A cluster of meningococcal disease on a school bus following epidemic influenza.

An outbreak of meningococcal disease among children on a school bus offered the opportunity to study a proposed association between this infection and preceding influenza infection. Five students who rode the bus became ill with invasive group C meningococcus. Transmission was limited to the bus; there was no evidence for school transmission. All five students reported influenza-like symptoms within several weeks before the development of meningococcal disease. School absenteeism, principally due to upper respiratory tract illness, was higher during the 3 weeks before the outbreak of meningococcal disease than during any period in the preceding 3 1/2 years, suggesting an unusually severe outbreak of respiratory illness. A case-control study comparing students with and without influenza symptoms revealed that the outbreak of respiratory disease was due to B/Ann Arbor/1/86 influenza (geometric mean titers, 86 for 80 patients and 33 for 47 controls [P = .0007]). These data add to the evidence suggesting that influenza respiratory infection predisposes to meningococcal disease.

The roles of vaccination and amantadine prophylaxis in controlling an outbreak of influenza A (H3N2) in a nursing home.

An outbreak caused by influenza A/Philippines/2/82 (H3N2)-like viruses occurred in a partially vaccinated nursing home population in January 1985. During the first six days of the outbreak, 14 (25%) of 55 residents developed influenzalike illness. The risk of illness was most strongly associated with undetectable levels of antibody against the epidemic strain, with unvaccinated case-patients having more severe illnesses and a higher rate of hospitalization than vaccinated case-patients (5/8 vs 0/6). During the period of amantadine hydrochloride prophylaxis (100 mg/d) from days 7 to 35, only two (5%) of the remaining 41 residents became ill, even though 11 (27%) had no detectable antibody. Serum amantadine levels obtained on day 35 ranged from 117 to 737 ng/mL (mean 309 ng/mL), similar to therapeutic levels documented in younger adults who have taken the standard regimen of 200 mg/d; there were few clinically significant side effects. These findings illustrate the benefits of influenza vaccination and support the use of amantadine hydrochloride at a dosage of 100 mg daily for outbreak control among elderly persons.

Hemodynamic and biochemical alterations in dogs with lymphoma after induction of chemotherapy.

BACKGROUND: Doxorubicin is a common antineoplastic agent with dose-dependent cardiotoxic adverse effects, and pre-existing myocardial dysfunction is a contraindication to its use. OBJECTIVES: To systematically define the hemodynamic and biochemical alterations in dogs undergoing chemotherapy for newly diagnosed lymphoma and assess the reversibility of these alterations with fluid administration. ANIMALS: Twenty-one client-owned dogs with newly diagnosed lymphoma were evaluated 1 week after induction of chemotherapy. Underlying degenerative valve disease was exclusionary. Eighteen healthy age- and weight-matched dogs were used as controls. METHODS: Physical examination, blood pressure by Doppler, echocardiography, and biochemical evaluation (routine serum biochemistry, plasma renin activity and aldosterone concentrations, plasma and urine osmolalities, and urine electrolyte concentrations) were measured in dogs with lymphoma and compared to controls. Dogs with lymphoma received crystalloids IV at 6 mL/kg/h for 24 hours. All variables were reassessed at 4 and 24 hours. Deuterium oxide dilution and bromide dilution were used to determine total body water and extracellular water space, respectively. RESULTS: Baseline echocardiograms showed significantly smaller chamber dimensions in dogs with lymphoma compared to controls. These changes were reversed by fluid administration. Systolic blood pressure and urine sodium concentration were significantly increased, and bromide dilution space, PCV, urine specific gravity, and urine potassium concentration were significantly decreased compared to controls. CONCLUSION AND CLINICAL IMPORTANCE: Echocardiographic and biochemical abnormalities in dogs with lymphoma appear consistent with volume depletion, and may be the result of systemic hypertension and subsequent pressure natriuresis.

Effect of opioids on CXCL-8 production in healthy cats.

The aim of this study was to evaluate the effect of opioid exposure on CXC chemokine ligand (CXCL)-8 production in cats using whole blood culture. Morphine, buprenorphine, fentanyl, and saline control were administered intravenously to five cats and whole blood pathogen associated molecular pattern motif-induced CXCL-8 production capacity was evaluated. Morphine potentiated CXCL-8 production. To further characterize this effect of morphine, morphine was incubated with whole blood ex vivo and pathogen associated molecular pattern motif-induced CXCL-8 production capacity was measured. There was a time and concentration dependent effect on CXCL-8 production, suggesting the proinflammatory effect of morphine is at least partially mediated by direct stimulatory effects on leukocytes. Additional investigation is indicated to assess the implications of the immunomodulatory actions of opioids in cats.

Human neonatal leukocyte interferon production and natural killer cytotoxicity in response to herpes simplex virus.

Human neonates are susceptible to severe herpes simplex virus (HSV) infection; however, the immunologic mechanisms explaining this lack of resistance remain to be defined. The ability of leukocytes from adults and neonates to produce interferon (IFN) in response to HSV challenge and to destroy HSV infected cells in the absence of antibody, termed natural killer cytotoxicity (NKC), were compared. The NKC activity of leukocytes from neonates (13.5 +/- 2.5%) was significantly lower (p less than 0.005, Mann Whitney test; p = 0.007, Students t-test) than the NKC activity of adult leukocytes (25.7 +/- 3.2%). In contrast, no significant difference was observed between IFN production by neonate (87.7 +/- 29.1 units) and adult (87.8 +/- 29.4 units) leukocytes.

Enzyme immunoassay for direct detection of influenza type A and adenovirus antigens in clinical specimens.

Detection of viral antigens in specimens without prior cultivation in cell culture provides the most rapid method for specific viral diagnosis. A solid-phase, double-antibody enzyme immunoassay was developed for this purpose and tested with clinical specimens containing influenza type A and adenovirus. Polystyrene microtiter wells were the solid phase and were coated with virus-specific guinea pig immunoglobulins. Specimens were added, and bound viral antigens were detected by addition of virus-specific rabbit immunoglobulins followed by enzyme-labeled goat antirabbit immunoglobulin G. Two methods of labeling goat anti-rabbit immunoglobulin G with horseradish peroxidase were investigated: covalent attachment and a noncovalent, immunological binding of antibody to enzyme, the peroxidase-antiperoxidase method. Both methods of labeling resulted in assays that could detect 10(3.5) 50% tissue culture infectious doses of influenza type A and 10(3.8) 50% tissue culture infectious doses of adenovirus. Equal sensitivity was noted with alkaline phosphatase-labeled goat anti-rabbit immunoglobulin G. An increase in sensitivity of three- to sixfold was achieved when virus-specific rabbit immunoglobulins and conjugate were diluted in 1% gelatin. The solid-phase, double-antibody enzyme immunoassay detected influenza type A and adenovirus in isolation-positive clinical specimens with 53% (21/40) and 62% (13/21) efficiency, respectively. The solid-phase, double-antibody enzyme immunoassay has considerable potential as a practical and rapid method for detection of respiratory viral antigens in nasal wash and throat swab specimens. For optimal value, however, greater sensitivity than was provided by the present methods is desirable.

Therapy of murine rabies after exposure: efficacy of polyriboinosinic-polyribocytidylic acid alone and in combination with three rabies vaccines.

A murine model simulating human street rabies virus infection was used to evaluate the efficacy of polyriboinosinic-polyribocytidylic acid (poly I-poly C), three rabies vaccines, and combinations of these modes of therapy administered after exposure. One or two doses of 100 mug of poly I-poly C, injected into the same intramuscular site as the challenge virus, significantly reduced the mortality rate when therapy was initiated 3 hr after challenge; however, the same quantity of poly I-poly C injected into the opposite leg did not reduce the mortality rate. The muscle injected with poly I-poly C invariably contained four to eight times more interferon than a similar noninjected muscle from the same animal. Mice treated 3 hr after challenge with each of the three vaccines produced significant levels of antibody but were not protected, whereas treatment with combinations of poly I-poly C and vaccine resulted in significant protection. These results suggest that the combination of induction of local interferon and an immune response contributes to the protection of mice after exposure to street rabies virus infection.

Effects of cytosine arabinoside, adenine arabinoside, and 6-azauridine on rabies virus in vitro and in vivo.

The antiviral agents cytosine arabinoside, adenine arabinoside, and 6-azauridine were shown to inhibit the replication of rabies virus in vitro but not the replication of Sindbis virus. These same drugs were not effective in reducing the mortality rate in mice challenged with street rabies virus.

Cytokine-stimulated human natural killer cytotoxicity: response to rotavirus-infected cells.

The ability of rotavirus-infected cells to stimulate leukocytes to release a cytokine which enhanced the subsequent leukocyte cytotoxicity to a second set of [51Cr] labeled rotavirus-infected cells was analyzed. Human interferon increased leukocyte cytotoxicity to Simian rotavirus (SA-11)-infected target cells. Similarly, 11 of 12 supernates of SA-11-stimulated peripheral blood leukocyte cultures increased the killing of SA-11-infected cells (P less than 0.005). This resulted in a calculated cytokine-dependent cellular cytotoxicity value of 9.6 +/- 1.9%. Three of five of the supernates tested contained measurable levels of interferon (12-48 unit/ml). In contrast, SA-11-stimulated colostral leukocyte culture supernates neither increased leukocyte cytotoxicity nor contained measurable levels of interferon.

Sensitive enzyme immunoassay with beta-D-galactosidase-Fab conjugate for detection of type A influenza virus antigen in clinical specimens.

The most sensitive method for diagnosis of type A influenza virus infection is isolation of the agent in cell culture. However, detection and identification may require several days to complete. This delay in diagnosis prevents effective use of the antiviral agents available for treatment of type A influenza infection. As a rapid diagnostic method, enzyme immunoassay (EIA) is attaining increased usage for direct detection of viral antigen in clinical specimens. Standard EIA techniques, however, are usually not sensitive enough for reliable detection of viral antigen in respiratory secretions. We developed a conjugate consisting of the antigen-binding fragment of goat antirabbit immunoglobulin G coupled to beta-d-galactosidase, using the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate. Other immunoreagents in our EIA consisted of guinea pig and rabbit antisera to influenza A/Brazil/11/78 (H1N1) for microtiter plate coating and primary antiserum, respectively. The sensitivity of this EIA was tested with 60 clinical specimens containing influenza A/England/333/80 (H1N1) which closely resembles A/Brazil. Of 31 initial specimens, collected within 24 h of the onset of symptoms, 27 (87%) were positive, using a fluorgenic substrate, and 18 of 29 (62%) specimens obtained 12 to 60 h after the initial specimens were positive, for a total of 75% (45 of 60). All positive reactions were specific, as shown in a confirmatory test with preimmune and hyperimmune guinea pig globulins. Clinical specimens negative for virus (n = 33) or containing heterologous respiratory viruses (n = 26) were negative in this system. These results indicate that EIA systems can be developed with a sensitivity approaching that required for clinical usefulness.

Detection of adenovirus by enzyme-linked immunosorbent assay.

A solid-phase direct enzyme-linked immunosorbent assay (ELISA) was developed for the detection of adenovirus antigen in extracts of infected cells by using antihexon serum. Results with simulated clinical specimens consisting of normal nasal wash specimens seeded with varying concentrations of adenovirus type 5 showed that antigen could be detected in extracts of HEp-2 cell cultures inoculated with 10(2.5) 50% tissue culture infective doses (TCID50) and 10(1.5) TCID50 after 2 and 4 days of incubation, respectively. Fifty-three clinical nasal wash specimens containing adenovirus type 5 (stored for 5 years at -70 degrees C) were used to evaluate antigen detection by ELISA in HEp-2 cell extracts and by manifestation of cytopathic effect in human embryonic kidney cells. After 2 days of incubation, 62% were positive by ELISA, whereas none was positive for cytopathic effect. After 4 days of incubation, 76% were ELISA positive and 47% were positive for cytopathic effect. The results according to infectivity titers indicated that clinical specimens containing 10(3.0) TCID50 or greater were all positive by ELISA after 2 days of incubation in HEp-2 cells, and by 4 days all but one specimen containing 10(2.0) TCID50 or greater were ELISA positive. ELISA and immunofluorescent methods for antigen detection were compared using 24 of the 53 clinical specimens containing adenovirus type 5. Nearly equivalent sensitivities were demonstrated. These results suggest that ELISA may provide an alternative method of detecting and identifying adenoviral infections in humans.

Effect of human nasal secretions on the antiviral activity of human fibroblast and leukocyte interferon.

Many normal human nasal secretions contain an inhibitor of human fibroblast IF. This inhibitor had no effect on human leukocyte IF. The amount of inhibition of fibroblast IF increased with increasing quantities of nasal secretions. Also, the inhibition could be overcome with increasing concentrations of IF.

Evidence of interferon production in the hamster lung after primary or secondary exposure to parainfluenza virus type 3.

Experimental infection of the hamster respiratory tract with parainfluenza virus type 3 has been used to study the pathogenesis of viral pneumonia and the host response to infection. In this study, hamsters inoculated intranasally with parainfluenza virus type 3 produced local interferon, which was detected in lung washes obtained by in situ lavage. Interferon activity was present as early as 2 days after infection, and titers correlated directly with the quantity of virus recovered in lung washes. Parainfluenza virus type 3 was sensitive to the antiviral state induced in vitro by the lung wash interferon. Infectious virus induced interferon in cultures of immune and nonimmune lung wash cells, primarily alveolar macrophages. A secondary response of immune, mixed cultures of lymphocytes and alveolar macrophages, stimulated with inactivated virus, produced low concentrations of interferon, perhaps type II. Lymphocyte-alveolar macrophage cultures produced a pH and temperature-sensitive interferon in response to mitogen induction, characteristics of type II or immune interferons in the human and murine systems. Interferon may be an early defense involved in recovery from primary infection with parainfluenza virus type 3, and may contribute to resistance to reinfection.

Post-exposure prophylaxis of murine rabies with polyinosinic-polycytidylic acid and chlorite-oxidized amylose.

Chlorite-oxidized amylose (COAM), polyinosinic-polycytidylic acid [poly(I:C)], and combinations of the two drugs were evaluated for their interferon-inducing properties and their ability to protect mice against rabies infection. Post-exposure administration of one or two doses (100 mug each) of poly(I:C) significantly protected mice against rabies infection. Pretreatment of mice with COAM 3 h before poly(I:C) stimulation resulted in an enhancement of the interferon response. However, the increased interferon titers were not reflected by increased protection against rabies infection over that achieved with poly(I:C) therapy alone. Therapy with COAM alone did not protect mice against rabies but, rather, was associated with enhanced mortality.

Activity of exogenous interferon in the human nasal mucosa.

The activity of exogenous human leukocyte IF has been studied in the human nasal mucosa and factors have been sought that would enhance its potential for clinical usefulness. Experiments have been performed which indicate that the nasal epithelial cells do respond to exogenous IF. In vivo dose requirements may be high because of mucociliary clearance mechanisms and because of the time and dose requirement for full antiviral activity. Future studies must define the IF sensitivity of different respiratory viruses in the human nasal epithelial cell and must evaluate more efficient methods of application.

Antibody response in humans to influenza virus type B host-cell-derived variants after vaccination with standard (egg-derived) vaccine or natural infection.

Hemagglutination inhibition (HI) and neutralization tests were used to determine antibody responses to egg-derived and Madin-Darby canine kidney (MDCK)-derived influenza B virus (B/England/222/82) in paired sera from persons naturally infected with influenza B and in persons vaccinated with standard egg-derived inactivated influenza vaccine. When tested by HI, the MDCK-derived antigen gave significantly higher (8- to 12-fold) geometric mean titers (GMT) in convalescent-phase sera from persons naturally infected during community outbreaks, as well as more 4-fold titer rises, than did tests with egg-derived antigen. When tested by neutralization, however, the convalescent-phase sera GMTs were only threefold higher with the MDCK-derived antigen and an equivalent number of fourfold titer rises were detected with both antigens. With postvaccine sera, the MDCK-derived antigen gave GMTs that were threefold higher than those obtained with egg-derived antigen in both the HI and neutralization tests and both antigens detected an equivalent number of fourfold titer rises in HI and neutralization tests. Sucrose gradient-fractionated egg-derived antigen showed a single peak of hemagglutinin activity corresponding to whole virions, whereas MDCK-derived antigen contained two distinct peaks of hemagglutinin activity, one of which had a lower sedimentation rate. The overall findings indicate that the egg-derived antigen in the vaccine induced HI and neutralizing antibody to both egg- and MDCK-derived variants and suggest that titers of antibody to MDCK-derived virus may be affected by the physical form of the hemagglutinin antigen.

Characterization and evaluation of monoclonal antibodies developed for typing influenza A and influenza B viruses.

Monoclonal antibodies that are broadly reactive with influenza A or influenza B viruses were produced as stable reagents for typing influenza viruses. Monoclonal antibodies to influenza A were specific for either matrix protein or nucleoprotein. The antibodies to influenza B were specific for nucleoprotein or hemagglutinin protein. In an enzyme immunoassay procedure, influenza A antibodies detected H1N1, H2N2, and H3N2 influenza A virus strains collected between 1934 and 1984. Each of the influenza B antibodies detected influenza B reference viruses collected between 1940 and 1984. Pools of either influenza A or influenza B monoclonal antibodies were used to detect influenza viruses reisolated from clinical specimens in tissue culture. At 48 h after inoculation, the influenza A monoclonal antibodies detected 64% of H1N1 and 94% of H3N2 influenza A specimens, and the influenza B monoclonal antibodies detected 79% of the influenza B specimens. The results of this study suggest that the monoclonal antibodies described should provide useful diagnostic reagents for workers in virology laboratories who wish to isolate and identify influenza virus but have been unable to obtain consistent supplies of animal sera specific for influenza A or B viruses.

Rapid detection of influenza virus by shell vial assay with monoclonal antibodies.

Of 45 influenza virus strains (43 type A and 2 type B) detected in conventional tube cell cultures (average time, 4 days), 25 (56%) were detected by immunofluorescence in the shell vial assay 24 h postinoculation. The specific fluorescence produced should allow this procedure to be readily adapted by laboratories with various degrees of experience with immunofluorescence methodology.

Isotype-specific enzyme immunoassay for influenza antibody with monoclonal antibodies to human immunoglobulins.

Mouse monoclonal antibodies specific for human immunoglobulin isotypes were investigated for use in an isotype-specific enzyme immunoassay for detection of antibody to influenza type A hemagglutinin (H1 and H3). The monoclonal antibody reagents were compared with isotype-specific, hyperimmune rabbit antisera from the National Institutes of Health. Endpoint titers for immunoglobulin G (IgG) obtained with the two reagents were within fourfold of each other 84% of the time (79 of 84) and within eightfold of each other 95% of the time (89 of 94). Regression analysis of the data gave a multiple correlation coefficient (r2) of 0.77 and a Spearman rank value of 0.83 (P less than 0.001). For IgA reagents, endpoint titers agreed within fourfold 77% of the time (88 of 114) and within eightfold 92% of the time (105 of 114). The r2 was 0.73, and Spearman rank was 0.83 (P less than 0.001). IgM antibody was detected in only 17 of 114 sera by either monoclonal or polyclonal reagents. Of these sera, 14 (82%) gave titers with the two reagents that were within fourfold of each other. A similar number of fourfold titer rises were detected with each reagent in paired sera showing hemagglutination inhibition titer rises. Monoclonal antibody reagents detected 27 IgA, 29 IgG, and 6 IgM rises, while polyclonal antisera detected 26 IgA, 31 IgG, and 7 IgM rises. These results show that monoclonal antibodies specific for human immunoglobulin isotypes are suitable as reagents for diagnostic assays. The advantages of monoclonal antibodies are their high degree of specificity and the ability to be standardized and produced in unlimited quantities. Moreover, the availability of immunoglobulin subclass- and allotype-specific monoclonal antibodies will enable a more detailed analysis of the antibody response to influenza as well as other infectious agents.

Trials of interferon in respiratory infections in man.

It is not yet clear from these recent volunteer studies (8) and from the results of Merigan et al. (14) what the optimal method and schedule for administering HuIFN should be. Taken together, the results with relatively low doses of interferon (1 to 4 X 10(6) units) plus antihistamine pretreatment, and with multiple doses of HuIFN alpha (total 14 X 10(6) units) indicate the need for better methods of delivery. However, it is of interest that protection was achieved by extremely small amounts of HuIFN alpha protein - Merigan (14) used only about 160 mcg. We anticipate that with better supplies of interferon from HuIFN genes cloned in bacteria, we may seek more efficient means of delivery and obtain better protection of volunteers against respiratory virus infections.