Specificity of antibodies against Neisseria gonorrhoeae that stimulate neutrophil chemotaxis. Role of antibodies directed against lipooligosaccharides.

Five strains each of Neisseria gonorrhoeae sensitive or resistant to complement (C) dependent killing by normal human serum (NHS) were examined for their ability to stimulate chemotaxis of polymorphonuclear leukocytes (PMNs) after preincubation with NHS; or IgM or IgG derived from NHS. Serum-sensitive N. gonorrhoeae stimulated C-dependent chemotaxis when opsonized with IgM, but not IgG, however, serum-resistant strains, taken as a whole, failed to promote chemotaxis when opsonized with either isotype. IgM titers in NHS against lipooligosaccharide (LOS) antigens from individual serum-sensitive, but not serum-resistant strains, correlated with the magnitude of chemotaxis generated by the corresponding opsonized strains (r = 0.99). Western blots demonstrated that IgM and IgG from NHS recognized different antigenic determinants on LOS from serum-sensitive gonococci. IgM from NHS immunopurified against serum-sensitive LOS accounted for two-thirds of the chemotaxis promoting activity present in whole serum. IgG titers in NHS against LOS antigens from individual serum-resistant strains also correlated with magnitude of chemotaxis generated by the corresponding opsonized strains (r = 0.87), although most opsonized serum-resistant strains did not generate significantly higher magnitudes of chemotaxis than controls. In contrast, a serum-resistant isolate from a patient with disseminated gonococcal infection (DGI) stimulated chemotaxis when opsonized with IgG obtained from the patient's convalescent serum. By Western blot, convalescent IgG antibody recognized an additional determinant on serum-resistant LOS not seen by normal IgG.

Gonococcal interactions with polymorphonuclear neutrophils: importance of the phagosome for bactericidal activity.

Gonococci are capable of attaching to the surface of polymorphonuclear leukocytes (PMN). In this location they resist phagocytosis and are not killed by PMN. To delineate the factors involved in the survival of these gonococci, we investigated the interaction of virulent gonococci, which adhere to cells and resist phagocytosis, and avirulent gonococci, which are phagocytized and killed by PMN. In the presence of serum, both virulent and avirulent gonococci associate equally well with PMN and stimulate increases in oxidative metabolism. In the absence of serum virulent gonococci attached to PMN and stimulated PMN oxidative metabolism to a greater extent than avirulent gonococci which did not attach to PMN (P = 0.0009). Therefore, the survival of virulent gonococci attached to the PMN surface is not a result of failure to activate oxidative and bactericidal mechanisms. Both virulent and avirulent gonococci stimulated equivalent PMN specific granule release as measured by the appearance of lactoferrin in the media. Phagocytosis of avirulent gonococci stimulated significantly greater beta-glucuronidase release (P = 0.01) and myeloperoxidase-mediated iodination of protein (P = 0.001) by PMN than attachment of virulent gonococci. In the absence of serum neither type of gonococci stimulated beta-glocuronidase release or protein iodination by PMN. Thus, virulent gonococci fail to stimulate primary granule release by PMN. To further assess the role of attachment versus ingestion on the survival of gonococci, PMN were treated with cytochalasin B to block ingestion. Cytochalasin B-treated PMN were unable to kill either virulent or avirulent gonococci despite normal degranulation stimulated by the latter. The failure of PMN to kill surface-attached gonococci appears to be a consequence of the failure of PMN to enclose the virulent gonococci within a phagosome. The phagocytic vacuole thus plays a critical role in normal PMN bactericidal activity by providing a closed space in which the proper concentration of substances may be achieved to generate microbicidal activity.

Two types of dysfunctional eighth component of complement (C8) molecules in C8 deficiency in man. Reconstitution of normal C8 from the mixture of two abnormal C8 molecules.

Restoration of hemolytic activity was examined in sera from seven unrelated eighth component of complement (C8)-deficient subjects. The sera fell into two groups, depending on whether hemolytic activity was restored by the addition of the beta-chain (group 1) or the alpha-gamma-subunit (group 2) purified from normal human C8. Antigenic analysis of these sera by double-immunodiffusion using anti-human C8 confirmed previous findings of a dysfunctional C8 in the four sera of group 1 and established the presence of a different dysfunctional C8 in one of the sera of group 2 when tested at a high concentration. Further characterization of the dysfunctional C8 molecules in the two sera by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that group 1 sera were missing the beta-subunit and group 2 sera were missing the alpha-gamma-subunit of the C8 molecule. Sera from either of these two groups alone did not produce hemolysis in hemolytic plates containing sheep erythrocytes coated with antibody and complement components up to C7 (EAC1-7) and C9. When sera from the two groups were added to adjacent wells in the hemolytic plates, a zone of hemolysis developed between the wells. The contribution of C8 alpha-gamma from the sera of group 1 and of C8 beta from those of group 2 to the lysis of EAC1-7 in the presence of C9 was confirmed by the inhibitory effect of specific antibodies against the two C8 subunits. In experiments in which hemolytic activity was reconstituted by mixing sera from group 1 with sera from group 2, the serum source of C8 beta (group 2) was the limiting reagent. The dysfunctional C8 molecule in this serum was able to bind to EAC1-7. Chromatographic analysis demonstrated that the generation of hemolytic activity in the mixture of the two sera resulted from the reconstitution of the C8 molecule rather than the sequential action of the two C8 subunits.

Group G streptococcal bacteremia. The clinical experience at Boston University Medical Center and a critical review of the literature.

Twenty-six patients with bacteremia caused by group G streptococci were seen during a 29-month period. Our findings suggest that the epidemiology of these infections has not changed appreciably during the last 15 years. The disease occurs most commonly as an acute community-acquired infection in older persons with underlying conditions predisposing to skin breakdown. Patients with these bacteremias fall into three categories: those with associated cutaneous infection only; those with serious focal infection; and those with probable infection. Underlying neoplastic conditions were relatively uncommon (25%) in our series compared with others (65%). In contrast to recent reports emphasizing this complication, endocarditis was uncommon in our patients. A prompt response to therapy with beta-lactam antibiotics occurred in most of our patients.

Beta chain deficiency in three patients with dysfunctional C8 molecules.

Structural and functional studies were performed on a dysfunctional C8 molecule present in the serum of two siblings and an unrelated individual. The C8 in these three sera exhibited a pattern of partial immunologic identity with C8 in normal serum but was devoid of functional activity. The C8 was immunoprecipitated from the three sera and from a control serum with an antihuman C8 antiserum and analyzed by SDS-PAGE using highly purified human C8 as a reference. A selective absence of a band of 62,000 mol. wt was observed in the immunoprecipitates from the sera containing dysfunctional C8. Experiments performed with the purified alpha-gamma and beta subunits showed that the hemolytic activity of the C8 deficient sera could be reconstituted by the addition of the beta chain but not the alpha-gamma dimer. Binding of the dysfunctional C8 to C567 was excluded by the following observations: (1) EAC1-7 treated with the C8 deficient sera and then washed could not be lysed after the addition of the beta subunit and C9; and (2) the abnormal molecules did not interfere with the consumption of normal C8 by the soluble complex SC5b-7.

Complement deficiency states and infection: epidemiology, pathogenesis and consequences of neisserial and other infections in an immune deficiency.

Inherited deficiencies of the complement proteins are rare in unselected populations. Examination of patients with the clinical correlates of complement deficiency (autoimmune disease and certain bacterial infections) shows the frequency of inherited complement deficiency to rise enormously (5.9% of patients with systemic lupus erythematosus, 10 to 25% of adults with sporadic meningococcal disease). Autoimmune diseases of all types, but especially systemic lupus erythematosus, discoid lupus and glomerulonephritis, are seen in all categories of complement deficiency, most typically in those of the early classical pathway (C1, C4, C2). Pneumococcal infections are characteristic of deficiencies of the early classical pathway, as well. Deficiencies of C3 are associated with severe disease including autoimmune phenomena, pneumococcal and neisserial infections. C3-deficient patients become ill substantially earlier in life. Infections with N. meningitidis and N. gonorrhoeae are most typical of the late component deficiencies, with over 40% of homozygotes affected. Despite the presence of this deficiency from birth and the peak age-specific incidence of meningococcal disease in the general population at ages 3-8 months, the median age of first infection in the late component-deficient patients is 17 years. Relapse of infection is ten times more common in these patients, and discrete recurrences are seen in 45% of affected individuals. An unusual and unexplained predilection for infection with serogroup Y N. meningitidis exists. Despite an immune deficiency, and problems with ascertainment bias, it appears that persons with late component complement deficiency enjoy less mortality than normals who contract meningococcal disease. Attempts to explain the pathogenesis of neisserial infection in late component deficiencies have focused on the concept that normally non-pathogenic serum-sensitive bacteria are etiologic in the absence of serum bactericidal activity. Data to support this concept remain to be developed and contrary data exist. A separate mechanism may predispose properdin-deficient patients to meningococcal infection, since they appear to develop fulminant infections with high mortality.

Complement deficiency states and meningococcal disease.

Analysis of complement deficiency states has supported the role of complement in host defense and elucidated diseases associated with defective complement function. Although neisserial infection plays a prominent role in these deficiency states, examination of individuals with late complement component deficiency (LCCD) reveals a particular propensity for recurrent meningococcal disease and provides important clues to the role of complement in neisserial infections. In response to meningococcal disease, LCCD individuals produce significantly greater amounts of antilipooligosaccharide (LOS) antibody which can kill group B meningococcus in a complement-sufficient in vitro system. Further studies of antibody cross-reactivity to other meningococci has led to a clearer understanding of its epitopic specificity. Nevertheless, epidemiologic evidence is consistent with the relative absence of protective immunity in LCCD persons following an episode of infection and supported by quantitation of antibody to capsular polysaccharide. However, compared to anti-LOS antibodies, anticapsular antibodies can offer immune protection to LCCD individuals via complement-dependent opsonophagocytosis--the only form of complement-mediated killing available to these persons. Thus vaccination of LCCD persons with capsular antigens is considered an important means of protecting these high-risk individuals against meningococcal disease.

Activation of leukocytes with complement C5a is associated with prostanoid-dependent constriction of large arteries in atherosclerotic monkeys in vivo.

Activated leukocytes release a variety of substances which have been shown in vitro to modulate vascular tone. The chemotactic peptide complement C5a is a physiological activator of leukocytes. We injected human recombinant complement C5a (10 and 100 micrograms) into the blood-perfused hind limb of normal and atherosclerotic cynomolgus monkeys and examined vascular responses. In both normal and atherosclerotic monkeys, the high dose of C5a produced about 65% decrease in leukocyte cell count in venous blood drainage from the hind limb. Injection of C5a produced a pronounced increase in resistance of large arteries (segment from iliac artery to dorsal pedal artery) in atherosclerotic, but not in normal monkeys. The constrictor effect of C5a in atherosclerotic monkeys was abolished by the thromboxane A2 receptor antagonist SQ 29,548 (2 mg/kg i.v.). The platelet-activating factor antagonist WEB 2086 (5 mg/kg, i.v.) did not alter vascular responses to C5a. We conclude that activation of leukocytes produces constriction of large arteries in atherosclerotic, but not normal, monkeys in vivo. This response may be mediated in part by release of thromboxane A2.

A sandwich ELISA for properdin in clinical specimens.

We have developed a symmetrical sandwich ELISA for measuring human properdin (P) in serum by using the globulin fraction from a commercial antiserum as the capture antibody adsorbed on the plastic. The detecting reagent was a glutaraldehyde conjugate of this Ig fraction with alkaline phosphatase. Two types of inhibition were observed in this study. First, inhibition was observed when greater than 2.5 micrograms/ml of the globulin fraction was used to coat the plates. A second type of inhibition was observed for serum dilutions less than 1/400; it was independent of the concentration of capture Ab and did not occur when purified P was assayed. The data generated with this assay could be fitted in log-log mode by a quadratic equation. The coefficient of the linear term in this equation was found to be the same for serum and purified P, within the limits of experimental error. The results for different samples run on the same plate were expressed in terms of the relative concentration of each sample required to produce an OD405 = 0.2. A sample of pooled normal human serum was run on each plate as a reference; it was assigned a titer of 100 ELISA units/ml (EU/ml). The titers of the unknown samples were expressed in terms of EU/ml by reference to this standard. For purified P, the assay could readily detect 10 ng/ml. By comparing purified P with our reference serum pool, we found that 1 EU equals 0.57 microgram. Day-to-day variation for a group of nine normal sera showed a mean difference of -0.85 EU/ml, SD 5.85 EU/ml. The mean titer for these normal sera was 78.9 EU/ml, SD 15.7 EU/ml. In three recovery experiments in which purified P was mixed with pooled normal serum, the recoveries ranged from 96 to 117%. We conclude that the sandwich ELISA constitutes an adequate immunochemical assay for human P in serum specimens.

Antineutrophil antibodies in infectious mononucleosis.

Antineutrophil antibodies were detected in the serum of 12 of 14 patients with acute Epstein-Barr virus-induced infectious mononucleosis. These antibodies were present in two patients with mononucleosis and severe neutropenia, and in 10 patients with mononucleosis uncomplicated by severe neutropenia. Antineutrophil antibodies were not detected in serum from five patients with cytomegalovirus-induced mononucleosis, or five renal allograft recipients with primary or reactivation Epstein-Barr virus infection. This study suggests that antineutrophil antibodies are among those produced by the polyclonal activation induced by Epstein-Barr virus during acute infectious mononucleosis.

Posthospital needs of elderly people at home: findings from an eight-month follow-up study.

In a study of 737 elderly hospital patients discharged to their homes in suburban areas northwest of Chicago, Illinois, 60 percent were assessed as needing help with personal care or housekeeping. Only 19 percent were referred by the hospital to community service agencies and, in the immediate postdischarge period, a large proportion of help in both personal care and housekeeping was given by relatives. Eight months after discharge, however, the proportion of care provided by relatives had decreased and the proportion of paid help had increased. The use of help at both points in time was strongly related to limitations in the basic activities of daily living (ADL) at time of hospital discharge. Many patients were unaware of available community services, and 64 percent said that no one in the hospital had talked with them about managing at home. These findings indicate the need for rethinking criteria for hospital discharge planning, more effective communication between service providers and patients, and community focus of attention on elders coming home from the hospital.

Use of out-of-plan services by Medicare members of HIP.

Use of out-of-plan services in 1972 by Medicare members of the Health Insurance Plan of Greater New York (HIP) is examined in terms of the demographic and enrollment characteristics of out-of-plan users, types of services received outside the plan, and the relationship of out-of-plan to in-plan use. Users of services outside the plan tended to be more seriously ill and more frequently hospitalized than those receiving all of their services within the plan. The costs to the SSA of providing medical care to HIP enrollees are compared with analogous costs for non-HIP beneficiaries, and the implications for the organization and financing of health services for the aged are discussed.

Student health policies of U.S. medical schools.

BACKGROUND: Medical students are at risk of exposure to bloodborne pathogens, yet few data are available about U.S. medical schools' policies to protect students. METHOD: A cross-sectional survey of the student affairs deans at the 126 U.S. medical schools was conducted in May 1994. A confidential questionnaire inquired about policies regarding vaccination for hepatitis B virus (HBV), blood and body-fluid exposures, universal precautions training, and health and disability insurance for students. RESULTS: A total of 108 (86%) of the schools participated in the survey. Most (99, 92%) required either HBV vaccination, evidence of immunity, or a signed waiver refusing vaccination. Nearly all (94, 87%) required health insurance, and almost all (101, 94%) offered a plan (at a mean cost of $690 annually), but fewer schools (69, 64%) offered disability insurance. The schools frequently held students responsible for the costs of HBV vaccination (73, 68%), postexposure serologic testing (22, 20%), and treatment of training-related medical problems (43, 40%). CONCLUSION: Most medical schools comply with current recommendations for preventing training-related exposures to bloodborne pathogens, illness, and injury, but students face a substantial financial responsibility for these services at a time when many have large debts. Many schools do not have disability insurance readily available for students. Medical schools should review their student health policies to protect students adequately.

A generalizability study of a new standardized rating form used to evaluate students' clinical clerkship performances.

PURPOSE: To investigate the measurement characteristics of standardized clinical evaluation forms (CEFs) used to assign grades for clerkship performance. METHOD: In 1996-97, the authors reviewed 5,168 CEFs completed for 175 students in eight clerkships. Limiting their analysis to the three clerkships that produced the most CEFs, the authors conducted a generalizability study to determine the five variance components for each clerkship. A decision study then calculated the generalizability coefficients and standard errors of measurement in each clerkship for varied numbers of raters and CEF items. RESULTS: The generalizability study found large variance components attributable to rater and rating context. The decision study found that, when three or more raters completed CEFs for a student, the generalizability coefficient and standard error of measurement reached levels acceptable for grading. Increasing the number of items on the CEF had no significant effect. CONCLUSION: The reliability of assigning students clerkship grades based on single CEFs is unacceptably low. However, CEFs can accurately measure students' clerkship performances if completed by three or more raters.

Teaching a screening musculoskeletal examination: a randomized, controlled trial of different instructional methods.

PURPOSE: To develop and test a program to teach a rapid screening musculoskeletal examination. METHOD: In 1995, 191 medical and physician assistant students were randomized to four intervention groups: written materials only (n = 47), written materials and videotape (n = 46), written materials and small-group sessions facilitated by fourth-year medical students (n = 55), and all three methods (n = 43). Assessments, in the form of a written test and standardized patient examinations, were conducted before the interventions (n = 40 randomly selected students), seven to ten days and again three months after the interventions (n = all 191 students), and 16 months after the interventions (n = 103 students). RESULTS: While the four intervention groups' written test scores were approximately equal, their scores on the standardized patient examination differed significantly. The students taught in small groups demonstrated significantly superior examination skills compared with the students taught with written material or videotape at seven to ten days and retained this relative superiority after three and 16 months (p < .0001). CONCLUSION: Small-group instruction with hands-on supervised practice is superior to more passive instructional methods for teaching musculoskeletal examination skills and can be successfully delivered by trained senior medical student facilitators with minimal direct expenditure of faculty time.

Is the AIDS epidemic ending?

In summary, current projections appear to underestimate the magnitude and course of the HIV epidemic because they are based on the end result of the infection rather than new infections. This concern is true at the local and the national level. Accurate planning requires that the level of and changes in the prevalence of HIV seropositivity in a random sample of the state and/or national population be determined at periodic intervals. Coupling these data to an assessment of the prevalence of affected individuals at various stages of HIV infection and the rate of progression from one stage to the next is critical to projections of the future magnitude and timing of expenditures for the various health care services.