Complement-independent adherence of Escherichia coli to complement receptors in vitro.

We studied adherence to human cells by a strain of Escherichia coli. Adherence to erythrocytes was assessed directly by phase-contrast microscopy and indirectly by hemagglutination; adherence to peripheral blood leukocytes, using radiolabeled bacteria and subsequent determination of leukocyte-associated radioactivity; and adherence to renal glomeruli, by microscopy of fluoresceinated bacteria and of Gram-stained nonfluoresceinated bacteria. In serum-free systems, E. coli of this strain adhered to human erythrocytes, which have surface receptors for the third component of complement (C3), but not to erythrocytes from species lacking this receptor. 1 mM trypan blue, a reagent that inhibits complement receptor function, inhibited adherence to human erythrocytes, as well as adherence to leukocytes and glomeruli. Preincubation of erythrocytes and leukocytes with complement-coated zymosan particles partially blocked subsequent bacterial adherence. Incubation of human erythrocytes with aging human serum, with trypsin-cleaved C3, or with C3 cleaved by the classical pathway convertase (EAC142)-all of which treatments deposited C3 on the erythrocyte surface, presumably at C3 receptors-inhibited subsequent E. coli adherence. Finally, incubation of E. coli with rabbit antiserum to human C3 blocked adherence to erythrocytes.Bacterial hemagglutination and erythrocyte adherence were not inhibited by mannose in concentrations up to 2.5%. And this strain of E. coli did not adhere to or agglutinate guinea pig erythrocytes, the usual test particle used for demonstration of common pili. Finally, electron microscopy of adherent bacteria showed only rare surface pili. In contrast, adherence to and agglutination of guinea pig erythrocytes by a stock piliated E. coli was inhibited by mannose but not by trypan blue. We conclude that organisms of this strain of E. coli adhere to human erythrocytes, leukocytes, and glomeruli at complement receptors. Complement is not required for this interaction. Adherence apparently involves a C3-like structure on the bacterial surface, but bacterial surface pili play no role. The physiological or pathological role of this adherence is not apparent, but study of this phenomenon may elucidate functions of complement receptors on various cells.

An objective measure of clinical performance.

Studies repeatedly have shown the clinical performance of students and residents to be less than expected by faculty. Because evaluation methods substantially influence education, poor performance can be improved with better clinical evaluation methods. This study evaluated a standardized method to measure clinical performance in which trained actual and simulated patients were organized in a multiple-station format for efficient testing of examinees on 17 cases in less than four hours. Specific checklists completed by patients and predetermined scoring protocols yielded reliable data and reduced faculty time. Data from 204 students in three clerkships were consistent with previous research showing case specificity and substantial case-to-case variability. As a group however, the students' overall total scores were very similar. This suggests that clinical education is inconsistent and that a profile of an examinee's performance is more accurate than a single overall score. Validity of this standardized clinical examination was supported by significant but moderate correlations with faculty ratings of ward performance and the medicine subtest of the National Board of Medical Examiners test, part II. Direct per-student costs were $21.00. This standardized objective examination of clinical skills is feasible for use in training programs and will provide reliable and valid data on clinical performance not available through typical methods.

Physician work force in Texas: planning for the future.

A number of proposals have been advanced to solve the shortage of primary care physicians in the United States. We analyzed the supply of primary care and nonprimary care physicians in Texas and contrasted work force supply with projected need. Texas has a serious shortage of primary care physicians and an adequate supply of nonprimary care specialists only in major urban areas. We also analyzed attitudes of medical students in and graduates of The University of Texas medical schools to assess factors that influenced their career choices. The most influential factors were remarkably similar among all groups and were related primarily to personal attributes and experience. This study assesses the current physician work force in Texas and suggests means by which policy can be decided to affect the supply and distribution of physicians in Texas.

Effect of pneumococci on blood clotting, platelets, and polymorphonuclear leukocytes.

Infections due to Streptococcus pneumoniae and products from the organism have been associated with alterations in blood clotting and function of platelets. Pneumococci and pneumococcal polysaccharide shortened the clotting times of whole blood, platelet-rich plasma (PRP), and platelet-poor plasma (PPP) in vitro. Clotting times of PPP and PRP from C6-deficient animals were likewise decreased. The bacteria had no effect on the one-stage prothrombin time or the partial thromboplastin time when the organisms were used as activating agents. Platelets aggregated in the presence of pneumococci, but aggregation was prevented by the addition of cyclic adenosine 3', 5'-monophosphate (cAMP). Furthermore, cAMP corrected the shortened clotting time of PRP in the presence of pneumococci. The clumping and release of polymorphonuclear coagulant that was induced by pneumococci was not prevented by cAMP. Thus, pneumococci exert several dose-dependent thromboplastic effects: (i) release of platelet thromboplastic substances; (ii) a direct thromboplastic effect; and (iii) release of polymorphonuclear coagulant.

Coagulopathy in experimental sepsis with Streptococcus pneumoniae in rabbits: effect of drug therapy and splenectomy.

The cause of death in bacteremia due to Streptococcus pneumoniae remains unclear. The role of intravascular coagulation and splenectomy was investigated in rabbits with lethal pneumococcal bacteremia. The staphylococcal clumping titer in serum, a measure of fibrin degradation products, increased early and persisted until death. This titer correlated with the level of bacteremia. The partial thromboplastin time and platelet-rich plasma clotting time also increased as the disease worsened. However, the prothrombin time remained normal. 125I-labeled fibrinogen was cleared normally from the plasma of infected rabbits, whether intact or splenectomized. Similarly, the concentration of fibrogen in plasma remained normal, even though the level of fibrin degradation products increased, and no difference in these parameters was noted between intact and splenectomized rabbits. Fibrin deposition could not be detected in any of the organs studied. Neither the level of fibrin degradation products nor survival was affected by treatment with hydrocortisone, hexadimethrine, cytochrome c, carboxypeptidase B, epsilon-aminocaproic acid, or heparin. These data suggest that intravascular coagulation occurs in this experimental infection prior to the onset of shock but probably plays only a minor role in lethality.

Trypan blue inhibits complement-mediated phagocytosis by human polymorphonuclear leukocytes.

Trypan blue completely inhibited attachment of human polymorphonuclear leukocytes (PMN) to Sepharose beads coated with C3 ant to sheep erythrocytes coated with IgM plus C3, but it did not inhibit attachment to erythrocytes coated with IgG. These results suggested that trypan blue inhibited C-mediated attachment to PMN membranes. Corroborative studies were performed with a strain of Staphylococcus aureus that requires C but not antibody, for opsonization and that activates the alternative pathway. Trypan blue was not toxic to PMN or bacteria, did nto interfere with immunoglobulin or C interactions, and did not affect attachment of opsonins to bacteria. However, the dye impaired PMN killing of S. aureus in normal nonimmune serum by inhibiting bacterial attachment to and ingestion by PMN. Further evidence that the inhibition was at the C3 receptor level came from the observations that, 1) once staphylococci were attached to PMN at either 37 degrees C or 0 degrees C, addition of trypan blue did not inhibit killing; and 2) trypan blue inhibited killing of bacteria opsonized with serum sufficient in C but previously absorbed at 0 degrees C with the same strain of organism to deplete specific antibody. Further studies with this agent may elucidate the roles of opsonic receptors on human phagocytes.

The role of opsonins in recovery from experimental pneumococcal pneumonia.

The opsonization of type 1 and type 25 pneumococci in the rabbit lung early in the course of experimental pneumonia was studied. Bacteria washed from the lungs of animals with type 1 pneumonia were coated with IgG, IgA, and complement within 24 hr of infection; these bacteria were not phagocytized. Both type 1 and type 25 pneumococci, when incubatd in concentrates of nonimmune bronchopulmonary washings (BPC), adsorbed IgG, IgA, and C3, but neither type of pneumococci was opsonized. Killing of pneumococci by surface phagocytosis, with or without the use of nonimmune BPC, was not demonstrated. When nonimmune serum was used, opsonization of type 25 pneumococci depended on the presence of heat-labile adsorbable factors, and when immune serum and immune BPC were used, opsonization was dependent on adsorbable factors. Depletion of serum complement with cobra venom factor before infection with type 25 pneumococci resulted in severe bacteremic pneumonia in the rabbit, but immunization before complement depletion allowed animals to clear type 25 pneumococci from the blood and lungs within 24 hr. Thus, opsonization is required for efficient clearance of pneumococci from the lung, and in the nonimmune animal, complement is also required for clearance.

A comparative evaluation of text formats in a medical school course.

Information mapping, a system of instruction design for categorizing, sequencing and graphically presenting printed information for highly technical communication in business, seemed ideally suited for presenting the technical procedures of the general screening physical examination to medical students. To evaluate the impact of this new text format one half of the class received a conventional text for those procedures and the other half used an information mapped text. Cognitive test scores were not significantly different. However, the information mapped text was rated significantly more favorably on six dimensions. Students using the mapped text also spent significantly more time studying it than those using the conventional text.

Evidence for quantitative variability of bacterial opsonic requirements.

We studied human serum opsonins by using combinations of heat inactivation and chelation to inhibit complement, adsorption to remove antibody, and trypan blue to inactivate the C3 receptor of human polymorphonuclear leukocytes. Streptococcus pneumoniae, serotype 25, required both complement and immunoglobulin for opsonization, even though that strain activated the alternative complement pathway. Both strains of Escherichia coli required antibody and complement, but varied in the degree of dependence on the C3 opsonin, since trypan blue moderately inhibited the killing of E. coli-1 and markedly inhibited the killing of E. coli-2. Serratia marcescens was opsonized in heat-inactivated serum (limited complement) or serum absorbed at 0 degrees C with S. marcescens (limited antibody), but depended on the C3 receptor in absorbed serum. S. marcescens activated the alternative pathway. Thus, opsonic requirements varied with the availability of opsonins. Requirements for bacterial opsonization vary with species and strains within species, perhaps reflecting quantitative relationships among alternative and classical pathway activation of C3, efficiency of adsorption of C3 or immunoglobulin G to bacterial surfaces, and efficiency of attachment of these ligands to polymorphonuclear leukocyte receptors. Furthermore, although not always sufficient for opsonization, the C3 opsonin (activated through either the classical or alternative pathway) appears necessary for effective phagocytosis and killing of all strains studied.

Opsonization of pneumococci. II. Metabolic effects of a "third" human serum activity that mediates intracellular killing.

We investigated the mechanisms by which a serum activity, neither complement nor immunoglobulin, mediates killing of pneumococci by polymorphonuclear leukocytes (PMN). Electron microscopy revealed type 25 pneumococci to be within PMN when incubated in normal serum, in serum absorbed twice at 0 degrees C with type 25 pneumococci, or in absorbed plus heat-inactivated serum. Uptake of radiolabeled bacteria, and activation of oxygen consumption and of the hexose monophosphate shunt by PMN with pneumococci, were similar in normal serum, absorbed serum, or the combination of absorbed and heat-activated serum. Reduction of nitroblue tetrazolium (NBT) and of cytochrome c by PMN in the presence of type 25 pneumococci and absorbed serum, with or without heat-inactivated serum were one-third and one-half, respectively, of those in normal serum. Likewise, protein iodination was one-half that in normal serum. Reduction of cytochrome c by cytochalasin B-treated PMN was the same in normal, absorbed, or absorbed plus heat-inactivated serum. Furthermore, release of beta-glucuronidase from PMN after ingestion of pneumococci in 10% normal, absorbed, or absorbed plus heat-inactivated serum was identical. These data indicate that the "third" serum activity is not necessary for attachment of pneumococci to or ingestion by PMN, nor is it necessary for stimulation of the plasma membrane oxidase. Rather, it functions somehow in intracellular killing.

Therapy with hyperbaric oxygen for experimental osteomyelitis due to Staphylococcus aureus in rabbits.

Hyperbaric oxygen (HBO) is used as adjunctive therapy of chronic osteomyelitis, but its efficacy remains controversial. A recently developed rabbit model for osteomyelitis due to Staphylococcus aureus was used to compare the results of treatment with HBO, cephalothin, a combination of both, or no treatment. Cultures of bone were positive in 10 (91%) of 11 control animals (untreated), five (36%) of 14 animals treated with HBO, eight (47%) of 17 treated with cephalothin, and six (40%) of 15 treated with HBO plus cephalothin. All three treatment groups differed significantly from untreated controls in the number of positive cultures obtained (P less than 0.01), but there were no significant differences among treatment groups. In vitro growth and killing curves (1.0 microgram of cephalothin/ml) constructed after exposure to HBO revealed no change from parallel control studies in ambient air. These data demonstrate that therapy with HBO is at least as effective as antibiotic therapy. The therapeutic effectiveness of HBO does not appear to be related to antibacterial activity.

Trypan blue inhibition of complement receptor function on various cells.

Trypan blue has previously been shown to inhibit complement-mediated phagocytosis by interaction with the C3 receptor but not with the Fc receptor. In studies reported here, trypan blue inhibited EAC3 rosette formation with human polymorphonuclear leucocytes (PMN) and mononuclear cells, rabbit alveolar macrophages (AM) and peritoneal PMN, and guinea-pig spleen cells. Trypan blue also inhibited C3-mediated bacterial adherence to the same receptor-bearing cells and to human glomerular cells. EAC3bi rosette formation was also inhibited, but EAC3d rosettes were not detected in our assay system. Although the precise molecular nature of C3b fragments deposited on antibody-sensitized erythrocytes has not been fully determined, trypan blue probably inhibits all C3 receptors from different species and may prove useful in vivo and in vitro for the definition of C3-receptor function in various aspects of the immune response.

Analysis of humoral and phagocytic defenses against Streptococcus pneumoniae serotypes 1 and 3.

To better define relationships among pneumococcal anticapsular antibodies, opsonophagocytosis, and in vivo mouse protection, we measured these functions in sera from healthy individuals who had not received pneumococcal vaccine. For serotype 1 pneumococci, the level of antibody measured by radioimmunoassay did not predict mouse protection, as has been noted by others. For some sera, opsonic requirements for antibody and complement could be clearly demonstrated and a strong correlation obtained between concentration of antibody and degree of phagocytic killing. However, for most sera, antibody concentration did not correlate with opsonic activity, as measured by phagocytic bactericidal assay or uptake of radiolabeled bacteria. Sera with high concentrations of anticapsular antibody did not always support in vitro bacterial killing by leukocytes. Conversely, highly opsonic sera did not necessarily have substantial levels of measurable antibody. Moreover, in vitro opsonophagocytic activity failed to predict in vivo protection; sera could be opsonic in vitro but not protective in vivo and vice versa. For serotype 3 pneumococci, antibody concentrations correlated strongly with mouse protective titers, as has been noted by others for type 3. Opsonophagocytosis, as measured by leukocyte bactericidal activity, required both complement and heat-stable substance(s) present in high-antibody sera, presumably antibody. Furthermore, increasing concentrations of serum enhanced phagocytic killing in a fashion that could be correlated with anticapsular antibody content. However, correlation with opsonophagocytosis was not so strong as with mouse protection, and there was no correlation between antibody concentration and opsonization as measured by uptake of radiolabeled bacteria. These observations suggest that opsonophagocytosis (with the definitive end point of bacterial killing) cannot be the standard against which to measure antibody concentrations. Furthermore, host protective mechanisms against pneumococci remain to be clearly defined. Even if opsonization by anticapsular antibody is the primary mechanism, there is need for development of improved functional assays of protection.

A mechanism for the amelioration by hyperbaric oxygen of experimental staphylococcal osteomyelitis in rabbits.

Although hyperbaric oxygen (HBO) is as effective as cephalothin against osteomyelitis due to Staphylococcus aureus in the rabbit, the effect is not by directing killing. To investigate the mechanism, argon washouts (perfusion) and oxygen tensions were measured by intramedullary probes placed in the metaphyses of infected and uninfected tibias. In vitro phagocytic killing activity for S. aureus was determined at oxygen tensions found in these bones under ambient and HBO conditions. Mean tibial oxygen tensions (mm Hg) under ambient conditions were 21 (infected) and 45 (uninfected); under HBO conditions, 104 (infected) and 321 (uninfected). Perfusion was decreased in osteomyelitic bone and was not acutely increased by HBO in either normal or infected bone. Phagocytic killing of S. aureus was markedly decreased at 23 mm Hg of O2, significantly improved at 45 and 109 mm Hg, and most effective at 150 mm Hg. Thus, in osteomyelitic bone, HBO increased intramedullary oxygen to tensions consistent with normal phagocytic function.