Lymphocyte predominant Hodgkin lymphoma, antigen-driven after all?

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) was suggested as an entity separate from other types of Hodgkin lymphoma 40 years ago and recognized in the WHO classification in 2001. Based on its relatively benign course with late distant relapses, relation with lymph node hyperplasia with progressively transformed germinal centers, presence of clonal immunoglobulin gene rearrangements with somatic hypermutations and ongoing mutations, and relation with a number of inherited defects affecting the immune system, it has been suspected that NLPHL might be antigen-driven. Recent evidence has shown that cases of IgD-positive NLPHL are associated with infection by Moraxella catarrhalis, a common bacterium in the upper respiratory tract and in lymph nodes. This review summarizes the evidence for NLPHL as a B-cell lymphoma involving follicular T-lymphocytes normally found in germinal centers, its molecular features and relation to inherited immune defects, and its relation and differential diagnosis from similar entities. Finally, it discusses the evidence that in many cases a watch and wait policy might be a viable initial management strategy. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Inflammatory Mediator Profiles in Secretory Otitis Media in Relationship to Viable Bacterial Pathogens and Bacterial and Viral Nucleic Acids.

Secretory otitis media (SOM) is characterized by persistence of fluid in the middle ear, often following an episode of acute otitis media. Our hypothesis is that failure to eliminate bacterial or viral pathogens may result in persistent low-grade inflammation. In this study, we analyzed inflammatory mediators in middle ear fluids from 67 children with SOM. This was combined with determinations of viable bacteria by culture along with detection of bacterial and viral genetic material by real-time polymerase chain reaction (PCR). The inflammatory mediators found at the highest concentrations (>30 ng/mL) were stem cell growth factor-ß (median 110 ng/mL), CXCL1, IL-16, IL-8, migration inhibitory factor, CXCL10, and CXCL9. Among bacterial pathogens, Moraxella catarrhalis and Haemophilus influenzae dominated, regardless of detection methods, while rhinovirus dominated among viral pathogens. Middle ear fluid levels of interleukin (IL)-1α, IL-17, IL-1ß, fibroblast growth factor basic, and tumor necrosis factor correlated strongly with presence of bacteria detected either by culture or PCR, while IL-1RA, IL-3, IL-6, IL-8, CCL3, CCL4, and granulocyte-colony stimulating factor correlated significantly with real-time PCR values. CXCL10, CXCL9, CCL2, and TRAIL correlated significantly with viral nucleic acid levels. To conclude, persistence of viral and bacterial pathogens may fuel persistent inflammation in SOM. Bacteria caused a broad inflammatory response, while viruses chiefly elicited the interferon-induced chemokines CXCL9 and CXCL10.

Lymphocyte predominant cells detect Moraxella catarrhalis-derived antigens in nodular lymphocyte-predominant Hodgkin lymphoma.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare lymphoma of B-cell origin with frequent expression of functional B-cell receptors (BCRs). Here we report that expression cloning followed by antigen screening identifies DNA-directed RNA polymerase beta' (RpoC) from Moraxella catarrhalis as frequent antigen of BCRs of IgD+ LP cells. Patients show predominance of HLA-DRB1*04/07 and the IgVH genes encode extraordinarily long CDR3s. High-titer, light-chain-restricted anti-RpoC IgG1/κ-type serum-antibodies are additionally found in these patients. RpoC and MID/hag, a superantigen co-expressed by Moraxella catarrhalis that is known to activate IgD+ B cells by binding to the Fc domain of IgD, have additive activation effects on the BCR, the NF-κB pathway and the proliferation of IgD+ DEV cells expressing RpoC-specific BCRs. This suggests an additive antigenic and superantigenic stimulation of B cells with RpoC-specific IgD+ BCRs under conditions of a permissive MHC-II haplotype as a model of NLPHL lymphomagenesis, implying future treatment strategies.

Synchrony in serum antibody response to conserved proteins of Moraxella catarrhalis in young children.

Conserved Moraxella catarrhalis (Mcat) proteins, oligopeptide permease (Opp)A, hemagglutinin (Hag), outer membrane protein (OMP) CD, Pilin A clade 2 (PilA2), and Moraxella surface protein (Msp) 22 have been studied as vaccine candidates. Children who experience frequent acute otitis media (AOM) confirmed with pathogen identification by tympanocentesis are referred to as stringently-defined otitis prone (sOP). Synchrony of serum antibody responses against 5 Mcat proteins, OppA, Hag, OMP CD, PilA2, and Msp22 resulting from nasopharyngeal colonization and AOM was studied for 85 non-otitis prone (NOP) children and 34 sOP children. Changes in serum IgG were quantitated with ELISA. Serum IgG antibody levels against OppA, Hag, OMP CD, and Msp22 rose in synchrony in NOP and sOP children; that is, the proteins appeared equally and highly immunogenic in children at age 6 to 22-25 months old and then leveled off in their rise at 22-25 to 30 months old. In contrast, rises of PilA2 were slow from 6 months old and kept constant and did not level off significantly before 30 months old. OppA, Hag, OMP CD, and Msp22 elicited a synchronous acquisition of naturally-induced serum antibody in young children. A multi-valent Mcat protein vaccine combining OppA, Hag, OMP CD, and Msp22 may exhibit less antigen competition when administered as a combination vaccine in young children.

Complement evasion by the human respiratory tract pathogens Haemophilus influenzae and Moraxella catarrhalis.

All infective bacterial species need to conquer the innate immune system in order to colonize and survive in their hosts. The human respiratory pathogens Haemophilus influenzae and Moraxella catarrhalis are no exceptions and have developed sophisticated mechanisms to evade complement-mediated killing. Both bacterial species carry lipooligosaccharides preventing complement attacks and attract and utilize host complement regulators C4b binding protein and factor H to inhibit the classical and alternative pathways of complement activation, respectively. In addition, the regulator of the terminal pathway of complement activation, vitronectin, is hijacked by both bacteria. An array of different outer membrane proteins (OMP) in H. influenzae and M. catarrhalis simultaneously binds complement regulators, but also plasminogen. Several of the bacterial complement-binding proteins are important adhesins and contain highly conserved regions for interactions with the host. Thus, some of the OMP are viable targets for new therapeutics, including vaccines aimed at preventing respiratory tract diseases such as otitis media in children and exacerbations in patients suffering from chronic obstructive pulmonary disease.

Immunological characterisation of truncated lipooligosaccharide-outer membrane protein based conjugate vaccine against Moraxella catarrhalis and nontypeable Haemophilus influenzae.

Moraxella catarrhalis and nontypeable Haemophilus influenzae are important bacterial causes of otitis media in children and respiratory diseases in adults. Lipooligosaccharide (LOS) from M. catarrhalis and outer membrane protein 26 (OMP26) from NTHi are major surface antigens identified as potential vaccine components against these organisms. We previously constructed M. catarrhalis in which LOS is truncated, but contains a structure common to the three known serotypes of M. catarrhalis. OMP26 is known to enhance clearance of NTHi following vaccination in animal models, so was chosen as the carrier protein. In this study, we conjugated wild-type and truncated M. catarrhalis detoxified-LOS to a recombinant modified OMP26, rOMP26VTAL. Vaccination of mice with these conjugates resulted in a significant increase in anti-LOS and anti-rOMP26VTAL IgG levels. Importantly, mouse antisera showed complement-mediated bactericidal activity against all M. catarrhalis serotype A and B strains and a NTHi strain tested. Serotypes A & B make up more than 90% of isolates. These data suggest that the LOS and OMP based conjugate can be used as vaccine components and require further investigation in animal models.

Potential impact of a Moraxella catarrhalis vaccine in COPD.

Moraxella catarrhalis is the second most common cause of exacerbations in adults with COPD, resulting in enormous morbidity and mortality in this clinical setting. Vaccine development for M. catarrhalis has lagged behind the other two important causes of exacerbations in COPD, nontypeable Haemophilus influenzae and Streptococcus pneumoniae. While no licensed vaccine is currently available for M. catarrhalis, several promising candidate vaccine antigens have been identified and characterized and are close to entering clinical trials. Key steps that are required to advance vaccines for M. catarrhalis along the translational pipeline include standardization of assay systems to assess candidate antigens, identification of a reliable correlate of protection and expansion of partnerships between industry, academia and government to overcome regulatory hurdles. A vaccine to prevent M. catarrhalis infections in COPD would have a major impact in reducing morbidity, mortality and healthcare costs in COPD.

Serum antibody response to Moraxella catarrhalis proteins in stringently defined otitis prone children.

BACKGROUND: Moraxella catarrhalis (Mcat) is a frequent pathogen of acute otitis media (AOM) in young children. Here we prospectively assessed naturally-induced serum antibodies to four Mcat vaccine candidate proteins in stringently defined otitis prone (sOP) and non-otitis prone (NOP) children age 6-36months old following nasopharyngeal (NP) colonization, at onset of AOM and convalescence from AOM. METHODS: Serum IgG and IgM antibody against recombinant Mcat proteins, oligopeptide permease A (OppA), outer membrane protein (OMP) CD, hemagglutinin (Hag), and PilA clade 2 (PilA2), were quantitated by ELISA. RESULTS: During NP colonization by Mcat all four antigens were immunogenic in both sOP and NOP children. However, sOP children had lower antibody responses than NOP children across age 6-36months, similar to our findings for protein vaccine candidates of Streptococcus pneumoniae (Spn) and Nontypeable Haemophilus influenzae (NTHi). sOP children displayed a later and lower peak of antibody rise than NOP children for all four antigens during NP colonization of Mcat. The age-dependent increase of antibody ranked as OppA>Hag5-9>OMP CD>PilA2 in both sOP and NOP children. Lower serum antibody levels to the Mcat antigens were measured in sOP compared to NOP children at the onset of AOM. We did not find a consistent significant increase of antibody at the convalescence phase after an AOM event. CONCLUSIONS: sOP children is a highly vulnerable population that mount lower serum antibody responses to Mcat candidate vaccine proteins compared to NOP children during asymptomatic NP carriage and at onset of AOM.

Short Leucine-Rich Proteoglycans Modulate Complement Activity and Increase Killing of the Respiratory Pathogen Moraxella catarrhalis.

The respiratory pathogen Moraxella catarrhalis is a human-specific commensal that frequently causes acute otitis media in children and stimulates acute exacerbations in chronic obstructive pulmonary disease patients. The exact molecular mechanisms defining host-pathogen interactions promoting pathogenesis are not clearly understood. Limited knowledge hampers vaccine and immunotherapeutic development required to treat this emerging pathogen. In this study, we reveal in detail a novel antibacterial role displayed by short leucine-rich proteoglycans (SLRPs) in concert with complement. We show that fibromodulin (FMOD), osteoadherin (OSAD), and biglycan (BGN) but not decorin (DCN) enhance serum killing of M. catarrhalis. Our results suggest that M. catarrhalis binding to SLRPs is a conserved feature, as the overwhelming majority of clinical and laboratory strains bound all four SLRPs. Furthermore, we resolve the binding mechanism responsible for this interaction and highlight the role of the ubiquitous surface protein (Usp) A2/A2H in mediating binding to host SLRPs. A conserved immune evasive strategy used by M. catarrhalis and other pathogens is the surface acquisition of host complement inhibitors such as C4b-binding protein (C4BP). We observed that FMOD, OSAD, and BGN competitively inhibit binding of C4BP to the surface of M. catarrhalis, resulting in increased C3b/iC3b deposition, membrane attack complex (MAC) formation, and subsequently decreased bacterial survival. Furthermore, both OSAD and BGN promote enhanced neutrophil killing in vitro, both in a complement-dependent and independent fashion. In summary, our results illustrate that SLRPs, FMOD, OSAD, and BGN portray complement-modulating activity enhancing M. catarrhalis killing, defining a new antibacterial role supplied by SLRPs.

Neuropeptides SP and CGRP Diminish the Moraxella catarrhalis Outer Membrane Vesicle- (OMV-) Triggered Inflammatory Response of Human A549 Epithelial Cells and Neutrophils.

Neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP) play both pro- and anti-inflammatory activities and are produced during infection and inflammation. Moraxella catarrhalis is one of the leading infectious agents responsible for inflammatory exacerbation in chronic obstructive pulmonary disease (COPD). Since the airway inflammation in COPD is connected with activation of both epithelial cells and accumulated neutrophils, in this study we determined the in vitro effects of neuropeptides on the inflammatory potential of these cells in response to M. catarrhalis outer membrane vesicle (OMV) stimulant. The various OMV-mediated proinflammatory effects were demonstrated. Next, using hBD-2-pGL4[luc2] plasmid with luciferase reporter gene, SP and CGRP were shown to inhibit the IL-1ß-dependent expression of potent neutrophil chemoattractant, hBD-2 defensin, in transfected A549 epithelial cells (type II alveolar cells) upon OMV stimulation. Both neuropeptides exerted antiapoptotic activity through rescuing a significant fraction of A549 cells from OMV-induced cell death and apoptosis. Finally, CGRP caused an impairment of specific but not azurophilic granule exocytosis from neutrophils as shown by evaluation of gelatinase-associated lipocalin (NGAL) or CD66b expression and elastase release, respectively. Concluding, these findings suggest that SP and CGRP mediate the dampening of proinflammatory action triggered by M. catarrhalis OMVs towards cells engaged in lung inflammation in vitro.

How bacteria hack the matrix and dodge the bullets of immunity.

Haemophilus influenzae, Moraxella catarrhalis and Pseudomonas aeruginosa are common Gram-negative pathogens associated with an array of pulmonary diseases. All three species have multiple adhesins in their outer membrane, i.e. surface structures that confer the ability to bind to surrounding cells, proteins or tissues. This mini-review focuses on proteins with high affinity for the components of the extracellular matrix such as collagen, laminin, fibronectin and vitronectin. Adhesins are not structurally related and may be lipoproteins, transmembrane porins or large protruding trimeric auto-transporters. They enable bacteria to avoid being cleared together with mucus by attaching to patches of exposed extracellular matrix, or indirectly adhering to epithelial cells using matrix proteins as bridging molecules. As more adhesins are being unravelled, it is apparent that bacterial adhesion is a highly conserved mechanism, and that most adhesins target the same regions on the proteins of the extracellular matrix. The surface exposed adhesins are prime targets for new vaccines and the interactions between proteins are often possible to inhibit with interfering molecules, e.g heparin. In conclusion, this highly interesting research field of microbiology has unravelled host-pathogen interactions with high therapeutic potential.

Update on non-typeable Haemophilus influenzae-mediated disease and vaccine development.

INTRODUCTION: Non-typeable Haemophilus influenzae (NTHi) has attracted more interest in recent years due to an increased prevalence of infections caused by the pathogen. This upsurge is at least partly ascribed to the introduction of the pneumococcal conjugated vaccines that has resulted in an aetiological shift in NTHi's favor with respect to upper respiratory tract infections. Moreover, an increased antimicrobial resistance has been associated with the pathogen, a fact that further strengthens the case for novel vaccine development. AREAS COVERED: A background to NTHi-mediated diseases and pathogenesis is outlined. The literature in the field of NTHi vaccine antigens and clinical trials is reviewed with focus on data added to scientific databases in the last two years. Various vaccine development strategies are conceptually discussed. EXPERT COMMENTARY: Several promising vaccine antigens have been defined in recent years. A multicomponent protein-based vaccine, potentially boosted with extracellular vesicles, would constitute a suitable path going forward. Of note, however, a clinical trial investigating the efficacy of a combined NTHi/Moraxella catarrhalis vaccine to prevent infections in chronic obstructive pulmonary disease (COPD) patients has been initiated. But, as this clinical trial has not yet concluded, and its results are thus unknown, investigations of NTHi pathogenesis must determinedly continue.

Are lactoferrin receptors in Gram-negative bacteria viable vaccine targets?

A number of important Gram-negative pathogens that reside exclusively in the upper respiratory or genitourinary tract of their mammalian host rely on surface receptors that specifically bind host transferrin and lactoferrin as a source of iron for growth. The transferrin receptors have been targeted for vaccine development due to their critical role in acquiring iron during invasive infection and for survival on the mucosal surface. In this study, we focus on the lactoferrin receptors, determining their prevalence in pathogenic bacteria and comparing their prevalence in commensal Neisseria to other surface antigens targeted for vaccines; addressing the issue of a reservoir for vaccine escape and impact of vaccination on the microbiome. Since the selective release of the surface lipoprotein lactoferrin binding protein B by the NalP protease in Neisseria meningitidis argues against its utility as a vaccine target, we evaluated the release of outer membrane vesicles, and transferrin and lactoferrin binding in N. meningitidis and Moraxella catarrhalis. The results indicate that the presence of NalP reduces the binding of transferrin and lactoferrin by cells and native outer membrane vesicles, suggesting that NalP may impact all lipoprotein targets, thus this should not exclude lactoferrin binding protein B as a target.

Antibody responses against Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis in children with acute respiratory infection with or without nasopharyngeal bacterial carriage.

BACKGROUND: We studied Immunoglobulin G (IgG) antibody responses against Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis in young children with acute viral type respiratory infection and analyzed the findings in a multivariate model including age, nasopharyngeal carriage of the tested bacteria and pneumococcal vaccination. METHODS: We included 227 children aged 6-23 months with acute respiratory infection. Nasopharyngeal aspirates were tested for bacterial carriage through detection of messenger RNA (mRNA) transcript with nCounter analysis. Acute and convalescent serum samples were tested for IgG antibody response against eight pneumococcal proteins, three proteins from H. influenzae and five proteins from M. catarrhalis in a fluorescent multiplex immunoassay. RESULTS: A two-fold or greater increase in antibodies to S. pneumoniae, H. influenzae and M. catarrhalis was detected in 27.8, 9.7 and 14.1%, respectively. Nasopharyngeal carriage of each of the studied bacteria was not associated with antibody response detection against each respective bacterium. Furthermore, neither age nor pneumococcal vaccination were independently associated to detection of antibody response against the studied bacteria. Children who carried H. influenzae had higher frequency of colonization by M. catarrhalis (175 [80.3%] vs. 2 [22.2%]; p < .001) than those without H. influenzae. Also, children with acute otitis media tended to have higher frequency of antibody response to S. pneumoniae. CONCLUSION: Nasopharyngeal colonization by S. pneumoniae, H. influenzae and M. catarrhalis did not induce significant increases in antibody levels to these bacteria. Carriage of pathogenic bacteria in the nasopharynx is not able to elicit antibody responses to protein antigens similar to those caused by symptomatic infections.

Virulence factors of Moraxella catarrhalis outer membrane vesicles are major targets for cross-reactive antibodies and have adapted during evolution.

Moraxella catarrhalis is a common human respiratory tract pathogen. Its virulence factors associated with whole bacteria or outer membrane vesicles (OMVs) aid infection, colonization and may induce specific antibodies. To investigate pathogen-host interactions, we applied integrated bioinformatic and immunoproteomic (2D-electrophoresis, immunoblotting, LC-MS/MS) approaches. We showed that OMV proteins engaged exclusively in complement evasion and colonization strategies, but not those involved in iron transport and metabolism, are major targets for cross-reacting antibodies produced against phylogenetically divergent M. catarrhalis strains. The analysis of 31 complete genomes of M. catarrhalis and other Moraxella revealed that OMV protein-coding genes belong to 64 orthologous groups, five of which are restricted to M. catarrhalis. This species showed a two-fold increase in the number of OMV protein-coding genes relative to its ancestors and animal-pathogenic Moraxella. The appearance of specific OMV factors and the increase in OMV-associated virulence proteins during M. catarrhalis evolution is an interesting example of pathogen adaptation to optimize colonization. This precisely targeted cross-reactive immunity against M. catarrhalis may be an important strategy of host defences to counteract this phenomenon. We demonstrate that cross-reactivity is closely associated with the anti-virulent antibody repertoire which we have linked with adaptation of this pathogen to the host.

Infection by Streptococcus pneumoniae in children with or without radiologically confirmed pneumonia / Infecção por Streptococcus pneumoniae em crianças com ou sem pneumonia radiologicamente confirmada

Abstract Objective: Community-acquired pneumonia is an important cause of morbidity in childhood, but the detection of its causative agent remains a diagnostic challenge. The authors aimed to evaluate the role of the chest radiograph to identify cases of community-aquired pneumonia caused by typical bacteria. Methods: The frequency of infection by Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis was compared in non-hospitalized children with clinical diagnosis of community acquired pneumonia aged 2-59 months with or without radiological confirmation (n = 249 and 366, respectively). Infection by S. pneumoniae was diagnosed by the detection of a serological response against at least one of eight pneumococcal proteins (defined as an increase ≥2-fold in the IgG levels against Ply, CbpA, PspA1 and PspA2, PhtD, StkP-C, and PcsB-N, or an increase ≥1.5-fold against PcpA). Infection by H. influenzae and M. catarrhalis was defined as an increase ≥2-fold on the levels of microbe-specific IgG. Results: Children with radiologically confirmed pneumonia had higher rates of infection by S. pneumoniae. The presence of pneumococcal infection increased the odds of having radiologically confirmed pneumonia by 2.8 times (95% CI: 1.8-4.3). The negative predictive value of the normal chest radiograph for infection by S. pneumoniae was 86.3% (95% CI: 82.4-89.7%). There was no difference on the rates of infection by H. influenzae and M. catarrhalis between children with community-acquired pneumonia with and without radiological confirmation. Conclusions: Among children with clinical diagnosis of community-acquired pneumonia submitted to chest radiograph, those with radiologically confirmed pneumonia present a higher rate of infection by S. pneumoniae when compared with those with a normal chest radiograph.

Resumo Objetivo: Avaliar o papel do raios X de tórax na identificação de casos de pneumonia adquirida na comunidade (PAC) causada por agentes bacterianos. Métodos: A frequência de infecção por Streptococcus pneumoniae, Haemophilus influenzae e Moraxella catarrhalis em crianças com PAC não hospitalizadas foi comparada com a presença de confirmação radiológica da pneumonia (n = 249 crianças com pneumonia radiologicamente confirmada e 366 crianças com raios X de tórax normal). Infecção por S. pneumoniae foi diagnosticada com base na resposta sorológica a pelo menos uma dentre oito proteínas pneumocócicas investigadas (aumento ≥ 2 vezes nos níveis de IgG em relação a Ply, CbpA, PspA1 e 2, PhtD, StkP-C e PcsB-N ou aumento≥ 1,5 vez em relação aPcpA). Infecção por H. influenzae e M. catarrhalis foi definida por aumento ≥ 2 vezes nos níveis de IgG específica a antígenos de cada agente. Resultados: Crianças com pneumonia radiologicamente confirmada apresentaram maior taxa de infecção pelo pneumococo. Além disso, a presença de infecção pneumocócica foi um fator preditor de pneumonia radiologicamente confirmada, o que aumenta sua chance de detecção em 2,8 vezes (IC 95%: 1,8-4,3). O valor preditivo negativo do raios X normal para a infecção por S. pneumoniae foi 86,3% (IC95%: 82,4%-89,7%). Não houve diferença nas frequências de infecção por H. influenzae e M. catarrhalis entre crianças com PAC com ou sem confirmação radiológica. Conclusão: Crianças com diagnóstico clínico de PAC submetidas a um raios X de tórax que apresentam confirmação radiológica têm maior taxa de infecção por S. pneumoniae comparadas com as crianças com raios X normal.

[Preparation and identification of polyclonal antibodies against Moraxella catarrhalis UspA1].

To prepare polyclonal antibodies (PcAb) against UspA1 of Moraxella catarrhalis (Mc), we used bioinformatic analysis to determine the surface exposed region in this protein that holds the antigen epitopes. Then the corresponding coding sequences for this fragment was artificially synthesized according to the codon usage of Escherichia coli. The gene fragment was then subcloned into the prokaryotic expression vector pET-28a(+) and expressed in E. coli rosseta (DE3), and then the recombinant UspA1-His proteins were purified. Two New Zealand white rabbits were immunized with this protein to prepare antiserum. The resulting PcAb was then purified from the antiserum with Protein A affinity column. The results of fluorescence antibody assay, enzyme linked immunosorbent assay and Western blotting analysis showed that the PcAb could specifically recognize the surface exposed region of UspA1 on Mc. The preparation of the PcAb laid a foundation of further development of rapid detection technique for M. catarrhalis.

Infection by Streptococcus pneumoniae in children with or without radiologically confirmed pneumonia.

OBJECTIVE: Community-acquired pneumonia is an important cause of morbidity in childhood, but the detection of its causative agent remains a diagnostic challenge. The authors aimed to evaluate the role of the chest radiograph to identify cases of community-aquired pneumonia caused by typical bacteria. METHODS: The frequency of infection by Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis was compared in non-hospitalized children with clinical diagnosis of community acquired pneumonia aged 2-59 months with or without radiological confirmation (n=249 and 366, respectively). Infection by S. pneumoniae was diagnosed by the detection of a serological response against at least one of eight pneumococcal proteins (defined as an increase ≥2-fold in the IgG levels against Ply, CbpA, PspA1 and PspA2, PhtD, StkP-C, and PcsB-N, or an increase ≥1.5-fold against PcpA). Infection by H. influenzae and M. catarrhalis was defined as an increase ≥2-fold on the levels of microbe-specific IgG. RESULTS: Children with radiologically confirmed pneumonia had higher rates of infection by S. pneumoniae. The presence of pneumococcal infection increased the odds of having radiologically confirmed pneumonia by 2.8 times (95% CI: 1.8-4.3). The negative predictive value of the normal chest radiograph for infection by S. pneumoniae was 86.3% (95% CI: 82.4-89.7%). There was no difference on the rates of infection by H. influenzae and M. catarrhalis between children with community-acquired pneumonia with and without radiological confirmation. CONCLUSIONS: Among children with clinical diagnosis of community-acquired pneumonia submitted to chest radiograph, those with radiologically confirmed pneumonia present a higher rate of infection by S. pneumoniae when compared with those with a normal chest radiograph.

Mapping Protective Regions on a Three-Dimensional Model of the Moraxella catarrhalis Vaccine Antigen Oligopeptide Permease A.

A vaccine against Moraxella catarrhalis would reduce tremendous morbidity, mortality, and financial burden by preventing otitis media in children and exacerbations of chronic obstructive pulmonary disease (COPD) in adults. Oligopeptide permease A (OppA) is a candidate vaccine antigen that is (i) a nutritional virulence factor expressed on the bacterial cell surface during infection, (ii) widely conserved among strains, (iii) highly immunogenic, and (iv) a protective antigen based on its capacity to induce protective responses in immunized animals. In the present study, we show that the antibodies to OppA following vaccination mediate accelerated clearance in animals after pulmonary challenge. To identify regions of OppA that bind protective antibodies, truncated constructs of OppA were engineered and studied to map regions of OppA with surface-accessible epitopes that bind high-avidity antibodies following vaccination. Protective epitopes were located in the N and C termini of the protein. Immunization of mice with constructs corresponding to these regions (T5 and T8) induced protective responses. Studies of overlapping peptide libraries of constructs T5 and T8 with OppA immune serum identified two discrete regions on each construct. These potentially protective regions were mapped on a three-dimensional computational model of OppA, where regions with solvent-accessible amino acids were identified as three potentially protective epitopes. In all, these studies revealed two regions with three specific epitopes in OppA that induce potentially protective antibody responses following vaccination. Detection of antibodies to these regions could serve to guide vaccine formulation and as a diagnostic tool for monitoring development of protective responses during clinical trials.

Immunomodulatory role for membrane vesicles released by THP-1 macrophages and respiratory pathogens during macrophage infection.

BACKGROUND: During infection, inflammation is partially driven by the release of mediators which facilitate intercellular communication. Amongst these mediators are small membrane vesicles (MVs) that can be released by both host cells and Gram-negative and -positive bacteria. Bacterial membrane vesicles are known to exert immuno-modulatory and -stimulatory actions. Moreover, it has been proposed that host cell-derived vesicles, released during infection, also have immunostimulatory properties. In this study, we assessed the release and activity of host cell-derived and bacterial MVs during the first hours following infection of THP-1 macrophages with the common respiratory pathogens non-typeable Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, and Pseudomonas aeruginosa. RESULTS: Using a combination of flow cytometry, tunable resistive pulse sensing (TRPS)-based analysis and electron microscopy, we demonstrated that the release of MVs occurs by both host cells and bacteria during infection. MVs released during infection and bacterial culture were found to induce a strong pro-inflammatory response by naive THP-1 macrophages. Yet, these MVs were also found to induce tolerance of host cells to secondary immunogenic stimuli and to enhance bacterial adherence and the number of intracellular bacteria. CONCLUSIONS: Bacterial MVs may play a dual role during infection, as they can both trigger and dampen immune responses thereby contributing to immune defence and bacterial survival.