Biomedical subjects

E J Miller

Publications and source records attributed to E J Miller.

At least 37 records · Page 2Linked to original sources

PubMed1997

Binding of SPAAT, the 44-residue C-terminal peptide of alpha 1-antitrypsin, to proteins of the extracellular matrix.

SPAAT (short piece of alpha 1-antitrypsin [AAT]), the 44-residue C-terminal peptide of AAT, was originally isolated from human placenta [Niemann et al. (1992): Matrix 12:233-241]. It was shown to be a competitive inhibitor of serine proteases [Niemann et al. (in press): Biochem Biophys Acta]. The binding of SPAAT to one or more proteins of the extracellular matrix (ECM) was initially suggested on the basis of its recovery from tissue residues following a series of extractions designed to remove easily solubilized proteins [Niemann et al. (1992): Matrix 12:233-241]. Our binding studies with the model ECMs, Matrigel and Amgel, suggested that SPAAT might be bound by a specific collagen type as well as one or more non-collagenous ECM proteins. Individual ECM components were screened for their ability to bind SPAAT. When the four commonly occurring fiber-forming collagens (types I, II, III, and V) were evaluated, type III was found to be preferred. In addition, although SPAAT bound to preformed type III collagen fibers in a concentration dependent fashion, it did not bind to type III collagen molecules undergoing fibril formation. This is consistent with a physiological mode of interaction between SPAAT and type III collagen in vivo. Of the non-collagenous ECM macromolecules (laminin-1, fibronectin, entactin, and heparan sulfate) tested, laminin-1 was preferred. The binding of radiolabelled SPAAT to type III collagen and laminin-1 was competitively inhibited by unlabelled SPAAT as well as an unrelated protein, human serum albumin (HSA), to establish binding specificity. The kinetics of the release of the bound radiolabelled SPAAT were also examined to substantiate the non-covalent and reversible nature of this association. These results support the view that susceptible proteins of the ECM may actually be coated with SPAAT in vivo, possibly affording protection against inappropriate protease digestion.

Collagen↗

PubMed1997

Inhibition of human serine proteases by SPAAT, the C-terminal 44-residue peptide from alpha1-antitrypsin.

SPAAT has previously been shown to be a competitive inhibitor of the model serine protease, chymotrypsin. We now present evidence that SPAAT is likewise a competitive inhibitor of human neutrophil elastase and cathepsin G with Ki's of 15-20 and 40 microM, respectively. The mechanism of this inhibition was investigated by comparing the relative effectiveness of the 23-residue N-terminal fragment of SPAAT (N-SPAAT) to inhibit chymotrypsin and human neutrophil elastase. N-SPAAT, which does not contain the primary chymotrypsin cleavage site, was approximately 10-fold less effective as an inhibitor of chymotrypsin than SPAAT (Ki of 65 microM versus 7.5 microM). In contrast, this fragment, which contains the primary human neutrophil elastase cleavage site, was found to competitively inhibit human neutrophil elastase with a Ki of 24 microM which was comparable to that of SPAAT (Ki = 15-20 microM). Thus it appears that SPAAT is a reversible inhibitor of these enzymes rather than an irreversible, stoichiometric one like its parent protein, AAT. Such fragmentation of AAT, however, might provide a mechanism whereby a cascade of decreasingly potent, but increasingly specific SPAAT-related inhibitory peptides could be generated. These results further substantiate the view that SPAAT may play a role in vivo in the protection of extracellular proteins from inappropriate attack by proteases which are elevated during various pathophysiological conditions.

Amino Acid Sequence↗

PubMed1997

A synthetic peptide inhibitor for alpha-chemokines inhibits the growth of melanoma cell lines.

Melanoma growth stimulatory activity (MGSA/GROalpha) is a 73 amino acid peptide sharing sequence characteristics with the alpha-chemokine superfamily. MGSA/GROalpha is produced by diverse melanoma cell lines and reported to act as an autocrine growth factor for the cells. We tested the binding of MGSA/GROalpha to melanoma cell lines, Hs 294T and RPMI7951, and found that these cells could bind to MGSA/GROalpha but not to interleukin-8. Recently, we defined a novel hexapeptide, antileukinate, which is a potent inhibitor of binding of alpha-chemokines to their receptors on neutrophils. When antileukinate was added to melanoma cells, it inhibited the binding of MGSA/ GROalpha. The growth of cells from both melanoma cell lines was suppressed completely in the presence of 100 microM peptide. The cell growth inhibition was reversed by the removal of the peptide from the culture media or by the addition of the excess amount of MGSA/GROalpha. The viability of Hs 294T cells in the presence of 100 microM peptide was > 92%. These findings suggest that MGSA/GROalpha is an essential autostimulatory growth factor for melanoma cells and antileukinate inhibits their growth by preventing MGSA/GROalpha from binding to its receptors.

Cell Division↗

PubMed1997

Adenomatoid odontogenic tumor presenting as periapical disease.

Adenomatoid odontogenic tumor commonly occurs in association with the crowns of unerupted teeth. An extrafollicular variant, radiographically in relationship to root apices, has been reported. However, clear association with the root apices at surgery has not been demonstrated. We report a case of adenomatoid odontogenic tumor in the anterior mandible in a 21-year-old woman that presented radiographically at the root apices and at surgery as a radicular cyst. We believe this represents the first reported case of adenomatoid odontogenic tumor presenting as periapical disease both clinically and radiographically. The diagnosis of adenomatoid odontogenic tumor should be considered when the clinician is presented with a corticated radiolucency in the anterior jaw, especially in teens and young adults.

Adult↗

PubMed1997

Low-cost system for real-time monitoring of luciferase gene expression.

In some mammalian cells transfected with luciferase reporter genes, the luciferase/luciferin reaction in a cell monolayer produces a very small light flux. While the low light levels are often measurable with single-photon counting cameras, these devices are expensive and may require long averaging times to acquire an image. We describe an approach for real-time monitoring of light produced by luciferase gene expression in intact, cultured cells using readily available and relatively inexpensive components. The system uses a single-photon counting photomultiplier tube with built-in high voltage supply and photon counting circuitry to rapidly measure average light output from growing cells in a 35 mm culture dish. The fast, accurate and highly sensitive response of the system makes it useful for studying the dynamics of gene expression over time periods ranging from minutes to days.

Antineoplastic Agents, Hormonal↗

PubMed1996

Anti-IL-8 autoantibodies in alveolar fluid from patients with the adult respiratory distress syndrome.

IL-8 is a potent neutrophil attractant and activator. IL-8 has been reported to be involved in the pathogenesis of several diseases, including rheumatoid arthritis, sepsis, psoriasis, and the adult respiratory distress syndrome (ARDS). Our previous studies demonstrated that high concentrations of IL-8 were present in alveolar fluids from patients with ARDS and were associated with increased mortality. In this study we report that a major portion of IL-8 in bronchoalveolar fluids from patients with ARDS is associated with anti-IL-8 autoantibody (anti-IL-8:IL-8 complexes). Free autoantibodies that recognize IL-8 were also detected in these fluids. Next, we examined the properties of anti-IL-8 autoantibodies present in lung fluids from ARDS patients and compared them with autoantibodies from normal plasma and arthritic synovial fluids. The anti-IL-8 autoantibody was polyclonal, and IgG3 and IgG4 were the primary IgG subclasses. Anti-IL-8:IL-8 complexes consisted of one IgG and one IL-8 molecule. In addition, anti-IL-8 autoantibody bound IL-8 with a high affinity (approximately 10(-12) M) and inhibited IL-8 interaction with its specific receptors on neutrophils. The results suggest that anti-IL-8 autoantibodies may regulate IL-8 activity.

Antibody Specificity↗

PubMed1996

Effect of physical crosslinking methods on collagen-fiber durability in proteolytic solutions.

We previously demonstrated that ultraviolet (UV) or dehydrothermal (DHT) crosslinking partially denatured fibers extruded from an insoluble type I collagen dispersion. In this study denaturation effects were evaluated by measuring collagen-fiber sensitivity to trypsin. Shrinkage-temperature measurements and sensitivity to collagenase served as indices of crosslinking. UV or DHT crosslinking increased the collagen-fiber shrinkage temperature, resistance to degradation in collagenase, and durability under load in collagenase. However, in trypsin solutions, solubility was significantly increased for UV (approximately 11%) or DHT (approximately 15%) crosslinked fibers compared with uncrosslinked fibers (approximately 4%). Size-exclusion chromatography indicated that no intact collagen alpha-chains were present in the soluble fraction of fibers exposed to trypsin (MW < 1 kD). Interestingly, UV-crosslinked collagen fibers remained intact an order of magnitude longer (4840 +/- 739 min) than DHT-crosslinked (473 +/- 39 min) or uncrosslinked (108 +/- 53 min) fibers when placed under load in trypsin solutions. These data indicate that mechanical loading during incubation in a trypsin solution measures denaturation effects not detected by the trypsin-solubility assay. Our results suggest that DHT-crosslinked collagen fibers should not be used as load-bearing implants. UV-crosslinked fibers may retain more native structure and should exhibit greater resistance to nonspecific proteases in vivo.

Animals↗

PubMed1996

Interleukin-8 (IL-8) is a major neutrophil chemotaxin from human alveolar macrophages stimulated with staphylococcal enterotoxin A (SEA).

Since Staphylococcus aureus is an important human pathogen, and infection of the lungs is characterized by neutrophil infiltration we studied the role of a staphylococcal toxin, enterotoxin A (SEA) on the synthesis and secretion of IL-8 by human alveolar macrophages. As SEA concentration was increased, the IL-8 accumulation in the macrophage conditioned medium increased. The concentration of mRNA encoding IL-8 was also elevated in the macrophage in response to increases in SEA concentration. Although the monocytic cell line U937 was able to respond to SEA and secrete IL-8, treatment with PMA prior to SEA stimulation increased the IL-8 accumulation around fifty fold indicating that maturation of the undifferentiated cell to a more macrophage-like cell facilitated IL-8 accumulation. Stimulating human alveolar macrophages with high concentrations of SEA caused an increase in IL-1 accumulation. However, when the cells were incubated with SEA in the presence of IL-1 receptor antagonist, there was no decrease in IL-8 accumulation. Addition of a neutralizing anti-IL-8 monoclonal antibody to the culture medium of SEA-stimulated macrophages significantly reduced the neutrophil chemotactic activity of the medium. These studies showed that IL-8 is a major neutrophil chemotaxin from human alveolar macrophages stimulated with SEA.

Blotting, Northern↗

PubMed1996

Peptide inhibitor of interleukin-8 (IL-8) reduces staphylococcal enterotoxin-A (SEA) induced neutrophil trafficking to the lung.

Staphylococcal enterotoxin A (SEA) is a superantigen, produced by some strains of Staphylococcus aureus (S. aureus), which can cause a variety of clinical manifestations ranging from food poisoning to shock. SEA can also stimulate human alveolar macrophages to produce interleukin-8 (IL-8), a member of the alpha-chemokine subfamily that activates and is chemotactic for neutrophils. In these studies we showed that in rabbits, intravenous SEA significantly decreased the circulating white blood cell population from a baseline value of 6409 +/- 2027 x 10(3) cells/ml to 1943 +/- 862 x 10(3) cells/ml in 7 h. There was a concommitent increase in IL-8 in the circulating plasma (baseline: 60 +/- 34 pg/ml, 7 h post SEA: 109 +/- 64 pg/ml). The increase in circulating IL-8 was accompanied by a much greater increase in the IL-8 concentration of the epithelial lining fluid (ELF) where the IL-8 increased from 0.05 +/- 0.08 ng/ml (control) to 13.8 +/- 9.3 ng/ml (SEA treated). The increase in IL-8 concentration in the alveolar spaces was paralleled by an increase in both the percentage of neutrophils (1.4 +/- 0.9% (control) to 26.0 +/- 10.8% (SEA treated)) and total number of neutrophils (0.04 +/- 0.02 x 10(6)/ml (control) to 4.8 +/- 3.3 10(6)/ml (SEA treated) in the airspaces, and the numbers of neutrophils in the ELF correlated with the IL-8 concentration r = 0.62, p = 0.006). When antileukinate, a hexapeptide which inhibits the binding of IL-8 to neutrophils, was administered to animals receiving SEA, the IL-8 concentration in the ELF (14.8 +/- 10.7 ng/ml) was not significantly different from the concentration of IL-8 in those animals receiving SEA alone). However, both the percentage of neutrophils (9.5 +/- 3.2%), and the total number of neutrophils (1.3 +/- 1.0 x 10(6)/ml) in the ELF following SEA and antileukinate administration was significantly lower than in animals which only received SEA (p < 0.05). The findings suggest that SEA released into the circulation during a Staphylococcal infection can cause an inflammatory reaction in the lung. Since this reaction is at least partially mediated by IL-8, antileukinate may have pharmacologic potential in reducing the inflammatory reaction.

Animals↗

PubMed1996

Trypsin interaction with the senile plaques of Alzheimer disease is mediated by beta-protein precursor.

In this study we demonstrate by in situ binding that trypsin interacts with the senile plaques found in Alzheimer disease. Characterization of various potential trypsin binding proteins shows that trypsin binding is mediated by beta-protein precursor (beta PP)-the progenitor of amyloid-beta in senile plaques. Using specific antisera against various proteins to sterically block trypsin blocking, we found that only those antibodies raised against proteins or peptides containing the Kunitz protease inhibitor domain were able to abolish binding. By analogy with other protease/inhibitor interactions, we speculate that the binding of trypsin to beta PP could involve concomitant beta PP cleavage. Therefore, beta PP in protecting against potentially damaging proteolysis could simultaneously liberate beta PP fragments or intermediate precursors of amyloid-beta deposits.

Alzheimer Disease↗

PubMed1996

Increased interleukin-8 concentrations in the pulmonary edema fluid of patients with acute respiratory distress syndrome from sepsis.

OBJECTIVE: To test the hypothesis that significantly higher concentrations of interleukin-8 (IL-8) are found in the pulmonary edema fluid and plasma of patients with a septic vs. a nonseptic etiology of acute respiratory distress syndrome (ARDS). DESIGN: Prospective measurement of IL-8 concentrations in previously collected edema fluid and plasma. SETTING: Adult intensive care units at a university medical center. PATIENTS: There were 27 patients with ARDS (16 patients with a septic etiology and nine patients with a nonseptic etiology) plus eight control patients with hydrostatic pulmonary edema. MEASUREMENTS AND MAIN RESULTS: IL-8 was present in the pulmonary edema fluid of all patients with ARDS, but the median IL-8 concentration was higher in the edema fluid of patients with ARDS associated with sepsis (84.2 ng/mL, n = 16) compared with the ARDS patients without sepsis (14.8 ng/mL, n = 11) (p < .05). In patients with cardiogenic edema, IL-8 concentration (5.0 ng/mL,n = 8, p < .05) was significantly lower than those values in patients with ARDS. Median plasma concentration of IL-8 was increased in septic individuals (1.3 ng/mL), but these concentrations were not significantly higher than in patients with a nonseptic etiology of ARDS (0.35 ng/mL) (p = .14) or those patients with cardiac failure (0.21 ng/mL). CONCLUSIONS: The high concentrations of IL-8 in pulmonary edema fluid, coupled with the relatively low concentrations of IL-8 in the plasma, suggest that the lung was the primary source of IL-8 in the patients with ARDS. The markedly increased concentrations of IL-8 in the pulmonary edema fluid of patients with ARDS from sepsis suggests that this group of patients may be particularly suitable for potential trials directed at inhibiting the activity of this important chemokine.

Adolescent↗

PubMed1996

Polymerization of fibrinogen in murine bleomycin-induced lung injury.

Bleomycin lung injury in mice leads to an acute alveolitis followed by a fibroproliferative response characterized by the accumulation of extracellular matrix. Because distinct regions of the fibrin(ogen) molecule have unique in vitro biological effects on cells, we quantified, localized, and biochemically characterized the molecular form of extravascular fibrin(ogen) in methoxyflurane anesthetized, bleomycin-injured mice. Bleomycin or saline (controls) was administered intratracheally, and lung tissue was harvested and analyzed at several times thereafter. Immunoreactive fibrin tissue content increased to a maximal 50-fold over controls in a temporal and spatial pattern paralleling that of alveolitis and maximal fibroproliferation. The generation of gamma-gamma-chain dimers and alpha-chain polymers, together with the loss of free alpha- and gamma-chains, indicates that the fibrin is predominantly covalently cross-linked. In fibroproliferative-phase lungs, the fibrin fibrils are branched and colocalize with those of collagen at the electron microscopic level. These observations strongly suggest that fibrin is a significant molecular effector of the in vivo fibroproliferative response after lung injury.

Animals↗

PubMed1995

Conditional lethality of null mutations in RTH1 that encodes the yeast counterpart of a mammalian 5'- to 3'-exonuclease required for lagging strand DNA synthesis in reconstituted systems.

A 5'- to 3'-exonuclease of about 45 kDa has been purified from various mammalian sources and shown to be required for the completion of lagging strand synthesis in reconstituted DNA replication systems. RTH1 encodes the yeast Saccharomyces cerevisiae counterpart of the mammalian enzyme. To determine the in vivo biological role of RTH1-encoded 5'- to 3'-exonuclease, we have examined the effects of an rth1 delta mutation on various cellular processes. rth1 delta mutants grow poorly at 30 degrees C, and a cessation in growth occurs upon transfer of the mutant to 37 degrees C. At the restrictive temperature, the rth1 delta mutant exhibits a terminal cell cycle morphology similar to that of mutants defective in DNA replication, and levels of spontaneous mitotic recombination are elevated in the rth1 delta mutant even at the permissive temperature. The rth1 delta mutation does not affect UV or gamma-ray sensitivity but enhances sensitivity to the alkylating agent methyl methanesulfonate. The role of RTH1 in DNA replication and in repair of alkylation damage is discussed.

DNA Damage↗

PubMed1995

Synthetic hexa- and heptapeptides that inhibit IL-8 from binding to and activating human blood neutrophils.

IL-8 is a member of the chemokine alpha subfamily that activates and is chemotactic for neutrophils. In these studies, we have synthesized and characterized a hexapeptide inhibitor of IL-8. This peptide, with an acetylated amino terminus and an amidated carboxyl terminus (Ac-RRWWCR-NH2), inhibited the specific binding of 125I-IL-8 to neutrophils. The inhibition was biphasic and apparent Ki was estimated to be approximately 2.7 microM and 13 microM for two different IL-8 binding sites. The peptide inhibited neutrophil chemotaxis, beta-glucuronidase release from neutrophils, and rabbit skin edema induced by IL-8 with an EC50 of 90 microM, 0.8 microM, respectively. Ac-RRWWCR-NH2 also suppressed the binding of macrophage inflammatory protein (MIP) 2 beta to neutrophils. However, it did not inhibit the binding of MIP-1 alpha, C5a, or leukotriene B4 to neutrophils, chemotaxis induced by FMLP, or beta-glucuronidase release induced by FMLP, C5a, or leukotriene B4. Additional peptides were analyzed to identify a better inhibitor. Inhibition of binding by Ac-rrwwcrc-NH2 synthesized with all D-amino acids was almost four times more potent than Ac-RRWWCR-NH2. Small peptide homologues of the amino-terminal end of IL-8 failed to inhibit IL-8 binding to neutrophils. These studies have identified several peptides that significantly inhibit IL-8 function. Because IL-8 seems to be an important inflammatory mediator of several human illnesses, these peptides may have pharmacologic potential.

Amino Acid Sequence↗

PubMed1995

Physical crosslinking of collagen fibers: comparison of ultraviolet irradiation and dehydrothermal treatment.

The strength, resorption rate, and biocompatibility of collagenous biomaterials are profoundly influenced by the method and extent of crosslinking. We compared the effects of two physical crosslinking methods, ultraviolet irradiation (UV) (254 nm) and dehydrothermal (DHT) treatment, on the mechanical properties and molecular integrity of collagen fibers extruded from an acidic dispersion of type I bovine dermal collagen. Collagen fibers exposed to UV irradiation for 15 min had ultimate tensile strength (54 MPa) and modulus (184 MPa) values greater than or equivalent to values for fibers crosslinked with DHT treatment for 3 or 5 days. UV irradiation is a rapid and easily controlled means of increasing the mechanical strength of collagen fibers. Characterization of collagen extracted from the crosslinked samples by dilute acetic acid and limited pepsin digestion indicate that both UV and DHT treatments cause fragmentation of at least a portion of the collagen molecules. Partial loss of the native collagen structure may influence attachment migration, and proliferation of cells on collagen fiberbased ligament analogs. These issues are currently being addressed in our laboratory.

Acetates↗

PubMed1995

High yields of interleukin-8 produced by a synthetic gene expressed in Escherichia coli and purified with a single antibody affinity column.

We developed a highly efficient expression system for the production of interleukin-8 (IL-8) in Escherichia coli. A synthetic gene used in the vector was designed to code for the 72-amino-acid form of IL-8 and incorporate additional new restriction sites. IL-8 was expressed in very large amounts in the periplasmic space and extracted by a gentle method which did not utilize denaturants. About 69% of the protein extracted from the periplasmic space was properly processed IL-8. A single anti-IL-8 monoclonal antibody affinity chromatography column yielded homogeneous IL-8 as determined by HPLC molecular sieve chromatography and amino-terminal sequencing. Between 14 and 22 mg of IL-8 was purified per liter of bacterial culture, in which the wet weight of E. coli was 7.6 g/liter. The recombinant IL-8 was fully active compared to published data and a commercially available preparation of recombinant IL-8. Our IL-8 and the commercial product had identical neutrophil binding isotherms, chemotactic activities, and enzyme release properties.

Amino Acid Sequence↗

PubMed1995

The intrinsic and extrinsic ligaments of the wrist. A correlation of collagen typing and histologic appearance.

20 fresh frozen human wrist specimens were dissected. Gross and histological examination and biochemical evaluation were performed on the intrinsic ligaments (scapho-lunate and luno-triquetral) and extrinsic ligaments (radio-scapho-capitate and radio-lunate). All ligaments were observed to have longitudinal collagen bundles. The intrinsic ligaments had large amounts of fibrocartilage near insertions and no elastin, while the extrinsic ligaments had little fibrocartilage and sparse amounts of elastin. The intrinsic ligaments were noted to have significantly more collagen Type 3 (41%) than the extrinsic ligaments (19%). These findings suggest that structural differences between the intrinsic and extrinsic ligaments of the wrist may in part account for the biomechanical observations that the intrinsic ligaments are stronger and elongate further prior to failure than the extrinsic ligaments.

Cadaver↗

PubMed1995

An anti-interleukin 8 monoclonal antibody that interferes with the binding of interleukin 8 to cellular receptors and the activation of human blood neutrophils.

Interleukin 8 (IL-8) is a proinflammatory cytokine produced by a wide variety of cells. Interleukin 8 acts as a neutrophil activator and chemotactic factor. In the current studies, we examined the properties of a monoclonal antibody against human IL-8. The estimated affinity of the antibody was 1.74 x 10(7) liters/mol. The antibody interfered with the binding of radiolabeled recombinant human IL-8 (rhIL-8) to human blood neutrophils (IC50 = 3 x 10(-7) M, at an IL-8 concentration of 2.4 nM). Neutrophil degranulation elicited by 5 x 10(-6)-4 x 10(-8) M rhIL-8 was blocked by the antibody at three-fold molar excess. However, a higher concentration of anti-IL-8 antibody was needed to suppress the chemotactic activity of rhIL-8. The inhibition of neutrophil chemotaxis triggered by 2 x 10(-7)-2 x 10(-9) M rhIL-8 required 6 x 10(-5) M antibody. Similarly, a 300-fold molar excess of anti-IL-8 antibody [10(-5) M] was necessary to abrogate the increase in cytosolic free calcium in neutrophils stimulated with 4 x 10(-8) M rhIL-8. In addition, epitope analysis using synthetic peptides corresponding to different regions of the IL-8 molecule showed that peptide consisting of residues 44-72 (corresponding to the C-terminal of the IL-8 molecule) competed with the antibody for binding to rhIL-8. Because IL-8 is an important inflammatory mediator in several human diseases, anti-IL-8 antibodies may have pharmacological potential.

Antibodies, Monoclonal↗