Biomedical subjects
Publications and source records attributed to Q N Myrvik.
Whether or not bacterial populations are massively enclosed in slime, it appears that antibiotic resistance, when compared to suspension organisms, is related to surface adhesion and to the specific material of the substratum. These findings are of significance in the understanding and treatment of biomaterial-localized infections.
Osteomyelitis and intra-articular infection are septic diseases that present pathogenic features characteristic of molecular mechanisms involving adhesion to substrata. In this review, mechanisms of microbial adhesion to bone and cartilage as substrata are presented and related to host tissue response and to antibiotic treatment.
The comparative abilities of various reagents to prime rabbit alveolar macrophages (AM) to produce reactive oxygen intermediates (ROI) in a chemiluminescent (CL) assay were investigated. It was noted that AM from normal rabbits cultured in a serum-free medium for 18 hr exhibited a "spontaneous" priming response following a challenge with phorbol myristate acetate (PMA); however, "spontaneous" priming was not evident when the AM were cultured for only 3 hr. It was further established that pretreatment of normal AM for 3 or 18 hr with MIF/MAF preparations (serum-free), fetal bovine serum (FBS), or bovine serum albumin (BSA) exhibited marked increases in their CL responses following challenge with PMA. When FBS was used in the culture medium, the priming activity of MIF/MAF was masked because of the high CL responses of controls due to the priming effects of FBS. BSA at concentrations approximately equivalent to the amount in FBS also displayed marked priming activity. Bacterial products (lipopolysaccharide and muramyl dipeptide), latex particles, rabbit IgG, PMA, and opsonized as well as nonopsonized zymosan and bacteria (BCG and Staphylococcus epidermidis) were inactive as priming agents. In comparison, AM from BCG-immune rabbits that were primed in vivo yielded a very large CL response when challenged with PMA. Opsonized zymosan and bacteria produced twofold increases in the CL responses in BCG-immune AM compared to nonopsonized preparations. The marked priming effect of serum on AM cultured for even a short period (3 hr) indicates that normal AM undergo marked changes in culture that complicate the interpretation of AM function when AM are cultured in vitro in media containing serum.
The effect of L-fucose on the priming of AM from normal adult rabbits and their subsequent chemiluminescent (CL) responses to phorbol myristate acetate (PMA) was investigated. It was observed that 12.5 mM L-fucose, but not D-fucose, blocked the "spontaneous" priming of normal AM during an 18-hr incubation period in serum-free RPMI 1640 medium by about 40% (P less than 0.01) as detected by their CL responses to a PMA challenge. In addition, the optimal concentration of L-fucose (12.5 mM) blocked MIF/MAF priming during the 3- or 18-hr incubation period by 71 or 93% (P less than 0.05), respectively, as determined by their CL responses following PMA challenge. It is of particular significance that D-fucose was inactive. These results, together with previously published data, indicate that L-fucose (a) blocks priming of AM for an oxidative response, (b) stimulates random migration of AM, and (c) reverses migration inhibition produced by migration inhibition factor.
The MBCs of nafcillin, vancomycin, gentamicin and daptomycin (LY146032) were determined for three clinical isolates of coagulase-negative staphylococci grown in suspension and adherent to biomaterials. Strains studied were the slime-producing strain Staphylococcus epidermidis RP-12 (ATCC 35983), S. hyicus SE-360, and the non-slime-producing strain S. hominis SP-2 (ATCC 35982). All three strains were allowed to colonize surgical-grade disks of stainless steel, polymethylmethacrylate, and ultrahigh-molecular-weight polyethylene for 24 h, and the disks were then exposed to various concentrations of antibiotics for 24 h. Surviving adherent bacteria were mechanically dislodged from the disks and quantitated by standard broth dilution plating techniques. Biomaterial-adherent RP-12 and SE-360 yielded approximately 10 times more CFU per disk than non-slime-producing SP-2 did. For all organisms, 10 times more bacteria bound to polymethylmethacrylate disks than to the other biomaterials. In general, bacteria adherent to biomaterials exhibited greater resistance to antibiotics than the same strains in suspension did. Resistance was independent of bacterial slime-producing characteristics and was related to the biomaterial colonized.
We have investigated the possible role of pulmonary surfactant in mediating a deficiency in alveolar macrophages (AM) from infant animals to produce chemiluminescent (CL) responses elicited by phorbol myristate acetate (PMA) or opsonized zymosan (Op-Zym). Freshly harvested AM from rabbits 7 to 28 days of age produced much reduced PMA-elicited CL responses compared to 5- to 6-month-old rabbits. Initially we observed that AM from both infant and adult rabbits exhibited increasing PMA- or Op-Zym-elicited CL responses in a time-dependent manner when they were incubated in RPMI 1640 medium without serum for more than 3 h. In addition, AM from infant rabbits acquired the capacity to become primed with macrophage activation factor (MAF) containing supernatants or fetal bovine serum (FBS) during an 18-h incubation period that was basically equivalent to that of AM from adult rabbits. Our studies reveal that natural and artificial surfactant inhibited "spontaneous" priming as well as priming of AM induced by MAF-containing supernatant or FBS for producing PMA- or OP-Zym-elicited CL responses from both normal infant and adult animals. In addition, these surfactant preparations also suppressed the response of AM from adult BCG-sensitized rabbits during the course of an 18-h incubation period. It is especially noteworthy that AM from infant rabbits appeared to be more susceptible to the inhibitory activity of surfactant than AM from adult animals. Our data indicate that freshly harvested AM from infant rabbits are under greater suppression than AM from adult rabbits in terms of their capacities to produce oxidative responses.(ABSTRACT TRUNCATED AT 250 WORDS)
Bacterial slime produced in mass cultures of the RP 12 strain of Staphylococcus epidermidis was extracted with 4 M guanidine-HCl plus 0.05 M sodium acetate and 0.5% CHAPS, concentrated, dialyzed, and subjected to separation on DEAE sephacel columns. Three fractions, I-2A, I-2B, and I-4, were eluted with linear gradients of NaCl. Fractions I-2A and I-2B were alcian blue positive, whereas I-4 was alcian blue negative but the most electronegative fraction. The crude polysaccharide fraction and the three purified fractions were incubated individually for 2.5 or 20 h with normal rabbit alveolar macrophages (AM) to determine their effect on a subsequent PMA-induced oxidative burst. The crude fraction (50-200 micrograms/mL) and I-2B (50-200 micrograms/mL) primed the AM to give approximately a threefold increase in the PMA-induced burst after 2.5 h incubation. In contrast, a 20-h incubation resulted in a 30-40% inhibition of the PMA-induced burst with AM incubated with the same concentrations of the crude, I-2A, or I-2B fractions. Fraction I-4 had no detectable effect. The fractions also were tested to determine if they could elicit an oxidative burst in BCG-immune AM. None of the fractions (up to 500 micrograms/mL) elicited a significant oxidative burst even though BCG-immune AM yielded a PMA-induced burst 100 times that observed with normal resident AM. These data suggest that slime from S. epidermidis can impair the PMA-induced oxidative burst of normal AM during a 20-h incubation period and could explain in part why host defenses are compromised by slime-producing S. epidermidis.
The data presented in this communication demonstrate preferential colonization of certain biomaterials by Staphylococcus epidermidis. Using a laminar flow biomaterial colonization chamber and surgical-grade biomaterials (stainless steel, aluminium ceramic, methyl methacrylate and high-density polyethylene), the pattern of colonization was quantitated using plate count techniques and electron microscopy. Under comparable conditions, methyl methacrylate was colonized by S. epidermidis in greater numbers than the other biomaterials. Increased bacterial colonization and slime production on methyl methacrylate was time-dependent and 15 times higher than on stainless steel and aluminium and four times higher than on high-density polyethylene. The data reveal that certain biomaterials may promote infection by favouring colonization by potential pathogens. This variable should be explored extensively in an in vivo setting because of its implication in clinical infections.
Chemiluminescence (CL) response of alveolar macrophages (AM) exposed to either phagocytosable or nonphagocytosable particles of polymethylmethacrylate (PMMA) or titanium in vitro was monitored. The results were correlated to known in vivo bone tissue compatibility of the materials. Moreover, the quantity and morphology of adherent AM on titanium and PMMA disks were assessed using scanning electron microscopy (SEM). Our results with PMMA indicated that a high CL response to phagocytosable as well as nonphagocytosable particles of the biomaterial in question, correlated with poor bone tissue compatibility. By contrast, a low CL response to phagocytosable particles and no response to nonphagocytosable particles correlated with a satisfactory bone tissue compatibility, as in the case of titanium. SEM showed 48% more AM adherent to PMMA than titanium per unit area. Also, the percentage of cells with membrane damage was three times higher on PMMA than on titanium. It appears that the CL response of AM exposed to biomaterials in vitro thus can be used as an easy to do screening test for potential orthopaedic implant materials before in vivo tests are considered.
While medical scientific knowledge pertaining to bacterial adhesion to biomaterials has become a rapidly growing field in most areas of medicine, its significance in ophthalmic infections has not been emphasized. Corneal bacterial ulceration in patients wearing extended-wear contact lenses has become a problem of epidemic proportions. The designation of the contact lens itself as a suitable substratum for bacterial colonization and as a source of subsequent inoculum to compromised epithelial cells are important factors in the pathophysiology of corneal ulcer formation. We demonstrate polysaccharide- (biofilm-)mediated adhesion to two ophthalmic pathogens (Pseudomonas aeruginosa and Staphylococcus epidermidis) to the surface of a typical extended-wear contact lens in vitro using cytochemistry and scanning and transmission electron microscopic techniques. This interaction between the biomaterial and bacterial organisms, which represents a favorable self-protective environment for propagation and inoculation, is a previously overlooked area of importance in the mechanism of corneal ulceration associated with hydrophilic (soft) contact lenses.
The effect of selected monosaccharides on the random migration of normal adult rabbit alveolar macrophages (AM) was investigated. It was observed that 10 mM of L-fucose, L-galactose, or D-mannose stimulated AM migration 1.5-2.0 times. In addition, derivatives of L-fucose and D-mannose occupying the carbon-6 position such as L-fucosyl-lactose, D-mannose-6-phosphate, D-mannitol, and mannan enhanced the migration of AM, whereas derivatives of L-fucose and D-mannose in the carbon-1 position produced no migration enhancement. Macrophage migration enhancement activity that was produced spontaneously by spleen cell cultures from normal young rabbits was destroyed by treatment with L-fucosidase. Accordingly, the migration enhancement factor (MEF) found in spleen cell culture supernatants appeared to depend on L-fucose conjugated to some protein carrier because MEF was non-dialyzable. When normal adult AM were treated with L-fucosidase, they lost their responsiveness to migration inhibitory factor (MIF) but retained their responsiveness to MEF. We have interpreted this to mean that the MIF and MEF receptors are distinct. Synthetic MEFs were prepared by conjugating L-fucose, D-mannose, of L-galactose to bovine serum albumin (BSA). It was noted that these sugar-BSA conjugates were about 200 times more effective than the corresponding free sugars in producing migration enhancement. In addition, these sugar-BSA conjugates neutralized MIF activity in a migration inhibition test.
Our data establish that migration inhibition factor (MIF) and migration enhancement factor (MEF) mutually neutralize the effect of each other in a concentration-dependent manner. The monosaccharides L-fucose and D-mannose were also shown to reverse MIF and additionally to stimulate alveolar macrophage (AM) migration in the absence of MIF. The specific activity of these sugars was increased 200-fold when conjugated to bovine serum albumin (BSA). Macrophage activation is usually observed concurrently with migration inhibition when macrophages are incubated with MIF preparations. Migration inhibition occurred also when AM were incubated in the presence of known metabolic activators (MDP, PMA, and LPS). It was found that L-fucose, D-mannose, L-fucosyl BSA, and D-mannosyl BSA could reverse migration inhibition caused by MIF as well as by these metabolic activators. These observations suggest that reversal of MIF by L-fucose is unexplained solely on the basis that L-fucose is functioning as a competitive inhibitor; instead, they suggest that MEF and the above sugars and their conjugates stimulate AM through a receptor system different from the MIF receptor. These observations support the concept that MEF is an important macrophage modulator in CMI responses.
This study addresses the problem of antibiotic resistance in adhesive, biomaterial-centred infections. It is suggested that this anionic, extracapsular, polysaccharide slime produced by bacteria protects them from antibiotics and sequesters critical ions from the surface of biomaterials. Biofilm-enclosed bacteria on the surface of stainless steel substrata in a test chamber were challenged with incremental levels of tobramycin. In this setting, the minimum inhibitory concentration and minimum bactericidal level of tobramycin for Staphylococcus epidermis were well above normal.
Macrophage endocytosis is part of the immune response to foreign material and includes the processes of phagocytosis, ingestion of particulate material, and pinocytosis, the uptake of liquid droplets. This work reports the development of a quantitative method to measure in vitro uptake of neutral red by fish macrophages, based on the spectrophotometric measurement of ingested neutral red dye. We have used this method to monitor the rates of uptake in three species of fish: spot, Leiostomus xanthurus; hogchoker, Trinectes maculatus; and summer flounder, Paralichthys dentatus. For the species tested, there appeared to be an intrinsic pattern of uptake which was highest in flounder, lowest in hogchoker and intermediate in spot. The excellent reproducibility of this assay should make it useful for detecting toxicological effects in the environment.
Macrophage migration enhancement factor (MEF), a lymphokine produced in the spleen by suppressor-like lymphoid cells, may be an important immunoregulatory molecule of macrophage function. MEF appears to be a potent positive chemokinetic factor and is unusual in that it lacks chemotactic activity. To aid in the development of a purification scheme for MEF we have employed biophysical characterization techniques to define its physical properties. Using the technique of velocity sedimentation in isokinetic sucrose gradients, the S20w for MEF was determined to be 2.25. The Stoke's radius for MEF was determined by Sephadex G-100 gel filtration to be 28.9 A. From these measurements the D20w was calculated to be 7.55 x 10(-7) cm2/sec, the mol. wt was calculated to be 28,000, the frictional ratio (f/f0) was calculated to be 1.45, the axial ratio was calculated to be 1:8, and the dimensions of the molecule were estimated to be 20 x 160 A. Using the technique of isoelectric focusing in liquid density gradients, the isoelectric point for MEF was estimated to be 8.8. We have also determined by enzyme treatment that MEF is resistant to DNase and RNase and susceptible to proteinase K and L-fucosidase. In addition, MEF partitioned to the aqueous phase during methanol-chloroform extraction procedures. MEF was inactivated at pH 12; at 100 degrees C MEF was stable for 10 min but was inactive after 1 hr. Collectively, these data will facilitate the development of a purification scheme for MEF which will ultimately permit the analysis of the molecule and its function.
Monoclonal antibodies (McAb), designated AMH1 (IgM, lambda) and AMH2 (IgG1, Kappa), against specific surface antigens of human lung macrophages were produced by the fusion of the NS-1 plasmacytoma cell line with spleen cells from BALB/c mice immunized with bronchoalveolar lavaged (BAL) cells obtained from selected smoking subjects. The screening and characterization of these McAb were carried out employing cellular radioimmunoassay, flow cytofluorography, and immunohistochemical methods. These two antibodies specifically reacted with macrophages in the alveolar spaces and BAL fluids. AMH1 did not react with peripheral blood cells including freshly separated monocytes, cultured monocytes, lymphocytes, granulocytes, and platelets. In addition, AMH1 did not react with peritoneal exudate cells or pleural exudate cells. On the other hand AMH2 showed the dull-positive reaction with some monocytes and pleural exudate cells among above-mentioned cells. These two McAb seemed to detect cell surface antigens that are expressed by highly differentiated or mature macrophages compared to OKM1. These antibodies will allow not only better characterization of immune cells but also assessment of maturity of lung macrophages.
Luminol-dependent chemiluminescence (CL) responses of alveolar macrophages (AM) from normal and Mycobacterium bovis BCG-vaccinated infant and adult rabbits were compared. AM from 1-, 7-, and 14-day-old normal rabbits exhibited much lower peak CL responses than did AM from 28- and 42-day-old normal animals as well as rabbits 2 to 3 or 5 to 6 months and 1 to 2 years of age. The most striking differences among AM from infant and adult rabbits were noted when AM were obtained from 28-day-old and 5- to 6-month old rabbits 21 days after the rabbits were immunized with 200 micrograms of BCG intravenously. In this case, AM from 5- to 6-month-old animals gave peak counts per minute of 400,000 to 500,000 whereas AM from 28-day-old rabbits vaccinated with BCG (harvested at 49 days of age) gave peak counts per minute of only 40,715 +/- 2,688. These data reveal that AM from neonatal animals are grossly deficient as responders to phorbol myristate acetate-induced CL. This deficiency, which improved with age, is still apparent in AM from 28-day-old animals. The data also reveal that BCG vaccination of 28-day-old animals yields AM that are poor responders to phorbol myristate acetate compared with AM from BCG-vaccinated animals 2 to 3 and 5 to 6 months of age. AM from animals vaccinated with BCG at 28 days of age contained fewer and smaller electron-dense lysosomelike structures than did AM from adult rabbits similarly vaccinated. These findings provide an explanation for the difficulties infants have in developing effective cell-mediated immune responses against intracellular parasites.
This study was undertaken to define the age-related alterations in lipid composition that resident rabbit alveolar macrophages (AM) undergo during postnatal development. The eventual goal is to correlate these changes with the functional maturation of these cells. The number of AM recorded from total lung lavages rose markedly during the first 14 days of life, from 4.9 X 10(5) to 1.1 X 10(7). Adult lungs yielded 1.1 X 10(8) AM. A gradual but significant increase in fluorescence polarization (P) was observed during development when purified AM plasma membranes were tagged with the probe 1,6-diphenyl-1,3,5 hexatriene trimethyl ammonium. The rise ranged from a mean P value of less than or equal to 0.22 to 0.24 (p less than 0.001) for AM plasma membranes from rabbits 1- or 7-day-old to 30- or 150-day-old rabbits, respectively. This finding suggests that the fluidity of the AM plasma membrane decreased during postnatal development. Palmitic, stearic, oleic, and linoleic acids were the most prevalent fatty acids found in the neutral lipid fraction of the AM plasma membrane throughout development. The content of stearic acid rose from 10 to 16%, arachidonic acid rose from 2.8 to 9%, myristic acid decreased from 3.2 to 1.3%, palmitic acid decreased from 42 to 36%, and oleic and linoleic acids changed relatively little during the first 30 days of life. The levels of docosatetraenoic and docosapentaenoic increased gradually during the first 14 days of life, and by 30 days of life the levels declined to that observed at birth. The sum of these changes resulted in an increase in the ratio of unsaturated to saturated fatty acids (1 to 1.15) in the neutral lipid fraction. During the first month of life, the neutral lipid fatty acid pool in the total lipid fraction of AM plasma membrane increased from 12 to 18 mole %, cholesterol increased from 7 to 14 mole %, and total phospholipids decreased from 81 to 67 mole %. These changes resulted in increasing the cholesterol to phospholipid ratio from 0.09 at birth to 0.23 by 150 days of life. The levels of all three major lipid fractions were comparable at 30 days and 150 days of life. Adult levels of choline phosphoglycerides, the predominant phospholipid, were observed by 7 days of life to have decreased from 47 to 34.5 mole %, and the levels of ethanolamine phosphoglycerides and sphingomyelin increased from 17.5 to 25 mole % and from 9 to 13 mole %, respectively. Adult levels of lyso-bis-phosphatidic acid were reached by 30 days of life increasing from 8.2 to 17.8 mole %.(ABSTRACT TRUNCATED AT 400 WORDS)