IgG1+ B-cell immunity predates IgE responses in epicutaneous sensitization to foods.

BACKGROUND: The generation of IgE-mediated food allergy in humans is silent and only diagnosed upon manifestation of clinical symptoms. While experimental models have been used to investigate some mechanisms of allergic sensitization, the generation of humoral immunity and memory remains to be elucidated. Here, we defined the evolution of allergen-specific B-cell responses during epicutaneous sensitization to foods. METHODS: Wild-type and genetic knockout animals, and drug or antibody strategies for cell depletion and immunoglobulin signaling blockade were used to investigate epicutaneous sensitization and disease progression; we analyzed allergen-specific germinal centers and IgG1+ memory B cells by flow cytometry, evaluated humoral responses, and determined clinical reactivity (anaphylaxis). RESULTS: Epicutaneous sensitization caused microscopic skin damage, inflammation, and recruitment of activated dendritic cells to the draining lymph nodes. This process generated allergen-specific IgG1+ germinal center B cells, serum IgG1, and anaphylaxis that was mediated by the alternative pathway. Whether we used peanut and/or ovalbumin from the egg white for sensitization, the allergen-specific IgG1+ memory compartment predominantly exhibited an immature, pro-germinal center phenotype (PDL-2- CD80- CD35+ CD73+ ). Subsequent subclinical exposures to the allergen induced IgE+ germinal center B cells, serum IgE, and likely activated the classical pathway of anaphylaxis. CONCLUSIONS: Our data demonstrate that IgG1+ B-cell immunity against food allergens in epicutaneous sensitization precedes the generation of IgE responses. Therefore, the assessment of allergen-specific cellular and humoral IgG1+ immunity may help to identify individuals at risk of developing IgE-mediated food allergy and hence provide a window for therapeutic interventions.

Neuroprotection by a caspase inhibitor in acute bacterial meningitis.

Half of the survivors of bacterial meningitis experience motor deficits, seizures, hearing loss or cognitive impairment, despite adequate bacterial killing by antibiotics. We demonstrate that the broad-spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone (z-VAD-fmk) prevented hippocampal neuronal cell death and white blood cell influx into the cerebrospinal fluid compartment in experimental pneumococcal meningitis. Hippocampal neuronal death was due to apoptosis derived from the inflammatory response in the cerebrospinal fluid. Apoptosis was induced in vitro in human neurons by inflamed cerebrospinal fluid and was blocked by z-VAD-fmk. As apoptosis drives neuronal loss in pneumococcal meningitis, caspase inhibitors might provide a new therapeutic option directed specifically at reducing brain damage.

Prokaryotic peptides that block leukocyte adherence to selectins.

Pertussis toxin binds target cells through the carbohydrate recognition properties of two subunits, S2 and S3, which share amino acid sequence similarity with the lectin domains of the eukaryotic selectin family. Selectins appear on inflamed endothelial cells and promote rolling of leukocytes by reversibly binding carbohydrates. S2, S3, and synthetic peptides representing their carbohydrate recognition domains competitively inhibited adherence of neutrophils to selectin-coated surfaces and to endothelial cells in vitro. These proteins and peptides also rapidly upregulated the function of the leukocyte integrin CD11b/CD18. These findings implicate mimicry of eukaryotic selectins by prokaryotic adhesive ligands and link the mechanisms underlying leukocyte trafficking to microbial pathogenesis.

Integrin-mediated localization of Bordetella pertussis within macrophages: role in pulmonary colonization.

The adherence of Bordetella pertussis to human respiratory cilia is critical to the pathogenesis of whooping cough but the significance of bacterial attachment to macrophages has not been determined. Adherence to cilia and macrophages is mediated by two large, nonfimbrial bacterial proteins, filamentous hemagglutinin (FHA), and pertussis toxin (PT). PT and FHA both recognize carbohydrates on cilia and macrophages; FHA also contains an Arg-Gly-Asp (RGD) sequence which promotes bacterial association with the macrophage integrin complement receptor 3 (CR3). We determined that virulent B. pertussis enter and survive in mammalian macrophages in vitro and that CR3 is important for this uptake process. We then determined the relative contribution of CR3 versus carbohydrate-dependent interactions to in vivo pulmonary colonization using a rabbit model. B. pertussis colonized the lung as two approximately equal populations, one extracellular population attached to ciliary and macrophage surface glycoconjugates and another population within pulmonary macrophages. Loss of the CR3 interaction, either by mutation of FHA or treatment with antibody to CR3, disrupted accumulation of viable intracellular bacteria but did not prevent lung pathology. In contrast, elimination of carbohydrate-bound bacteria, either by a competitive receptor analogue or an anti-receptor antibody, was sufficient to prevent pulmonary edema. We propose that CR3-dependent localization of B. pertussis within macrophages promotes persistence of bacteria in the lung without pulmonary injury. On the other hand, the presence of extracellular bacteria adherent to cilia and macrophages in carbohydrate-dependent interactions is associated with pulmonary pathology.

Reduction of inflammation, tissue damage, and mortality in bacterial meningitis in rabbits treated with monoclonal antibodies against adhesion-promoting receptors of leukocytes.

We tested if specific inhibition of recruitment of leukocytes across the blood brain barrier from the vascular compartment to the cerebrospinal fluid (CSF) space reduced tissue damage and improved the outcome of infection in a rabbit model of experimental meningitis. The CD11/CD18 complex of receptors on leukocytes promotes adhesion of these cells to endothelia, a process required for egress of cells into the extravascular space. Intravenous injection of the anti-CD18 mAb IB4 effectively blocked the development of leukocytosis in the CSF of animals challenged intracisternally with living bacteria, bacterial endotoxin, or bacterial cell wall. This effect was associated with protection from blood brain barrier injury as measured by exclusion of serum proteins from CSF in mAb-treated animals. The densities of bacteria in CSF and the degrees of bacterial killing due to ampicillin were not affected by the antibody. Animals receiving the antibody experienced a delay in the development of bacteremia and a significantly reduced inflammatory response during ampicillin-induced bacterial killing. Therapy with mAb IB4 prevented development of brain edema and death in animals challenged with lethal doses of Streptococcus pneumoniae. These studies indicate that the major mechanism of leukocyte migration across the blood brain barrier involves the CD11/CD18 receptors and that inflammatory leukocytes recruited by this mechanism are a major cause of blood brain barrier injury and cerebral edema during meningitis.

Penetration of the blood-brain barrier: enhancement of drug delivery and imaging by bacterial glycopeptides.

The blood-brain barrier restricts the passage of many pharmacological agents into the brain parenchyma. Bacterial glycopeptides induce enhanced blood-brain barrier permeability when they are present in the subarachnoid space during meningitis. By presenting such glycopeptides intravenously, blood-brain barrier permeability in rabbits was enhanced in a reversible time- and dose-dependent manner to agents < or = 20 kD in size. Therapeutic application of this bioactivity was evident as enhanced penetration of the antibiotic penicillin and the magnetic resonance imaging contrast agent gadolinium-diethylene-triamine-pentaacetic acid into the brain parenchyma.

Receptor analogs and monoclonal antibodies that inhibit adherence of Bordetella pertussis to human ciliated respiratory epithelial cells.

The adherence of Bordetella pertussis to human respiratory cilia is critical to the pathogenesis of whooping cough. To explore the development of agents that could interrupt adherence, the structure of the receptor on the ciliary surface was investigated. Using an in vitro adherence assay to human ciliated epithelial cells, galactose, lactose, and complex carbohydrates containing lactose eliminated adherence when preincubated with the bacteria. 10(-2) M galactose eluted adherent bacteria from cilia. B. pertussis and its two purified adhesins bound specifically to natural lactose-containing glycolipids in a TLC assay. mAbs to eukaryotic glycoconjugates with specificity for substituted galactose-glucose moieties blocked adherence when preincubated with ciliated cells. The carbohydrates that serve as receptors for B. pertussis on human cilia are galactose-glucose-containing glycolipids. Receptor analogs and anti-receptor antibodies effectively block adherence of B. pertussis to cilia and thus should be considered candidates for therapeutic intervention against disease.

The role of cytokines in the generation of inflammation and tissue damage in experimental gram-positive meningitis.

Cytokines mediate many host responses to bacterial infections. We determined the inflammatory activities of five cytokines in the central nervous system: TNF-alpha, IL-1 alpha, IL-1 beta, macrophage inflammatory protein 1 (MIP-1), and macrophage inflammatory protein 2 (MIP-2). Using a rabbit model of meningeal inflammation, each cytokine (except IL-1 beta) induced enhanced blood brain barrier permeability, leukocytosis in cerebrospinal fluid, and brain edema. Homologous antibodies to each mediator inhibited leukocytosis and brain edema, and moderately decreased blood brain barrier permeability. In rabbits treated with anti-CD-18 antibody to render neutrophils dysfunctional for adhesion, each cytokine studied lost the ability to cause leukocytosis and brain edema. After intracisternal challenge with pneumococci, antibodies to TNF or IL-1 prevented inflammation, while anti-MIP-1 or anti-MIP-2 caused only a 2-h delay in the onset of inflammation. We suggest these cytokines have multiple inflammatory activities in the central nervous system and contribute to tissue damage during pneumococcal meningitis.

Coordinate regulation of beta-lactamase induction and peptidoglycan composition by the amp operon.

The amp operon, which is located on the Escherichia coli chromosome, modulates the induction of plasmid-borne beta-lactamase genes by extracellular beta-lactam antibiotics. This suggests that the gene products AmpD and AmpE may function in the transduction of external signals. beta-Lactam antibiotics are analogs of cell wall components that can be released during cell wall morphogenesis of enterobacteria. The amp operon was studied to determine its importance in signal transduction during cell wall morphogenesis. The peptidoglycan compositions of amp mutants were determined by high-performance liquid chromatography and fast atom bombardment mass spectrometry. When a chromosomal or plasmid-borne copy of ampD was present, the amount of pentapeptide-containing muropeptides in the cell wall increased upon addition of the cell wall constituent diaminopimelic acid to the growth medium. These results suggest that beta-lactamase induction and modulation of the composition of the cell wall share elements of a regulatory circuit that involves AmpD. Escherichia coli requires AmpD to respond to extracellular signaling amino acids, such as diaminopimelic acid, and this signal transduction system may regulate peptidoglycan composition in response to cell wall turnover products.

A structure-activity relationship for induction of meningeal inflammation by muramyl peptides.

Components of bacterial peptidoglycans have potent biological activities, including adjuvant effects, cytotoxicity, and induction of sleep. Mixtures of peptidoglycan components also induce inflammation in the lung, subarachnoid space, and joint, but the structural requirements for activity are unknown. Using a rabbit model for meningitis, we determined the biological activities of 14 individual muramyl peptides constituting > 90% of the peptidoglycan of the gram-negative pediatric pathogen Haemophilus influenzae. Upon intracisternal inoculation, most of the muropeptides induced leukocytosis in cerebrospinal fluid (CSF), influx of protein into CSF, or brain edema, alone or in combination. The disaccharide-tetrapeptide, the major component of all gram-negative peptidoglycans, induced CSF leukocytosis and protein influx at doses as low as 0.4 microgram (0.42 nM). Modification of the N-acetyl muramic acid or substitution of the alanine at position four in the peptide side chain decreased leukocytosis but enhanced brain edema. As the size of the muropeptide increased, the inflammatory activity decreased. Muropeptide carrying the diaminopimelyl-diaminopimelic acid cross-link specifically induced cytotoxic brain edema. These findings significantly expand the spectrum of biological activities of natural muramyl peptides and provide the basis for a structure-activity relationship for the inflammatory properties of bacterial muropeptides.

Pneumococcal trafficking across the blood-brain barrier. Molecular analysis of a novel bidirectional pathway.

Although Streptococcus pneumoniae is a major cause of meningitis in humans, the mechanisms underlying its traversal from the circulation across the blood-brain barrier (BBB) into the subarachnoid space are poorly understood. One mechanism might involve transcytosis through microvascular endothelial cells. In this study we investigated the ability of pneumococci to invade and transmigrate through monolayers of rat and human brain microvascular endothelial cells (BMEC). Significant variability was found in the invasive capacity of clinical isolates. Phase variation to the transparent phenotype increased invasion as much as 6-fold and loss of capsule approximately 200-fold. Invasion of transparent pneumococci required choline in the pneumococcal cell wall, and invasion was partially inhibited by antagonists of the platelet-activating factor (PAF) receptor on the BMEC. Pneumococci that gained access to an intracellular vesicle from the apical side of the monolayer subsequently were subject to three fates. Most opaque variants were killed. In contrast, the transparent phase variants were able to transcytose to the basal surface of rat and human BMEC in a manner dependent on the PAF receptor and the presence of pneumococcal choline-binding protein A. The remaining transparent bacteria entering the cell underwent a previously unrecognized recycling to the apical surface. Transcytosis eventually becomes a dominating process accounting for up to 80% of intracellular bacteria. Our data suggest that interaction of pneumococci with the PAF receptor results in sorting so as to transcytose bacteria across the cell while non-PAF receptor entry shunts bacteria for exit and reentry on the apical surface in a novel recycling pathway.

Differing roles for platelet-activating factor during inflammation of the lung and subarachnoid space. The special case of Streptococcus pneumoniae.

Although well-characterized in the lung, the role of platelet-activating factor (PAF) in inflammation in the central nervous system is undefined. Using rabbit models of meningitis and pneumonia, PAF was found to induce significant blood-brain barrier permeability and brain edema at doses five times lower than those required to generate leukocyte recruitment to the subarachnoid space. Both leukocytosis and increased vascular permeability occurred in response to PAF in the lung. Antibody to the CD-18 family of leukocyte adhesion molecules inhibited leukocyte recruitment in response to PAF in the brain (greater than 80%); a similar level of inhibition in the lung required treatment with a combination of a PAF receptor antagonist (L-659,989) and anti-CD18 antibody. Treatment with L-659,989 decreased abnormal cerebrospinal fluid cytochemical values induced by intracisternal challenge with pneumococci but not Haemophilus influenzae, indicating a special role for PAF in pneumococcal disease. Antibodies directed at phosphorylcholine, a unique, shared determinant of bioactivity of PAF and pneumococcal cell wall, obviated the inflammatory potential of both agents. However, no evidence for a direct PAF-like activity of pneumococcal cell wall components was detected in vitro by bioassay using platelets or neutrophils. It is concluded that PAF can induce inflammation in the subarachnoid space. In brain, PAF effects appear to be mediated through CD-18-dependent events, while in lung, PAF effects independent of CD-18 are also evident. At both sites, PAF is of particular clinical importance during inflammation induced by pneumococci apparently due to a unique proinflammatory relationship between the pneumococcal cell wall teichoic acid and PAF.

Molecular and cellular biology of pneumococcal infection.

The complete pneumococcal genome sequence was released in November, 1997. This advance has been combined with new understanding of physiology and pathogenesis including characterization of a family of surface proteins adducted to choline, regulation of virulence, and the regulon for natural DNA transformation. These developments in our understanding of molecular and cellular biology of pneumococcal infection will allow the development of new vaccines and antibiotics against this community acquired pathogen.

Entry of pathogens into the central nervous system.

The blood-brain barrier (BBB) is formed by the tight junctions of the cerebral capillary endothelium and the choroid plexus epithelium. The molecular anatomy of the tight junction resembles that of a polarized, transporting epithelium, suggesting some model cell culture systems can provide insight into traffic into the central nervous system. Pathogens target both the endothelium, causing encephalitis, and the choroid plexus, leading to meningitis. Routes of entry are diverse including paracellular and transcellular penetration. In addition, circulating microbial products can induce loss of BBB function. Understanding the heterogeneous molecular interactions between pathogens and the BBB may provide avenues to interrupt the devastating neurological sequelae that accompany central nervous system infections.

Attachment factors of Bordetella pertussis: mimicry of eukaryotic cell recognition molecules.

Bacteria localize to specific target organs during infection by recognizing eukaryotic cell surface addresses. This address system exists to facilitate normal trafficking of host cells such as leukocytes. The mechanisms by which bacteria subvert host targeting codes provide a unique link between microbial pathogenesis and eukaryotic cell trafficking, and can point to a new array of antibodies and peptides of potential therapeutic value.

Responsiveness to a pandemic alert: use of reverse genetics for rapid development of influenza vaccines.

BACKGROUND: In response to the emergence of severe infection capable of rapid global spread, WHO will issue a pandemic alert. Such alerts are rare; however, on Feb 19, 2003, a pandemic alert was issued in response to human infections caused by an avian H5N1 influenza virus, A/Hong Kong/213/03. H5N1 had been noted once before in human beings in 1997 and killed a third (6/18) of infected people. The 2003 variant seemed to have been transmitted directly from birds to human beings and caused fatal pneumonia in one of two infected individuals. Candidate vaccines were sought, but no avirulent viruses antigenically similar to the pathogen were available, and the isolate killed embryonated chicken eggs. Since traditional strategies of vaccine production were not viable, we sought to produce a candidate reference virus using reverse genetics. METHODS: We removed the polybasic aminoacids that are associated with high virulence from the haemagglutinin cleavage site of A/Hong Kong/213/03 using influenza reverse genetics techniques. A reference vaccine virus was then produced on an A/Puerto Rico/8/34 (PR8) backbone on WHO-approved Vero cells. We assessed this reference virus for pathogenicity in in-vivo and in-vitro assays. FINDINGS: A reference vaccine virus was produced in Good Manufacturing Practice (GMP)-grade facilities in less than 4 weeks from the time of virus isolation. This virus proved to be non-pathogenic in chickens and ferrets and was shown to be stable after multiple passages in embryonated chicken eggs. INTERPRETATION: The ability to produce a candidate reference virus in such a short period of time sets a new standard for rapid response to emerging infectious disease threats and clearly shows the usefulness of reverse genetics for influenza vaccine development. The same technologies and procedures are currently being used to create reference vaccine viruses against the 2004 H5N1 viruses circulating in Asia.

Mechanisms of antibiotic resistance and tolerance in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major pathogen causing potentially life-threatening community-acquired diseases in both the developed and developing world. Since 1967, there has been a dramatic increase in the incidence of penicillin-resistant and multiply antibiotic-resistant pneumococci worldwide. Prevention of access of the antibiotic to the target, inactivation of the antibiotic and alteration of the target are mechanisms that S. pneumoniae has developed to resist antibiotics. Recent studies on antibiotic-tolerant pneumococcal mutants permitted development of a novel model for the control of bacterial cell death.

Molecular pathogenesis of pneumococcal pneumonia.

The past two decades have witnessed an explosion of data on the molecular pathogenesis of pneumonia caused by Streptococcus pneumoniae, one of the most important pathogens currently plaguing man. Identification and functional analysis of genes and their proteins, elucidation of mechanisms involved in adherence, colonization, inflammation, and invasion, and an understanding of interactions with the host and with external factors have provided knowledge that can be used to attack this organism with small molecule or vaccine based strategies. Study of the pneumococcus has also led to insights into other pathogens that share a unique spectrum of respiratory disease. In this review we will discuss recent advances in our understanding of the pathogenesis of pneumonia due to S. pneumoniae, highlighting emerging themes common to other organisms such as Haemophilus influenzae and Neisseria meningitidis.

The impact of dexamethasone on hearing loss in experimental pneumococcal meningitis.

Bacterial meningitis, particularly that resulting from Streptococcus pneumoniae, is a common cause of acquired profound sensorineural deafness in children. The pathogenesis of meningogenic hearing loss has been investigated in an experimental rabbit model. In this study significant deafness was documented within the first 15 hours of infection. Initiation of antibiotic therapy at this time diminished the severity of hearing loss in most animals. The addition of dexamethasone to antibiotic therapy prevented the development of profound deafness. These results suggest this model will be useful in developing antiinflammatory strategies to improve the outcome of bacterial meningitis.

Hearing loss and pneumococcal meningitis: an animal model.

Clinical studies of predisposing factors in the development of hearing loss secondary to bacterial meningitis have produced conflicting results. An animal model of meningogenic labyrinthitis was developed for more precise study of these parameters. Rabbits were inoculated intrathecally with 10(5) pneumococci to induce meningitis. Hearing thresholds were measured using auditory-evoked responses to 1 kHz, 10 kHz, and click stimuli before infection and every 12 hours thereafter. Profound deafness occurred in all subjects at an average of 48 hours following infection. The incidence and severity of hearing loss was strongly correlated with the duration of meningitis. Temporal bone histology revealed acute inflammation of all perilymphatic spaces including the cochlear aqueduct. This model demonstrated that the risk and severity of hearing loss increase with the duration of meningitis and suggested that the cochlear aqueduct is an anatomic pathway for the extension of infection from the cerebrospinal fluid to the cochlea. The implications for therapy in humans is discussed.