence microscopy, focal deposits of immunoglobulins and complement components Clq and C3 were found. The results suggest that immune complexes, elicited by the underlying malignant process, may play important role in the pathogenesis of the thrombus formation in NBTE.

Adenocarcinoma↗
PubMed1985

Problems in the detection of complement-fixing immune complexes.

In the application of the anti-Clq, the anti-C3, or the conglutinin-binding assay for the detection of complement-fixing circulation immune complexes in patients with ankylosing spondylitis or healthy blood donors, antibodies cross-reacting with the solid-phase bound agent were observed in several sera, resulting in erroneous interpretation of the tests. Corrections for the interfering antibodies were made by testing for the binding to conglutinin both in the presence and in the absence of Ca++, while the anti-Clq and the anti-C3 assays included the application of F(ab')2 fragments of normal IgG to check for anti-F(ab)2 anti-bodies.

Antigen-Antibody Complex↗
PubMed1986

Immunohistological skin investigations in patients with the acquired immune deficiency syndrome.

Twenty-one male patients with acquired immune deficiency syndrome (AIDS), 6 male patients with AIDS-related complex (ARC) and 23 controls' had a punch biopsy taken from clinically unaffected skin of the buttock. Vertical skin sections were examined by immunofluorescence for in vivo deposits of immunoglobulins (Ig) and other plasma proteins, as well as for morphology and distribution of Langerhans cells (LC). Deposits of IgM in the dermo-epidermal junction zone (DEJ) were found in one patient with ARC. Apart from this single finding, no in vivo deposits of IgG, IgM, IgA, IgD, IgE, complement C1q and C3, fibrinogen or albumin were demonstrated in epidermis, DEJ or dermal vessel walls, either in patients or in controls. Epidermal LC were more superficially situated in patients as compared to controls. No differences in number and localisation of dermal LC were seen. We concluded that it was not possible to confirm a recent report of in vivo Ig deposits in the epidermis of patients with AIDS. The abnormalities observed in epidermal LC should be interpreted together with the recent finding of reduced numbers of epidermal LC; they thus support the hypothesis that LC are involved in the pathological processes in AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1988

A reference procedure to study chemiluminescence induced in polymorphonuclear leukocytes by Neisseria meningitidis.

Luminol-enhanced chemiluminescence (CL) was used to study the ability of various strains of Neisseria meningitidis (MC) to induce oxidative metabolism of polymorphonuclear leukocytes (PMNL); an indirect measure of phagocytic activity. To circumvent variations related to different PMNL donors, a MC serogroup X strain was used as a control for indexing the CL responses induced by other MC strains. This procedure, with pooled serum from healthy blood donors to standardize opsonising conditions, gave reproducible and comparable results, irrespective of PMNL donors. Under these conditions, there was a highly significant difference between pathogenic and non-pathogenic MC strains as regards their ability to induce CL responses (p less than 0.001). The results indicated that the differences were due partly to opsonizing antibodies, partly to other differences related to pathogenicity of tested MC strains. These differences in leukocyte/MC interaction were also confirmed by phagocytic-killing experiments. The index procedure of CL measurements may be a suitable method to study the appearance of natural immunity to MC disease, as well as the pathogenicity of particular MC strains.

Antibodies, Bacterial↗
PubMed1990

Branhamella catarrhalis: an organism gaining respect as a pathogen.

Branhamella catarrhalis was formerly regarded as a common, essentially harmless inhabitant of the pharynx. This misapprehension was caused, in part, by confusion with another pharyngeal resident, Neisseria cinerea. The two organisms can now be differentiated by the positive reactions of B. catarrhalis in tests for nitrate reduction and hydrolysis of tributyrin and DNase. B. catarrhalis is currently recognized as the third most frequent cause of acute otitis media and acute sinusitis in young children. It often causes acute exacerbations of chronic bronchopulmonary disease in older or immunocompromised adults and is incriminated occasionally in meningitis, endocarditis, bacteremia, conjunctivitis, keratitis, and urogenital infections. Virulence-associated factors, such as pili, capsules, outer membrane vesicles, iron acquisition proteins, histamine-synthesizing ability, resistance to the bactericidal action of normal human serum, and binding to the C1q complement component, have been identified in some strains. beta-Lactamase producing strains, first detected in 1976, have risen to approximately 75% worldwide. Thus far, however, practically all American strains of B. catarrhalis remain susceptible to alternative antibiotics. A possible selective advantage of recent isolates is their reportedly heightened tendency for adherence to oropharyngeal cells from patients with chronic bronchopulmonary disease.

Bacterial Infections↗
PubMed2001

BrkA protein of Bordetella pertussis inhibits the classical pathway of complement after C1 deposition.

Bordetella pertussis produces a 73-kDa protein, BrkA (Bordetella resistance to killing), which inhibits the bactericidal activity of complement. In this study we characterized the step in the complement cascade where BrkA acts, using three strains: a wild-type strain, a strain containing an insertional disruption of brkA, and a strain containing two copies of the brkA locus. Following incubation with 10% human serum, killing was greatest for the BrkA mutant, followed by that for the wild-type strain, while the strain with two copies of brkA was the most resistant. Complement activation was monitored by enzyme-linked immunosorbent assay (ELISA) or Western blotting. ELISAs for SC5b-9, the soluble membrane attack complex, showed that production of SC5b-9 was greatest with the brkA mutant, less with the wild type, and least with the strain containing two copies of brkA. Deposition of complement proteins on the bacteria was monitored by Western blotting. A decrease in deposition on the bacteria of C4, C3, and C9 corresponded with decreased complement sensitivity. Deposition of C1, however, was not affected by the presence of BrkA. These studies show that BrkA inhibits the classical pathway of complement activation and prevents accumulation of deposited C4.

Bacterial Outer Membrane Proteins↗
PubMed2004

Proteus mirabilis ZapA metalloprotease degrades a broad spectrum of substrates, including antimicrobial peptides.

The 54-kDa extracellular metalloprotease ZapA is an important virulence factor of uropathogenic Proteus mirabilis. While ZapA has the ability to degrade host immunoglobulins (Igs), the dramatic attenuation of virulence in ZapA mutants suggests that this enzyme may have a broader spectrum of activity. This hypothesis was tested by in vitro assays with purified ZapA and an array of purified protein or peptide substrates. The data reveal that many proteins found in the urinary tract are substrates of ZapA proteolysis, including complement (C1q and C3), cell matrix (collagen, fibronectin, and laminin), and cytoskeletal proteins (actin and tubulin). Proteolysis of IgA and IgG was significantly enhanced by conditions that denatured the Igs. It was discovered that the antimicrobial peptides human beta-defensin 1 (hBD1) and LL-37 are readily cleaved by the enzyme. To the best of our knowledge, this is the first report of a bacterial protease capable of cleaving hBD1, a component of the human renal tubule innate immune response. Proteolysis of hBD1 resulted in ca. six peptides, while proteolysis of LL-37 resulted in at least nine products. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of the molecular masses of the reaction products indicated that ZapA preferred no distinct peptide bond. The antimicrobial activity of hBD1 and LL-37 was significantly reduced following ZapA treatment, suggesting that proteolysis results in inactivation of these peptides. The data suggest that a function of ZapA during urinary tract infections is the proteolysis of antimicrobial peptides associated with the innate immune response.

Amino Acid Sequence↗
PubMed2005

Killing of dsrA mutants of Haemophilus ducreyi by normal human serum occurs via the classical complement pathway and is initiated by immunoglobulin M binding.

Previously, we showed that serum resistance in Haemophilus ducreyi type strain 35000HP required expression of the outer membrane protein DsrA because the isogenic dsrA mutant FX517 is highly serum susceptible. In this study, we confirmed this finding by construction of additional serum-susceptible dsrA mutants in more recently isolated serum-resistant strains. We also demonstrated that killing of dsrA mutants required an intact classical complement cascade but not the alternative or mannan-binding lectin pathways. Between 5- and 10-fold more purified human immunoglobulin M (IgM) but not IgG was deposited onto dsrA mutant FX517 than onto parent strain 35000HP, consistent with IgM initiation of the classical cascade. Depletion of IgM, but not IgG, from complement-intact serum inhibited killing of FX517. As predicted from the amounts of IgM bound, more of the individual complement components were bound by FX517 than by parent strain 35000HP. Examination of the binding of negative regulators of complement as an explanation for serum resistance indicated that parent strain 35000HP bound more C4 binding protein and vitronectin than FX517 but not factor H. However, the degree and pattern of complement component binding observed suggested that IgM binding to the serum-susceptible mutant FX517 was responsible for the activation of the classical pathway and the observed killing of FX517 as opposed to binding of negative regulators of complement by the serum-resistant parent. We speculate that an undefined neo-epitope, possibly carbohydrate, is exposed in the dsrA mutant that is recognized by naturally occurring bactericidal IgM antibodies present in human sera.

Bacterial Outer Membrane Proteins↗
PubMed2001

Structural flexibility and functional valence of CD4-IgG2 (PRO 542): potential for cross-linking human immunodeficiency virus type 1 envelope spikes.

CD4-immunoglobulin G2 (CD4-IgG2) incorporates four copies of the D1D2 domains of CD4 into an antibody-like molecule that potently neutralizes primary human immunodeficiency virus type 1. Here electron microscopy was used to explore the structure and functional valence of CD4-IgG2 in complex with gp120. CD4-gamma2, a divalent CD4-immunoglobulin fusion protein, was evaluated in parallel. Whereas CD4-gamma2-gp120 complexes adopted a simple Y-shaped structure, CD4-IgG2-gp120 complexes consisted of four gp120s arrayed about a central CD4-IgG2 molecule, a structure more reminiscent of complement C1q. Molecular modeling corroborated the electron microscopy data and further indicated that CD4-IgG2 but not CD4-gamma2 has significant potential to cross-link gp120-gp41 trimers on the virion surface, suggesting a mechanism for the heightened antiviral activity of CD4-IgG2.

CD4 Immunoadhesins↗
PubMed1983

Protein composition of urinary casts from healthy subjects and patients with glomerulonephritis.

Urinary casts from 46 healthy volunteers and 60 patients with glomerulonephritis were examined for the presence of Tamm-Horsfall glycoprotein and other proteins. All samples gave immunofluorescence evidence of Tamm-Horsfall protein in casts. Casts from 59 of the patients but only three of the controls contained other proteins in addition (p less than 0.001). Immunoglobulins (IgG, IgM, IgA) were detected in casts from 53 of the patients but none of the healthy volunteers. Examination of urinary casts for immunoglobulins, complement, and fibrin provides a non-invasive method for distinguishing patients with active glomerular disease.

Complement Activating Enzymes↗
PubMed1982

Immunohistological study of human lungs by immunoperoxidase technique.

An unlabelled antibody peroxidase-antiperoxidase method for the detection of IgG, IgM, complement (C3 and Clq), fibrinogen and albumin was applied to routinely processed paraffin sections of lung from 27 cases. The results in 11 cases were compared with those obtained by immunofluorescence using frozen sections. Tissue was obtained from surgical specimens of cases with interstitial pneumonia comprising 10 of the usual type (UIP) and three of the desquamative type (DIP). Tissue was also obtained from the specimens of cases with sarcoidosis (two cases) and granulomatous inflammation of unknown cause (one case). There were 11 control cases, nine with primary carcinoma of the lung and two with metastatic tumours of the lung. Immunoglobulins of various types and complement were seen in diseased lung tissue. Although most of these deposits were probably due to a non-immunological mechanism there was evidence of the possible implication of immune complexes in three cases of UIP and in the interstitial pneumonia present in the two cases of sarcoidosis. The immunoperoxidase technique is a more sensitive method than immunofluorescence and has the additional advantage of the easy identification of the precise sites of the various deposits.

Adult↗
PubMed1985

Isolated arteritis of the epididymis.

The clinical, histological, and immunohistochemical features of two cases of apparently isolated arteritis of the epididymis are presented. The aetiology and pathogenesis of the condition are discussed. Immunoglobulin and complement were shown in the acute arterial lesions, but this is not conclusive evidence that isolated arteritis is either an immune complex disease or a forme fruste of polyarteritis nodosa.

Adolescent↗
PubMed1989

Circulating immune complexes after splenectomy.

Circulating immune complexes were evaluated in 25 patients (age range 10 to 46 years) who had undergone splenectomy for non-malignant conditions by studying a polyethylene glycol insoluble serum fraction. Although the extent of binding to Clq was within normal limits, these patients had increased concentrations of factor B in the immune complex serum fraction. These findings indicate that an unusual type of circulating immune complex may be detected after splenectomy, suggesting a possible role for the spleen in the removal of circulating immune complexes.

Adolescent↗
PubMed1989

Immunofluorescence of immunoglobulins and complement in kidneys taken at necropsy.

The immunofluorescence of immunoglobulins and complement components in kidney specimens taken at necropsy was investigated to determine the persistence of antigenicity of immune reactants. Of 74 consecutive necropsies, 12 cases had positive glomerular fluorescence. The pattern and intensity were followed up for up to 15 days. Along with necropsy specimens, tissue samples of normal looking kidney from 14 nephrectomies were also studied. Two of these specimens turned out to be positive in the immediate immunofluorescence study. To rule out possible false positive staining after death immunofluorescence findings in all nephrectomy specimens were followed up for up to 19 days. The presence of immunoglobulins and complement could be shown for between 12 and 15 days after death; no changes in immunofluorescence findings occurred during this period. It is concluded that immunofluorescence provides valuable information when immunologically mediated reactions need to be clarified in necropsy kidneys.

Autopsy↗