Eighty-five patients with both chronic sinusitis and chronic asthma (41 females and 44 males, ranging from 19 to 58 years with a mean age of 33.54 years) were selected for this study. Eighty-one out of 85 treated patients completed the one-year follow-up visit. On admission the written consent was obtained from each parent whom was requested to complete a questionnaire about recent or recurrent hospitalization, visits to the health care facilities, antibiotic therapy, and surgery within the previous 6 months. Diagnosis of chronic sinusitis was made on the basis of at least a one-year history of clinical symptoms including purulent nasal and postnasal secretions, middle meatus purulent secretions, and CT scan showing evidence of maxillary sinusitis or/and ethmoid sinusitis. All patients tried different medical treatments and did not obtain satisfactory results and required surgical treatment. These patients also underwent routine physical examinations upon admittance. This study was approved by the Ethic Committee of the First Hospital, Shanxi Medical University.

All patients also suffered from perennial asthma. Among them, 51 patients' asthma symptoms occurred after their sinusitis. The diagnosis of asthma was based on the guidelines provided in ref ₁₂. The history of asthmatic symptoms included episodic dyspnea and wheezing. These symptoms could be controlled by inhaling β2-adrenoreceptor agonists, or theophyline preparations. Lung function tests were performed for patients with asthma before and after sinus surgery as well as at follow-up visits. Exercise-induced bronchoconstriction was determined at every visit by a 6-min run with sub-maximal exercise load, on a motor-driven treadmill. FEV1 was measured before running, immediately after and 3, 6, 10, 15 min after running. Maximum percentage falls in FEV1 after exercise was calculated using the formula: [(pre-exercise FEV1 – minimum post-exercise FEV1) ÷ pre-exercise FEV1] × 100.

Twenty-five healthy volunteers participated in this study as normal control subjects. None of them had a history of asthma or chronic sinusitis, nor acute upper respiratory infections within the last month.

The intradermal allergen test was employed to determine allergic status. Antigens in this study included house dust, dust mite and inhalant allergens. Total serum IgE levels and serum specific IgE (to dust mite, all the patients of this study showed positive skin test results to dust mite) levels were determined by radioimmunoassay. The reagents for IgE level determination were purchased from Pharmacia Diagnostics (Uppsada, Sweden).

The clinical symptoms of both sinusitis and asthma were scored on a four-point scale: 0 for no symptoms, 1 for mild, 2 for mild severe and 3 for severe. The symptom scores were recorded before and after sinus surgery, as well as at every follow-up visit.

All patients in this group were treated with functional endoscopic sinus surgery. The technique of this surgery is referred to in Kennedy's report. ₁₃ The patients were asked to follow up in the clinic 0.5, 1, 2, 4, 6 and 12 months after the surgical procedure. Necessary treatments were done in the follow-up visits or upon re-admitted into the hospital if required.

Tissue from surgery was rinsed with PBS and then snap frozen by immersion into liquid nitrogen and stored at –70° C until use. After weighing, tissue was homogenized with a triple detergent lysis buffer. Following lysis, samples were centrifuged at 12000 rpm for 15 min at 4°C. The BCA assay (Pierce; Rockford, IL, USA) was employed to determine protein concentration of the tissue lysate supernatant. Nasal cavity lavage was done using 5mL saline for each subject. The content of SEB in the tissue lysate and lavage solution was measured using ELISA (Sigma). Total IgE, specific IgE to dust mite and SEB and cytokines including IL-4 and IFN-γ were determined with serum samples. Measurement of cytokines was done with enzymatic-linked immunoassay (ELISA, reagents purchased from Sigma, St. Louise, Missouri, USA). Evaluations were done before and after sinus surgery, as well as at every follow-up visit.

Surgical removed tissue was obtained from each patient (a nasal lateral wall swab was obtained from each healthy control instead) that was sent to our Microbiological Laboratory within 30 minutes. The cultures were performed aerobically on blood agar plates. Colonies of different morphology growing on the staphylococcal agar were separately enumerated, subcultured on blood agar, Gram stained, and tested for catalase production. Isolates with a typical Gram-stained appearance and a positive catalase test were tested for coagulase production, and positive isolates were regarded as S. aureus. The template DNA was extracted from the positive cultured colony of each subject with the procedures reported by Riffon.₁₄ PCR was performed with the samples from each subject. The nucleotide sequence is available at the GenBank data library (accession number, X 72700). The primers were designed using the software Promer3 and specificity was tested with Blast. The primers were: 5'- tcaattgcattgcttttgct-3' and 5'- gcccttcattttcgttcaaa-3'. Amplification was performed on a Perkin-Elmer (Norwalk, Conn.) thermocycler in 25-µl reaction mixtures. The program consisted of an initial denaturation step at 94°C for 2 min, followed by 22 cycles of 1 s at 94°C, 2 s at 58°C, and 10 s at 72°C, with a final extension step at 72°C for 5 min. Amplification products were separated on a 1% agarose gel and stained with ethidium bromide before being analyzed on a UV bench by using GelDoc2000 (Bio-Rad).

Data is expressed as means ± SD. Statistical significance was determined using the χ²tests, or ANOVA. Significant difference was accepted when p values were less than 0.05.

Surgically removed tissue and nasal lavage samples were measured for SEB. Healthy control subjects demonstrate a small amount of SEB in their lavage solution (17.7 ± 9.5 pg/ml), whereas patients with rhinosinusitis demonstrated significantly higher levels of SEB in their nasal lavage solution, patients with sinusitis showed much more SEB content in their nasal lavage solution. The surgical removed tissue contained SEB as high as 134.5 ± 47.12 pg/g tissue (Fig 1).

Figure 1

S. aureus was identified in the surgical removed tissues. All the samples from the patients with serum anti-SEB > 1 IU/ml showed positive results in the culture and further confirmed with PCR (Fig 2). Only 3 (8.8%) samples from the 34 patients with serum anti-SEB < 1 IU/ml showed positive results (p<0.05, compared with those patients with serum anti-SEB > 1 IU/ml). The results also showed positive culture in the samples from 4 (16%) healthy controls.

Figure 2

Five (5/34, 14.7%) patients had nasal polyps in the group with serum anti-SEB IgE less than 1 IU/ml. 24 (24/51, 47.1%) patients had nasal polyps in the group patient with serum anti-SEB antibody greater than 1 IU/ml (p<0.01 with χ² test, comparing with the group with anti-SEB less than 1 IU/ml). The group patient with anti-SEB greater than 1 IU/ml was further divided into two sub-groups, sinus infection only group and multi-site infection group. 6 patients had nasal polyps in the patients with multi-sites infection (6/16, 37.5%, p<0.05, comparing with the patients with serum anti-SEB less than 1 IU/ml). Of the patients with sinus infection only, 18 patients had nasal polyps (18/35, 51.4%, p<0.01, comparing with the patients with multi-sites infection, and the patients with serum anti-SEB less than 1 IU/ml respectively). Correlation analysis was performed with the data of nasal polyp and serum anti-SEB levels. Positive correlation was determined in the patients with serum anti-SEB greater than 1 IU/ml (r=0.63, p<0.01, not included the patients with multi-site infections), but not in the patients with serum anti-SEB less than 1 IU/ml (r=0.27, p>0.05). Nasal polyps were radically removed during FESS. No nasal polyps relapsed during the follow-up period.

Upon admittance, serum IgE antibodies including total IgE, antigen specific IgE and anti-SEB antibodies were evaluated. The results are shown in Fig 3. Total IgE and antigen specific IgE antibodies were general higher in the patients of the present study than those healthy controls (p<0.05). SEB-specific antibodies in serum were significantly high in 51 of 85 patients. After sinus surgery, the serum anti-SEB antibody and nasal lavage SEB levels in 35 patients out of the 51 patients decreased significantly and closed to the levels of the normal control subjects.

Figure 3

Before sinus surgery, serum IL-4 was significantly higher; IFN-γ levels were significantly lower in all patients with asthma and chronic sinusitis compared with normal controls; after sinus surgery, serum levels of IL-4 significantly decreased and IFN-γ level increased in 35 of 85 patients (Fig 5). Correlative analysis showed a positive correlation of anti-SEB antibody with levels of serum IL-4 (r=0.63, p<0.01) and a negative correlation with levels of IFN-γ (r=0.76, p<0.01).

Figure 4

Figure 5

Nasal blockage and nasal purulent secretions of patients were significantly improved after sinus surgery. Before and after sinus surgery, lung function test data was obtained from all patients. The FEV1 value fall percentage after exercise was not as good as nasal symptom improvement in every patient after sinus surgery. Thirty-five patients who showed improvements in anti-SEB antibody and serum IL-4 and INF-γ levels demonstrated significant decreases in asthma symptom scores after sinus surgery (p<0.05); the fall of FEV1 value after exercise in these 35 patients was significantly improved compared with test results obtained before sinus surgery and approaching to the levels of those normal controls. (Fig 6).

Figure 6

Serum anti-SEB antibody levels of the patients in this study ranged from 0.3 IU/mL to 4.5 IU/mL before sinus surgery. Asthmatic symptom scores correlated with the levels of serum anti-SEB antibody (r = 8.12, p < 0.01). Patients with serum anti-SEB higher than 1.0 IU/mL before sinus surgery obtained better results of asthmatic symptom relief and pulmonary function improvement than those whose serum anti-SEB was less than 1.0 IU/mL.

Fifty (58.82%) patients did not obtain satisfactory improvement in their asthma one month after sinus surgery. Three of these perceived their asthma to be worse after surgery. A review of these case files was done, we found that 5 patients had chronic tonsillitis, 6 patients had residual adenoiditis, 2 patients had chronic otitis media, and 3 patients had dacryocystitis. These 16 patients with additional infections showed high levels of serum anti-SEB, IL-4 and low levels of IFN-γ before sinus surgery and neither showed little effect on decreasing anti-SEB antibodies, serum IL-4 and IFN-γ levels and almost no improvement on their clinical asthma symptoms although the sinusitis clinical symptoms were significantly improved following sinus surgery.

All of these 16 patients underwent surgical treatments for these disorders within two months after the sinus surgery, with subsequent improvement in their asthmatic symptoms. These patients no longer require any anti-asthma medications. Laboratory results showed decreased serum anti-SEB antibodies, IL-4, with increased IFN-γ levels compared with the data before surgery (p<0.01), with values approaching those of normal controls (p>0.05). SEB was also evaluated with these surgical removed tissues.

The remaining 34 patients showed little or no improvement in their asthmatic symptom scores and continued needing to use anti-asthma medications sometimes or regularly. Laboratory results showed they had low levels serum anti-SEB antibody levels that were close to the normal control values (p>0.05) and much less than the anti-SEB antibody levels (before sinus surgery) of the patients who achieved satisfactory results after sinus surgery.