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CD3(-)CD4(+)CD45(+) inducer cells are required for the initiation of mucosa-associated organogenesis of both nasopharynx-associated lymphoid tissues (NALT) and Peyer's patches (PP) in the aerodigestive tract. CXCL13(-/-) mice and mice carrying the paucity of lymph node T cell (plt) mutation and lacking expression of CCL19 and CCL21 accumulate CD3(-)CD4(+)CD45(+) cells at the site of NALT but not of PP genesis. Although NALT was observed to develop in adult CXCL13(-/-) and plt/plt mice, the formation of germinal centers in CXCL13(-/-) mice was affected, and their population of B cells was much lower than in the NALT of CXCL13(+/-) mice. Similarly, fewer T cells were observed in the NALT of plt/plt mice than in control mice. These findings indicate that the initiation of NALT organogenesis is independent of CXCL13, CCL19, and CCL21. However, the expression of these lymphoid chemokines is essential for the maturation of NALT microarchitecture.

Initiation of nasopharyngeal-associated lymphoid tissue (NALT) development is independent of the programmed cytokine cascade necessary for the formation of Peyer's patches (PP) and peripheral lymph nodes (PLN), a cytokine cascade which consists of IL-7R, LTalpha1beta2/LTbetaR, and NIK. However, the subsequent organization of NALT seems to be controlled by these cytokine signaling cascades since the maturation of NALT structure is generally incomplete in those cytokine cascade-deficient mice. NALT as well as PP and PLN are completely absent in Id2(-/-) mice. NALT organogenesis is initiated following the adoptive transfer of CD3(-)CD4(+)CD45(+) cells into Id2(-/-) mice, constituting direct evidence that CD3(-)CD4(+)CD45(+) inducer cells can provide an IL-7R-, LTalpha1beta2/LTbetaR-, and NIK-independent tissue organogenesis pathway for secondary lymphoid tissue development.

To clarify the role of viral infection in otitis media, we intranasally inoculated mice with influenza A virus and examined histologic changes in the nasopharyngeal mucosa using a battery of lectins. Additionally, live Haemophilus influenzae or Streptococcus pneumoniae was injected into the nasopharynx after virus inoculation, and the clearance of bacteria from the nasopharynx was examined. Staining of the mucous blanket and epithelial cell surfaces in the nasopharynx with peanut agglutinin, succinyl wheat-germ agglutinin, and Bandeiraea simplicifolia agglutinin was significantly enhanced with intranasal virus inoculation when compared with that in control animals. The nasopharynx was moderately stained with Maachia amurensis agglutinin and wheat-germ agglutinin in control animals, and the staining was enhanced after virus inoculation. These findings were most remarkable 5 and 9 days after virus inoculation. The numbers of bacteria cultured from the nasopharynx were significantly increased when bacteria were injected 5 days after virus inoculation. These results suggest that an alteration in the glycoconjugate structure lining the nasopharyngeal mucosa caused by the influenza virus might be associated with the reduction in bacterial clearance.

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.

Nasopharyngeal colonization with nontypeable Haemophilus influenzae (NTHi) and immune responses in the adenoid were studied in 18 patients with otitis media with effusion (OME) who underwent adenoidectomy. NTHi in excised adenoid tissue and in nasopharyngeal secretions was examined by quantitative culture assay. NTHi was detected in 13 adenoid samples and 12 nasopharyngeal secretions. In 11 cases, NTHi was cultured from both samples of nasopharyngeal secretion and adenoids, and the concentration of NTRi in nasopharyngeal secretions was significantly correlated with that in adenoids. A significant negative correlation was observed between anti-NTHi IgA antibody titers and the number of NTHi in nasopharyngeal secretions. The number of NTRi in nasopharyngeal secretions and adenoids was decreased in the cases having a large number of NTHi-specific IgA and IgG antibody-producing cells in their adenoids. The results suggest that mucosal immunity in the nasopharynx is responsible for reducing the colonization of NTHi, and that the adenoid plays an important role in regulating the immune responses.

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.

Adherence of nontypable Haemophilus influenzae and Streptococcus pneumoniae to nasopharyngeal epithelial cells was investigated in vitro. Both strains had higher affinity to the epithelial cells of children than to those of adults. In children, the adherence was significantly greater in patients with otitis media with effusion than in normal subjects. Secretory IgA in nasopharyngeal secretions was found to have antibody activity against the bacteria. Adherence of both bacteria was significantly smaller in the group having secretory IgA antibody activity than in the group having no activity. These results suggest that bacterial adherence to the nasopharynx may play an important role in the pathogenesis of otitis media with effusion in children, and that secretory IgA in nasopharyngeal secretions may be related to the decrease of adherence.

In order to clarify the role of the microflora of the nasopharynx and adenoids as causative factors of otitis media with effusion (OME), bacteriologic species in middle ear effusions (MEEs), nasopharyngeal smears, and adenoid tissues of children with OME were cultured. The change in the microflora of the nasopharynx after adenoidectomy, and the degree of bacterial agreement between the microflora of the nasopharynx and adenoid tissue were investigated. Nasopharyngeal bacterial flora were investigated in 259 children with OME from November 1984 to November 1987. The patients were divided into two groups: group A (43 patients) underwent adenoidectomy, and group B (216 patients) had no adenoidectomy. Hemophilus influenzae, Streptococcus pneumoniae, Branhamella catarrhalis, and Staphylococcus aureus were cultured in 66.7% of nasopharyngeal smears from group B, and in 46.5% from group A. Adenoid specimens for bacteriologic investigation were obtained from 38 adenoidectomy patients at surgery. Of these, H influenzae were cultured from adenoid specimens taken from 16 of the 26 patients with OME (group C), while only three of the 12 adenoidectomy patients without OME (group D) were found to have H influenzae in their adenoid specimens. The findings of this study suggest that adenoid vegetation plays an important role in the etiology of OME.

The adherence of Haemophilus influenzae to nasopharyngeal mucosal cells was investigated in vitro. Secretory IgA antibody activity and the content of fibronectin in nasopharyngeal secretion were determined by enzyme-linked immunosorbent assay. Adherence was greater in children than in adults; and was significantly greater in children with otitis media with effusion than in control children. Secretory IgA and fibronectin in nasopharyngeal secretion were considered to influence adherence of H. influenzae to nasopharyngeal mucosa.