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Background: Recurrent tonsillitis is one of the most common otolaryngological disorders caused by cell-invading bacteria, such as Streptococcus pyogenes (S. pyogenes) and Haemophilus influenzae. The aim of this study was to investigate the effect of antibacterial agents against cell-invading bacteria., Methods: The intracellular invasion of Detroit 562 cells by five strains of nontypeable Haemophilus influenzae (NTHi) and four strains of S. pyogenes was investigated. The antibacterial agents used were garenoxacin (GRNX), clarithromycin (CAM), amoxicillin (AMPC), cefditoren pivoxil (CDTR-PI), and levofloxacin (LVFX)., Results: Both NTHi and S. pyogenes fully invaded Detroit 562 cells in 6 h and were less sensitive to CAM. GRNX, CAM, and LVFX were effective against bacteria invading the cells, but AMPC and CDTR-PI were not effective. GRNX was the most effective., Conclusion: GRNX was the most effective agent against bacteria invading cells.

Objective: Phosphorylcholine (PC) is a structural component of Streptococcus pneumoniae (Spn) and nontypeable Haemophilus influenzae (NTHi), and is known to be associated with adherence through the platelet activating factor receptor (PAF-R). Furthermore, high PC expression is considered to be involved in Spn and NTHi virulence. In this study, we examined the influence of PC expression on the adherence of Spn and NTHi to epithelial cells in order to clarify the potential effectiveness of a vaccine targeting PC., Methods: Twenty-seven strains of Spn and twenty-two strains of NTHi were used, cultured overnight, and PC expression was evaluated by fluorescence activated cell sorting; the strains were divided into two groups: PC low expression (PC-low) and PC high expression (PC-high) groups. Bacterial adherence was then examined using Detroit 562 cells and BALB/c mice. Bacterial invasion was then examined in Detroit 562 cells., Results: The adherence of Spn and NTHi and invasion of NTHi in the PC-high group was significantly reduced by pretreatment with a monoclonal anti-PC antibody (TEPC-15), PAF-R antagonist (ABT-491), and PC-keyhole limpet hemocyanin (PC-KLH). However, such findings were not observed in the PC-low group., Conclusion: The present study suggests that PC is involved in the mucosal adhesion of Spn and NTHi, and the mucosal invasion of NTHi with PC-high strains, but not PC-low strains. These results suggest that a PC-targeting mucosal vaccine only affects PC-high Spn and NTHi strains and does not disturb commensal bacterial flora in the upper respiratory tract, which comprises nonpathogenic PC-low bacteria., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Phosphorylcholine (PC) is a structural component of a wide variety of pathogens including Streptococcus pneumoniae and Haemophilus influenzae, and anti-PC immune responses are known to protect mice against invasive bacterial diseases. The present study tested the capability of PC as an intranasal plurispecific vaccine against upper airway infections. BALB/c mice immunized with intranasal PC-keyhole limpet hemocyanin (KLH) plus cholera toxin (CT) as a mucosal adjuvant showed increased PC-specific IgM in serum, IgA in nasal wash and saliva, and numbers of PC-specific nasal and splenic antibody producing cells. Enhanced production of IL-4 and IFN-gamma by CD4+ T cells indicated the participation of Th2- and Th1-type cells. Salivary IgA antibodies produced by intranasal immunization with PC-KLH plus CT reacted to most strains of S. pneumoniae and H. influenzae. Further we demonstrated that the clearance of S. pneumoniae and H. influenzae from the nasal tract was significantly enhanced by nasal immunization with PC-KLH and CT. Thus, intranasal vaccination to induce PC-specific immune responses might help to prevent upper airway infections caused by S. pneumoniae and H. influenzae.

To determine the efficacy of a mucosal vaccine against nontypeable Haemophilus influenzae (NTHi), mice were immunized nasally, orally, intratracheally, or intraperitoneally with NTHi antigen together with cholera toxin. Antigen-specific IgA antibody titers in nasal washes and the numbers of antigen-specific IgA-producing cells in nasal passages showed the greatest increases in mice immunized nasally. Cytokine analysis showed that interferon-gamma, interleukin (IL)-2, IL-5, IL-6, and IL-10 were induced by nasal immunization, suggesting that Th2- and Th1-type cells were generated. Furthermore, bacterial clearance of a homologous strain of NTHi from the nasal tract was significantly enhanced in the nasal immunization group. These findings suggest that nasal immunization is an effective vaccination regimen for the induction of antigen-specific mucosal immune responses, which reduce the colonization of NTHi in the nasal tract.

BALB/c mice were orally immunized with liposomes containing outer membrane proteins (OMPs) isolated from nontypeable Haemophilus influenzae (NTHi) and GM-53 as an adjuvant daily for 14 days. Anti-OMP IgA antibody titers in nasal wash, saliva, and fecal extract were significantly increased after the immunization. Although anti-OMP IgM and IgG antibodies were detected in serum, serum IgA antibodies specific to OMPs were not found. Enzyme-linked immunospot assay showed an increased number of OMP-specific IgA-secreting cells in nasal passages, intestinal lamina propria lymphocytes, and the spleen. Following oral immunization, a suspension of live NTHi was injected into the nose; nasal washes were collected 12 h after the inoculation. The number of NTHi in nasal washes was significantly reduced in mice immunized with liposomes containing OMPs and GM-53 compared to that in mice immunized with liposomes containing GM-53 alone. There was a significant negative correlation between the number of NTHi and anti-OMP IgA antibody titers in nasal washes. These findings suggest that antigen-specific IgA responses in the nose can be induced by oral immunization with OMPs and might be associated with the ability to clear NTHi from the nose.

Objectives: Investigate immune responses of adenoidal lymphocytes against outer-membrane protein P6 purified from nontypeable Haemophilus influenzae (HI). Clarify the role of adenoids in regulating the colonization of HI in the nasopharynx., Study Design: Microbiological and immunological examinations of adenoids obtained from 21 children, 15 boys and five girls, from 1 to 13 years of age (median, 5 y), suffering from adenoidal hypertrophy complicated by otitis media with effusion (OME)., Methods: The incidence of HI in adenoids was compared with the number of P6-specific immunoglobulin (Ig) A-secreting cells in adenoids, determined by enzyme-linked immunoassay., Results: Quantitative culture assay showed significant correlation between the numbers of HI in adenoids and those in nasopharyngeal secretions (NS). In children aged 5 years and younger, the numbers of P6-specific IgA-secreting cells in adenoids were significantly correlated with IgA antibody titers in NS (r = 0.68, P < .05). The numbers of P6-specific IgG- and IgA-secreting cells were lower in children aged 6 years and older than in children aged 5 years and younger. Furthermore, the number of P6-specific IgA-secreting cells was significantly increased in HI-negative subjects when compared with HI-positive subjects (P < .05)., Conclusions: Adenoids play an important role as an effector site of the mucosal immune system in the upper respiratory tract. IgA immune responses in adenoids are responsible for the clearance of HI from the nasopharynx.

BALB/c mice were immunized orally or subcutaneously with formalin-killed nontypeable Haemophilus influenzae (NTHi). Salivary immunoglobulin A (IgA) antibody titers against NTHi were significantly increased by oral immunization, but not by subcutaneous immunization. Both immunization procedures remarkably increased the levels of serum antibody activities of both IgA and immunoglobulin G. Live NTHi were then inoculated into the naso-pharynx, and the clearance of the pathogen from the nasopharynx was observed. Significantly fewer bacteria were present in the nasopharynx of the orally immunized mice than in the control mice. However, there was no significant difference between the subcutaneously immunized mice and the control mice. The results indicate that oral immunization can enhance the ability of mice to clear NTHi from the nasopharynx.

Haemophilus influenzae in the nasopharynx is thought to be related to the pathogenesis of otitis media with effusion (OME). However, the positive rate measured by the conventional culture method is not high enough for bacterial infection to be considered the chief pathogenic factor. Polymerase chain reaction (PRC) and the conventional culture method were applied to detect H. influenzae in the adenoids and nasopharyngeal secretions of 17 children with OME (2 to 7 years of age). The percentage of P6-positive adenoid samples was 82.4% while 70.6% were culture positive. In nasopharyngeal secretions, 88.2% were P6-positive and 64.7% were culture positive. All samples in which H. influenzae was cultured were positive with PCR. The positive rate was significantly greater with PCR than with the culture method, and it showed a significant concordance between adenoids and nasopharyngeal secretions (p = 0.0027). These findings suggest that the presence of H. influenzae in the nasopharynx is more common in children with OME than previously reported, and that H. influenzae in adenoids might play an important role in OME.

PCR was used to detect Haemophilus influenzae in samples of nasopharyngeal secretion and middle ear effusion (MEE). Nasopharyngeal secretions were collected from 102 patients with otitis media with effusion and from 111 healthy subjects. Eighty samples of MEE were collected from patients with otitis media with effusion. A pair of primers was designed to amplify a DNA segment of the gene encoding P6 outer membrane protein of H. influenzae. The amplified PCR product was detected with an internal probe that hybridized specifically to the P6 DNA of H. influenzae. Samples of MEE and nasopharyngeal secretion were also examined by a conventional culture method. The incidence of P6 gene DNA in nasopharyngeal secretions detected by PCR was about two times higher than that of H. influenzae detected by the conventional culture. Culture-positive samples were all positive in the PCR test. In MEEs, the rate of detection of the P6 gene DNA target was about five times higher than that of H. influenzae detected by the culture method. All patients who had P6 gene DNA in MEEs were found to have the DNA in nasopharyngeal secretions. These findings suggest that the presence of H. influenzae in MEEs and in nasopharyngeal secretions is more common than previously reported.

Nontypeable Hemophilus influenzae organisms were inoculated into the nasopharynx of BALB/c mice immunized by oral administration of formalin-killed bacteria. Salivary antibodies and the colonization of H influenzae in the nasopharynx were investigated in order to clarify the effect of oral immunization. Salivary immunoglobulin A antibody titers against H influenzae were significantly increased by oral immunization, but salivary immunoglobulin G antibody titers were not. The bacteria inoculated into the nasopharynx were more rapidly eliminated in immunized mice than in control mice. The results suggest that oral immunization might be useful in preventing otitis media with effusion by inhibiting the colonization of the nasopharynx by pathogenic bacteria.

The role of secretory immunoglobulin (Ig) A in nasopharyngeal secretions in the adherence of Streptococcus pneumoniae and Hemophilus influenzae to nasopharyngeal epithelial cells was investigated in vitro. The adherence was remarkably reduced by treating bacteria with nasopharyngeal secretions, and the antiadhesive activity was significantly greater in nasopharyngeal secretions having secretory IgA antibody activity against bacteria than in those having no activity. Noticeable changes were not observed in the antiadhesive activity caused by absorption of IgG from nasopharyngeal secretions. Results suggest that secretory IgA in nasopharyngeal secretions is related to bacterial adherence and adds to the prevention of nasopharyngeal infections.