Flow cytometric analysis of group B streptococci phagocytosis and oxidative burst in human neutrophils and monocytes.

Group B streptococci (GBS) are a major cause of meningitis and septicemia in neonates and numerous invasive diseases in adults. Host defense against GBS infections relies upon phagocytosis and killing by phagocytic cells. To better understand the importance of this defense mechanism a flow cytometric assay was developed to study phagocytosis and oxidative burst of leukocytes stimulated by bacteria. GBS labeled with fluorescein isothiocyanate were used for phagocytosis experiments and the extracellular fluorescence was quenched by ethidium bromide to differentiate intracellular from extracellular bacteria. The intracellular oxidative burst was determined by using 2',7'-dichlorofluorescein diacetate to measure hydrogen peroxide production and hydroethidine for superoxide anion production. We found that for GBS serotypes Ia, Ib/c, II, and III phagocytosis was greater in neutrophils than monocytes. Hydrogen peroxide production and superoxide anion production were also greater for neutrophils than monocytes in all serotypes tested. A comparison of seven type III strains revealed greater phagocytosis and superoxide anion production by neutrophils than monocytes but no difference in hydrogen peroxide production. Therefore, monocytes react similarly as neutrophils in response to GBS but at a reduced level. This methodology of measuring both phagocytosis of GBS and oxidative burst simultaneously in neutrophils and monocytes should be very useful in further studies on the importance of factors such as complement and IgG receptors for the killing of bacteria.

Enhancement of bismuth antibacterial activity with lipophilic thiol chelators.

The antibacterial properties of bismuth are greatly enhanced when bismuth is combined with certain lipophilic thiol compounds. Antibacterial activity was enhanced from 25- to 300-fold by the following seven different thiols, in order of decreasing synergy: 1,3-propanedithiol, dimercaprol (BAL), dithiothreitol, 3-mercapto-2-butanol, beta-mercaptoethanol, 1-monothioglycerol, and mercaptoethylamine. The dithiols produced the greatest synergy with bismuth at optimum bismuth-thiol molar ratios of from 3:1 to 1:1. The monothiols were generally not as synergistic and required molar ratios of from 1:1 to 1:4 for optimum antibacterial activity. The most-active mono- or dithiols were also the most soluble in butanol. The intensity of the yellow formed by bismuth-thiol complexes reflected the degree of chelation and correlated with antibacterial potency at high molar ratios. The bismuth-BAL compound (BisBAL) was active against most bacteria, as assessed by broth dilution, agar diffusion, and agar dilution analyses. Staphylococci (MIC, 5 to 7 microM Bi3+) and Helicobacter pylori (MIC, 2.2 microM) were among the most sensitive bacteria. Gram-negative bacteria were sensitive (MIC, < 17 microM). Enterococci were relatively resistant (MIC, 63 microM Bi3+). The MIC range for anaerobes was 15 to 100 microM Bi3+, except for Clostridium difficile (MIC, 7.5 microM). Bactericidal activity averaged 29% above the MIC. Bactericidal activity increased with increasing pH and/or increasing temperature. Bismuth-thiol solubility, stability, and antibacterial activity depended on pH and the bismuth-thiol molar ratio. BisBAL was stable but ineffective against Escherichia coli at pH 4. Activity and instability (reactivity) increased with increasing alkalinity. BisBAL was acid soluble at a molar ratio of greater than 3:2 and alkaline soluble at a molar ratio of less than 2:3. In conclusion, certain lipophilic thiol compounds enhanced bismuth antibacterial activity against a broad spectrum of bacteria. The activity, solubility, and stability of BisBAL were strongly dependent on the pH, temperature, and molar ratio. Chelation of bismuth with certain thiol agents enhanced the solubility and lipophilicity of this cationic heavy metal, thereby significantly enhancing its potency and versatility as an antibacterial agent.

Salicylate-enhanced exposure of Klebsiella pneumoniae subcapsular components.

The capsular polysaccharide (CPS) of Klebsiella pneumoniae is an important virulence factor. Salicylate, which inhibits CPS production, was used to expose subcapsular antigens and components that may play an important role in host defense. Salicylate treatment greatly increased phagocytosis of five O1 serotypes by human polymorphonuclear leukocytes with normal rabbit serum and rabbit antisera against purified O1 lipopolysaccharide (O1LPS) as opsonins (p < 0.01 or < 0.05). Similar results were obtained with rabbit antiserum against a non-encapsulated isogenic strain. To further determine how salicylate increases susceptibility to phagocytosis, the binding of monoclonal antibodies against O1LPS or the LPS core and the binding of complement component C3b were measured by ELISA. The data indicate that salicylate reduced the barrier of CPS in serotypes O1:K1, O1:K10, and O1:K16 and unmasked subcapsular antigenic components in serotypes O1:K2 and O1:K66 so that bound opsonins could react with receptors on phagocytes. Serum bactericidal assays supported this conclusion. Therefore, decapsulating agents such as salicylate accentuate phagocytosis of K. pneumoniae by making subcapsular antigens and components accessible to immune and nonimmune host defences and vaccination with subcapsular antigens may exhibit optimal protection against lethal infection when combined with salicylate therapy.

Polysaccharide capsule-mediated resistance to opsonophagocytosis in Klebsiella pneumoniae.

The polysaccharide capsule of Klebsiella pneumoniae is an important virulence factor that confers resistance to phagocytosis. The treatment of encapsulated bacteria with salicylate to inhibit capsule expression was found to enhance the phagocytosis of encapsulated bacteria by human neutrophils only in the presence of cell surface-specific antibodies. Both type-specific rabbit antisera and anticapsular human hyperimmune globulin were employed as opsonins. Salicylate significantly enhanced phagocytosis with homologous, but not heterologous, whole-cell antisera. Antisera, diluted 1:40, no longer opsonized fully encapsulated bacteria but promoted the uptake of multiple salicylate-treated bacteria in > 90% of neutrophils. Salicylate (0.25 to 1.0 mM) also enhanced opsonization with globulin against homologous bacteria. Higher salicylate levels (1 to 2.5 mM) enhanced the opsonization of heterologous serotypes with human globulin. The nature of antibody attachment to encapsulated bacteria was determined by immunofluorescence. Even after the addition of purified capsular polysaccharide to prevent phagocytosis, K-specific antibodies attached in large amounts to bacteria. K-specific antibodies reacted with antigens throughout the capsule and showed a predilection for a denser inner layer of the capsule, indicating that many of the K-specific antibodies may be masked underneath the capsule surface. K-specific antibodies can also be rendered nonfunctional by soluble, cell-free capsular antigen. In culture, large quantities of soluble capsular polysaccharide extrude from bacteria after overnight growth. The reduction in capsule expression caused by salicylate largely affected the soluble, cell-free fraction. Purified capsular polysaccharide was shown to retard the opsonophagocytosis of salicylate-treated bacteria in a concentration-dependent manner. However, extensive washing of encapsulated bacteria to remove loosely attached capsular material did not significantly enhance opsonophagocytosis. In conclusion, cell-free capsule and cell-associated capsule are antiphagocytic; both act to neutralize K-specific antibodies by binding or concealment. Salicylate-mediated inhibition of capsule expression, particularly of the cell-free fraction, improved K-specific opsonization dramatically.

Salicylate or bismuth salts enhance opsonophagocytosis of Klebsiella pneumoniae.

After treatment of encapsulated Klebsiella pneumoniae with salicylate or bismuth compounds, phagocytic uptake by human peripheral white blood cells or rat alveolar macrophages was assessed. Without salicylate pretreatment of bacteria, a 30-60% net increase in viable bacteria resulted in phagocytic assays after a 1 hour incubation. With salicylate pretreatment, dose-related decreases in bacterial counts were seen, achieving a maximal reduction of 60% with 240 microM salicylate pretreatment. Bacterial variants producing less capsule were more serum sensitive and more readily phagocytosed. Micrographs of Giemsa-stained cells revealed phagocytic uptake of multiple bacteria after salicylate pretreatment, but virtually no uptake of untreated bacteria. Opsonization with polyclonal antiserum decreased bacterial cell counts by 20% without and by 90% with salicylate pretreatment of bacteria. Pretreatment of bacteria with bismuth salts also enhanced opsonophagocytosis of encapsulated bacteria. Thus, agents known to reduce capsule expression in K. pneumoniae also enhance phagocytic uptake of bacteria.

Evaluation of five monoclonal antibody-based kits or reagents for the identification and culture confirmation of herpes simplex virus.

Five immunofluorescence (IF) kits or reagents (Bartels [Bartels Immunodiagnostic Supplies, Inc., Bellevue, Wash.], Imagen [CellTech Diagnostics, Ltd., distributed by Analytab Products, Plainview, N.Y.], Ortho [Ortho Diagnostics Systems, Inc., Raritan, N.J.], Syva [Syva Co., Palo Alto, Calif.], Whittaker [Whittaker Bioproducts, Walkersville, Md.]) were evaluated for typing and laboratory confirmation of herpes simplex virus (HSV). Of 101 clinical isolates tested by each kit or reagent, results for 97 of them were in agreement. Identification of the four isolates with discordant results was performed by restriction endonuclease analysis of the viral DNA. The sensitivity and specificity of the Imagen and Bartels kits were 100%. For the Ortho, Syva, and Whittaker kits or reagents, the HSV type 1 (HSV-1) and HSV type 2 (HSV-2) sensitivities were 97.4 and 100%, 100 and 100%, and 97.4 and 100%, respectively, and the specificities were 100 and 97.4%, 100 and 92.4%, and 100 and 97.4%, respectively. There was one false-positive HSV-2 isolate identified by each of the Ortho and Whittaker kits or reagents. Three false-positive HSV-2 isolates occurred by staining with Syva, giving the erroneous indication of dual isolates. Several isolates stained with Imagen and Whittaker reagents displayed dull IF patterns. A dull green background occurred in ca. one-third of the HSV-2 isolates tested with the Ortho kit. The intensities of IF staining by the Bartels and Syva kits were satisfactory; however, the latter displayed a specificity of 92.7%. A total of 38 and 63 specimens were finally designated as HSV-1 and HSV-2, respectively. Identification of each isolate with the Bartels kit was consistently interpretable and is recommended as the typing and confirmatory assay of choice.

Occurrence of nonspecific reactions among stool specimens tested by the Abbott TestPack rotavirus enzyme immunoassay.

Sixty-five stool specimens obtained from children suffering from gastroenteritis were tested for the presence of antigen to rotavirus by the Abbott TestPack Rotavirus (TestPack) enzyme immunoassay kit. The Kallestad Pathfinder enzyme immunoassay, polyacrylamide gel electrophoresis, immune electron microscopy, and virus isolation were utilized as reference assays. Fifty-four specimens were in accord by TestPack and Kallestad Pathfinder. Among 11 discordant specimens positive with TestPack but negative by Kallestad Pathfinder, rotavirus was not identified by polyacrylamide gel electrophoresis, immune electron microscopy, or isolation in primary African green monkey kidney cell cultures. TestPack displayed a performance specificity of 83%. The inordinately high number of stool specimens reported as false-positive by TestPack precludes the incorporation of this antigen detection kit into our routine regimen of diagnostic virologic testing.

Enhanced killing of group B streptococci in vitro by penicillin and opsonophagocytosis with intravenous immunoglobulin.

Combined effects of intravenous immunoglobulin (IVIG) and antibiotics in killing bacteria are of interest with broadening clinical use of IVIG. Since the kinetics of killing by these agents differ and each may influence the outcome of the other, it is difficult to evaluate combination effects in vitro. Conditions were developed to measure killing of group B streptococci (GBS), type III strain M732, by an opsonic mixture with IVIG, fresh serum, and human polymorphonuclear leukocytes (PMNL) with or without penicillin. Bacterial killing was observed with the opsonic IVIG mixture, penicillin, and the opsonic IVIG mixture plus penicillin at 1 h. The effect of the combination was greater than the sum of the effects of two separate incubations. The enhanced killing was evident for up to 18 h. By 24 h, the killing by the combination was no greater than that by penicillin alone. A similar pattern of GBS killing was observed with cord blood PMNL, six different GBS type III strains, and pretreatment of GBS with either IVIG or penicillin. These effects suggest that the combination of IVIG and penicillin has potential for use in the treatment of neonatal GBS infections.

Effect of interferon on herpes simplex virus replication in murine macrophage-like cell lines.

The effect of interferon on the replication of herpes simplex virus types 1 and 2 was studied in two murine macrophage-like cell lines which differ in their ability to synthesize interferon. Higher titers of herpes simplex virus type 1 occurred in PU5-1.8 cell cultures where interferon was not produced than in J774A.1 cell cultures where low amounts of interferon were produced. Herpes simplex virus type 2 replicated poorly in both types of cell cultures. Interferon synthesis was induced in J774A.1 cell cultures but not in PU5-1.8 cell cultures. Exogenously added interferon was shown to inhibit virus replication, however the restrictiveness of these cells to HSV replication was not relieved by treating cell cultures with anti-interferon serum. These results show that factors other than induced interferon regulate the replication of herpes simplex viruses in these cells and suggest that induced interferon synthesis does not affect herpes simplex virus replication in macrophages.

Effect of prostaglandins on interferon synthesis in murine macrophage-like cell lines.

Murine macrophage-like cell lines were used to determine whether exogenously added prostaglandins and endogenous prostaglandins suppress interferon (IFN) synthesis in macrophages. The amount of IFN produced by J774A.1 cells induced with bacterial lipopolysaccharide (LPS) was reduced by 0.1 and 1 microM PGE1 or PGE2. These prostaglandins also inhibited Newcastle disease virus (NDV) induced IFN production, but only at a concentration of 1 microM. Thromboxane B2 at 0.01 to 1 microM had no effect on IFN production. Cells treated before, during, or before and during IFN synthesis with 0.15 to 4.8 microM indomethacin to inhibit prostaglandin synthesis did not increase IFN yields. Indomethacin also had no effect on NDV-induced IFN production by P388D1 and PU5-1.8 cells, and these cells remained nonresponsive to LPS for IFN production. These results indicate that endogenous levels of cyclooxygenase-dependent metabolites of arachidonic acid do not regulate IFN synthesis in macrophages.

Monoclonal antibodies to Mojave toxin and use for isolation of cross-reacting proteins in Crotalus venoms.

Hybridomas secreting monoclonal antibodies against Mojave toxin were established. The antibodies were used for identifying cross-reacting proteins in individual C. s. scutulatus and other Crotalus venoms and to isolate Mojave toxin. The antibodies recognized five bands with a pI range from 5.1 to 6.1 in immunoblots of electrofocused crude venom and Mojave toxin purified by immunoaffinity chromatography. The specificity of the antibodies was for the basic subunit of the toxin, which resolved into four bands of pI between 9.3 and 9.6. Individual C. s. scutulatus venoms of snakes from Texas and southern Arizona had multiple bands with pI's ranging from 4.9 to 6.3. Cross-reacting proteins were also recognized by the antibodies in the electrophoresed venoms of C. basiliscus, C. d. durissus, C. d. terrificus, C. h. horridus and C. v. concolor, and may be isolated by immunoaffinity chromatography with the monoclonal antibodies.

Interferon production in murine macrophage-like cell lines.

Five murine macrophage (M phi)-like cell lines were examined to determine their suitability for the characterization of M phi interferons (IFNs). The J774A.1, RAW 309 Cr.1, and RAW 264.7 cell lines produced 30-800 international units (IU)/10(6) cells when treated with 5-200 micrograms of bacterial lipopolysaccharide (LPS). No IFN was detected in LPS-treated P388D1 or PU5-1.8 cell cultures. All cell lines produced IFN when inoculated with Newcastle disease virus (NDV); however, only 15 IU/10(6) cells of acid stable IFN were produced in PU5-1.8 cell cultures in comparison to 4.2 X 10(3)-1.7 X 10(4) IU/10(6) cells in the other cell lines. Most of the IFN was produced within 4 h in LPS-treated cell cultures and within 12 h in NDV-infected cell cultures. All IFNs were stable at pH 2.0 and were neutralized with antiserum against mouse L cell IFN. These cell lines appear competent for use in studying the synthesis, molecular weights, and regulatory functions of M phi IFNs.

Hepatitis B surface antigen (HBSAg) in peritoneal fluid of HBSAg carriers undergoing peritoneal dialysis.

Samples of ascitic fluid and outflow dialysate were collected from HBSAg carriers undergoing peritoneal dialysis and tested for HBSAg by solid-phase radioimmunoassay. The surface antigen was detected in every sample from HBSAg carriers. This finding was not dependent upon the presence of occult blood in the sample. Surface antigen particles and possibly Dane particles were also observed in HBSAg-positive samples by immunoelectron microscopy. These results identify the outflow dialysate of HBSAg carriers undergoing peritoneal dialysis as a potential source of hepatitis B virus transmission.

Inhibition of the replication of parvovirus X14 by 5-iodo-2'-deoxyuridine pre-treatment of cell cultures.

Pre-treatment of rat embryo cell cultures with 5-iodo-2'-deoxyuridine (IdUrd) inhibits the replication of parvovirus X14. Reduced yields of haemagglutinating and infectious particles were observed. Adsorption of virus to cells was not affected, but both virus protein and DNA synthesis were inhibited. Fewer cells were capable of supporting protein or antigen synthesis as determined by immunofluorescence. Virus-specific DNA was detected in IdUrd pre-treated cells, but the amount synthesized was considerably less than that from control cultures. Cellular DNA synthesis was also inhibited in IdUrd pre-treated cells. Therefore, the replication of parvoviruses appears dependent upon host cell factors involved in cellular DNA synthesis.

Inactivation of enteroviruses by ascorbic acid and sodium bisulfite.

Poliovirus type 1, coxsackievirus type A9, and echovirus type 7 were inactivated by sodium bisulfite and ascorbic acid. Inactivation rates depended upon concentration, temperature, and pH. RNA infectivity was lost during inactivation; the capsid was also altered by these inactivating agents, as determined by enzyme sensitivity assays and by tests of adsorption to cells. Structural modifications of the virus particles were not identical, suggesting that the mechanism of inactivation by ascorbic acid differs from that of sodium bisulfite.

Isoelectric focusing of parvoviruses.

Adenovirus-associated virus type 4 and X14 migrate during electrophoresis to pH 2.6 in sucrose-stabilized, pH 2.5-6.0 gradients. Naturally occurring empty capsids appear to have the same isoelectric point as complete virus particles.

Effect of acid pH, salts, and temperature on the infectivity and physical integrity of enteroviruses.

At 2 degrees and 30 degrees C, enteroviruses are more stable on the acid than on the alkaline side of neutrality. In the range from pH 3 to 9, temperature is so influential that the fastest inactivation rate at 2 degrees C is slower than the slowest inactivation rate at 30 degrees C. Specific ions or salts also affect the rate of inactivation of enteroviruses. NaCl and other chloride salts enhance the inactivation of poliovirus at pH 3. NaCl is considerably less effective against poliovirus in the range of pH 4.5 to 7.0 than at pH less than 4.5. Loss of RNA infectivity of the virus particle proceeds as rapidly as the loss of infectivity of the particle itself, except at pH 3 in the presence of MgCl2. Inactivation results in alterations to the physical integrity of enteroviruses. At pH 5 and 7, RNA hydrolysis of poliovirus particles occurs; and at pH3, 5,6, and 7 the nucleic acid becomes susceptible to ribonuclease. Only virus particles inactivated at pH 3 show a sensitivity to chymotrypsin. The hemagglutinins of echovirus type 7 are destroyed during inactivation at pH 3,4,5, and 6; but at pH 6 this alteration precedes the loss of infectivity. The pH of the suspension is a primary determinant of the mechanism of virus destruction and possibly of the loss of infectivity at these temperatures.