Technological Aspects of Lithium-Titanium Ferrite Synthesis by Electron-Beam Heating.

Solid-phase synthesis of lithium-titanium ferrite by electron-beam heating of a Fe2O3-Li2CO3-TiO2 initial reagents mixture with different history (powder, compact, mechanically activated mixture) was studied using X-ray diffraction, thermomagnetometric and specific saturation magnetization analyses. Ferrite was synthesized using an ILU-6 pulsed electron accelerator; it generated electrons with electron energy of 2.4 MeV to heat samples to temperatures of 600 and 750 °C. The isothermal holding time upon reaching the synthesis temperature was 0-120 min. The efficiency of ferrite synthesis by electron-beam heating was evaluated via comparison of the characteristics of the obtained samples with those synthesized by conventional ceramic technology under similar temperature-time conditions. It was found that the rate of ferrite formation depends on the heating method, temperature, synthesis time, density, and activity of the initial mixture. It was shown that sample compaction provides the preferential formation of unsubstituted lithium ferrite of Li0.5Fe2.5O4 composition with a Curie temperature of at ca. 630 °C in both synthesis methods. High-energy electron-beam heating of the mechanically activated mixture significantly accelerates synthesis of Li0.6Fe2.2Ti0.2O4 substituted ferrite, for which the Curie temperature and specific saturation magnetization were recorded as 534 °C and 50 emu/g, respectively. Therefore, LiTi ferrites can be obtained at a lower temperature (750 °C) and with a shorter synthesis time (120 min) compared to traditional ceramic technology.

Quantitative Meta-analyses of Cognitive Abilities in Children With Pediatric-onset Multiple Sclerosis.

Pediatric-onset multiple sclerosis (POMS), is the manifestation of multiple sclerosis in individuals before 18 years of age. About a third of children with POMS show some form of lower cognitive performance. The purpose of this study is to examine using quantitative meta-analyses the effect size of altered performance between children with and without POMS on overall intelligence quotient (IQ), information processing speed, and language functions. We searched the literature for studies that reported scores on cognitive tests administered to children with and without POMS. Studies were systematically reviewed using PRISMA guidelines. We analyzed data from 14 studies that examined 1283 children with and without POMS when cognitive categories consisted of five or more studies. Effect sizes, publication bias and potential confounds were considered. Significant cognitive differences are revealed for all categories with the strongest effect observed for overall IQ. A moderate effect is observed for information processing speed, and small effects for verbal fluency and verbal memory. Cognitive abilities present differently in children with POMS and a better understanding of this manifestation will inform intervention and remediation tools that can improve clinical and educational practice for the benefit of children with POMS.

Neuronal genetic rescue normalizes brain network dynamics in a lysosomal storage disorder despite persistent storage accumulation.

Although neurologic symptoms occur in two-thirds of lysosomal storage disorders (LSDs), for most we do not understand the mechanisms underlying brain dysfunction. A major unanswered question is if the pathogenic hallmark of LSDs, storage accumulation, induces functional defects directly or is a disease bystander. Also, for most LSDs we do not know the impact of loss of function in individual cell types. Understanding these critical questions are essential to therapy development. Here, we determine the impact of genetic rescue in distinct cell types on neural circuit dysfunction in CLN3 disease, the most common pediatric dementia and a paradigmatic neurodegenerative LSD. We restored Cln3 expression via AAV-mediated gene delivery and conditional genetic rescue in a CLN3 disease mouse model. Surprisingly, we found that low-level rescue of Cln3 expression in neurons alone normalized clinically relevant electrophysiologic markers of network dysfunction, despite the presence of substantial residual histopathology, in contrast to restoring expression in astrocytes. Thus, loss of CLN3 function in neurons, not storage accumulation, underlies neurologic dysfunction in CLN3 disease. This impliesies that storage clearance may be an inappropriate target for therapy development and an ineffectual biomarker.

Neuronal network dysfunction precedes storage and neurodegeneration in a lysosomal storage disorder.

Accumulation of lysosomal storage material and late-stage neurodegeneration are hallmarks of lysosomal storage disorders (LSDs) affecting the brain. Yet, for most LSDs, including CLN3 disease, the most common form of childhood dementia, it is unclear what mechanisms drive neurologic symptoms. Do deficits arise from loss of function of the mutated protein or toxicity from storage accumulation? Here, using in vitro voltage-sensitive dye imaging and in vivo electrophysiology, we find progressive hippocampal dysfunction occurs before notable lysosomal storage and neuronal loss in 2 CLN3 disease mouse models. Pharmacologic reversal of lysosomal storage deposition in young mice does not rescue this circuit dysfunction. Additionally, we find that CLN3 disease mice lose an electrophysiologic marker of new memory encoding - hippocampal sharp-wave ripples. This discovery, which is also seen in Alzheimer's disease, suggests the possibility of a shared electrophysiologic signature of dementia. Overall, our data describe new insights into previously unknown network-level changes occurring in LSDs affecting the central nervous system and highlight the need for new therapeutic interventions targeting early circuit defects.

Modulating membrane fluidity corrects Batten disease phenotypes in vitro and in vivo.

The neuronal ceroid lipofuscinoses are a class of inherited neurodegenerative diseases characterized by the accumulation of autofluorescent storage material. The most common neuronal ceroid lipofuscinosis has juvenile onset with rapid onset blindness and progressive degeneration of cognitive processes. The juvenile form is caused by mutations in the CLN3 gene, which encodes the protein CLN3. While mouse models of Cln3 deficiency show mild disease phenotypes, it is apparent from patient tissue- and cell-based studies that its loss impacts many cellular processes. Using Cln3 deficient mice, we previously described defects in mouse brain endothelial cells and blood-brain barrier (BBB) permeability. Here we expand on this to other components of the BBB and show that Cln3 deficient mice have increased astrocyte endfeet area. Interestingly, this phenotype is corrected by treatment with a commonly used GAP junction inhibitor, carbenoxolone (CBX). In addition to its action on GAP junctions, CBX has also been proposed to alter lipid microdomains. In this work, we show that CBX modifies lipid microdomains and corrects membrane fluidity alterations in Cln3 deficient endothelial cells, which in turn improves defects in endocytosis, caveolin-1 distribution at the plasma membrane, and Cdc42 activity. In further work using the NIH Library of Integrated Network-based Cellular Signatures (LINCS), we discovered other small molecules whose impact was similar to CBX in that they improved Cln3-deficient cell phenotypes. Moreover, Cln3 deficient mice treated orally with CBX exhibited recovery of impaired BBB responses and reduced autofluorescence. CBX and the compounds identified by LINCS, many of which have been used in humans or approved for other indications, may find therapeutic benefit in children suffering from CLN3 deficiency through mechanisms independent of their original intended use.

Screening of Conditionally Reprogrammed Patient-Derived Carcinoma Cells Identifies ERCC3-MYC Interactions as a Target in Pancreatic Cancer.

PURPOSE: Even when diagnosed prior to metastasis, pancreatic ductal adenocarcinoma (PDAC) is a devastating malignancy with almost 90% lethality, emphasizing the need for new therapies optimally targeting the tumors of individual patients. EXPERIMENTAL DESIGN: We first developed a panel of new physiologic models for study of PDAC, expanding surgical PDAC tumor samples in culture using short-term culture and conditional reprogramming with the Rho kinase inhibitor Y-27632, and creating matched patient-derived xenografts (PDX). These were evaluated for sensitivity to a large panel of clinical agents, and promising leads further evaluated mechanistically. RESULTS: Only a small minority of tested agents was cytotoxic in minimally passaged PDAC cultures in vitro Drugs interfering with protein turnover and transcription were among most cytotoxic. Among transcriptional repressors, triptolide, a covalent inhibitor of ERCC3, was most consistently effective in vitro and in vivo causing prolonged complete regression in multiple PDX models resistant to standard PDAC therapies. Importantly, triptolide showed superior activity in MYC-amplified PDX models and elicited rapid and profound depletion of the oncoprotein MYC, a transcriptional regulator. Expression of ERCC3 and MYC was interdependent in PDACs, and acquired resistance to triptolide depended on elevated ERCC3 and MYC expression. The Cancer Genome Atlas analysis indicates ERCC3 expression predicts poor prognosis, particularly in CDKN2A-null, highly proliferative tumors. CONCLUSIONS: This provides initial preclinical evidence for an essential role of MYC-ERCC3 interactions in PDAC, and suggests a new mechanistic approach for disruption of critical survival signaling in MYC-dependent cancers. Clin Cancer Res; 22(24); 6153-63. ©2016 AACR.

AAV gene transfer delays disease onset in a TPP1-deficient canine model of the late infantile form of Batten disease.

The most common form of the childhood neurodegenerative disease late infantile neuronal ceroid lipofuscinosis (also called Batten disease) is caused by deficiency of the soluble lysosomal enzyme tripeptidyl peptidase 1 (TPP1) resulting from mutations in the TPP1 gene. We tested whether TPP1 gene transfer to the ependyma, the epithelial lining of the brain ventricular system, in TPP1-deficient dogs would be therapeutically beneficial. A one-time administration of recombinant adeno-associated virus (rAAV) expressing canine TPP1 (rAAV.caTPP1) resulted in high expression of TPP1 predominantly in ependymal cells and secretion of the enzyme into the cerebrospinal fluid leading to clinical benefit. Diseased dogs treated with rAAV.caTPP1 showed delays in onset of clinical signs and disease progression, protection from cognitive decline, and extension of life span. By immunostaining and enzyme assay, recombinant protein was evident throughout the brain and spinal cord, with correction of the neuropathology characteristic of the disease. This study in a naturally occurring canine model of TPP1 deficiency highlights the utility of AAV transduction of ventricular lining cells to accomplish stable secretion of recombinant protein for broad distribution in the central nervous system and therapeutic benefit.

Tolerance of a phage element by Streptococcus pneumoniae leads to a fitness defect during colonization.

The pathogenesis of the disease caused by Streptococcus pneumoniae begins with colonization of the upper respiratory tract. Temperate phages have been identified in the genomes of up to 70% of clinical isolates. How these phages affect the bacterial host during colonization is unknown. Here, we examined a clinical isolate that carries a novel prophage element, designated Spn1, which was detected in both integrated and episomal forms. Surprisingly, both lytic and lysogenic Spn1 genes were expressed under routine growth conditions. Using a mouse model of asymptomatic colonization, we demonstrate that the Spn1(-) strain outcompeted the Spn1(+) strain >70-fold. To determine if Spn1 causes a fitness defect through a trans-acting factor, we constructed an Spn1(+) mutant that does not become an episome or express phage genes. This mutant competed equally with the Spn1(-) strain, indicating that expression of phage genes or phage lytic activity is required to confer this fitness defect. In vitro, we demonstrate that the presence of Spn1 correlated with a defect in LytA-mediated autolysis. Furthermore, the Spn1(+) strain displayed increased chain length and resistance to lysis by penicillin compared to the Spn(-) strain, indicating that Spn1 alters the cell wall physiology of its host strain. We posit that these changes in cell wall physiology allow for tolerance of phage gene products and are responsible for the relative defect of the Spn1(+) strain during colonization. This study provides new insight into how bacteria and prophages interact and affect bacterial fitness in vivo.

Comparative analysis of measures of viral reservoirs in HIV-1 eradication studies.

HIV-1 reservoirs preclude virus eradication in patients receiving highly active antiretroviral therapy (HAART). The best characterized reservoir is a small, difficult-to-quantify pool of resting memory CD4(+) T cells carrying latent but replication-competent viral genomes. Because strategies targeting this latent reservoir are now being tested in clinical trials, well-validated high-throughput assays that quantify this reservoir are urgently needed. Here we compare eleven different approaches for quantitating persistent HIV-1 in 30 patients on HAART, using the original viral outgrowth assay for resting CD4(+) T cells carrying inducible, replication-competent viral genomes as a standard for comparison. PCR-based assays for cells containing HIV-1 DNA gave infected cell frequencies at least 2 logs higher than the viral outgrowth assay, even in subjects who started HAART during acute/early infection. This difference may reflect defective viral genomes. The ratio of infected cell frequencies determined by viral outgrowth and PCR-based assays varied dramatically between patients. Although strong correlations with the viral outgrowth assay could not be formally excluded for most assays, correlations achieved statistical significance only for integrated HIV-1 DNA in peripheral blood mononuclear cells and HIV-1 RNA/DNA ratio in rectal CD4(+) T cells. Residual viremia was below the limit of detection in many subjects and did not correlate with the viral outgrowth assays. The dramatic differences in infected cell frequencies and the lack of a precise correlation between culture and PCR-based assays raise the possibility that the successful clearance of latently infected cells may be masked by a larger and variable pool of cells with defective proviruses. These defective proviruses are detected by PCR but may not be affected by reactivation strategies and may not require eradication to accomplish an effective cure. A molecular understanding of the discrepancy between infected cell frequencies measured by viral outgrowth versus PCR assays is an urgent priority in HIV-1 cure research.

Within-host competition drives selection for the capsule virulence determinant of Streptococcus pneumoniae.

For many opportunistic pathogens, it is unclear why their virulence determinants and expression of pathogenic behavior have evolved when damage or death of their host offers no obvious selective advantage to microbial growth or survival. Many pathogens initiate interactions with their host on mucosal surfaces and must compete with other members of the microflora for the same niche. Here we explore whether competitive interactions between microbes promote the acquisition of virulence characteristics. During model murine nasal colonization, Haemophilus influenzae outcompetes another member of the local flora, Streptococcus pneumoniae, by recruiting neutrophils and stimulating the killing of complement-opsonized pneumococci. For S. pneumoniae, resistance to opsonophagocytic killing is determined by its polysaccharide capsule. Although there are many capsule types among different S. pneumoniae isolates that allow for efficient colonization, virulent pneumococci express capsules that confer resistance to opsonophagocytic clearance. Modeling of interspecies interaction predicts that these more virulent S. pneumoniae will prevail during competition with H. influenzae, even if production of a capsule is otherwise costly. Experimental colonization studies confirmed the increased survival of the more virulent S. pneumoniae type during competition. Our findings demonstrate that competition between microbes during their commensal state may underlie selection for characteristics that allow invasive disease.

Recognition of peptidoglycan from the microbiota by Nod1 enhances systemic innate immunity.

Humans are colonized by a large and diverse bacterial flora (the microbiota) essential for the development of the gut immune system. A broader role for the microbiota as a major modulator of systemic immunity has been proposed; however, evidence and a mechanism for this role have remained elusive. We show that the microbiota are a source of peptidoglycan that systemically primes the innate immune system, enhancing killing by bone marrow-derived neutrophils of two major pathogens: Streptococcus pneumoniae and Staphylococcus aureus. This requires signaling via the pattern recognition receptor nucleotide-binding, oligomerization domain-containing protein-1 (Nod1, which recognizes meso-diaminopimelic acid (mesoDAP)-containing peptidoglycan found predominantly in Gram-negative bacteria), but not Nod2 (which detects peptidoglycan found in Gram-positive and Gram-negative bacteria) or Toll-like receptor 4 (Tlr4, which recognizes lipopolysaccharide). We show translocation of peptidoglycan from the gut to neutrophils in the bone marrow and show that peptidoglycan concentrations in sera correlate with neutrophil function. In vivo administration of Nod1 ligands is sufficient to restore neutrophil function after microbiota depletion. Nod1(-/-) mice are more susceptible than wild-type mice to early pneumococcal sepsis, demonstrating a role for Nod1 in priming innate defenses facilitating a rapid response to infection. These data establish a mechanism for systemic immunomodulation by the microbiota and highlight potential adverse consequences of microbiota disruption by broad-spectrum antibiotics on innate immune defense to infection.

Natural antibody to conserved targets of Haemophilus influenzae limits colonization of the murine nasopharynx.

Nasopharyngeal colonization represents the initial interaction between Haemophilus influenzae and its human host. Factors that influence bacterial carriage likely affect transmission and incidence of infection. Therefore, we investigated host factors involved in limiting H. influenzae colonization in BALB/c mice, as colonization can be established in this genetic background. Unlike what is observed in the C57BL/6 background, initial colonization of BALB/c mice was mainly limited by adaptive immune components. This effect on colonization did not require either CD4- or CD8-positive T cells. Instead, initial colonization by genetically diverse strains was limited by preexisting natural antibody with a lesser contribution of complement activity and the presence of neutrophils. Natural serum immunoglobulin from mice was able to bind to the bacterial surface and exhibited complement-dependent bactericidal activity against these genetically diverse H. influenzae strains. Moreover, natural immunoglobulin G (IgG) recognizing these strains was detected at the nasopharyngeal mucosal surface. This antibody-mediated effect required exposure to the normal mouse microbial flora, since mice raised under germfree (GF) conditions showed increased levels of H. influenzae colonization that were not limited by adaptive immunity. In addition, serum IgG from GF mice exhibited less surface binding to H. influenzae, suggesting that natural antibody, induced through prior exposure to the microbial flora, mediated the observed reduction in initial colonization. The broad effect of natural IgG against genetically diverse isolates suggests the presence of conserved species-wide protective targets of antibody.

Early bacterial colonization induces toll-like receptor-dependent transforming growth factor beta signaling in the epithelium.

Colonization of the upper respiratory tract is an initial step that may lead to disease for many pathogens. To prevent compromise of the epithelial barrier, the host must monitor and tightly control bacterial levels on the mucosa. Here we show that innate immune functions of respiratory epithelial cells control colonization by Streptococcus pneumoniae and Haemophilus influenzae in a Toll-like receptor (TLR)-dependent manner. Activation of inflammatory pathways, including mitogen-activated protein kinase signaling, in respiratory epithelial cells was accompanied by the induction of the transforming growth factor beta signaling cascade during early colonization. Thus, colonization resulted in upregulation of factors involved in a proinflammatory response (e.g., interleukin-6) as well as factors known to modulate the epithelial barrier (e.g., Snail-1). These in vivo data provided a link between inflammation control and maintenance of the mucosal barrier function during infection and emphasized the importance of TLR-dependent inflammatory responses of the respiratory epithelium.

Mucosal clearance of capsule-expressing bacteria requires both TLR and nucleotide-binding oligomerization domain 1 signaling.

Expression of capsular polysaccharide by bacterial pathogens is associated with increased resistance to host clearance mechanisms, in particular by evading opsonization and uptake by professional phagocytes. The potential for rapid progression of disease caused by encapsulated bacteria points to the importance of innate immunity at the mucosal surface where infection is initiated. Using a murine model of nasopharyngeal colonization, host immune components that contribute to the mucosal clearance of capsule-expressing bacteria were investigated. Clearance of encapsulated Haemophilus influenzae (Hi) required both TLR and nucleotide-binding oligomerization domain (NOD) signaling pathways, whereas individual deficiencies in each of these signaling cascades did not affect clearance of nonencapsulated strains. Moreover, clearance of Hi-expressing capsular polysaccharide required the recruitment of neutrophils to the site of infection, and ex vivo phagocytic bacterial killing required expression of the NOD1 signaling pathway. Conversely, redundancies within these innate immune pathways of non-neutrophil cells were sufficient to promote mucosal clearance of nonencapsulated Hi. Our findings reveal a role for NOD1 in protection from encapsulated pathogens. In addition, this study provides an example of a microbial virulence determinant that alters the requirements for host signaling to provide effective protection.

Nod1 signaling overcomes resistance of S. pneumoniae to opsonophagocytic killing.

Airway infection by the Gram-positive pathogen Streptococcus pneumoniae (Sp) leads to recruitment of neutrophils but limited bacterial killing by these cells. Co-colonization by Sp and a Gram-negative species, Haemophilus influenzae (Hi), provides sufficient stimulus to induce neutrophil and complement-mediated clearance of Sp from the mucosal surface in a murine model. Products from Hi, but not Sp, also promote killing of Sp by ex vivo neutrophil-enriched peritoneal exudate cells. Here we identify the stimulus from Hi as its peptidoglycan. Enhancement of opsonophagocytic killing was facilitated by signaling through nucleotide-binding oligomerization domain-1 (Nod1), which is involved in recognition of gamma-D-glutamyl-meso-diaminopimelic acid (meso-DAP) contained in cell walls of Hi but not Sp. Neutrophils from mice treated with Hi or compounds containing meso-DAP, including synthetic peptidoglycan fragments, showed increased Sp killing in a Nod1-dependent manner. Moreover, Nod1(-/-) mice showed reduced Hi-induced clearance of Sp during co-colonization. These observations offer insight into mechanisms of microbial competition and demonstrate the importance of Nod1 in neutrophil-mediated clearance of bacteria in vivo.

Nod1 mediates cytoplasmic sensing of combinations of extracellular bacteria.

During mucosal colonization, epithelial cells are concurrently exposed to numerous microbial species. Epithelial cytokine production is an early component of innate immunity and contributes to mucosal defence. We have previously demonstrated a synergistic response of respiratory epithelial cells to costimulation by two human pathogens, Streptococcus pneumoniae and Haemophilus influenzae. Here we define a molecular mechanism for the synergistic activation of epithelial signalling during polymicrobial colonization. H. influenzae peptidoglycan synergizes with the pore-forming toxin pneumolysin from S. pneumoniae. Radiolabelled peptidoglycan enters epithelial cells more efficiently in the presence of pneumolysin, consistent with peptidoglycan gaining access to the cytoplasm via toxin pores. Other pore-forming toxins (including anthrolysin O from Bacillus anthracis and Staphylococcus aureus alpha-toxin) can substitute for pneumolysin in the generation of synergistic responses. Consistent with a requirement for pore formation, S. pneumoniae expressing pneumolysin but not an isogenic mutant expressing a non-pore-forming toxoid prime epithelial responses. Nod1, a host cytoplasmic peptidoglycan-recognition molecule, is crucial to the epithelial response. Taken together, these findings demonstrate a role for cytosolic recognition of peptidoglycan in the setting of polymicrobial epithelial stimulation. We conclude that combinations of extracellular organisms can activate innate immune pathways previously considered to be reserved for the detection of intracellular microorganisms.

The role of innate immune responses in the outcome of interspecies competition for colonization of mucosal surfaces.

Since mucosal surfaces may be simultaneously colonized by multiple species, the success of an organism may be determined by its ability to compete with co-inhabitants of its niche. To explore the contribution of host factors to polymicrobial competition, a murine model was used to study the initiation of colonization by Haemophilus influenzae and Streptococcus pneumoniae. Both bacterial species, which occupy a similar microenvironment within the nasopharynx, persisted during colonization when given individually. Co-colonization, however, resulted in rapid clearance of S. pneumoniae from the upper respiratory tract, associated with increased recruitment of neutrophils into paranasal spaces. Systemic depletion of either neutrophil-like cells or complement was sufficient to eliminate this competitive effect, indicating that clearance was likely due to enhanced opsonophagocytic killing. The hypothesis that modulation of opsonophagocytic activity was responsible for host-mediated competition was tested using in vitro killing assays with elicited neutrophil-like cells. Components of H. influenzae (but not S. pneumoniae) stimulated complement-dependent phagocytic killing of S. pneumoniae. Thus, the recruitment and activation of neutrophils through selective microbial pattern recognition may underlie the H. influenzae-induced clearance of S. pneumoniae. This study demonstrates how innate immune responses may mediate competitive interactions between species and dictate the composition of the colonizing flora.

Host and bacterial factors contributing to the clearance of colonization by Streptococcus pneumoniae in a murine model.

Nasopharyngeal colonization is the first step in the interaction between Streptococcus pneumoniae (the pneumococcus) and its human host. Factors that contribute to clearance of colonization are likely to affect the spread of the pneumococcus and the rate of pneumococcal disease in the population. To identify host and bacterial factors contributing to this process, we examined the time course of colonization using genetically modified mice and pneumococci. Severe combined immunodeficient mice remained persistently colonized (>6 weeks). Major histocompatibility complex II-deficient mice, but not microMT mice, were unable to clear colonization and showed a diminished T helper 1 response. Thus, CD4+ T cells, rather than the generation of specific antibody, appear to be required for effective Th1-mediated clearance. In addition, the microbial pattern recognition receptor toll-like receptor 2 (TLR2), but not TLR4, was necessary for efficient clearance of colonization. In contrast, no role of complement component 3, inducible nitric oxide synthetase, interleukin 12 (IL-12), or IL-4 could be demonstrated. Expression of the pneumococcal toxin pneumolysin enhanced acute localized inflammatory responses and promoted clearance of colonization in a TLR4-independent manner. We conclude that both innate and CD4+ T-cell-mediated immunity and proinflammatory bacterial factors, rather than a humoral adaptive immune response, are important for clearance of S. pneumoniae from the murine nasopharynx.

Synergistic proinflammatory responses induced by polymicrobial colonization of epithelial surfaces.

The epithelial surfaces of the upper respiratory tract are continuously exposed to a wide variety of commensal microorganisms. In addition to acting as a physical barrier, epithelial cells respond to specific microbial products with the generation of signals, such as cytokines, that trigger inflammation. Because they are common components of the nasopharyngeal flora that share the potential to cause disease, we investigated the effects of Haemophilus influenzae and Streptococcus pneumoniae, alone and in combination, on human respiratory epithelial cells in culture and in a murine model of nasopharyngeal colonization. Exposure of A549 or Detroit 562 epithelial cells to both S. pneumoniae and H. influenzae led to a synergistic increase in production of IL-8, the major neutrophil chemokine in the airway, through an NF-kappaB-dependent mechanism. Likewise, nasal cocolonization of mice caused a synergistic rise in local production of macrophage inflammatory protein 2 in nasal lavage fluid and subsequent recruitment of neutrophils. This synergistic effect depended on production of the pore-forming cytolytic toxin, pneumolysin, by S. pneumoniae and activation of host p38 mitogen-activated protein kinase. Although both H. influenzae and S. pneumoniae have ligands for Toll-like receptors (TLRs) TLR2 and TLR4, synergistic activation was TLR2- and TLR4-independent. Thus, epithelial surfaces are capable of amplifying proinflammatory responses during concurrent stimulation by multiple microbial species. These synergistic responses, demonstrated both in vitro and in vivo, may contribute to inflammation of heavily colonized mucosal barriers.

Multiple mechanisms for choline transport and utilization in Haemophilus influenzae.

Haemophilus influenzae obtains choline from either its growth medium or host cell membrane lipids and expresses it on its lipopolysaccharide (LPS) in the form of phosphorylcholine (ChoP), which contributes to its pathogenesis by mimicry of host cell molecules. Two genes (licB and betT) revealed by whole genomic analysis as encoding potential choline transporters were tested for their role in LPS-ChoP synthesis. The betT gene in H. influenzae is similar to betT in Escherichia coli, which functions in choline transport for the generation of betaine in osmoprotection. The licB gene has homology to bacterial permeases including betT and is encoded in the lic1 locus, which is essential for the expression of LPS-ChoP. In the presence of high concentrations of choline, neither licB nor betT were necessary for expression of LPS-ChoP raising the possibility that other unidentified choline uptake mechanisms may exist in this species. However, under choline limiting conditions, including growth in human nasal airway surface fluid, the licB, but not betT, gene was required for choline transport and synthesis of LPS-ChoP suggesting that LicB functions as a high affinity choline permease. The betT, but not licB, gene was shown to function in osmoprotection in H. influenzae, similar to the role of betT in E. coli. Further analysis demonstrated growth condition dependent differences in the regulation of transcription of the licB and betT genes. We conclude that H. influenzae may have multiple mechanisms for choline uptake and distinct pathways for choline utilization in LPS-ChoP biosynthesis and osmoregulation.