A molecular mucosal adjuvant to enhance immunity against pneumococcal infection in the elderly.

Streptococcus pneumoniae (the pneumococcus) causes a major upper respiratory tract infection often leading to severe illness and death in the elderly. Thus, it is important to induce safe and effective mucosal immunity against this pathogen in order to prevent pnuemocaccal infection. However, this is a very difficult task to elicit protective mucosal IgA antibody responses in older individuals. A combind nasal adjuvant consisting of a plasmid encoding the Flt3 ligand cDNA (pFL) and CpG oligonucleotide (CpG ODN) successfully enhanced S. pneumoniae-specific mucosal immunity in aged mice. In particular, a pneumococcal surface protein A-based nasal vaccine given with pFL and CpG ODN induced complete protection from S. pneumoniae infection. These results show that nasal delivery of a combined DNA adjuvant offers an attractive potential for protection against the pneumococcus in the elderly.

Brain regions responsible for tinnitus distress and loudness: a resting-state FMRI study.

Subjective tinnitus is characterized by the perception of phantom sound without an external auditory stimulus. We hypothesized that abnormal functionally connected regions in the central nervous system might underlie the pathophysiology of chronic subjective tinnitus. Statistical significance of functional connectivity (FC) strength is affected by the regional autocorrelation coefficient (AC). In this study, we used resting-state functional MRI (fMRI) and measured regional mean FC strength (mean cross-correlation coefficient between a region and all other regions without taking into account the effect of AC (rGC) and with taking into account the effect of AC (rGCa) to elucidate brain regions related to tinnitus symptoms such as distress, depression and loudness. Consistent with previous studies, tinnitus loudness was not related to tinnitus-related distress and depressive state. Although both rGC and rGCa revealed similar brain regions where the values showed a statistically significant relationship with tinnitus-related symptoms, the regions for rGCa were more localized and more clearly delineated the regions related specifically to each symptom. The rGCa values in the bilateral rectus gyri were positively correlated and those in the bilateral anterior and middle cingulate gyri were negatively correlated with distress and depressive state. The rGCa values in the bilateral thalamus, the bilateral hippocampus, and the left caudate were positively correlated and those in the left medial superior frontal gyrus and the left posterior cingulate gyrus were negatively correlated with tinnitus loudness. These results suggest that distinct brain regions are responsible for tinnitus symptoms. The regions for distress and depressive state are known to be related to depression, while the regions for tinnitus loudness are known to be related to the default mode network and integration of multi-sensory information.

PspA family distribution, antimicrobial resistance and serotype of Streptococcus pneumoniae isolated from upper respiratory tract infections in Japan.

BACKGROUND: The protection against pneumococcal infections provided by currently available pneumococcal polysaccharide conjugate vaccines are restricted to the limited number of the serotypes included in the vaccine. In the present study, we evaluated the distribution of the pneumococcal capsular type and surface protein A (PspA) family of pneumococcal isolates from upper respiratory tract infections in Japan. METHODS: A total of 251 S. pneumoniae isolates from patients seeking treatment for upper respiratory tract infections were characterized for PspA family, antibiotic resistance and capsular type. RESULTS: Among the 251 pneumococci studied, the majority (49.4%) was identified as belonging to PspA family 2, while most of the remaining isolates (44.6%) belonged to family 1. There were no significant differences between the distributions of PspA1 versus PspA2 isolates based on the age or gender of the patient, source of the isolates or the isolates' susceptibilities to penicillin G. In contrast, the frequency of the mefA gene presence and of serotypes 15B and 19F were statistically more common among PspA2 strains. CONCLUSION: The vast majority of pneumococci isolated from the middle ear fluids, nasal discharges/sinus aspirates or pharyngeal secretions represented PspA families 1 and 2. Capsular serotypes were generally not exclusively associated with certain PspA families, although some capsular types showed a much higher proportion of either PspA1 or PspA2. A PspA-containing vaccine would potentially provide high coverage against pneumococcal infectious diseases because it would be cross-protective versus invasive disease with the majority of pneumococci infecting children and adults.

[A new scoring system for assessing the severity of adult acute rhinosinusitis].

Treating acute rhinosinusitis requires assessing severity and selecting appropriate antimicrobial agents. In 2006, we developed clinical scoring system for diagnosing and treating acute rhinosinusitis based on three clinical symptoms of rhinorrhea, fever, and facial pain and three nasal findings of characteristics and nasal discharge amount, nasal mucosal swelling and nasal mucosal redness. To verify and update scoring, we studied score-based diagnosis of adult acute rhinosinusitis severity. Prevalence of symptoms such as fever and serous nasal discharge in 95 subjects was low as 8.4% and 3% indicating less useful as evaluation items on the diagnosis. Mucopurulent nasal discharge (r = 0.67), facial pain (r = 0.51), rhinorrhea (r = 0.47), and swelling (r = 0.45) correlated significantly with severity evaluated by attending otolaryngological specialists. Nasal mucosal swelling caused discrepancy between clinical scoring and specialists' assess- ment. Evaluated by multivaliate analysis, factors affecting severity assessment were mucopurulent nasal discharge, facial pain, and rhinorrhea (p < 0.0001), but not swelling (p = 0.49). We concluded that mucopurulent nasal discharge scored 0, 2, or 4, facial pain scored 0, 1, or 2, and rhinorrhea scored 0, 1, or 2 should be used in evaluation in new clinical scoring, classified by severity as mild scored 1-3, moderate scored 4-6, and severe scored 7-8 by evaluating consistency with specialist assessment.

Haemophilus influenzae and Haemophilus haemolyticus in tonsillar cultures of adults with acute pharyngotonsillitis.

OBJECTIVE: The aim of this study was to evaluate the clinical implication of Haemophilus haemolyticus, one of the closest relative of Haemophilus influenzae, on acute pharyngotonsillitis. METHODS: We applied polymerase chain reaction (PCR) for 16S ribosomal DNA (rDNA) and IgA protease gene (iga) to distinguish H. haemolyticus and H. influenzae. RESULTS: Among the 199 Haemophilus spp. isolated from 214 patients with acute pharyngotonsillitis, 52 (24.3%) H. influenzae strains and 23 (10.7%) H. haemolyticus strains were identified by polymerase chain reaction (PCR) for 16S rDNA and IgA protease gene (iga). All H. haemolyticus strains showed hemolysis on horse blood agar and there were no other Haemophilus spp., nonhemolytic H. haemolyticus and H. influenzae variant strains that had absent iga gene. H. hemolyticus showed close genetic relationship with H. influenzae evaluated by pulsed field gel electrophoresis (PFGE). The cases of acute pharyngotonsillitis showing WBC=7000/mm(3) or CRP=8 mg/dl were frequently found among cases with H. influenzae rather than cases with H. haemolyticus. CONCLUSION: H. haemolyticus is a pharyngeal commensal that is isolated frequently from adults with acute pharyngotonsillitis.

Nontypeable Haemophilus influenzae isolated from intractable acute otitis media internalized into cultured human epithelial cells.

OBJECTIVES: The aim of this study is to examine the internalization of nontypeable Haemophilus influenzae (NTHi) into human epithelial cells. METHODS: Bactericidal assay was applied to examine the effects of antibiotics against cell-adherent NTHi using HEp-2 cells. A trans-well chamber assay was applied to examine the internalization and penetration of NTHi using Detroit562 cells. RESULTS: The adherence of NTHi to HEp-2 cells was noted after 2h of incubation. Azithromycin had a strong bactericidal effect against both cell-associated and non-adherent NTHi, while ceftriaxone did not show bactericidal effects on NTHi adhered to the HEp-2 cells. Three (60.0%) out of five NTHi isolates from the nasopharynx of children with intractable acute otitis media (AOM) internalized into and subsequently penetrated through the epithelial cells at various degrees. Azithromycin had a strong bactericidal effect against the cell-internalized NTHi, while ceftriaxone was bactericidal only against extracellular NTHi. CONCLUSION: The potential of NTHi as the intracellular pathogen may contribute to the persistent existence of this pathogen that result in the prolonged and intractable clinical course of AOM. Azithromycin may be a therapeutically significant antibiotic for patients with prolonged respiratory tract infections due to NTHi.

Distribution of fibronectin-binding protein genes (prtF1 and prtF2) and streptococcal pyrogenic exotoxin genes (spe) among Streptococcus pyogenes in Japan.

Two hundred and seventy-two strains of Streptococcus pyogenes isolated from patients with invasive and noninvasive infections in Japan were evaluated for the prevalence of fibronectin-binding protein genes (prtF1 and prtF2). The possible associations of the genes with streptococcal pyrogenic exotoxin genes, macrolide resistance genes, and emm types were also evaluated. Overall, about 50% of S. pyogenes isolates carried fibronectin-binding protein genes. The prevalence of the prtF1 gene was significantly higher among isolates from noninvasive infections (71.4%) than among isolates from invasive infections (30.8%; P = 0.0037). Strains possessing both the prtF1 and prtF2 genes were more likely to be isolates from noninvasive infections than isolates from invasive infections (50.6% vs 15.4%; P = 0.019). S. pyogenes isolates with streptococcus pyrogenic exotoxin genes (speA and speZ) were more common among isolates without fibronectin-binding protein genes. The speC gene was more frequently identified among isolates with fibronectin-binding protein genes (P = 0.05). Strains belonging to emm75 or emm12 types more frequently harbored macrolide resistance genes than other emm types (P = 0.0094 and P = 0.043, respectively). Strains carrying more than one repeat at the RD2 region of the prtF1 gene and the FBRD region of the prtF2 gene were more prevalent among strains with macrolide resistance genes than among strains negative for macrolide resistance genes. These genes (i.e., the prtF1, prtF2, and spe genes) may enable host-bacteria interaction, and internalization in the host cell, but may not enable infection complications such as invasive diseases.

A recombinant P4 protein of Haemophilus influenzae induces specific immune responses biologically active against nasopharyngeal colonization in mice after intranasal immunization.

Outer membrane protein P4, together with P6, is highly conserved among all typeable and nontypeable strains of Haemophilus influenzae (H. influenzae). Thus, the protein is an attractive antigen for the inclusion in a vaccine against nontypeable H. influenzae (NTHi). However, the ability of P4 to induce antibodies protective against NTHi infections is still controversial. In this study, we investigated the specific mucosal immune responses against NTHi induced by intranasal immunization with the lipidated form of recombinant P4 protein (rP4) and non-fatty acylated recombinant P6 protein (rP6) with or without cholera toxin (CT) in BALB/c mice model. Intranasal immunization with either rP4+CT, a mixture of rP4 and rP6+CT, or rP4 and rP6 without CT elicited anti-rP4 specific IgG antibody in serum of mice. Intranasal immunization with either rP4+CT or a mixture of rP4, rP6+CT elicited anti-rP4 specific IgA antibody in nasopharyngeal washing (NPW), while intranasal immunization with rP4 and rP6 without CT did not induced anti-rP4 specific IgA antibody responses in NPWs. Sera from mice intranasally immunized with rP4+CT and a mixture of rP4, rP6+CT also showed bactericidal activity. Significant clearance of NTHi in nasopharynx was seen 3 days after the inoculation of live NTHi in mice intranasally immunized with rP4+CT. The current findings suggested that P4 would be a useful antigen as the component of the vaccine to induce protective immune responses against NTHi. The use of an intranasal vaccine composed of the different surface protein antigens is an attractive strategy for the development of a vaccine against NTHi.

Treatment and outcome of severe and non-severe acute otitis media.

UNLABELLED: To determine outcomes in acute otitis media (AOM) according to severity of disease and to assess different initial treatment regimens, 308 with AOM were enrolled and divided into severe (n = 277; 89.9%) and non-severe (n = 31; 10.1%) groups based on symptoms and tympanic membrane changes. Children in the severe group were initially managed with amoxicillin (AMPC) whereas children in the non-severe group were initially managed without antibiotics. Children were monitored on days 1, 5, 10, 14 and 28. Five outcome measures were assessed: disappearance of symptoms at day 5, resolution of tympanic membrane changes by day 28, disappearance of middle ear effusions by day 28, recurrence of acute symptoms prior to day 28, and need to change treatment regimens. Children with severe disease were more often male (57% versus 36%, P < 0.05) and more often colonized with pathogens (77% versus 55%, P < 0.05 than children with non-severe disease. The two groups were similar with respect to age and day care attendance. Despite differences in initial treatment regimens between the two groups, symptoms improved at the same rate for severe and non-severe disease, 94% by day 5. In contrast, tympanic membranes returned to normal in 69% of the severe and 81% of the non-severe groups by day 28; however, as early as day 5, 10% of the severe and 55% of the non-severe groups demonstrated normal tympanic membranes. Middle ear effusions similarly disappeared more slowly in the severe group, 52% versus 74% by day 14 and 76% versus 84% by day 28. Recurrence rates of acute symptoms occurred with equal frequency in the severe, 15%, and non-severe groups, 10%. Failure of the symptoms or the tympanic membranes to improve led to antibiotic changes in 59.9% of the severe group and to the addition of antibiotics in 51.6% of the non-severe group. Children in the severe group who failed to improve with an initial course of amoxicillin were younger (40.2 months versus 45.8 months, P < 0.05), had higher tympanic membrane scores (4.5 versus 4.1, P < 0.05), and were more often colonized with penicillin-resistant Streptococcus pneumoniae (33.8% versus 6.5%, P < 0.01) than children who responded to AMPC. In a similar manner, children with non-severe disease who failed to improve without antibiotics were younger (40.7 months versus 54.8 months, P < 0.05) and more often colonized with pathogens (75.0% versus 33.4%, P < 0.05). CONCLUSION: Severe disease occurred more often among males and among children colonized with pathogens. Response to treatment was impaired in younger children and in children colonized with pathogens, especially penicillin-resistant Streptococcus pneumoniae.

Factors associated with clinical outcomes in acute otitis media.

Acute otitis media (AOM) is a common disease in childhood. If predictors of outcome in AOM were known, it would be possible to individualize therapy. Our aim was to identify factors that predict the outcome in AOM. We enrolled 368 children with AOM (ages, 10 to 86 months). The severity of symptoms and the severity of tympanic membrane changes were graded with a scoring system. Nasopharyngeal colonization with middle ear pathogens was determined on day 1. Three outcomes were assessed: persistence of symptoms at day 5, persistence of tympanic membrane changes at day 28, and recurrence of acute symptoms prior to day 28. Persistence of symptoms at day 5 was associated with younger age (35 versus 44 months; p < .001), higher symptom score on day 1 (3.5 versus 2.9; p < .05), and colonization with Streptococcus pneumoniae (61% versus 41%; p < .05). Persistence of tympanic membrane changes at day 28 was associated with younger age (39 versus 45 months; p < .01), higher tympanic membrane score on day 1 (4.1 versus 3.6; p < .01), and nasopharyngeal colonization with S. pneumoniae, especially drug-resistant S. pneumoniae (33% versus 13%; p < .05). Recurrence of acute symptoms prior to day 28 occurred in 14% of the children. Streptococcus pneumoniae was the only pathogen associated with an increased recurrence rate (23%) as compared to the group without pathogens (7%; p < .05). Age, severity of disease at presentation, and nasopharyngeal colonization patterns were proven to be important determinants of outcome in AOM.

Intranasal immunization with recombinant outer membrane protein P6 induces specific immune responses against nontypeable Haemophilus influenzae.

OBJECTIVE: Nontypeable Haemophilus influenzae (NTHi) is one of the leading causative pathogens for otitis media. The outer membrane protein P6 of NTHi is highly conserved among the strains and is an attractive candidate for a preventive vaccine. However, for the production of a relatively small amount P6 containing lipopolysaccharides, the development of a recombinant version of this protein is required. This study was designed to investigate the specific mucosal immunity induced by intranasal immunization of recombinant P6 (rP6) with cholera toxin (CT). METHODS: BALB/c mice were immunized with of rP6 (30 microg) and CT (2 microg) intranasally every 2 days for 2 weeks. Anti-rP6 specific IgG, IgA and IgM antibodies and the subclass of anti-rP6 specific IgG antibody were determined by enzyme linked immunosorbent assay (ELISA). Anti-rP6 specific IgA in nasopharyngeal washings were also determined by ELISA. Nasopharyngeal clearance of inoculated NTHi after the intranasal immunization were assessed. All statistical differences between the two groups were assessed by ANOVA parametric test. RESULTS: Intranasal immunization with rP6 and CT evoked rP6-specific mucosal IgA immune response as well as the systemic IgG immune response against rP6 and enhanced nasopharyngeal clearance of inoculated live NTHi. CONCLUSION: These results indicate the good immunogenicities of rP6 to induce specific immune responses against NTHi. Intranasal immunization with rP6 will be an effective approach to protect infections of NTHi.