Traumatic myositis ossificans simulating soft tissue infection.
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Accidents, Traffic↗
Biomedical subjects
J F Bohnsack
Publications and source records attributed to J F Bohnsack.
Mechanisms of beta 1 integrin-dependent adherence of granulocytic HL60 to fibronectin.
We investigated the mechanism of beta 1 integrin-mediated adherence of stimulated granulocytic HL60 cells to fibronectin using a monoclonal antibody (15/7) that recognizes beta 1 integrins only when the receptors are active for ligand binding. Phorbol myristate acetate (PMA) stimulated expression of the 15/7 epitope on granulocytic HL60 by nearly fivefold but had an insignificant effect on the expression of the epitope on undifferentiated HL60 cells. These results paralleled the effect of PMA on HL60 and granulocytic HL60 adhesion to fibronectin, indicating that activation of beta 1 integrins is important for beta 1-mediated adherence of granulocytic HL60 cells to fibronectin. Agonists that stimulate alpha 5 beta 1-dependent human polymorphonuclear leukocyte (PMN) adhesion to fibronectin (C5a and PMA) also upregulated the 15/7 epitope on purified human PMNs. Although PMA rapidly induces increased levels of filamentous actin (F-actin) in granulocytic HL60 cells and a decrease in F-actin levels in undifferentiated HL60 cells, depolymerization of the actin cytoskeleton with cytochalasin B did not affect increased expression of the 15/7 epitope on granulocytic HL60 cells. Cytochalasin B did, however, inhibit granulocytic HL60 adherence to fibronectin by 50%, demonstrating that actin polymerization is important for optimal beta 1-dependent granulocytic adherence.
Actins↗
Effects of cytomegalovirus infection on growth factor production in endothelial cells and fibroblasts.
To determine whether cytomegalovirus (CMV) infection alters growth factor production from endothelial cells (EC) or fibroblasts, we infected human umbilical vein EC with CMV VHL/E, a strain of CMV with affinity for human EC, and we infected human foreskin fibroblasts with CMV AD169. CMV caused cytopathic effect and positive CMV staining by immunofluorescence within 5 d, effects not seen in cells infected with UV-irradiated CMV or in uninfected (control) cells. The supernatants from the EC were assayed for platelet-derived growth factor (PDGF)-like protein using a radioreceptor inhibition assay, and EC and fibroblasts were assayed for basic fibroblast growth factor (bFGF) by Western blot analysis. There were no significant differences in PDGF production between groups of EC: CMV-infected EC, 13.5 +/- 2.6; UV-irradiated infected EC, 12.1 +/- 3.6; control EC, 12.9 +/- 1.7 fmol/10(6) EC (mean +/- SD, n = 10, p = NS). There were also no significant differences in bFGF production between CMV-infected EC, UV-irradiated infected EC, and control EC as evidenced by similar intensity of migration of bFGF as a single band at approximately 18 kD (n = 5). In contrast, CMV infection of fibroblasts induced a shift in production of bFGF to higher molecular weight forms migrating at 24 and 26 kD molecular mass. alpha-Interferon failed to alter bFGF production. We conclude that CMV VHL/E infection of EC does not directly alter PDGF or bFGF production from EC. However, CMV infection of cultured human fibroblasts qualitatively alters bFGF by inducing a shift to higher molecular weight forms.
3T3 Cells↗
Effects of fibronectin and group B streptococci on tumour necrosis factor-alpha production by human culture-derived macrophages.
Group B streptococci (GBS) are an important cause of sepsis and shock in the new-born. We have previously reported that GBS induce the production of tumour necrosis factor-alpha (TNF-alpha) by human monocytes and culture-derived macrophages. We have also shown that fibronectin (FN) promotes interaction between GBS and human phagocytes. In the present study, we investigated the effect of FN and GBS on the production of TNF-alpha by adult and neonatal culture-derived macrophages. We report that soluble FN alone was a strong stimulus for the production of TNF-alpha by culture-derived macrophages (FN 50 micrograms/ml = 623.33 +/- 47 pg/ml TNF, versus media alone 3 +/- 1.5 pg/ml; P < 0.0001). While GBS also induce the production of TNF-alpha by macrophages, the addition of FN to GBS had more than an additive effect on TNF-alpha levels. FN-mediated TNF-alpha production by macrophages was inhibited by both soluble arginine-glycine-aspartic acid (RGD) peptide (71%; P < 0.0001) and anti-beta 3-integrin monoclonal antibody 7G2 (54%; P < 0.0001). Neonatal culture-derived macrophages produced significantly more TNF-alpha in response to GBS (356.4 pg/ml +/- 27.7) than adult cells did (222.0 pg/ml +/- 21.0; P = 0.037), and dramatically more in response to FN alone (neonatal 1931.0 pg/ml +/- 23.0 versus adult 463.5 43.5 pg/ml; P < 0.0001). FN may contribute to the high levels of TNF-alpha production implicated in the pathophysiology of GBS sepsis and shock.
Adult↗
Activation of beta 1 integrin fibronectin receptors on HL60 cells after granulocytic differentiation.
Phorbol esters upregulate the functional affinity of beta 1 integrin receptors for fibronectin on human neutrophils and other leukocytes. We investigated the ability of phorbol myristate acetate (PMA) to stimulate the human promyelocytic cell line HL-60 to adhere to fibronectin, either in its undifferentiated state (HL60) or after dimethylsulfoxide-induced differentiation along the granulocytic pathway (dHL60). PMA stimulated little adherence of undifferentiated HL60 to fibronectin or to the 120-kD chymotryptic cell-binding domain (CBD) of fibronectin. In contrast, PMA stimulated dHL60 cells to rapidly adhere to both fibronectin- and to CBD-coated plastic. PMA-stimulated dHL60 adherence to fibronectin was largely mediated by both alpha 4 beta 1 and alpha 5 beta 1, whereas PMA-stimulated dHL60 adherence to CBD was largely mediated by alpha 5 beta 1. There was little contribution from beta 2 integrins to PMA-stimulated dHL60 adherence to fibronectin or CBD. The inability of undifferentiated HL60 to adhere to fibronectin and CBD did not result from lack of expression of alpha 4 beta 1 or alpha 5 beta 1 because HL60 and dHL60 express similar amounts of both alpha 4 beta 1 and alpha 5 beta 1 on their surface. In addition, 1 mmol/L Mn2+ induced similar amounts of alpha 5 beta 1-dependent adherence of both HL60 and dHL60, showing that alpha 5 beta 1 on undifferentiated HL60 is capable of binding to its ligand. These data suggest that activation of protein kinase C cannot functionally upregulate these beta 1 integrins on undifferentiated HL60 cells. The development of PMA-stimulated beta 1-dependent adherence after granulocytic differentiation of HL60 cells suggests that the differentiated HL60 cell is a useful model for investigating functional coupling of protein kinase C to beta 1 integrin in myeloid cells.
Animals↗
Effects of fibronectin on actin organization and respiratory burst activity in neutrophils, monocytes, and macrophages.
Previous studies have shown that fibronectin (Fn) enhances phagocytosis and killing of antibody-coated bacteria by neutrophils and macrophages. In an attempt to understand the mechanism of this enhancement, we have investigated the effects of Fn on phagocytosis-related actin organization as well as respiratory burst activity in neutrophils, monocytes and culture-derived macrophages. Employing an NBD-phallacidin flow cytometric analysis of filamentous actin formation, we found that Fn promotes rapid actin polymerization within 30 seconds in neutrophils, monocytes, and macrophages, but not lymphocytes. Enhancement of actin polymerization by Fn was concentration-dependent and mediated by a pertussis toxin- but not cholera toxin-sensitive G protein. Inhibition of protein kinase C by sphingosine (20 microM), calcium influx by verapamil (0.1 mM), or intracellular calcium mobilization by 8-(N,N-diethyl-amino) octyl-3,4,5-trimethoxybenzoate HCl (TMB-8; 0.1 mM) did not block Fn-enhanced actin polymerization in phagocytes. Incubation of neutrophils and macrophages on microtiter plates precoated with Fn suppressed superoxide (O2-) production induced by IgG- and IgA- opsonized group B streptococci. In contrast, Fn significantly enhanced IgA- and IgG-mediated O2- production by freshly isolated monocytes. These data suggest that Fn enhances phagocytosis, presumably through G protein-coupled cytoskeleton reorganization and augments O2- production by circulating monocytes. In contrast, it appears to suppress O2- production by the active phagocytic cells, neutrophils and macrophages. This may result in enhanced phagocytosis and intracellular killing of microorganisms without damaging interstitial tissues.
Actins↗
A gene for distal arthrogryposis type I maps to the pericentromeric region of chromosome 9.
Club foot is one of the most common human congenital malformations. Distal arthrogryposis type I (DA-1) is a frequent cause of dominantly inherited club foot. Performing a genomewide search using short tandem repeat (STR) polymorphisms, we have mapped a DA-1 gene to the pericentromeric region of chromosome 9 in a large kindred. Linkage analysis has generated a positive lod score of 5.90 at theta = 0, with the marker GS-4. Multiple recombinants bracketing the region have been identified. Analysis of an additional family demonstrated no linkage to the same locus, indicating likely locus heterogeneity. Of the autosomal congenital contracture disorders causing positional foot deformities, this is the first to be mapped.
Arthrogryposis↗
Production of tumor necrosis factor by human cells in vitro and in vivo, induced by group B streptococci.
Tumor necrosis factor alpha (TNF alpha) has been implicated as one of the major mediators of the gram-negative septic shock syndrome. In our studies, group B streptococci (GBS) induced the production of TNF alpha by human mononuclear cells in a dose- and time-dependent manner. Human mixed mononuclear cell cultures exposed to an encapsulated (657.6 +/- 71.3 pg/ml; n = 30 preparations) or an unencapsulated transposon mutant of type III GBS (755.8 +/- 54.7 pg/ml; n = 9) produced similar amounts of TNF alpha. Isolated monocytes and culture-derived macrophages produced higher amounts of TNF alpha (1565 +/- 211 and 1790 +/- 928 pg/ml respectively) in response to GBS than did mixed mononuclear cell cultures. In response to GBS, mixed mononuclear cells from neonates produced significantly more TNF alpha (729.1 +/- 45 vs 520.3 +/- 47.2 pg/ml; p = 0.004) than did cells from adults. Examination of specimens from patients with neonatal GBS disease revealed detectable levels of TNF alpha (7 to 424 pg/ml) in the serum of 5 of 10 patients with sepsis, in 5 of 5 urine samples from infants with sepsis, and in the cerebrospinal fluid of 1 patient with meningitis. These results suggest both a major role for TNF alpha in the pathogenesis of human neonatal GBS sepsis and shock and a potential role for immunotherapy directed against this cytokine in this fulminant neonatal bacterial infection.
Adult↗
Restricted ability of group B streptococcal C5a-ase to inactivate C5a prepared from different animal species.
Most strains of group B streptococci (GBS) elaborate a cell surface-associated enzyme that rapidly inactivates the human complement-derived chemoattractants C5a and C5adesarg by cleaving the His-Lys bond at positions 67 and 68 in the C5a molecule. We have suggested that rapid inactivation of C5a and C5adesarg by this enzyme, called C5a-ase, can hinder the inflammatory response at sites of GBS infection. We tested the ability of GBS C5a-ase to inactivate C5a preparations from various animal species to determine the proper species for studying the role of GBS C5a-ase in the pathogenesis of GBS infections. Exposure of C5a preparations from humans, monkeys, and cows to GBS caused inhibition of C5a functional activity as measured by the ability of C5a to stimulate human polymorphonuclear leukocyte (PMN) adherence and human PMN chemotaxis. Bovine PMN chemotaxis to bovine C5a was also abolished after exposure of bovine C5a to GBS. In contrast, mouse, rat, guinea pig, rabbit, pig, and sheep C5a preparations retained full functional activity after exposure to GBS as measured by chemotaxis of human PMNs, PMNs from the same animal species, or both. These data suggest that there are structural differences between C5a proteins from different species which alter their susceptibility to GBS C5a-ase and indicate that most commonly used animal models of human GBS infection are inadequate for detection of a contribution of GBS C5a-ase to GBS virulence.
Amino Acid Sequence↗
Mechanism of fibronectin enhancement of group B streptococcal phagocytosis by human neutrophils and culture-derived macrophages.
In previous studies, we reported that fibronectin (FN) markedly enhances phagocytic uptake of antibody-coated group B streptococci (GBS) by human polymorphonuclear leukocytes. Furthermore, administration of FN along with a GBS type-specific monoclonal or polyclonal antibody to infected neonatal rats significantly enhances survival. In this study, we have examined the molecular mechanism of this enhancement through phagocyte receptors which recognize the Arg-Gly-Asp (RGD) peptide sequences contained within the FN molecule. Incubation of human polymorphonuclear leukocytes or culture-derived macrophages on coverslips coated with GRGDSP but not GRGESP markedly enhanced uptake of immunoglobulin G-coated GBS. The enhancing effect of the RGD-containing peptides was blocked by monoclonal antibodies B6H12 (directed against the integrin-associated protein) and 7G2 (directed against the beta 3-integrin receptor for RGD). These data suggest that FN enhancement of antibody-coated GBS uptake is mediated by the critical RGD sequence. Furthermore, this active peptide sequence may have an important role in immunotherapy of bacterial infections, especially in patients with decreased plasma FN concentrations.
Amino Acid Sequence↗
Aberrant glycosylation of L-selectin on the lymphocytes of chronic lymphocytic leukemia.
Abnormal trafficking of chronic lymphocytic leukemia (CLL) cells may account for the differences in accumulation of malignant lymphocytes within the bone marrow and lymphoid tissues of this lymphoproliferative disorder. We therefore hypothesized that CLL cells aberrantly express one or more receptors involved in lymphocyte trafficking. Leukemia cells from patients with B-cell CLL showed no quantitative difference in surface expression of L-selectin, LFA-1, or CD44 by flow cytometry compared to normal B cells. Analysis of L-selectin by dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, however, demonstrated a consistent, reproducible approximately 3.7 kDa decrease in the M(r) of L-selectin on CLL cells compared to normal B cells. In contrast, Western blot analysis revealed no obvious qualitative abnormality in either CD11a (the alpha-chain of LFA-1) or CD44 on CLL cells. Analysis of L-selectin cDNA by polymerase chain reaction revealed identically sized products for both normal and CLL cells, suggesting that the abnormality in M(r) does not result from a difference in primary structure. Inhibition of N-linked glycosylation by tunicamycin resulted in the production of identical-sized nascent L-selectin by normal and CLL cells. These studies demonstrate that L-selectin on CLL cells is aberrantly glycosylated compared to normal peripheral blood lymphocytes. The functional importance of this aberrant glycosylation is unclear, however, since L-selectin is shed normally from phorbol myristate acetate (PMA)-stimulated CLL cells and since normal and CLL lymphocytes bind equally well in vitro to high endothelial venules. Understanding the mechanism that accounts for the aberrance may provide important insights into the molecular basis of CLL.
Binding Sites↗
Fibronectin in host defense: implications in the diagnosis, prophylaxis and therapy of infectious diseases.
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Animals↗
Divalent cation substitution reveals CD18- and very late antigen-dependent pathways that mediate human neutrophil adherence to fibronectin.
We investigated the mechanisms by which Mn2+ alters human neutrophil (PMN) adherence to various connective tissue proteins. Substitution of Mn2+ for Ca2+ and Mg2+ significantly increased adhesion of human PMN to plastic well coated with fibronectin, fibrinogen, and laminin but not gelatin. Anti-CD18 mAb almost completely blocked adherence to laminin, partly blocked adherence to fibrinogen, but did not inhibit adhesion to fibronectin at all. In contrast, anti-very late antigen (VLA)-5 mAb antibodies significantly reduced Mn(2+)-mediated PMN adherence to fibronectin, but not to laminin or fibrinogen, demonstrating that VLA-5-mediated PMN adherence to fibronectin, but not to fibrinogen or laminin. This was supported by experiments in which synthetic GRGDSP peptide significantly inhibited Mn(2+)-mediated adherence to fibronectin, but not to laminin or fibrinogen. Activation of PMN with phorbol ester or C5a stimulated VLA-5-mediated adhesion to fibronectin, but the contribution of VLA-5 to the forces mediating adherence could only be detected when CD18 function was either blocked with mAb, or when CD18 was congenitally absent. VLA-5 mediated adhesion was also more transient than CD18-dependent adhesion. These data further confirm the presence of PMN VLA integrins and demonstrate that PMN VLA-5 contributes to stimulated PMN adherence to fibronectin.
Antigens, CD↗
CD11/CD18-independent neutrophil adherence to laminin is mediated by the integrin VLA-6.
Regulated adherence of polymorphonuclear leukocytes (PMNs) to endothelium and subendothelial matrix is a critical event for PMN localization at and migration into inflammatory sites. We previously reported that human PMNs stimulated in vitro adhere to laminin, the major glycoprotein of mammalian basement membrane, by both CD11/CD18 (beta 2 integrin)-dependent and CD11/CD18-independent mechanisms. This CD11/CD18-independent adherence is inhibited by monoclonal antibodies (MoAbs) directed against the beta 1 subunit of integrins (very late antigens [VLA]). The specific PMN VLA receptor responsible for stimulated CD11/CD18-independent PMN adherence to laminin was not elucidated. We show here that this CD11/CD18-independent adherence is mediated by a member of the beta 1 integrins, VLA-6. MoAbs GoH3 and 450-30, which bind the alpha 6 subunit of VLA-6, significantly reduced adherence of phorbol myristate acetate-stimulated PMNs to laminin-coated surfaces when CD11/CD18-independent adherence was blocked with anti-CD11/CD18 MoAbs. Furthermore, GoH3 completely inhibited stimulated adherence of CD11/CD18-deficient PMNs to laminin. Analysis by flow cytometry showed that human PMNs express VLA-6. The PMN alpha 6 is identical in size and pl to the platelet alpha 6, but the PMN beta 1 exhibits considerable heterogeneity in molecular weight compared with the platelet beta 1. This activation-dependent adherence receptor for laminin may play a role in PMN interaction with basement membrane laminin during PMN movement through vascular walls.
Animals↗
Comparison of the opsonic and complement triggering activity of human monoclonal IgG1 and IgM antibody against group B streptococci.
We have compared the opsonic and complement-triggering activity of transfectoma-derived, class-switched human IgG1 and IgM mAb (HumAb) against types Ia, II and III group B streptococci (GBS). These antibodies appear to be directed against the common group B cell wall Ag of these organisms. The HumAb IgM promotes uptake of type Ia and II GBS at concentrations as low as 37 ng/ml and type III GBS at concentrations of 150 ng/ml in the presence of human neonatal complement. In contrast, the IgG1 GBS HumAB showed no detectable opsonic activity in concentrations up to 600 ng/ml. When the concentration of HumAb IgG1 is raised to 2.5 micrograms/ml, significant opsonic activity against GBS is detected and when the concentration is approximately 40 micrograms/ml, the opsonic activity peaked at a slightly higher level than that with the HumAb IgM. Thus, approximately 100- fold higher concentrations of the IgG1 than the IgM HumAb are required for optimal opsonization. The opsonic activity of the IgM and IgG1 HumAb are closely related to their ability to consume complement and deposit C3 on the surface of type Ia, II, and III GBS (r = 0.959). We believe that the marked opsonic and protective activity of the IgM GBS HumAb is due to its enhanced avidity and ability to trigger the complement system. Further studies are indicated to determine the feasibility of employing human IgM antibody preparations in the immunotherapy of neonatal GBS disease.
Antibodies, Bacterial↗
Bacterial evasion of the antibody response: human IgG antibodies neutralize soluble but not bacteria-associated group B streptococcal C5a-ase.
Most strains of group B streptococci (GBS) possess an enzyme that inactivates the human anaphylatoxin C5a by cleaving a heptapeptide from the carboxyl terminus of C5a. This enzyme, called GBS C5a-ase, has been purified to homogeneity and cleaves and inactivates C5a in physiologic buffer. The enzymatic activity of soluble C5a-ase is completely inhibited, however, in the presence of plasma or serum from normal human adults. The neutralization of soluble C5a-ase by plasma and serum results largely from naturally occurring IgG antibodies directed against C5a-ase. IgG does not neutralize C5a-ase present on intact encapsulated type III GBS but does neutralize the C5a-ase activity associated with a transposon-induced mutant strain of type III GBS that lacks capsule. The location of GBS C5a-ase on the surface of encapsulated type III GBS permits the C5a-ase to inactivate C5a while evading neutralization by IgG antibodies.
Adhesins, Bacterial↗
Purification of the proteinase from group B streptococci that inactivates human C5a.
We previously reported that group B streptococci (GBS) possess a cell-associated activity that inactivates the chemotactic activity generated in zymosan-activated serum by cleaving a specific site within the carboxy termini of C5a and C5adesarg. This inactivates the major chemoattractants for neutrophils that are generated when serum complement is activated. We now report the isolation of the enzyme responsible for the proteolytic cleavage of C5a. Treatment of GBS with mutanolysin, an endo-N-acetyl muramidase, released activity from GBS which destroyed the functional activity of C5a. The soluble activity was purified to homogeneity by hydroxyapatite, ion-exchange and gel-filtration chromatography. Analysis by SDS-PAGE showed that the enzyme (GBS C5a-ase) has an Mr of approx. 120,000. The GBS C5a-ase appears to be a serine esterase on the basis of its sensitivity to di-isopropyl fluorophosphate. This enzyme is distinct from the C5a-cleaving enzyme produced by group A streptococci, since the two bacterial products migrate differently on SDS-PAGE, and lack antigenic cross reactivity. This enzyme may play a role in the pathogenesis of group B streptococcal disease through its ability to rapidly inactivate the potent neutrophil agonist, C5a, at sites of infection.
Adhesins, Bacterial↗
Group B streptococci inactivate complement component C5a by enzymic cleavage at the C-terminus.
Incubation of recombinant human C5a (rC5a) with the 7360 strain of group B streptococci (GBS) destroyed the ability of rC5a to stimulate chemotaxis or adherence of purified human polymorphonuclear leucocytes (PMNs). Treatment of 125I-labelled rC5a with GBS 7360 correspondingly decreased rC5a binding to human PMNs. This also resulted in an approx. 600 Da decrease in the molecular mass of rC5a as determined by SDS/PAGE. Incubation of rC5a with the GBS strain GW, which only minimally altered the ability of rC5a to activate human PMNs, did not affect rC5a binding to PMNs and did not alter the molecular mass of rC5a on SDS/PAGE. Plasma-desorption m.s. of rC5a inactivated by GBS 7360 showed that the GBS cleaved the rC5a between histidine-67 and lysine-68 near the C-terminus of rC5a. This mechanism of inactivation of C5a by proteolytic cleavage at the C-terminus of C5a is consistent with the known critical role of the C-terminus in C5a activation of human PMNs. This C5a-cleaving proteinase activity may contribute to the pathophysiology of GBS infections.
Adult↗