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.

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.

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.

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.

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.

PAf receptor anchors Streptococcus pneumoniae to activated human endothelial cells.

Streptococcus pneumoniae can produce asymptomatic colonization or aggressive sepsis. We sought to differentiate the molecular mechanisms of these disparate courses. Cytokine or thrombin activation of human vascular endothelial cells and type II pneumocytes enhanced pneumococcal adherence relative to resting cells. Adherence and subsequent invasion was dramatically reduced by PAF receptor antagonists. Cells transfected with the PAF receptor gained the ability to support pneumococcal adherence. PAF or PAF receptor antagonists inhibited attachment and invasion. Adherence involved phosphorylcholine on the pneumococcal teichoic acid. Virulent pneumococci target the PAF receptor on activated human cells, a necessary step to facilitate subsequent invasion.

Pathogenesis of pneumococcal inflammation: otitis media.

Pneumococci cause damage to the ear in otitis media and in association with bacterial meningitis. The pathogenesis of injury involves host response to cell wall and pneumolysin. Release of cell wall, particularly during antibiotic-induced bacterial lysis, leads to an influx of leukocytes and subsequent tissue injury. The signal transduction cascade for this response is becoming defined and includes CD14, Toll-like receptor 2, NFkB, and cytokine production. The second source of injury is the cytotoxicity of the pore forming toxin, pneumolysin. Decreasing the sequelae of otitis can be achieved by an increased understanding of the site-specific mechanisms of pneumococcal-induced inflammation.

The biology of pneumococcal infection.

For 100 y, the study of the molecular mechanism of pneumococcal infection has richly rewarded biomedical science and pediatrics. More recently, a framework has emerged for how the pathogen engineers colonization, invasion of the lung and bloodstream, and finally, entry into the brain. This trafficking is then followed by a separate set of events to generate the symptoms of disease. Understanding the ligand receptor interactions that dictate these events has suggested new concepts for how to control the course of an infectious process and improve the morbidity and mortality of encounters with this prevalent pathogen of children.

Receptor specificity of adherence of Streptococcus pneumoniae to human type-II pneumocytes and vascular endothelial cells in vitro.

The adherence of S. pneumoniae to human type-II pneumocytes and endothelial cells (EC) is critical to the pathogenesis of pneumococcal pneumonia and bacteremia. We established that the preferred target cell to which pneumococci adhere in the lung is the type-II lung cell (LC) and have developed an in vitro adherence assay to determine the molecular details of this interaction. Pneumococcal receptors on cultured human LC and EC appeared to be glycoproteins since treatment of the monolayers with tunicamycin significantly impaired bacterial adherence. Inhibition of adherence to LC and EC occurred following incubation with several carbohydrates including GalNAc, mannose and GalNAc beta-4Gal- and GalNAc beta 1-3Gal-containing glycoconjugates. Pneumococci could bind directly to these immobilized sugars and their addition to adherent pneumococci could elute the bacteria from LC and EC. Combinations of glycoconjugates indicated that two independent classes of pneumococcal receptor existed on both cell types. These were defined by the minimal receptor units GalNAc beta 1-4Gal and GalNAc beta 1-3Gal which participate in pneumococcal cell wall and protein-dependent mechanisms of adherence, respectively.

The pathophysiology of pneumococcal meningitis.

The interactions between pneumococcal surface components and host defence systems that initiate pneumococcal meningitis have been studied in considerable molecular detail over the past decade. In this sense, the pneumococcus has served as a prototype for the unravelling of the genesis of inflammation caused by gram-positive bacteria. This review outlines the progression of these early events involving the cytokine cascade, the coagulation cascade, and leukocyte migration, and relates these processes to the production of blood-brain barrier permeability, the hallmark of injury in meningitis. This new understanding has radically altered the therapy of disease with the promise of greatly improved outcome.

Adherence of Bordetella pertussis to human respiratory epithelial cells.

Adherence of Bordetella pertussis to ciliated respiratory tract mucosa is important in the pathogenesis of whooping cough. The adherence of B pertussis to human respiratory epithelial cells was investigated using cells obtained by brushing at bronchoscopy. B pertussis attached exclusively to the ciliary tufts of ciliated cells. A mean +/- SEM of 5.0 +/- 0.3 organisms attached per cell when bacteria in a concentration of 2 X 10(9)/ml were incubated with ciliated cells. Organisms examined by electron microscopy were found to adhere to the cilia both by direct apposition and by filaments coursing between bacteria and cilia. The specificity of the adherence of B pertussis to ciliary tufts may explain the unique ability of this organism to infect the human tracheobronchial mucosa.

Staphylococcal protein A adsorption of neonatal serum to facilitate early diagnosis of congenital infection.

The early diagnosis of congenital infection frequently depends on the ability to distinguish between infant IgM and maternal IgG antibodies. Staphylococcal protein A, which specifically binds IgG, removed maternal IgG from the serum of newborn infants. Residual IgM antibodies to CMV, rubella, toxoplasmosis, and syphilis were then identified by routine serologic techniques. Persistence of greater than or equal to 25% of the original antibody titer following SPA adsorption distinguished the sera of infants with congenital infection from those of healthy infants. No false negative results were encountered. Specificity of the serologic results of SPA-treated infant sera correlated with IgM-specific identification of the causative agent. Potentially false positive titers were identified by concurrent elevation of IgA or rheumatoid factor. Adsorption of cord or neonatal serum with SPA facilitates accurate serologic diagnosis of congenital infection.

Consequences of microbial attachment: directing host cell functions with adhesins.

We take the view that adherence is not just a static process of holding hands but rather elicits a response in the targeted cell. From this point of view, adherence is an active process with an outcome. This outcome or fate is predictable only when several parameters of the host cell-adhesin interaction are known: is the adhesin acting alone or in series with other products, is the receptor up- or down-regulated at the time of ligation, which domain of the receptor is bound, and finally, which intracellular response circuits are connected to the receptor in the cell type targeted? Variations in these parameters are the basis for the ability of the adhesins of pathogens to orchestrate outcomes as disparate as simple address recognition versus actin nucleation, cytokine induction, activation of plasmin, derangement of leukocyte migration, or deposition of antibody on host cell membranes. The recognition of the relatedness of some eukaryotic and prokaryotic adhesive domains and the shared use of existing eukaryotic cell-cell interaction systems between host and pathogen suggest that the cellular interactions of interest in eukaryotic cell biology can be revealed by taking clues from the pathogens, which have studied and adapted to them the longest.

The molecular basis of pneumococcal infection: a hypothesis.

New insight has been gained into the mechanisms underlying the tissue tropism and inflammation of pneumococcal infection. Virulence has been linked to a transparent colonial morphology. Adherence has been characterized at the molecular level, and the importance of receptors arising upon activation of eukaryotic cells in promoting the progression to disease has been established. The contribution of peptidoglycan and teichoic acid to the generation of inflammation has suggested the need to couple anti-inflammatory therapy with antibiotic treatment in order to improve the outcome of invasive disease. Elucidation of the pathogenesis of pneumococcal infection, including the identification of virulence determinants by recently developed genetic strategies, can provide a paradigm for new mechanisms that are active in gram-positive bacterial infections and that are clearly distinct from the familiar pathways triggered by endotoxin.

Pneumococcal cell wall activates NF-kappa B in human monocytes: aspects distinct from endotoxin.

The transcription factor NF-kappa B plays a central role in inflammation by controlling the transcription of multiple genes which participate in the acute phase response. Mice with a targeted disruption of the p50 subunit of NF-kappa B are hyper-susceptible to challenge with pneumococci but not endotoxin. We sought to clarify the role of NF-kappa B in the host response to the critical inflammatory component of pneumococci, the cell wall. Activation of NF-kappa B was monitored by expression of luciferase from cells transfected with an NF-kappa B dependent luciferase reporter construct. 70Z/3 murine pre-B cells and U937 human monocytes failed to produce luciferase in response to 10(7) pneumococci or 10 mu g cell wall; strong responses were obtained with lO mu g of LPS. In contrast, THP-1 human monocytes showed strong luciferase production with all three stimuli: LPS, intact pneumococci and cell wall. The response was time and dose dependent. Cell wall activity was retained despite alteration of the choline of the teichoic acid or protease treatment suggesting the glycopeptide backbone to be a critical determinant of bioactivity. We conclude that activation of NF-kappa B by pneumococci is restricted to certain cells and that this proinflammatory activity may be a specific feature of the pneumococcal cell wall glycopeptide backbone.