Objective To describe NP and AOM otopathogens during the time frame 2007-2009, six to eight years after the introduction of 7-valent pneumococcal conjugate (PCV7) in the US and to compare nasopharyngeal (NP) colonization and acute otitis media (AOM) microbiology in children 6 to 36 months of age having 1st and 2nd AOM episodes with children who are otitis prone. Methods Prospectively, the microbiology of NP colonization and AOM episodes was determined in 120 children with absent or infrequent AOM episodes. NP samples were collected at 7 routine visits between 6 and 30 months of age and at the time of AOM. For 1st and subsequent AOM episodes, middle ear fluid (MEF) was obtained by tympanocentesis. Eighty otitis prone children were comparatively studied. All 200 children received age-appropriate doses of PCV7. Results We found PCV7 serotypes were virtually absent: (0.9% isolated from both NP and MEF) in both study groups. However, non-PCV7 serotypes replaced PCV serotypes such that the frequency of isolation of S. pneumoniae (Spn) was nearly equal to that of non-typeable Haemophilus influenzae (NTHi). M. catarrhalis (Mcat) was less common and Staphylococcus aureus infrequent in the NP and MEF from the two groups. The proportion of Spn, NTHi and Mcat causing AOM was similar in children with 1st and 2nd AOM episodes compared to otitis prone children. However, oxacillin-resistant Spn isolated from the NP and MEF was 19% for the absent/infrequent and 58% for the otitis prone groups, p<0.0001. Beta-lactamase producing NTHi occurred more frequently in the otitis prone group, p=0.04. Conclusions Six to 8 years after widespread use of PCV7, Spn strains expressing vaccine-type serotypes have virtually disappeared from the NP and MEF of vaccinated children. NP colonization and AOM has changed to non-PCV7 strains of Spn. NTHi continues to be a major AOM pathogen. The otopathogens in 1st and 2nd AOM and in otitis prone children are very similar although Spn and NTHi are more often antibiotic resistant in the otitis prone.

B cell anergy represents an important mechanism of peripheral immunological tolerance for mature autoreactive B cells that escape central tolerance enforced by receptor editing and clonal deletion. While well documented in mice, the extent of its participation in human B cell tolerance remains to be fully established. In this study, we characterize the functional behavior of strictly defined human naïve B cells separated on the basis of their surface IgM (sIgM) expression levels. We demonstrate that cells with lower sIgM levels (IgMlo) are impaired in their ability to flux calcium in response to either anti-IgM or anti-IgD cross-linking, and contain a significantly increased frequency of autoreactive cells compared to naïve B cells with higher levels of sIgM. Phenotypically, in healthy subjects, IgMlo cells are characterized by the absence of activation markers, reduction of co-stimulatory molecules (CD19 and CD21) and increased levels of inhibitory CD22. Functionally, IgMlo cells display significantly weaker proliferation, impaired differentiation, and poor antibody production. In aggregate, the data indicates that hypo-responsiveness to BCR cross-linking associated with sIgM down-regulation is present in a much larger fraction of all human naïve B cells than previously reported, and is likely to reflect a state of anergy induced by chronic autoantigen stimulation. Finally, our results indicate that in SLE patients, naïve IgMlo cells display increased levels of CD95 and decreased levels of CD22, a phenotype consistent with enhanced activation of autoreactive naïve B cells in this autoimmune disease.

9G4+ IgG Abs expand in systemic lupus erythematosus (SLE) in a disease-specific fashion and react with different lupus Ags including B cell Ags and apoptotic cells. Their shared use of VH4-34 represents a unique system to understand the molecular basis of lupus autoreactivity. In this study, a large panel of recombinant 9G4+ mAbs from single naive and memory cells was generated and tested against B cells, apoptotic cells, and other Ags. Mutagenesis eliminated the framework-1 hydrophobic patch (HP) responsible for the 9G4 idiotype. The expression of the HP in unselected VH4-34 cells was assessed by deep sequencing. We found that 9G4 Abs recognize several Ags following two distinct structural patterns. B cell binding is dependent on the HP, whereas anti-nuclear Abs, apoptotic cells, and dsDNA binding are HP independent and correlate with positively charged H chain third CDR. The majority of mutated VH4-34 memory cells retain the HP, thereby suggesting selection by Ags that require this germline structure. Our findings show that the germline-encoded HP is compulsory for the anti–B cell reactivity largely associated with 9G4 Abs in SLE but is not required for reactivity against apoptotic cells, dsDNA, chromatin, anti-nuclear Abs, or cardiolipin. Given that the lupus memory compartment contains a majority of HP+ VH4-34 cells but decreased B cell reactivity, additional HP-dependent Ags must participate in the selection of this compartment. This study represents the first analysis, to our knowledge, of VH-restricted autoreactive B cells specifically expanded in SLE and provides the foundation to understand the antigenic forces at play in this disease.

Although B cell depletion therapy (BCDT) is effective in a subset of rheumatoid arthritis (RA) patients, both mechanisms and biomarkers of response are poorly defined. Here we characterized abnormalities in B cell populations in RA and the impact of BCDT in order to elucidate B cell roles in the disease and response biomarkers. In active RA patients both CD27+IgD- switched memory (SM) and CD27-IgD- double negative memory (DN) peripheral blood B cells contained significantly higher fractions of CD95+ and CD21- activated cells compared to healthy controls. After BCD the predominant B cell populations were memory, and residual memory B cells displayed a high fraction of CD21- and CD95+ compared to pre-depletion indicating some resistance of these activated populations to anti-CD20. The residual memory populations also expressed more Ki-67 compared to pre-treatment, suggesting homeostatic proliferation in the B cell depleted state. Biomarkers of clinical response included lower CD95+ activated memory B cells at depletion time points and a higher ratio of transitional B cells to memory at reconstitution. B cell function in terms of cytokine secretion was dependent on B cell subset and changed with BCD. Thus, SM B cells produced pro-inflammatory (TNF) over regulatory (IL10) cytokines as compared to naïve/transitional. Notably, B cell TNF production decreased after BCDT and reconstitution compared to untreated RA. Our results support the hypothesis that the clinical and immunological outcome of BCDT depends on the relative balance of protective and pathogenic B cell subsets established after B cell depletion and repopulation.

Background The 3 most commonly encountered bacteria in acute otitis media (AOM) are Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Conventional culture methods detect these pathogens in only 60% to 70% of cases of AOM. Alloiococcus otitidis, another potential pathogen, has often been ignored. Methods Tympanocentesis was performed in 97 children with AOM presenting with a bulging tympanic membrane (TM) producing 170 middle ear fluids (MEFs). S. pneumoniae, H. influenzae, M. catarrhalis, and A. otitidis were isolated in 21%, 32%, 8%, and 0% of MEFs, respectively; no otopathogen was isolated in 29% of MEFs. In nasopharyngeal cultures at the time of AOM diagnosis, 34%, 36%, 17%, and 0% and in oropharyngeal cultures, 7%, 31%, 11%, and 0% grew S. pneumoniae, H. influenzae, M. catarrhalis, and A. otitidis, respectively. No otopathogen was isolated in 23% of nasopharyngeal and 20% of oropharyngeal cultures. Multiplex polymerase chain reaction (PCR) was used to detect DNA of the 4 bacterial species in culture negative samples. Results All culture-positive MEF, nasopharyngeal and oropharyngeal samples tested were also multiplex-PCR positive, indicating the reliability of the method. Culture-negative samples of MEF from children with a bulging TM yielded S. pneumoniae, H. influenzae, M. catarrhalis, and A. otitidis DNA in 51%, 35%, 14%, and 32% of MEF, in 45%, 31%, 10%, and 9% of nasopharyngeal and in 31%, 23%, 0%, and 3% of oropharyngeal, respectively. In 9% of the cases A. otitidis DNA was found without detection of a second organism in MEF. Conclusions Conventional culture detected otopathogens in MEF of children with a bulging TM in 71%; using multiplex-PCR, otopathogens were detected in 88% of MEF (P < 0.01). Similar improved detection of otopathogens was noted with nasopharyngeal and oropharyngeal cultures.

Objective Streptococcus pneumoniae (Spn) and Haemophilus influenzae (Hflu) are major etiologic pathogens for acute otitis media (AOM). However, when Spn and Hflu strains are not identified by traditional culture methods, use of alternative PCR-based diagnosis becomes critical. This study aimed to develop a combined molecular method to accurately detect these otopathegens. Methods Middle ear fluid (MEF) samples were collected by tympanocentesis from children with AOM to isolate Spn and Hflu by standard culture procedures. Multiplex PCR (mPCR) and multi-locus sequence typing (MLST) techniques were used to detect Spn and Hflu in culture-negative MEF samples. Results We found 20 Spn or Hflu culture-positive MEF samples that were mPCR-positive and typeable by MLST. The sequences of the housekeeping genes and the MLST allelic profiles obtained from Spn or Hflu culture isolates matched exactly MEF samples that were tested directly without culture isolation. Of 63 MEF samples that were culture-negative for Spn, 38% (24/63) were mPCR-positive for Spn. Of 50 MEF samples that were culture-negative for Hflu, 24% (12/50) were mPCR-positive for Hflu. Among these culture-negative but mPCR-positive MEF samples, 25% (6/24) and 25% (3/12) were typeable by MLST for Spn and Hflu, respectively. Conclusions MEF samples may be analyzed with mPCR and MLST directly without culture isolation and the addition of mPCR and MLST may accurately identify Spn and Hflu in MEF of children with AOM when bacterial culture is negative.

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Moraxella catarrhalis is an important cause of respiratory infections in adults with chronic obstructive pulmonary disease (COPD) and of otitis media in children. Outer membrane protein (OMP) G1a is an ϳ29-kDa surface lipoprotein and is a potential vaccine candidate. The gene that encodes OMP G1a was expressed and purified using a novel plasmid vector. [ 3 H]palmitic acid labeling demonstrated that both native and recombinant OMP G1a contain covalently bound palmitic acid. To assess the expression of OMP G1a during human infection, paired sera and sputum supernatants from adults with COPD followed prospectively were studied by enzyme-linked immunosorbent assays with recombinant lipidated OMP G1a to detect antibodies made specifically during carriage of M. catarrhalis. Overall, 23% of patients developed either a serum immunoglobulin G (IgG) response (9%) or sputum IgA response (21%) to OMP G1a, following 100 episodes of acquisition and clearance of M. catarrhalis. Patients developed antibody responses at similar rates following episodes of clinical exacerbation compared to asymptomatic colonization. Serum IgG antibodies following natural infection were directed predominantly at OMP G1a epitopes that are not exposed on the bacterial surface. These data show that OMP G1a is expressed during infection of the human respiratory tract and is a target of systemic and mucosal antibodies. These observations indicate that OMP G1a, a highly conserved surface protein, should be evaluated further as a vaccine candidate.Over the past 2 decades, Moraxella catarrhalis has emerged as an important human respiratory pathogen. Acquisition of a new strain of M. catarrhalis is associated with the development of an exacerbation in patients with chronic obstructive pulmonary disease (COPD) (43). Indeed, M. catarrhalis is the second most common bacterial cause of exacerbations in adults with COPD after nontypeable Haemophilus influenzae (32,38,44). COPD is the fourth-leading cause of death in the United States and is projected to rank fifth in the world by 2020 (2,26,28,39). M. catarrhalis is also the third-most-common bacterial cause of otitis media in children after Streptococcus pneumoniae and nontypeable H. influenzae (25). Approximately 25 million episodes of otitis media occur annually in the United States, and $2 billion is spent annually on treatment (25). It is estimated that M. catarrhalis causes 10% to 20% of cases of acute otitis media. Recurrent otitis media in infants and young children affects speech development and cognitive abilities (25, 42). Finally, M. catarrhalis is also a common cause of sinusitis in children and adults (32). Since M. catarrhalis is an important human pathogen for infants and adults with COPD, there is a need for a vaccine. Infants could be immunized to prevent otitis media and adults with COPD could be immunized to prevent exacerbations caused by M. catarrhalis.One approach to vaccine development for gram-negative bacteria has been to use outer membrane proteins (OMPs) as vaccine antigens. Effectiv...