Biomedical subjects

K Reddi

Publications and source records attributed to K Reddi.

At least 19 recordsLinked to original sources

PubMed2001

Bradykinin B1 receptor up-regulation by interleukin-1beta and B1 agonist occurs through independent and synergistic intracellular signaling mechanisms in human lung fibroblasts.

Bradykinin B1 receptors (B1R) are rapidly induced after tissue trauma and are thought to be involved in maintaining the inflammatory response. Little is known about the intracellular signaling pathways mediating B1R induction in response to stress and inflammation. Here, we show that up-regulation of B1R by B1R agonist and interleukin-1beta (IL-1beta) occur through distinct but synergistic pathways in IMR-90 human lung fibroblasts. Incubation of cells with the B1R agonist desArg10kallidin (desArg10KD; 100 nM) and IL-1beta (500 pg/ml) resulted in a 3- and 4-fold increase, respectively, in B1R by 6 h, whereas coincubation of these factors produced up to a 20-fold increase. Furthermore, coincubation increased the potency of IL-1beta by 2-fold. Both the individual and the synergistic responses were sensitive to genistein, a general tyrosine kinase inhibitor. On the other hand, only the desArg10KD response and the synergistic response were sensitive to the p38 mitogen-activated protein kinase inhibitor SB 203580. Furthermore, only the synergistic response was sensitive to the nuclear factor-kappaB inhibitor pyrrolidine dithiocarbamate. Despite B1R up-regulation in A549 human lung epithelial cells by desArg10KD or IL-1beta individually, these factors did not act synergistically in this cell line. In conclusion, our results reinforce the view that kinins act in concert with proinflammatory cytokines to enhance selectively the inflammatory response of certain lung cells to kinins through distinct but synergistic intracellular signaling mechanisms. Thus, kinins may exert a pivotal role in maintaining and modulating feed-forward inflammatory processes in the lung.

Cells, Cultured↗

PubMed1999

Molecular chaperones stimulate bone resorption.

Molecular chaperones, also known as heat shock proteins (hsp), are intracellular proteins found in all cells that catalyze protein folding. We have discovered that one class of bacterial molecular chaperone, the chaperonins, are potent inducers of bone resorption. To address the question of whether the osteolytic activity of the chaperonins was unique to this protein class, or was a common attribute of molecular chaperones generally, we have examined a number of bacterial and mammalian molecular chaperones for activity in the murine calvarial bone resorption assay. All the Escherichia coli molecular chaperones (groEL, groES, and dnaK) were active. The osteolytic activity of groEL was inhibited by indomethacin and the natural antagonist of interleukin-1 receptor antagonist (IL-1ra) but was unaffected by neutralization of tumor necrosis factor (TNF) or inhibition of 5-lipoxygenase. Mammalian molecular chaperones of molecular mass 27, 47, 70, and 90 kDa were also tested and, with the exception of the 47 kDa protein, all showed activity in the murine calvarial assay. Molecular chaperones appear, therefore, to have the capacity to modulate the cellular processes in bone explant cultures, resulting in resorption of the calcified matrix. The possibility that these proteins could play a role in the normal or pathological remodeling of bone is discussed.

Animals↗

PubMed1998

Homogeneous Escherichia coli chaperonin 60 induces IL-1 beta and IL-6 gene expression in human monocytes by a mechanism independent of protein conformation.

Escherichia coli chaperonin (cpn) 60 (groEL) is a protein-folding oligomer lacking tryptophan residues that copurifies with tryptophan-containing proteins and peptides. Cpn 60 is a major immunogen in infectious diseases, and evidence suggests that groEL and mycobacterial cpn 60s can induce cytokine synthesis, stimulate cytokine-dependent bone resorption, and up-regulate expression of vascular endothelial cell adhesion molecules. Whether such activities are due to the cpn 60 or to the copurifying/contaminating proteins/peptides has not been determined. Here we report a method for removing the protein contaminants of groEL and demonstrate that this, essentially homogeneous, groEL remains a potent inducer of human monocyte IL-1beta and IL-6 production. Contaminating peptides had no cytokine-inducing activity and did not synergize with purified groEL. The LPS inhibitor polymyxin B and the CD14-neutralizing Ab MY4 had no inhibitory action on groEL demonstrating that activity is not due to LPS contamination. Heating groEL had no effect on its capacity to stimulate human monocytes to secrete IL-6. Proteolysis of groEL with trypsin, sufficient to produce low molecular mass peptides, also had no inhibitory effect. Thus, we conclude that groEL is a potent inducer of monocyte proinflammatory cytokine production, which acts through the binding of nonconformational peptide domains that are conserved after proteolysis. These data suggest that if groEL was released from bacteria it could induce prolonged tissue pathology by virtue of its cytokine-inducing activity and its resistance to proteolytic inhibition of bioactivity.

Antibodies, Monoclonal↗

PubMed1998

The Escherichia coli chaperonin 60 (groEL) is a potent stimulator of osteoclast formation.

Chaperonins (cpns) are intracellular oligomeric protein complexes that fold and refold proteins in a catalytic manner and aid in the transmembrane transport of cellular proteins. We reported previously that the lipopolysaccharide-free recombinant cpn60 of Escherichia coli (groEL) is able to stimulate the breakdown of murine calvarial bone in culture and showed that such resorption is potently inhibited by an inhibitor of the enzyme cyclo-oxygenase and to a lesser extent by inhibitors of 5-lipoxygenase. In this study, we have investigated the effects of groEL on the resorptive activity and formation of osteoclasts in culture. In low density, osteoclast-containing cultures from neonatal rats incubated for 24 or 96 h on dentine discs, groEL (1-1000 ng/ml) stimulated resorption pit formation up to 4-fold, but this effect was essentially dependent on cell number. Using 12-day cultures of mouse bone marrow to assess osteoclast recruitment, groEL (1-1000 ng/ml) caused a dramatic dose-dependent stimulation of the formation of tartrate-resistant acid phosphatase-positive multinucleated cells and the resorption of the dentine on which bone marrow cells were cultured. Osteoclast formation elicited by groEL was almost completely abolished by indomethacin, an inhibitor of cyclo-oxygenase, but was unaffected by inhibitors of 5-lipoxygenase, suggesting that prostaglandins but not leukotrienes may mediate the action of groEL on osteoclastogenesis. It is possible that bacterial cpn60s such as groEL may play a role in the osteolysis associated with bone infections. Whether endogenous ("self") chaperonins have a role in other bone loss disorders, such as osteoporosis, is an intriguing possibility.

Acid Phosphatase↗

PubMed1997

Mycobacterium tuberculosis chaperonin 10 stimulates bone resorption: a potential contributory factor in Pott's disease.

Pott's disease (spinal tuberculosis), a condition characterized by massive resorption of the spinal vertebrae, is one of the most striking pathologies resulting from local infection with Mycobacterium tuberculosis (Mt; Boachie-Adjei, O., and R.G. Squillante. 1996. Orthop. Clin. North Am. 27:95-103). The pathogenesis of Pott's disease is not established. Here we report for the first time that a protein, identified by a monoclonal antibody to be the Mt heat shock protein (Baird, P.N., L.M. Hall, and A.R.M. Coates. 1989. J. Gen. Microbiol. 135:931-939) chaperonin (cpn) 10, is responsible for the osteolytic activity of this bacterium. Recombinant Mt cpn10 is a potent stimulator of bone resorption in bone explant cultures and induces osteoclast recruitment, while inhibiting the proliferation of an osteoblast bone-forming cell line. Furthermore, we have found that synthetic peptides corresponding to sequences within the flexible loop and sequence 65-70 of Mt cpn10 may comprise a single conformational unit which encompasses its potent bone-resorbing activity. Our findings suggest that Mt cpn10 may be a valuable pharmacological target for the clinical therapy of vertebral tuberculosis and possibly other bone diseases.

Amino Acid Sequence↗

PubMed1997

Interactions between periodontopathogenic bacteria and cytokines.

Cytokines produced in response to plaque bacteria clearly play a key role in the periodontal diseases. However, we know very little about the interactions between cytokines and periodontopathogenic bacteria. The aims of this study were to determine whether the key pro-inflammatory cytokines interleukin-1 beta (IL-1 beta) and IL-6 could affect the growth of Actinobacillus actinomycetemcomitans or Porphyromonas gingivalis and to determine whether these organisms could hydrolyse IL-1 beta, IL-6 or the anti-inflammatory IL-1 receptor antagonist (IL-1ra). Culture medium containing up to 100 ng/ml of IL-1 beta or IL-6 was inoculated with A. actinomycetemcomitans (serotypes a, b and c) or P. gingivalis and growth was monitored by measuring changes in electrical conductivity every 3 min for up to 48 h. IL-1 beta, IL-6 or IL-1ra were added to culture supernatants and incubated for up to 24 h. Samples were taken at various times, analysed by SDS-PAGE and the separated proteins transferred by Western blotting to PVDF membranes and probed with anti-cytokine antibodies. None of the cytokines tested had any effect on the rate of growth or yield of A. actinomycetemcomitans or P. gingivalis. Supernatants from P. gingivalis cultures, but not those from A. actinomycetemcomitans, hydrolysed IL-1 beta, IL-6 and IL-1ra. The hydrolysate from the P. gingivalis supernatant-treated IL-1 beta was unable to stimulate the release of IL-6 from human gingival fibroblasts showing that it had lost biological activity. These results suggest that P. gingivalis can perturb the cytokine network, not only by stimulating the release of cytokines from host cells, but also by removing them from its local environment.

Aggregatibacter actinomycetemcomitans↗

PubMed1996

Surface-associated material from the bacterium Actinobacillus actinomycetemcomitans contains a peptide which, in contrast to lipopolysaccharide, directly stimulates fibroblast interleukin-6 gene transcription.

The oral commensal Gram-negative bacterium Actinobacillus actinomycetemcomitans is believed to be the causative organism of localized juvenile periodontitis, a disease in which there is rapid loss of alveolar bone supporting the teeth. Previously, we have reported that gentle saline extraction of this bacterium removed a loosely adherent proteinaceous fraction from the cell surface of the bacterium, which we have termed surface-associated material. This material contained potent bone-resorbing activity. We now report that surface-associated material is also a potent stimulator of cytokines, and in particular, interleukin-6 (IL-6) synthesis, while the lipopolysaccharide from this bacterium is only a weak stimulator of IL-6 synthesis by fibroblasts and monocytes. In contrast to enteric lipopolysaccharide (LPS), which induces fibroblast IL-1, IL-6 and tumour necrosis factor (TNF) alpha synthesis, surface-associated material stimulated gingival fibroblasts to synthesize only IL-6, with no induction of IL-1 or TNF (the normal inducers of IL-6 synthesis). Reverse transcriptase PCR also failed to detect mRNA for IL-1 or TNF in surface-associated-material-stimulated fibroblasts, although both mRNAs were present in Escherichia coli LPS-stimulated cells. Neutralizing antibodies to IL-1 and/or TNF or the natural IL-1 receptor antagonist (IL-1ra) inhibited enteric LPS-induced IL-6 synthesis, but did not inhibit surface-associated-material-induced synthesis. In addition, dexamethasone, which completely suppressed LPS-induced IL-6 synthesis, only inhibited surface-associated-material-induced IL-6 synthesis by 50%. This suggests that the active constituent in the surface-associated material stimulates IL-6 gene transcription by a transcriptional control mechanism distinct to that of E. coli LPS. The IL-6 stimulating activity of the surface-associated material is inhibited by both heat and trypsin, suggesting that it is proteinaceous. The activity has been isolated using anion-exchange, reverse-phase and size-exclusion HPLC. The active moiety is a peptide of molecular mass 2kDa which may be the product of a bacterial short open reading frame.

Aggregatibacter actinomycetemcomitans↗

PubMed1996

Comparison of the pro-inflammatory cytokine-stimulating activity of the surface-associated proteins of periodontopathic bacteria.

Saline extraction of the periodontopathic bacterium, Actinobacillus actinomycetemcomitans, releases surface-associated material (SAM), a complex mixture of proteins and carbohydrates with potent biological actions on isolated bone and on various mammalian cell populations. In this study, the relative ability of the SAM from 5 organisms, implicated in the pathology of periodontal disease, to stimulate human mesenchymal and myelomonocytic cells to synthesize the proinflammatory cytokines - interleukin (IL)-1 beta, IL-6 and tumour necrosis factor (TNF)alpha has been investigated. The bacteria investigated were Actinobacillus actinomycetemcomitans, Eikenella corrodens, Porphyromonas gingivalis, Prevotella intermedia and Campylobacter rectus. Human cells were exposed to a four log order range of concentrations of the SAM, or of Escherichia coli lipopolysaccharide, to provide full agonist dose responses in order to allow comparison of the potency and efficacy of each SAM. All SAMs demonstrated the capacity to stimulate human gingival fibroblasts (HGFs), human peripheral blood mononuclear cells (PBMCs) or the myelomonocytic cell line - Mono-Mac-6 to release one or all of the cytokines assayed. Activity was heat- and trypsin-sensitive suggesting that the active components were proteinaceous. However, there were substantial differences in the potency and efficacy of each SAM when compared on a concentration basis (w/v). The most active SAM was from A. actinomycetemcomitans with those from E. corrodens and P. gingivalis being slightly less active. The least active cytokine-stimulating SAMs were from C. rectus and Pr. intermedia. One major difference between the SAMs and E. coli LPS was the inability of the former to stimulate HGFs to release IL-1 beta or TNF alpha although they could stimulate PBMCs to release these cytokines. This may have relevance to the pathology of the periodontal diseases.

Aggregatibacter actinomycetemcomitans↗

PubMed1996

Cytokine-inducing components of periodontopathogenic bacteria.

Pro-inflammatory cytokines such as interleukin (IL)-1, IL-6, IL-8 and tumour necrosis factor (TNF) are believed to be the major pathological mediators of inflammatory diseases ranging from arthritis to the periodontal diseases. The stimuli inducing proinflammatory cytokine induction in the former disease is unclear but in the periodontal diseases it is obvious that the stimulus is the accumulation of bacteria in the subgingival region. As these bacteria do not invade the lesional tissues in large numbers, it is believed that their soluble components or products interact with host tissues to induce cytokine gene transcription. The paradigm is that lipopolysaccharide is the key bacterial component inducing pro-inflammatory cytokine gene expression. However, over the past decade a growing number of reports on non-oral bacteria have established that many other bacterial components, as well as secretory products, have the capacity to induce cytokine synthesis. Some of these, such as the protein pneumolysin from Streptococcus pneumoniae, are incredibly potent (in this case inducing cytokine synthesis at femtomolar concentrations). This review surveys the range of bacterial components and products which have been shown to stimulate cytokine synthesis with particular emphasis on the hypothesis that these components play a role in the pathology of the periodontal diseases.

Animals↗

PubMed1996

Bacterially induced bone destruction: mechanisms and misconceptions.

Normal bone remodelling requires the coordinated regulation of the genesis and activity of osteoblast and osteoclast lineages. Any interference with these integrated cellular systems can result in dysregulation of remodelling with the consequent loss of bone matrix. Bacteria are important causes of bone pathology in common conditions such as periodontitis, dental cysts, bacterial arthritis, and osteomyelitis. It is now established that many of the bacteria implicated in bone diseases contain or produce molecules with potent effects on bone cells. Some of these molecules, such as components of the gram-positive cell walls (lipoteichoic acids), are weak stimulators of bone resorption in vitro, while others (PMT, cpn60) are as active as the most active mammalian osteolytic factors such as cytokines like IL-1 and TNF. The complexity of the integration of bone cell lineage development means that there are still question marks over the mechanism of action of many well-known bone-modulatory molecules such as parathyroid hormone. The key questions which must be asked of the now-recognized bacterial bone-modulatory molecules are as follows: (i) what cell population do they bind to, (ii) what is the nature of the receptor and postreceptor events, and (iii) is their action direct or dependent on the induction of secondary extracellular bone-modulating factors such as cytokines, eicosanoids, etc. In the case of LPS, this ubiquitous gram-negative polymer probably binds to osteoblasts or other cells in bone through the CD14 receptor and stimulates them to release cytokines and eicosanoids which then induce the recruitment and activation of osteoclasts. This explains the inhibitor effects of nonsteroidal and anticytokine agents on LPS-induced bone resorption. However, other bacterial factors such as the potent toxin PMT may act by blocking the normal maturation pathway of the osteoblast lineage, thus inducing dysregulation in the tightly regulated process of resorption and replacement of bone matrix. At the present time, it is not possible to define a general mechanism by which bacteria promote loss of bone matrix. Many bacteria are capable of stimulating bone matrix loss, and the information available would suggest that each organism possesses different factors which interact with bone in different ways. With the rapid increase in antibiotic resistance, particularly with Staphylococcus aureus and M. tuberculosis, organisms responsible for much bone pathology in developed countries only two generations ago, we would urge that much greater attention should be focused on the problem of bacterially induced bone remodelling in order to define pathogenetic mechanisms which could be therapeutic targets for the development of new treatment modalities.

Alveolar Bone Loss↗

PubMed1995

Surface-associated proteins from Staphylococcus aureus demonstrate potent bone resorbing activity.

Staphylococcus aureus infections are associated with rapid bone destruction in conditions such as osteomyelitis, bacterial arthritis, and infected orthopedic implant failure. How this bacterium induces bone destruction has not been defined. In studies of the role of oral Gram-negative bacteria in periodontal pathology, we have established that cell surface-associated proteins (SAPs) are potent stimulators of bone resorption. The surface-associated components from S. aureus have now been isolated and demonstrated to be extremely potent stimulators of bone resorption in the murine calvarial bone resorption assay. Bone resorption appears to be due to proteins, is not the result of contamination with lipoteichoic acid or muramyl dipeptide, and is potently inhibited by indomethacin and can be completely blocked by high concentrations of interleukin-1 receptor antagonist or TN3-19.12, a neutralizing monoclonal antibody to murine TNF. The SAP fraction can stimulate fibroblasts or monocytes to release osteolytic cytokines, but only at high concentrations. Fractionation of the SAPs by high performance liquid chromatography demonstrated that a number of fractions were osteolytically active. The most active contained a heterodimeric protein of molecular weight 32-36 kD. The presence of this osteolytically active surface-associated fraction may account for the bone resorption associated with local infection with S. aureus.

Animals↗

PubMed1995

Interleukin 6 production by lipopolysaccharide-stimulated human fibroblasts is potently inhibited by naphthoquinone (vitamin K) compounds.

Naphthoquinone vitamins (vitamins K) are widely recognized for their role in the gamma-carboxylation of specific glutamyl residues in coagulation, anti-coagulation and extra-hepatic proteins. Recently, however, there have been reports that these compounds can exert actions other than those normally associated with protein gamma-carboxylation. These observations suggest that naphthoquinones may have effects on the production of inflammatory mediators including cytokines. Fibroblasts are now recognized as a rich source of cytokines and we have examined the effect of various naphthoquinones on the production of interleukin 6 (IL-6) by lipopolysaccharide-stimulated human gingival fibroblasts. Compounds examined in this study include: phylloquinone (K1), menaquinone-4 (K2), menadione (K3), 2,3-dimethoxy-1,4-naphthoquinone (DMK) and a synthetic product of vitamin K catabolism, 2-methyl, 3-(2'methyl)-hexanoic acid-1,4-naphthoquinone (KCAT). All of these compounds are capable of inhibiting IL-6 production with a rank order of potency: KCAT > K3 > DMK > K2 > K1. The most potent compound, KCAT, inhibited IL-6 production with an IC50 of 3 x 10(-7)M. The mechanism of action of these naphthoquinones on fibroblast IL-6 production is unknown. Given that K3 and KCAT are inactive in the gamma-carboxylation reaction, we suggest that this activity is not essential for the inhibition of IL-6 production and that activity may be related to the redox capacity of these naphthoquinones.

Cells, Cultured↗

PubMed1995

Relative cytokine-stimulating activities of surface components of the oral periodontopathogenic bacterium Actinobacillus actinomycetemcomitans.

The purpose of this study was to determine whether bacterial surface components other than lipopolysaccharide (LPS) could stimulate pro-inflammatory cytokine synthesis by mesenchymal and myelomonocytic cells in vitro. LPS, lipid A-associated proteins (LAP) and saline-extractable surface-associated material (SAM) were isolated from the periodontopathogenic bacterium Actinobacillus actinomycetemcomitans and added to cultures of human gingival fibroblasts (HGFs), human PBMCs and the human myelomonocytic MonoMac-6 cell line. Pro-inflammatory cytokine release into culture supernatants was determined by two-site ELISAs. Contrary to expectation, the highly purified LPS extracted from this bacterium was significantly less potent than the other surface extracts in stimulating release of IL-1 beta, IL-6 and TNF-alpha by all three cell types. The SAM was the most potent cytokine-stimulating agent showing equivalent activity to highly purified E. coli LPS in stimulating IL-6 release by PBMCs. LAP also had cytokine-stimulating activity although it was generally significantly less potent than the SAM. Thus in the case of this organism, which is involved in the pathology of chronic inflammatory diseases the LPS does not appear to be the major cytokine-stimulating component.

Aggregatibacter actinomycetemcomitans↗

PubMed1995

Lipid A-associated proteins from periodontopathogenic bacteria induce interleukin-6 production by human gingival fibroblasts and monocytes.

The aim of this study was to determine whether lipid A-associated proteins (LAP) from two periodontopathogenic species of bacteria were able to stimulate interleukin-6 (IL-6) release from human gingival fibroblasts and myelomonocytic cells. LAP and lipopolysaccharide (LPS) were extracted from Porphyromonas gingivalis and Prevotella intermedia and added to cultures of human gingival fibroblasts and mono-mac-6 monocytic cells. Release of IL-6 into the culture supernatants was determined by ELISA. LAP and LPS from Por. gingivalis, but not from Prev. intermedia, stimulated IL-6 release from both cell types in a dose-dependent manner although LPS was less potent than LAP in inducing IL-6 release from the fibroblasts. IL-6 was detectable in cultures of both cell types following stimulation with LAP from Por. gingivalis at a concentration as low as 10 ng/ml. In response to LAP from Prev. intermedia, IL-6 was produced by mono-mac-6 cells but not by fibroblasts. Our results show that bacterial cell wall components other than LPS can induce IL-6 release from cells of the periodontium in vitro. The production of such potent immunomodulatory agents in vivo may contribute to the connective tissue breakdown characteristic of chronic periodontitis.

Bacterial Proteins↗

PubMed1995

Comparison of the osteolytic activity of surface-associated proteins of bacteria implicated in periodontal disease.

OBJECTIVES: To compare the osteolytic activity of surface-associated material (SAM) and lipid A-associated proteins (LAPs) from periodontopathogenic bacteria. MATERIALS AND METHODS: Surface-associated material was extracted from the surface and LAPs from the cell walls of a range of periodontopathic bacteria including Actinobacillus actinomycetemcomitans and Eikenella corrodens. These bacterial fractions were assayed to determine their composition and their capacity to induce bone resorption was determined by use of the neonatal murine calvarial bone resorption assay. RESULTS: The SAMs from E. corrodens and A. actinomycetemcomitans demonstrated bone-resorbing capacity at concentrations as low as 1 ng ml-1 which, given the molecular weights of the active components, is in the picomolar range of activity. In contrast, the SAMs from the other three bacteria were significantly less potent and showed a lower efficacy. The LAPs all showed significant, and similar, capacities to induce bone breakdown. CONCLUSIONS: This is the first demonstration that LAP from periodontopathic bacteria can stimulate bone degradation. The LAPs from diverse bacteria all produced similar levels of bone-resorbing activity. In contrast, the SAM showed significant differences in potency and in efficacy (maximal stimulation). This may mean that in vivo certain periodontopathic bacteria have significantly more bone-resorbing capacity than others and should be therapeutic targets.

Aggregatibacter actinomycetemcomitans↗

PubMed1995

Characterization of an antiproliferative surface-associated protein from Actinobacillus actinomycetemcomitans which can be neutralized by sera from a proportion of patients with localized juvenile periodontitis.

The gentle agitation of suspensions of Actinobacillus actinomycetemcomitans serotype a, b, or c in saline resulted in the release of a proteinaceous surface-associated material (SAM) which produced a dose-dependent inhibition of tritiated thymidine incorporation by the osteoblast-like cell line MG63 in culture. This cell line was sensitive to low concentrations of SAM (50% inhibitory concentration, 200 ng/ml for serotype c). Immunoglobulin G antibodies to constituents of the SAM were found in the blood of patients with localized juvenile periodontitis (LJP). Sera from 9 of 16 patients with LJP significantly neutralized the antiproliferative activity of the SAM, while sera from 15 controls, with no evidence of periodontal disease, were unable to neutralize this activity. Neutralization was not directly related to the patient's antibody titer to the whole SAM. Characterization of the antiproliferative activity in the SAM demonstrated that it was not cytotoxic and was heat and trypsin sensitive. The active component separated in a well-defined peak in anion-exchange high-performance liquid chromatography (HPLC) which, when further analyzed by size exclusion HPLC, revealed a single active peak, which had an apparent molecular mass of approximately 8 kDa. The lipopolysaccharide from A. actinomycetemcomitans was only weakly active. SAM from Porphyromonas gingivalis W50 and Eikenella corrodens NCTC 10596 did not exhibit any antiproliferative activity with this cell line, even at concentrations as high as 10 micrograms/ml. This study has shown that SAM from A. actinomycetemcomitans contains a potent antiproliferative protein whose activity can be neutralized by antibodies in the sera from some patients with LJP.

Adolescent↗

PubMed1995

The potent bone-resorbing mediator of Actinobacillus actinomycetemcomitans is homologous to the molecular chaperone GroEL.

Actinobacillus actinomycetemcomitans is a Gram-negative bacterium implicated in the pathology of localized juvenile periodontitis, a condition involving rapid destruction of alveolar bone. We have established that gentle extraction of this bacterium in saline releases a proteinaceous fraction (which we have termed surface-associated material [SAM] which has potent osteolytic activity in the murine calvarial bone resorption assay. Fractionation of the SAM has now revealed that activity is associated with a 62-kD protein. This bone-resorbing activity can be blocked by a monoclonal antibody (raised to the whole bacterium) that is claimed to recognize a protein homologous to the Escherichia coli molecular chaperone GroEL. Purification of this bone-resorbing protein to homogeneity has been achieved by a combination of anion exchange, gel filtration, and ATP-affinity chromatography and the NH2-terminal sequence shows > 95% homology to E. coli GroEL. This GroEL homologue is found in the SAM of A. actinomycetemcomitans but is not found in the osteolytically active SAM from other Gram-negative or Gram-positive bacteria. The GroEL protein from E. coli, but not from Mycobacterium tuberculosis and Mycobacterium leprae, also showed activity in the bone resorption assay. We believe this to be the first observation that a molecular chaperone has the capacity to stimulate the breakdown of connective tissue.

Aggregatibacter actinomycetemcomitans↗

PubMed1991

Neutralization of kinin-releasing enzymes from viperid venoms by antivenom IgG fragments.

The activities of kinin-releasing enzymes in the venoms of Vipera xanthina xanthina, V. lebetina obtusa, V. aspis aspis, V. lebtina schweizeri, V. ammodytes ammodytes and V. berus berus were determined using a specific radioimmunoassay for kinin. The kinin-releasing activities of all the viperid venoms measured in vitro were neutralized, to varying extents, by two commercially available monospecific antivenoms in the form of F(ab')2 (Zagreb) and Fab (TAb) immunoglobin fragments, indicating a high degree of cross-neutralization of those enzymes.

Animals↗