Mechanisms of clearance from the peritoneal cavity.
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Animals↗
Biomedical subjects
D L Dunn
Publications and source records attributed to D L Dunn.
Lessons of the New Jersey DRG payment system.
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Cost Control↗
High-performance liquid chromatographic analysis of epinephrine enantiomers using a UV detector in series with an optical activity detector.
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Chromatography, High Pressure Liquid↗
Enhanced survival during murine gram-negative bacterial sepsis by use of a murine monoclonal antibody.
We developed a murine monoclonal antibody (5B10 MAb) that reacted in vitro specifically to lipopolysaccharide (LPS) obtained from Escherichia coli 0111:B4. Enzyme-linked immunosorbent assay (ELISA) titers to a variety of gram-negative bacterial whole cell and LPS antigens demonstrated that this antibody may react with the O antigen portion of 0111:B4 LPS. We then examined the ability of this antibody to protect mice in vivo against a challenge of either viable bacteria or purified LPS. One milligram of 5B10 MAb was administered intraperitoneally (IP) and protected against a lethal challenge of either viable E coli 0111:B4 or 0111:B4 LPS, but no other type of bacterial or LPS challenge. Protection occurred in an antibody dose-dependent manner, and as little as 0.01 mg of 5B10 MAb enhanced survival. We concluded that IP pretreatment with a single MAb would protect against lethal sepsis or endotoxemia in this animal model and that anti-LPS specificity was a sufficient condition for an antibody to protect during bacteremia, confirming the importance of LPS in the pathogenesis of gram-negative bacterial sepsis.
Animals↗
Metabolic effects of pretreatment with Escherichia coli J5 antiserum on guinea pig gram-negative bacterial sepsis.
Antiserum raised to a rough mutant Escherichia coli, termed J5 (anti-J5 RS), protected against lethal gram-negative bacterial sepsis in a guinea pig model when animals were pretreated with both antiserum and heparin. This same model was used to examine and compare the effects of pretreatment with anti-J5 RS, normal rabbit serum (NRS), or saline, each +/- heparin on physiologic and metabolic parameters during a septic insult. Results demonstrated that leukopenia and thrombocytopenia occurred to a similar degree in all pretreatment groups; no significant leuko- or thrombostasis was noted on examination of histologic specimens; complement activation was maximal in those animals receiving anti-J5 RS alone without heparin; the most abnormal amino acid profile was present in the NRS + heparin group; and only the anti-J5 RS + heparin group did not develop glomerular lesions indicative of disseminated intravascular coagulation. A complement-mediated cell aggregation-type injury does not appear to occur in this model. It is hypothesized that both excessive complement and coagulation system activation occur after bacterial challenge when antibody directed against the bacteria is present (anti-J5 RS) leading to antigen-antibody complex formation and complement and coagulation cascade activation. Heparin may block either or both these cascade systems allowing enhanced, antibody-mediated opsonization and clearance of blood-borne bacteria, thus preventing end-organ alterations and organ failure during sepsis when combined with anti-J5 RS.
Amino Acids↗
Effects of Escherichia coli and Bacteroides fragilis on peritoneal host defenses.
Escherichia coli and Bacteroides fragilis are common copathogens in clinical intra-abdominal sepsis, yet it is unclear how they interact synergistically in vivo. We sought to determine whether E. coli and B. fragilis, in combination but not alone, could exert a detrimental effect on the peritoneal host defenses of translymphatic absorption and bacterial phagocytosis. Our data indicated that nonviable E. coli (O18ab:K56/K7:- and O111:B4), Klebsiella pneumoniae, B. fragilis, and Bacteroides thetaiotaomicron were handled in a similar fashion by both host defenses of the peritoneal cavity. The use of 2 X 10(8) nonviable radiolabeled E. coli as a tracer and either 2 X 10(9) B. fragilis or 2 X 10(9) E. coli (either viable or nonviable) as a competing agent to inhibit host defenses demonstrated that although clearance and phagocytosis could be inhibited, the inhibition occurred to a similar degree with either E. coli or B. fragilis. Thus, B. fragilis did not compete to any greater extent than E. coli did for peritoneal clearance or opsonization and phagocytosis in vivo. These data indicate that bacterial synergy probably does not occur on the basis of reduced peritoneal clearance or by a reduction in the opsonization and phagocytosis of either organism by the copathogen. These results provide indirect support for the hypothesis that in bacterial synergy, one organism directly stimulates the growth of the other, perhaps by providing a growth factor.
Animals↗
Role of resident macrophages, peripheral neutrophils, and translymphatic absorption in bacterial clearance from the peritoneal cavity.
Microbial pathogens within the peritoneal cavity are thought to encounter three categories of host defense mechanisms: (i) removal mechanisms, which occur via diaphragmatic lymphatic absorption; (ii) killing mechanisms, in which host phagocytes act as effector cells; and (iii) sequestration mechanisms due to fibrin trapping and the formation of adhesions between visceral surfaces. We sought to define and quantitate the relative role of the first two components in an experimental rat model of Escherichia coli peritonitis in which fibrinous adhesions do not form. Intraperitoneal challenge with greater than or equal to 2 X 10(8) CFU of viable E. coli led to an initial decline in bacterial numbers followed by ongoing proliferation and greater than 50% mortality. With inocula of less than or equal to 5 X 10(7) CFU, elimination of bacteria occurred after moderate initial proliferation, and no mortality ensued. Nonviable, radiolabeled E. coli organisms were utilized to examine bacterial clearance via translymphatic absorption and phagocytosis. Both processes were extremely rapid, serving to eliminate free bacteria rapidly within the peritoneal cavity. Although macrophages and polymorphonuclear leukocytes within the peritoneal cavity demonstrated similar phagocytic capacities, the predominance of macrophages at the time of the initial bacterial insult led to the conclusion that these cells, in addition to translymphatic absorption, represent the first line of host defenses, acting to eliminate bacteria in the incipient stages of infection.
Animals↗
Efficacy of type-specific and cross-reactive murine monoclonal antibodies directed against endotoxin during experimental sepsis.
To study the role of antibodies in promoting survival during gram-negative bacterial sepsis, we have developed several murine monoclonal antibodies (MAbs). One MAb (5B10) reacted in an enzyme-linked immunosorbent assay with only a single organism (Escherichia coli 0111:B4), while the other (8A1) reacted to all gram-negative whole-cell and lipopolysaccharide (LPS) antigens examined. Either 5B10 MAb, 8A1 MAb, or sterile saline solution was administered intravenously to outbred male Swiss-Webster mice immediately before one of three challenges: (1) viable bacteria intravenously, (2) viable bacteria with hemoglobin intraperitoneally, or (3) intravenous actinomycin D plus LPS. 5B10 MAb provided significant protection against either an E. coli 0111:B4 bacterial or LPS challenge but not against any other organism or type of LPS. 8A1 MAb provided protection against several challenge bacteria (intravenously or intraperitoneally) and against all types of LPS studied except Pseudomonas aeruginosa LPS. A higher dose (2 mg) of cross-reactive antibody (8A1 MAb) was required to produce protection when compared with the type-specific protection produced with 5B10 MAb (0.1 mg). Although ideal antibody therapy would consist of directing a specific MAb against a single microorganism, the acute nature of the disease process and time required to prepare reagents may preclude the use of type-specific MAbs. We believe that the cross-reactive and cross-protective capacity of 8A1 MAb or a similar MAb may be useful in averting the lethal effects of clinical gram-negative bacterial sepsis and warrants testing in the clinical setting.
Animals↗
Fibrin in peritonitis. IV. Synergistic intraperitoneal infection caused by Escherichia coli and Bacteroides fragilis within fibrin clots.
We measured the rate of lethality and abscess formation in rats that underwent intraperitoneal implantation of fibrin clots contaminated with either Escherichia coli or Bacteroides fragilis alone or in combination, to determine whether the two organisms together would produce a synergistic infection. Ten-day mortality produced by 10(9) colony-forming units (CFU) of E coli was 33.3%. Encapsulated B fragilis led to 3.3% mortality. Escherichia coli (5 X 10(8) CFU) plus B fragilis (5 X 10(8) CFU) led to a sharp increase both in the rate and final ten-day mortality (80.0%). Eighty percent of the rats that received E coli (10(9) CFU within fibrin clots) had abscesses determined on the basis of grossly purulent material. All animals that received B fragilis and survived ten days contained abscesses. Synergy between E coli and B fragilis was noted to occur only when 5 X 10(8) CFU of each organism was present within the fibrin clot. Lower numbers did not produce significant synergy compared with controls that received either E coli or B fragilis. Quantitation of the number of organisms present at 24 hours within contaminated fibrin clots demonstrated a similar amount of growth of both organisms, either when added alone or in combination as copathogens.
Abscess↗
Comparative pulmonary effects of intraperitoneal inoculation of live v dead Escherichia coli.
We studied the effect of 2.5 X 10(9) live Escherichia coli per kilogram v 2.7 X 10(9) dead E coli per kilogram injected into the peritoneal cavity of sheep with chronic pulmonary lymph fistulas. The effects of dead E coli were compared with those of live E coli, with respect to (1) pulmonary hypertension, (2) hemodynamic failure, (3) damage to the pulmonary microvasculature, (4) systemic arterial hypoxemia, (5) neutropenia and lymphopenia, (6) thrombocytopenia and platelet aggregation, (7) plasma fibrinogen concentration, and (8) classic- and alternative-pathway hemolytic complement. The time after injection of the bacteria was divided into an early period (zero to two hours) and a late period (two to seven hours). We made two conclusions: (1) The early period effects, with the exception of the absolute neutrophil count and Pao2, were independent of bacterial viability, whereas the late period effects were strongly dependent on bacterial viability. (2) The early notable difference between the live v dead groups, with respect to the absolute neutrophil count and Pao2, could not be explained on the basis of an increase in bacterial numbers alone.
Animals↗
Association of concurrent herpes simplex virus and cytomegalovirus with detrimental effects after renal transplantation.
Herpes simplex virus (HSV) and cytomegalovirus (CMV) infections occur frequently after renal transplantation. To determine the effect of these infections on long-term prognosis, we reviewed the charts of 558 patients who underwent primary renal transplantation between Jan 1, 1974 and Dec 31, 1978, at the University of Minnesota Hospitals, Minneapolis, for cultural evidence of these infections. The presence of HSV alone appeared to have a minimal effect on either patient or allograft survival. Positive CMV cultures were associated with decreased patient and allograft survival. Patients with HSV and CMV infections had further reduced patient and allograft survival. Lethal CMV syndrome and associated severe bacterial and fungal infections were common and frequently were the immediate cause of death in patients who had concomitant positive HSV and CMV cultures. Concurrent HSV and CMV infections in renal transplant patients may be a grave prognostic indicator, being associated with enhanced mortality and renal allograft loss.
Cytomegalovirus Infections↗
The adjuvant effect of peritoneal fluid in experimental peritonitis. Mechanism and clinical implications.
At laparotomy, many surgeons routinely instill crystalloid solutions into the peritoneal cavity, presumably to dilute out necrotic debris, bacteria, and adjuvant substances which foster bacterial growth. We examined the effect on mortality, bacterial growth, clearance, and phagocytosis of various volumes of saline instilled into the peritoneal cavity of rats during Escherichia coli peritonitis. Minimal intraperitoneal bacterial growth was seen after the introduction of a nonlethal inoculum of viable E. coli in 1 ml of saline, while administration of an identical inoculum in 30 ml of saline intraperitoneally (i.p.) led to increased 48-hour mortality (p less than 0.01), and associated rapid bacterial proliferation (p less than 0.01). Clearance of nonviable radiolabelled E. coli from the peritoneal cavity was delayed, bacterial association with host peritoneal leukocytes was decreased, and blood uptake of radiolabelled bacteria was diminished in animals receiving 30 ml of saline i.p., compared to controls which received the identical inoculum in 1 ml of saline i.p. The clinical relevance of these studies is manifold: (1) they provide a possible explanation why patients with ascites due to cirrhosis or the nephrotic syndrome, or those patients undergoing peritoneal dialysis are more susceptible to primary and secondary bacterial peritonitis, possibly on the basis of impaired peritoneal clearance or diminished phagocytosis and, (2) although irrigation of the peritoneal cavity with crystalloid solution would seem prudent during laparotomy, these solutions must be removed prior to closure to prevent interference with normal peritoneal host defense mechanisms.
Adjuvants, Immunologic↗
Anticore endotoxin F(ab')2 equine immunoglobulin fragments protect against lethal effects of gram-negative bacterial sepsis.
Gram-negative bacterial sepsis and shock remain a cause of substantial morbidity and mortality in hospitalized patients despite appropriate antimicrobial therapy, fluid resuscitation, and monitoring. We sought to test the ability of equine antibody directed against core endotoxin, a portion of bacterial outer membrane lipopolysaccharide common to many gram-negative microorganisms, to bind to various gram-negative bacteria in vitro, to promote bacterial phagocytosis by leukocytes, and to protect against lethal gram-negative bacteremia in mice. The importance of the IgG Fc leukocyte attachment site was examined by comparing the ability of intact IgG and IgG F(ab')2 fragments to protect against lethality during murine sepsis. A single horse was immunized with Escherichia coli J5, an organism that expresses a portion of core endotoxin extensively on the cell surface. Preimmunization IgG and F(ab')2 possessed no titer as determined by enzyme-linked immunosorbent assay, did not promote in vitro phagocytosis, and did not protect in vivo. Postimmunization IgG and F(ab')2 possessed a significant titer to E. coli J5 whole cell and lipopolysaccharide antigens and provided significant (p less than 0.05) protection in vivo during lethal intravenous sepsis caused by either E. coli J5, E. coli 0111:B4, Klebsiella pneumoniae, or Pseudomonas aeruginosa. Only postimmunization IgG, but not F(ab')2, promoted in vitro phagocytosis of these same organisms. We therefore hypothesized that protection occurred as a result of antitoxin activity rather than opsonization and phagocytosis, as F(ab')2 fragments were as active as the intact molecule. Further studies must be done to determine the role of anticore endotoxin antibody in conjunction with antibiotics so that appropriate clinical studies may be undertaken.
Animals↗
Analysis of chlorobutanol in ophthalmic ointments and aqueous solutions by reverse-phase high-performance liquid chromatography.
A reverse-phase high-performance liquid chromatographic assay for chlorobutanol was developed and found suitable for the routine analysis of ophthalmic ointments and aqueous solutions. The method utilized a column packed with 10-microns octadecylsilane with a mobile phase of methanol-water (50:50). Peak detection was by UV absorption at 210 nm. In this chromatographic system, chlorobutanol had a capacity factor (K') of 4.1. Standard curves obtained in the presence of ointment vehicle containing an aminoglycoside were linear, intercepted at zero, and averaged 99.4% recovery. Degradation studies indicated that the method was stability indicating. The analytical results for a complete experimental ophthalmic ointment and an aqueous ophthalmic diluent are presented. This high-performance liquid chromatographic method of analysis represents an alternative to GC procedures for determining chlorobutanol.
Chlorobutanol↗
The role of heparin in guinea pig gram negative bacterial sepsis.
The ability of antibody directed against shared antigenic determinants of gram negative organisms to protect against a challenge of diverse gram negative bacterial species remains controversial in the experimental setting. Attention has focused, however, on the use as immunogens of rough mutants of Escherichia coli and Salmonella minnesota, which express a portion of core lipopolysaccharide (LPS) extensively on their cell surface. Core LPS is a structure present on the outer membrane of most, if not all, gram negative bacteria. In this study rabbits were immunized with E. coli J5, a rough mutant of E. coli, to produce anti-E. coli J5 rabbit antiserum (anti-J5 RS). Anti-J5 RS was found to cross react extensively by enzyme-linked immunosorbent assay with various gram negative bacterial whole cell or LPS antigens, compared to normal rabbit serum (NRS). Anti-J5 RS +/- heparin was also compared to NRS +/- heparin pretreatment in a guinea pig model of sepsis utilizing E. coli O111:B4 as the challenge organism. Anti-J5 RS +/- heparin augmented systemic bacterial clearance compared to NRS +/- heparin, but only the combination of anti-J5 RS and heparin enhanced survival 48 hr after bacterial challenge. It was concluded that pretreatment with anti-J5 RS was a necessary, but not sufficient condition for enhanced survival, and that the addition of heparin to anti-J5 RS pretreatment might diminish the otherwise lethal consequences of complement activation and disseminated intravascular coagulation in this model system.
Animals↗
The meaning of research in experimental peritonitis.
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Animals↗
Mechanisms of the adjuvant effect of hemoglobin in experimental peritonitis. VI. Effects of stroma-free hemoglobin and red blood cell stroma on mortality and neutrophil function.
Hemoglobin is known to increase the lethality of experimental E. coli peritonitis, but its mechanism of action has not been defined. Some evidence from this laboratory previously suggested that hemoglobin could interfere with local leukocyte function in the peritoneal cavity. In the present study we compared the effect of impure bovine hemoglobin, highly purified stroma-free hemoglobin, and red blood cell stroma on the mortality rate of rats with Escherichia coli peritonitis, as well as on human neutrophil function in vitro. Both hemoglobin preparations markedly increased the mortality rate at low bacterial concentrations. Red blood cell stromal elements increased the rate to a minimal degree and only at high bacterial concentrations. The character of the E. coli-induced peritoneal leukocytosis was not significantly affected by concomitant administration of either stroma-free hemoglobin or red blood cell stroma. Although crystalline bovine hemoglobin inhibited in vitro leukocyte chemotaxis, centrifugation of this preparation to remove debris abrogated this effect. Stroma-free hemoglobin had no effect on in vitro leukocyte chemotaxis. Red blood cell stromal elements in high concentration diminished phagocytosis and bacterial killing by human neutrophils, but stroma-free hemoglobin had no effect on these neutrophil functions. The results confirm that hemoglobin is a potent adjuvant in experimental E. coli peritonitis, but contrary to our previous conclusion does not owe this effect to direct interference either with neutrophil influx into the peritoneal cavity during bacterial peritonitis or with human leukocyte function in vitro. Such prior conclusions appear to have been based on insoluble material including stromal elements in the hemoglobin preparations used. Red cell stromal elements derived from as little as 1 ml of packed red cells can significantly interfere with the phagocytosis and killing of E. coli.
Animals↗
Mechanisms of the adjuvant effect of hemoglobin in experimental peritonitis. VII. Hemoglobin does not inhibit clearance of Escherichia coli from the peritoneal cavity.
Hemoglobin has been shown to be a potent adjuvant in experimental Escherichia coli peritonitis, although a satisfactory mechanistic rationale is still obscure. Hemoglobin has been thought to impair intraperitoneal neutrophil function, delay clearance of bacteria from the peritoneal cavity by the normal absorptive mechanisms, or directly enhance bacterial growth. Using highly purified stroma-free hemoglobin (SFHgb), we have largely discounted any direct effect of hemoglobin on peritoneal white blood cell function. In the present study, we confirmed that uncontrolled proliferation of bacteria takes place in the presence of hemoglobin in the peritoneal cavity. Nonviable 5-iododeoxyuridine 125I-labelled bacteria were then used to directly study peritoneal clearance kinetics, eliminating the problem of bacterial growth. SFHgb had no influence on the removal of intraperitoneal bacteria. The rate of bloodstream appearance of radiolabel was similar with or without intraperitoneal SFHgb. Thus, SFHgb does not prevent clearance of bacteria from the peritoneal cavity by interfering with normal host clearance mechanisms. SFHgb may act as a bacterial growth adjuvant, either by serving as a bacterial nutrient or by suitably modifying the environment so that extensive bacterial proliferation can occur. The latter hypothesis appears to be an area in which investigation concerning the adjuvant effect of hemoglobin may prove most fruitful.
Animals↗