Peter O'Hanley

Effects of anti-C5a antibodies on the adult respiratory distress syndrome in septic primates.

In vitro and in vivo studies have suggested that human complement component C5a plays a key role in neutrophil injury in the adult respiratory distress syndrome (ARDS). First, using leukocyte aggregometry, we demonstrated that the addition of a recently developed rabbit anti-human polyclonal antibody to C5a des arg to endotoxin-activated plasma prevented leukocyte aggregation in vitro. We then administered the anti-C5a des arg antibody to septic primates (Macaca fascicularis). Three groups of primates, control, septic, and anti-C5a antibody treated septic, were studied (n = 4 in each group). A 30-min infusion of Escherichia coli (1 X 10(10)/kg) resulted in severe sepsis and ARDS. Primates were killed 4 h after completion of the E. coli infusion. Septic animals not treated with anti-C5a antibody had 75% mortality (3/4), decreased oxygenation, severe pulmonary edema, and profound hypotension. Septic primates treated with anti-C5a antibodies did not die and did not develop decreased oxygenation (P less than 0.05) or increased extravascular lung water (P less than 0.05). They also had a marked recovery in their mean arterial blood pressure (P less than 0.05). This study demonstrates that treatment with rabbit anti-human C5a des arg antibodies attenuates ARDS and some of the systemic manifestations of sepsis in nonhuman primates.

Molecular basis of Escherichia coli colonization of the upper urinary tract in BALB/c mice. Gal-Gal pili immunization prevents Escherichia coli pyelonephritis in the BALB/c mouse model of human pyelonephritis.

Most human pyelonephritis Escherichia coli isolates express both mannose (MS)- and globoside (Gal-Gal)-binding pili. An ascending E. coli urinary tract infection model was established in the 16-wk-old female BALB/c mouse to compare the pathogenic significance of MS and Gal-Gal pili and their efficacy as vaccines for the prevention of pyelonephritis. The distribution and density of pilus receptor compounds in urogenital tissues and as soluble compounds in urine were determined with antibodies to the synthetic receptor analogues, alpha D-Gal(1----4) beta D-Gal and alpha D-Man(1----2) alpha D-Man. Both carbohydrates were detected in vagina, bladder, ureter, and renal pelvis epithelium and in collecting duct and tubular cells. A pilus receptor compound also was detected in urine. It competitively inhibited the binding capacity of MS pili and was found to be physically, chemically, and immunologically related to Tamm-Horsfall uromucoid. Infectivity and invasiveness were quantitatively and histologically characterized for four E. coli strains: J96, a human pyelonephritis strain that expresses both MS and Gal-Gal pili; two recombinant strains prepared from J96 chromosomal DNA encoding MS pili or Gal-Gal pili; and the nonpiliated K12 recipient. Intravesicular administration of J96 (10(6) colony-forming units [CFU]) resulted in renal colonization and invasion in each of nine mice. The Gal-Gal clone (10(6) CFU) colonized the kidneys in each of 10 mice but did not invade. In contrast, the MS clone (10(6) CFU) did not colonize renal epithelium or invade. This effect was superceded when larger doses (greater than or equal to 10(10) CFU) of the MS clone were administered in volumes that cause acute vesicoureteric reflux. The efficacy was determined of vaccines composed of pure MS or Gal-Gal pili or the lipopolysaccharide containing O somatic antigen of the challenge strain, J96. The Gal-Gal pilus vaccine blocked renal colonization in 19 of 22 mice and renal invasion in 10 of 11 mice. Gal-Gal pili may be useful immunogens for the prevention of pyelonephritis in anatomically normal urinary tracts.

Gal-Gal binding and hemolysin phenotypes and genotypes associated with uropathogenic Escherichia coli

To determine whether uropathogenic strains of Escherichia coli exhibit a distinctive constellation of phenotypes, we examined 44 urinary isolates from women with radiologically normal urinary tracts and pyelonephritis, cystitis, or asymptomatic bacteriuria and 73 fecal isolates from healthy control subjects. The strains were characterized by their O serogroup, by their binding specificity (as determined by adhesins), and by their production of hemolysin and colicin V. In addition, the strains were assessed for homologous gene sequences by means of DNA-hybridization probes prepared from cistrons that encode hemolysin and the Gal-Gal binding adhesin--two determinants of virulence, which cause tissue injury and promote bacterial colonization of uroepithelia, respectively. In contrast to most isolates from normal feces and from the urine of patients with asymptomatic bacteriuria, pyelonephritis strains belong to a small number of O serogroups; all express the Gal--Gal binding adhesin and 75 per cent are hemolytic. A gene probe for the Gal--Gal binding adhesin, derived from the chromosome of one strain from a patient with pyelonephritis, hybridized with the DNA of all other pyelonephritis strains. The probe for the hemolysin gene hybridized with DNA from all other hemolytic strains. These data indicate that most cases of pyelonephritis are due to a small number of pathogenic clones that express critical determinants of virulence, and that the nucleotide sequences for hemolysin and the Gal--Gal binding adhesin in heterologous strains share homology. We are tempted to speculate that the gene products of these shared regions of the genome might form the basis for a vaccine against pyelonephritis.

Complement levels in septic primates treated with anti-C5a antibodies

During gram-negative sepsis it is known that endotoxin activates complement by the alternate pathway. The complement anaphylatoxin C5a, a result of this activation, is thought to play a key role in attracting and activating neutrophils in the lungs, leading to the adult respiratory distress syndrome. Complement levels were measured in primates made septic by Escherichia coli infusions. Anti-human C5a antibodies were administered to study their effect on neutrophil-mediated lung injury. Control (I), septic (II) and septic + anti-C5a antibody (III) groups (n = 4) were studied. The antibody-treated group (III) demonstrated a significant attenuation of septic shock and pulmonary edema as has been previously reported. All complement profiles were corrected for varying hemoglobin concentrations. C3, C4, and C5 levels were measured by radial immunodiffusion and were depleted in both septic groups. Once the levels were depleted from the plasma, they did not recover. The depletion of C4 indicates that classical pathway activation also occurred. C3a, C4a, and C5a levels were measured by radioimmunoassay. Significantly increased peak levels were reached in the septic groups 15 min after initiation of the E. coli infusion. There were no significant differences in early peak C3a and C4a levels between groups II and III. However, the mean peak C5a level in group III (anti-C5a antibodies) was 42% lower than that in group II, and after this early peak, C5a levels were not elevated above control levels in group III. The antibody to human C5a was thus shown to be cross-reactive with primate C5a and was specific since C3a and C4a levels were not decreased in group III.(ABSTRACT TRUNCATED AT 250 WORDS)

Infectious disease management of adult leukemic patients undergoing chemotherapy: 1982 to 1986 experience at Stanford University hospital

PURPOSE The purpose of this study was to determine the recent incidence of infection and to evaluate antimicrobial usage among adult leukemic patients undergoing chemotherapy for acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) at Stanford University Hospital. PATIENTS AND METHODS The records of 142 adult patients from a consecutive series of 226 induction or consolidation/maintenance chemotherapy courses for AML or ALL between 1982 to 1986 were reviewed retrospectively. Data were analyzed to compare the infectious disease complications and antimicrobial usage for patients receiving identical chemotherapy for a specific phase of leukemia treatment. Evaluation for each chemotherapy course included assessments for the following: compliance with criteria for initiating antibiotics, incidence of infection that was documented by culture or clinical criteria, predictive value of surveillance cultures, incidence of superinfection, survival outcomes, antimicrobial usage, antibiotic-related adverse effects, and cost for antibiotics and diagnostic studies. RESULTS Antimicrobials were employed in 190 (84%) of 226 chemotherapy courses. Broad-spectrum antibiotics were regularly begun within the first five days of admission and they were continued for an average of 3.5 weeks until the granulocyte count was greater than 1,000/microL after discontinuation of chemotherapy. There were no differences in the types of infection or outcomes among the patient groups. There was only a 37% rate of documented infections by culture or clinical signs among these patients during their entire hospital stay. Bacterial infections, especially those caused by coagulase-negative staphylococci in patients with Hickman catheters, accounted for 93% of the episodes. Viral and fungal infections accounted for 4% and 3% of documented cases, respectively, and occurred more than 10 days after the institution of broad-spectrum antibiotic therapy. A total of 922 different antimicrobials were employed in 190 courses (average 4.9 per course). The rationale for excessive usage and multiple changes was a persistent or intermittent fever, rather than documented infection(s). This practice led to usage of more broad-spectrum and expensive antibiotics. Further analyses indicate that the greater number of antibiotics employed correlated with apparent increased toxicity, especially renal and hepatic adverse reactions. These toxicities were associated with higher rates of fatal outcomes, i.e., 12 (39%) of 31 patients died with antibiotic-associated hepatic and/or renal insufficiency, compared with 12 (7.5%) of 159 patients who died without antibiotic-associated organ damage. CONCLUSION Excessive antibiotic usage and multiple antibiotic changes among adult leukemic patients undergoing chemotherapy appear to increase the risks of adverse hepatic and renal effects and death. Furthermore, this practice leads to use of more broad-spectrum and expensive antibiotics...

Solubilization and characterization of functionally coupled Escherichia coli heat-stable toxin receptors and particulate guanylate cyclase associated with the cytoskeleton compartment of intestinal membranes

1. Particulate guanylate cyclase and receptors for E. coli heat-stable enterotoxin were solubilized from the rat intestinal cytoskeletal compartment using Lubrol-PX and KCl. 2. Thirty to forty percent of the ST receptor and guanylate cyclase activities were extracted from the lipid layer with Lubrol-PX alone. 2. Seventy percent of the remaining activities were solubilized from the cytoskeleton with Lubrol-PX and KCl. 3. Guanylate cyclase solubilized from either compartment exhibited similar reaction kinetics. 4. Both high- and low-affinity classes of ST receptors were solubilized from the lipid and cytoskeleton compartments. 5. In the presence of ATP gamma S, ST selectively activated the guanylate cyclase solubilized from the cytoskeleton compared to that solubilized from the lipid bilayer. 6. Crosslinking experiments demonstrated a preferential solubilization of the 130 kDa receptor subunit from the cytoskeleton and the 56 kDa subunit from the lipid bilayer. 7. Development of a procedure to solubilize ST receptors and guanylate cyclase from the intestinal membrane cytoskeleton will permit purification and further detailed studies of the coupling of these activities.

Rat intestinal cell atrial natriuretic peptide receptor coupled to guanylate cyclase

The present studies were initiated to determine if cells of intestinal origin possess the molecular components supporting a response to atrial natriuretic peptides. Specific binding in cultured rat ileal cells with 125I-labeled atrial natriuretic peptide was saturable and of high affinity. Scatchard analyses showed a single population of binding sites with a Kd of 2.1 nmol/L and a Bmax of 300 fmol/mg protein. Atrial natriuretic peptide activated particulate guanylate cyclase 5- to 10-fold in a concentration- and time-dependent fashion. The EC50 for activation of enzyme by atrial natriuretic peptide was 6 nmol/L. Accumulation of cyclic guanosine monophosphate stimulated by atrial natriuretic peptide was observed in the intracellular (25-fold) and extracellular (50-fold) compartments and was dependent on concentration and time. Half-maximum intracellular accumulation was observed with 10 nmol/L atrial natriuretic peptide. These data suggest a role for atrial natriuretic peptides in the gastrointestinal tract.