Evaluation of the virulence of nontypeable Haemophilus influenzae lipooligosaccharide htrB and rfaD mutants in the chinchilla model of otitis media.

Considerable evidence has implicated nontypeable Haemophilus influenzae (NTHi) lipooligosaccharide (LOS) in the pathogenesis of otitis media (OM); however, its exact role has not been conclusively established. Recently, two NTHi LOS-deficient mutants have been created and described. Strain 2019-DK1, an rfaD gene mutant, expresses a truncated LOS consisting of only three deoxy-D-manno-octulosonic acid residues, a single heptose, and lipid A. Strain 2019-B29, an isogenic htrB mutant, possesses an altered oligosaccharide core and an altered lipid A. Each strain's ability to colonize the nasopharynx and to induce OM subsequent to transbullar inoculation was evaluated in the chinchilla model. Nasopharyngeal colonization data indicate that the parent strain and both mutants are able to colonize the nasopharynx and exhibit comparable clearance kinetics. Compared with the parent and each other, however, the mutants demonstrated marked differences in virulence regarding their relative abilities to induce OM and persist in the middle ear post-transbullar inoculation. Strain B29 required a 3-log-greater dose to induce OM than the parent strain and did not exhibit evidence of sustained multiplication but persisted for the same duration as the parent. Conversely, strain-DK1, even when inoculated at a dose 4 logs greater than the parent dose, was eliminated from the middle ear 72 h after challenge. A comparison of the relative pathogenicities of these isolates provides the opportunity to address fundamental questions regarding the contribution of LOS to pathogenesis issues at the molecular level. Specifically, the impact of these LOS gene disruptions on OM pathogenesis can be defined and may thus provide potential new targets for future protection and intervention strategies.

Relative immunogenicity and efficacy of two synthetic chimeric peptides of fimbrin as vaccinogens against nasopharyngeal colonization by nontypeable Haemophilus influenzae in the chinchilla.

The OMP P5-homologous fimbriae of nontypeable Haemophilus influenzae (NTHi) are an adhesin and a virulence factor for otitis media in chinchilla models. We synthesized two peptides (LB1 and LB2) which incorporate determinants of the fimbrial subunit co-linearly synthesized with a "promiscuous" T-cell epitope from the fusion protein of measles virus. Sera obtained from immunized rabbits and chinchillas demonstrated significant reciprocal titers against both the homologous peptide and isolated fimbrial protein. Antisera also immunolabeled native fimbriae of whole unfixed NTHi. Immunization with LB1 or fimbrin resulted in elimination of NTHi from the chinchilla nasopharynx 2-3 weeks earlier than controls, respectively.

The effect of antibiotic treatment on the release of endotoxin during nontypable Haemophilus influenzae-induced otitis media in the chinchilla.

The gram negative bacteria, nontypable Haemophilus influenzae (NTHi) was used to induce otitis media in a total of 18 chinchillas. Three days post-inoculation, three cohorts of 6 chinchillas each were treated daily for four days with either ceftriaxone, chloramphenicol, or diluent without antibiotics. Middle ear fluid (MEF) was obtained daily, assayed for endotoxin content by means of the chromogenic limulus amebocyte lysate assay, and concentration of the NTHi/mL MEF determined by standard plate count. The endotoxin concentration per mL MEF from both the antibiotic treated cohorts decreased during the observation period, but increased in the MEF of the untreated control group. The data indicate that, unlike the dramatic increase in endotoxin concentration, after antibiotic treatment in the cerebrospinal fluid (CSF) during experimental Haemophilus influenzae-induced meningitis, there is no demonstrable sustained release of endotoxin in the middle ear subsequent to antibiotic treatment during experimental otitis media.

Immunization with outer membrane protein P6 from nontypeable Haemophilus influenzae induces bactericidal antibody and affords protection in the chinchilla model of otitis media.

The role of nontypeable Haemophilus influenzae (NTHi) outer membrane protein (OMP) P6 in the pathogenesis of otitis media (OM) has not been defined. OMPs, fimbriae, pili, and lipooligosaccharide are several types of surface antigens of NTHi that are currently being evaluated as potential vaccine candidates. P6 is antigenically conserved among both nontypeable and type b H. influenzae strains and elicits bactericidal as well as protective antibodies; however, initial evaluation of a vaccine mixture of P6 combined with other NTHi OMPs failed to induce bactericidal antibody or protection in the chinchilla model of OM. We undertook an assessment of the ability of immunization with isolated P6 lipoprotein alone to confer protection. Chinchillas were immunized with P6 and challenged 10 days after the final immunization with either 3 x 10(3) CFU of NTHi delivered directly into the middle ear to induce OM or 5 x 10(8) CFU of NTHi delivered intranasally to establish nasopharyngeal colonization. All immunized animals responded with elevated serum titers of anti-P6 antibody, which also demonstrated bactericidal activity against homologous as well as a heterologous NTHi isolate. By 14 days post-transbullar challenge, the number of chinchillas with middle ear fluid and the incidence of NTHi culture-positive middle ear fluids were reduced 48 and 51%, respectively, in the P6-immunized chinchillas relative to the sham-immunized cohort. Nasopharyngeal colonization levels were comparable in the two cohorts. These data demonstrate that active immunization with P6 results in the production of NTHi-specific bactericidal antibody in the chinchilla and also affords a reduction in the incidence of NTHi-induced OM; however, parenteral immunization does not appear to affect the extent or duration of nasopharyngeal colonization by NTHi.

Peptidoglycan isolated from nontypeable Haemophilus influenzae induces experimental otitis media in the chinchilla.

Bacterial cell wall components induce a number of biologic effects and promote inflammatory changes in a variety of hosts. Peptidoglycan isolated from Streptococcus pneumoniae can induce inflammation in the middle ear; however, an analogous role for peptidoglycan derived from gram-negative otitis media pathogens has not been described. Peptidoglycan isolated from nontypeable Haemophilus influenzae (NTHi), a major cause of otitis media, was evaluated in a chinchilla model. The direct injection of the middle ear with 3-300 micrograms of peptidoglycan resulted in tympanic membrane inflammation, abnormal pressure in the middle ear, leukocytosis, and histopathologic changes in the middle ear mucosa that included marked edema, osteoneogenesis, focal hemorrhage, and a mononuclear infiltration into the subepithelial space. These data indicate that NTHi peptidoglycan induced inflammation and histopathologic changes in the tympanic membrane and middle ear mucosal epithelium and may contribute to the pathogenesis of otitis media.

Attachment relationships in infant twins: the effect of co-twin presence during separation from mother.

In this study the roles of the mother and the co-twin in inhibiting emotional arousal and reducing manifest distress of a twin who had been isolated in a modified strange situation were compared. The subjects were 15 children, each a member of a twin pair. The subjects were placed in a playroom under three conditions in the following order: (a) mother and twins present; (b) twins together, mother absent; (c) subject isolated from both co-twin and mother. The episodes in which all partners were together were alternated with brief separations. The subjects' distress was minimal when they were separated from the mother with the co-twin present. Upon reunion, stable social behavior was quickly restored. However, separation from the mother and co-twin produced a high level of distress for the subjects. When reunited, the isolated twin initiated physical contact with the mother, soliciting and receiving comfort from her. Furthermore, the distress of the isolated twin was transmitted to the co-twin who had remained with the mother during the isolation period. The nonisolated twin also solicited comfort from the mother. The presence of the co-twin during the reunion following isolation had little effect in reducing the subject twin's distress.

Role of fimbriae expressed by nontypeable Haemophilus influenzae in pathogenesis of and protection against otitis media and relatedness of the fimbrin subunit to outer membrane protein A.

Nontypeable Haemophilus influenzae is a primary pathogen in both acute otitis media (OM) and chronic OM, yet the pathogenesis of this disease is not fully understood. Although fimbriae have been observed on all clinical OM isolates examined to date, their role in pathogenesis remains unclear. Therefore, the gene which codes for the fimbrial subunit protein (fimbrin) in nontypeable H. influenzae 1128 was isolated, cloned, and sequenced. The nucleotide sequence of the fimbrin gene was found to contain an open reading frame of 1,077 bp which would encode a mature fimbrin protein consisting of 338 amino acid with a calculated molecular mass of 36.4 kDa. The translated amino acid sequence was found to be homologous with various OmpA proteins of other gram-negative bacteria, and algorithmic analysis predicted that this protein is organized as a coiled coil. To directly test whether fimbriae are involved in pathogenesis, the fimbrin gene was disrupted, and the biological consequences of disruption were absence of both expression of the fimbrial appendage and the specific immunogold labeling thereof with antisera directed against isolated fimbrial protein, reduced adherence to human oropharyngeal cells in vitro, augmented clearance from the tympanum post-transbullar inoculation, and significantly reduced induction of OM post-intranasal inoculation in a chinchilla model compared with the fimbriated parent strain. We additionally find that either passive immunization or active immunization against isolated fimbrial protein confers partial protection against transbullar challenge. A Western blot (immunoblot) indicated a degree of serological relatedness among fimbrin proteins of 15 nontypeable and type b isolates. These data suggest that fimbrin could be useful as a component of a vaccine to protect against OM.

Human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus type 2 (HSV-2) can coinfect and simultaneously replicate in the same human CD4+ cell: effect of coinfection on infectious HSV-2 and HIV-1 replication.

Experiments were designed to determine whether HIV-1 and herpes simplex virus type 2 (HSV-2) coinfection leads to simultaneous replication of both viruses in the same human CD4+ cell (MT-4 cell line) and the possible effects of coinfection on infectious virus production. Results from transmission electron microscopy analysis revealed replication of typical HSV-2 nucleocapsids in the nucleus and budding of HIV-1 particles through the plasma membrane and through intracytoplasmic vacuoles containing enveloped HSV-2 particles in the same coinfected cell. Coinfection of HIV-1 persistently infected H9IIIB or promonocytic U1 cells with HSV-2 did not alter total production of infectious HSV-2 or the percentage of HSV-2 infectious centers compared with control H9 and U937 cells infected with HSV-2 alone. However, in coinfected promonocytic U1 cells HSV-2 induced infectious HIV-1 production measured by syncytial plaque assay. In summary, both HIV-1 and HSV-2 can coinfect and simultaneously replicate in the same human CD4+ cell. Interactions between HIV-1 and HSV-2 appear to be unidirectional, resulting in accelerated replication of HIV-1 as reported by Albrecht et al. (J Virol 1989;63:1861-1868), but not HSV-2 as shown by us.

Novel membrane-interactive ether lipid analogs that inhibit infectious HIV-1 production and induce defective virus formation.

A new class of membrane-active ether lipid (EL) analogs of platelet-activating factor were studied for in vitro anti-HIV-1 activity. Human T-cell (CEM-ss) monolayers or suspension cultures were used to determine effects of structural modifications of Type A phosphorus-containing and Type B nonphosphorus EL analogs on (a) the inhibitory concentration50 (IC50) for HIV-1 syncytial plaque formation and cell growth, and, (b) virus budding at the cell plasma membrane. Results indicate that representative Type A and Type B EL inhibit HIV-1 but not herpes simplex virus type 2 plaque formation when added before or up to 2 days after viral infection. Anti-HIV-1 activity does not involve direct inactivation of virus infectivity. Type A EL (IC50 range = 0.2-1.4 microM) with alkyoxy, alkylthio, or alkyamido substitution at glycerol position 1 and ethoxy or methoxy substitution at position 2, and Type B compounds (IC50 range = 0.33-0.63 microM) with an inverse choline or nitrogen heterocyclic substitution at position 3 have selective activity against HIV-1-infected T-cells. EL treatment of HIV-1-infected cells is associated with subsequent release of reverse transcriptase activity, but infectious virus production is inhibited with time after infection. Electron microscopic examination of HIV-1-infected and EL-treated cells revealed absence of detectable budding virus at the plasma membrane but presence of intracytoplasmic vacuolar virus particles. In summary, these data suggest that EL analogs are a novel class of agents that induce defective intracytoplasmic vacuolar HIV-1 formation in T-cells. Being membrane interactive, EL are ideally suited for combination chemotherapy with DNA-interactive anti-HIV nucleoside analogs.

Chemiluminescent responses of alveolar macrophages from normal and Mycobacterium bovis BCG-vaccinated rabbits as a function of age.

Luminol-dependent chemiluminescence (CL) responses of alveolar macrophages (AM) from normal and Mycobacterium bovis BCG-vaccinated infant and adult rabbits were compared. AM from 1-, 7-, and 14-day-old normal rabbits exhibited much lower peak CL responses than did AM from 28- and 42-day-old normal animals as well as rabbits 2 to 3 or 5 to 6 months and 1 to 2 years of age. The most striking differences among AM from infant and adult rabbits were noted when AM were obtained from 28-day-old and 5- to 6-month old rabbits 21 days after the rabbits were immunized with 200 micrograms of BCG intravenously. In this case, AM from 5- to 6-month-old animals gave peak counts per minute of 400,000 to 500,000 whereas AM from 28-day-old rabbits vaccinated with BCG (harvested at 49 days of age) gave peak counts per minute of only 40,715 +/- 2,688. These data reveal that AM from neonatal animals are grossly deficient as responders to phorbol myristate acetate-induced CL. This deficiency, which improved with age, is still apparent in AM from 28-day-old animals. The data also reveal that BCG vaccination of 28-day-old animals yields AM that are poor responders to phorbol myristate acetate compared with AM from BCG-vaccinated animals 2 to 3 and 5 to 6 months of age. AM from animals vaccinated with BCG at 28 days of age contained fewer and smaller electron-dense lysosomelike structures than did AM from adult rabbits similarly vaccinated. These findings provide an explanation for the difficulties infants have in developing effective cell-mediated immune responses against intracellular parasites.

Phagosomal membranes of Mycobacterium bovis BCG-immune alveolar macrophages are resistant to disruption by Mycobacterium tuberculosis H37Rv.

Data obtained in this study reaffirm that virulent Mycobacterium tuberculosis H37Rv has a potent phagosome-destroying capacity when ingested by normal alveolar macrophages. In contrast, Mycobacterium bovis BCG-immune alveolar macrophages are highly resistant to this virulence mechanism. BCG-immune sera incubated with BCG-immune alveolar macrophages did not increase resistance of BCG-immune alveolar macrophages as compared with the data obtained from experiments with normal sera. BCG-immune sera failed to confer resistance to normal alveolar macrophages against the phagosomal membrane-destroying H37Rv virulence mechanism.

Scanning and transmission electron microscopy as tools for the study of phagocytosis of bacteria adherent to hard surfaces.

A technique for studying the phagocytosis of bacteria colonizing hard surfaces is described. Rabbit peritoneal macrophages were allowed to settle on the surface of high-molecular-weight polyethylene which had been previously colonized by Escherichia coli. To ascertain the presence of bacteria on the surface of the polyethylene and the degree of spreading of the attached macrophages, the preparations were observed by scanning electron microscopy. The ingestion of E. coli by the macrophages was studied by transmission electron microscopy on ultrathin sections of resin-embedded monolayers after their separation from the polyethylene surface. Numerous intracellular bacteria were located near the area of attachment of the macrophages to the substrata, suggesting that the phagocytosis of bacteria adherent to the surface of the plastic had taken place.

Disruption of phagosomal membranes of normal alveolar macrophages by the H37Rv strain of Mycobacterium tuberculosis. A correlate of virulence.

The H37Rv and H37Ra strains of Mycobacterium tuberculosis were incubated with normal rabbit alveolar macrophages and examined by electron microscopy at 5 to 6 and 18 to 24 h of incubation. At the 18- to 24-h incubation interval, 60 to 100% of the endocytosed organisms of the H37Rv strain disrupted the phagosomal membranes and appeared free in the cytoplasm of the macrophages. In contrast, the H37Ra strain lacked this putative virulence characteristic, and greater than 99% of the organisms appeared within intact phagosomes. Heating the organisms of the H37Rv strain abrogated to a large extent, but not completely, their capacity to disrupt phagosomal membranes. In the course of the interaction of organisms of the virulent H37Rv strain with phagosomal membranes of normal rabbit alveolar macrophages, adherence of the phagosomal membrane to the surface of the organisms was a prominent feature that was followed by fragmentation and apparent disintegration of the membrane. This potential virulence characteristic could explain why there is essentially no resistance expressed in the lung during the early postinfectious period of primary infection to M. tuberculosis.

Toxicity of staphylococcal alpha toxin for rabbit alveolar macrophages.

Highly purified staphylococcal alpha toxin was toxic in vitro for rabbit alveolar macrophages. Cytotoxicity, manifested by loss of the ability to exclude trypan blue dye and by morphological evidence of cell necrosis and lysis, was observed after exposure for 4 h to 1 microgram of toxin preparation per ml and after exposure for 8 h to 0.1 microgram of toxin per ml. In addition, exposure to toxin under conditions which did not kill more than 10% of the cells (1 microgram/ml for 1.5 to 2 h) significantly reduced the phagocytic activity of the cells and their ability to respond to an activator of hexose monophosphate shunt activity.

Use of chemotaxis chambers for studying in vitro bacterial colonization of biomaterials.

Blind-well chemotaxis chambers were used to study the in vitro bacterial adhesion and colonization of biomaterials. Staphylococcus aureus and Pseudomonas aeruginosa were selected for the bacterial inocula. Abundant growth on the surfaces of methyl methacrylate, polyethylene, stainless steel, and Vitallium was detected by using scanning electron microscopy after 24 h of incubation. The culture technique employed proved to be of value for the study of surface bacterial colonization of inert materials.

Ultrasonic effects on alveolar macrophages in suspension.

Rabbit alveolar macrophages in suspension were exposed to 5 or 10 min of continuous 2-MHz ultrasound with 5, 10, and 15 W/cm2 spatial average intensities. Viability as determined by dye exclusion decreased with increasing intensity. Pressure experiments indicated that this was a result of acoustic cavitation. Ultrasound induced clumping of cells and often reduced membrane ruffling. Some cells were disintegrated. Cells that appeared to be otherwise intact had swollen mitochondria with ruptured cristae.