Arnold Vogt

Glomerular immune deposits in murine lupus models may contain histones

Two types of lupus mice, NZB/NZW F1 female hybrids and mice with graft‐rersus‐host disease (GVHD). were studied. Histones H3 and H2A were detected by immunofluorcscenee in glomeruli of 22/22 proteinuric GVHD and 8/12 proteinuric NZB/W F1 female mice; in non‐proteinuric animals. 3/5 GVHD and 2/27 NZB/W F1 female were positive. Using antibodies to histone peptides it was shown that mainly the N‐terminal regions of histones H3 and H2A were exposed in glomerular deposits. Western blot analysis revealed antibodies lo histone subfractions in sera of 33/34 lupus mice that developed proteinuria. This study provides evidence that histones are involved in the pathogenesis of lupus nephritis.

Purification and characterization of a tryptic peptide of Borrelia burgdorferi flagellin, which reduces cross-reactivity in immunoblots and ELISA

In man the early immune response in Lyme disease is primarily directed against the endoflagellin antigen. Isolated flagellar protein of Borrelia burgdorferi suggests itself as a suitable test antigen. However, cross-reactivity between flagellins of B. burgdorferi, Escherichia coli, Bacillus subtilis, Proteus mirabilis and Salmonella typhimurium was demonstrated by immunoblotting and ELISA with polyclonal rabbit-hyperimmune-sera. Tryptic cleavage of recombinant B. burgdorferi 41 kDa flagellin, expressed in E. coli, produced a peptide fragment which was recognized exclusively by antisera to Borrelia species. This peptide was designated as the 14 kDa fragment due to its migratory behaviour in SDS-PAGE. The fragment is part of the variable region of the flagellin, as proven by amino acid sequencing. The flagellin peptide was employed as an antigen in ELISA and immunoblot assays, testing the polyclonal sera mentioned above. The specificity was superior to that obtained with the intact recombinant flagellin.

Anti-DNA antibody derived from a systemic lupus erythematosus (SLE) patient forms histone-DNA-anti-DNA complexes that bind to rat glomeruli in vivo

Histone can mediate the binding of both free DNA and DNA complexed to anti‐DNA antibody to the glomerular capillary wall. We tested whether preformed histone–DNA–anti‐DNA immune complexes (IC) could bind to the glomerular capillary wall. The immune complex, generated with anti‐DNA antibody derived from an SLE patient and excess of 125I‐DNA followed by digestion with DNase, was mixed with histones. The complex containing 4 μg DNA was injected via the aorta into the left kidney of rats. At 15 min, 1.3% of the histone–DNA–anti‐DNA antibody complex bound (measured as 125I‐DNA), when histone was omitted less than 0.1% of the DNA–anti‐DNA antibody complex bound. By immunofluorescence human immunoglobulins and histones, representing the IC, could be observed in a capillary pattern; but no complement deposition was detected. Electron microscopy revealed discrete, electron dense deposits in a subendothelial, subepithelial and mesangial localization at 15 min. These results provide direct evidence that antibodies from serum of SLE patients can form soluble histone–DNA–anti‐DNA immune complexes that bind to the glomerular capillary wall in vivo

Antibodies to DNA, chromatin core particles and histones in mice with graft‐versus‐host disease and their involvement in glomerular injury

Chronic graft‐versus‐host disease (GVHD) was induced in female (C57 B10S/DBA/2)F1 hybrid mice with two successive injections of lymphoid cells from parental DBA/2 strain. Serial bleedings of 27 GVHD mice were screened with a panel of antigens including the five histones HI, H2A, H2B, H3 and H4, 15 histonc peptides. core particles. dsDNA. heat‐shock proteins hsp70 and ubiquitin, a branched peptide of ubiquitinaled H2A (U‐H2A), poly(ADP‐ribose) and SSB/La protein. The predominant IgG response to histone peptides was directed against regions 204‐218 of HI, 1‐25 of H2B and 1 29 of H4. GVHD mice also produced IgG antibodies lo dsDNA and chromatin core particles as reported previously. IgG antibodies reacting with dsDNA appeared before antibodies to core particles and histones. Raised levels of antibodies to U‐H2A, but not to monomeric ubiquitin, were also found. While the level of antibodies to dsDNA, histones and core particles decreased significantly before the appearance of proteinuria, suggesting their involvement in glomerular injury, the longitudinal pattern of anti‐U‐H2A peptide response was apparently not linked to the manifestation of lupus nephritis in GVHD mice.

Immunohistological staining of antigens on semithin sections of specimens embedded in plastic (GMA-Quetol 523)

Glycol methacrylate-Quetol 523, introduced by Kushida (1977) for combined light and electronmicroscopy studies at low magnification, also permits application of immunofluorescence methods to semithin sections. To recover the antigenicity of proteins fixed with formaldehyde, abrupt dehydration before embedding and subsequent treatment of the semithin sections with protease were essential. Post-staining with suitable histological stains allows exact correlation of antigen localization with tissue structure.

Glomerular injury induced by cationic 70‐kD staphylococcal protein; specific immune response is not involved in early phase in rats

A highly cationic staphylococcal protein (designated p70, MW 70 kD, pI>10) belongs to the groups of bacterial proteins that can bind immunoglobulin without specific antigen–antibody recognition; heparin inhibition tests indicated a charge interaction. This study evaluated the nephritogenicity of p70, which has affinity for the glomerular basement membrane (GBM), and the influence of various mediator systems on the induction of glomerulonephritis by p70. The left kidneys of intact rats, rats given cobra venom factor (complement‐depleted), or rats given anti‐adhesion molecules (ICAM‐1 and LFA‐1a) were perfused with p70. Proteinuria started within 24 h and persisted at day 5. Intraglomerular infiltration of cells was seen as early as 15 min, peaking at day 1. Deposits of rat IgG and C3 were seen in a subendothelial location 15 min after p70 perfusion in the left kidney and were found in a predominantly subepithelial location from 1 day onwards. Complement depletion and blockade of adhesion molecules suppressed proteinuria from day 2 onwards; these manipulations also prevented the recruitment of infiltrating cells and partially hindered the transfer of IgG across the GBM and the accumulation of IgG in the subepithelial region. In the non‐perfused right kidneys, deposits of IgG and C3 were comparable to those in the left kidneys, suggesting that p70–IgG complexes formed in the circulation may also contribute to the deposits in the GBM. Heparin inhibition tests indicated an electrostatic interaction between p70 and immunoglobulin. Complement and inflammatory mediator systems (granulocytes, monocytes/macrophages, and/or lymphocytes) were required to provoke glomerular injury. p70 might play a role in acute glomerulonephritis following Staphylococcus aureus infection.

A two-stage method for cross-linking antibody globulin to ferritin by glutaraldehyde. III. Size and antibody activity of the conjugates.

Immunoferritin conjugates consist of conjugates of different size. They can be separated in 4.5% polyacrylamide gel electrophoresis into at least 4 bands. The amount of the smallest 1:1 conjugate, in which one antibody molecule is linked to one ferritin molecule, is highest in coupling products with low overall yields of conjugated ferritin. Therefore relatively mild reaction conditions are recommended. With increasing size the antibody binding capacity of the conjugate is reduced.

Two novel cationic staphylococcal proteins induce IL‐2 secretion, proliferation and immunoglobulin synthesis in peripheral blood mononuclear cells (PBMC) of both healthy controls and patients with common variable immunodeficiency (CVID)

Two cationic proteins, a neutral phosphatase (NP‐tase) and a 70‐kD protein (p70) were isolated from Staphylococcus aureus by ion exchange chromatography. We compared their properties to those of the well established B cell mitogen of whole, fixed Staph. aureus strain Cowan I cells (SAC). Both purified proteins were able to induce immunoglobulin synthesis in PBMC cultures of healthy donors. NP‐tase and p70 also induced immunoglobulin synthesis of PBMC from those patienis with CVID who were also responsive to SAC plus IL‐2 stimulation. Immunoglobulin synthesis in response to NP‐tase and to p70 was time‐ and dose‐dependent and could be inhibited by addition of specific antibodies against the proteins. In contrast to SAC, no addition of exogenous IL‐2 was necessary to obtain maximal immunoglobulin synthesis induced by NP‐tase or p70. However, neither protein was able to induce immunoglobulin synthesis in B cell‐enriched cultures. High amounts of IL‐2 were found in supernatants of PBMC from healthy donors following stimulation with low concentrations of NP‐tase or p70, and this was associated with vigorous lymphocyte proliferation. Both proteins behave like typical antigens, and not like lectins or superantigens, since an NP‐tase‐stimulated T cell line showed an antigen‐specific, MHC‐restricted secondary response. In addition, no preferential T cell receptor Vβ chain usage was found with eight Vβ‐specific MoAb. It is likely that the two proteins induce antigen‐specific T cell activation, which is then followed by polyclonal activation of B cells via CD40 receptors and cytokine release

Isolation of an outer membrane protein complex from Borrelia burgdorferi by n-butanol extraction and high-performance ion-exchange chromatography

Borrelia burgdorferi, the causative agent of Lyme disease, expresses two major membrane proteins, designated outer surface proteins A and B, which are of antigenic relevance, especially in the chronic phase of Lyme disease. Both proteins exhibit strain-related molecular weight variation. A method is described for obtaining these proteins from the bacterial membrane, without the use of detergents, by a combination of n-butanol extraction and cation-exchange chromatography on a Mono S fast protein liquid chromatographic column. This method yields up to five times larger amounts of the proteins in aqueous solution than previously described protocols, which applied ionic or non-ionic detergents. A comparison of extracts obtained by this method from different Borrelia burgdorferi strains is reported.

Studies on the mesangial handling of protein antigens: influence of size, charge and biologic activity.

The influence of certain physicochemical and biologic properties of protein antigens on their handling by the glomerular mesangium was studied in rats. The following ferritin-based probes were employed: (a) naturally occurring ferritin isomers to examine the role of molecular size; (b) chemically cationized ferritins (pI 7.1 and 8.8) to test charge effects; (c) glutaraldehyde (GA-)coupled ferritin as an analogue of a toxic molecule. Molecular size was found to be a major determinant of the extent and rapidity of antigen uptake, but had only a minor influence on persistence. The rapidity of uptake and antigen persistence was charge dependent, but to a limited extent. Biologic activity of macromolecules appears to be a very important determinant of mesangial handling since GA-treated ferritin was taken up much more rapidly and to a greater extent than native ferritin, and could persist for very prolonged periods.