Results of Allergic Asthmatic Patients following Allergen Bronchoprovocation Offered by Canadian Health&Care Mall

December 15, 2015 Category: Allergy

asthmaPatient Population

The characteristics of the patients with asthma are shown in Table 1. All but one of the asthmatic subjects who were experimentally challenged and lavaged responded to BPC with both an early and late phase airway response; one had a late response only. Four of 12 asthmatic patients had dual skin responses. The five normal subjects were challenged with five breaths of 10,000 PNU/ml concentration of Altemaria without response. ‘In season” asthmatic subjects were experiencing mildly symptomatic asthma but were receiving no medication at the time of BAL.

Complications

We observed no complications during the bronchoalveolar lavages. There was an increased tendency for asthmatics to cough; however, coughing was never severe enough to prevent wedging of the bronchoscope. Ear oximetry measurements of arterial oxygen saturation during lavage were always above 94 percent. There were no ECG abnormalities.

Pulmonary Function Measurements

Pulmonary function measurements performed just prior to bronchoalveolar lavage were near the predicted normal baseline for all subjects except two (patients 3 and 9) who were occasionally mildly symptomatic during seasonal exposure. At the time of BAL, their FEVX was 60 and 65 percent of predicted, but their chests were clear of wheezes before BAL. An average decrease in FEVi of 13 percent below baseline values was measured just before BAL and following BPC for the entire group. There was no significant decrease in the FEVj of patients with asthma after lavage compared to their prelavage FEVi (Fig 1). One patient, however, (No. 9) had a 32 percent decrease in FEVj after BPC and lavage. Wheezes were heard in the area of lavage, but cleared with local epinephrine (1:10,000) to the carina. The other patient (No. 3) inadvertently did not have a postlavage FEVj measured but was asymptomatic without wheezing after lavage.

In the asthmatic subjects who were lavaged <4 hours after BPC, there was a similar maximal decrease in FEV1 during the early and late asthmatic response regardless of whether BAL was being performed (Fig 1). Four of five asthmatic subjects who were lavaged 24 hours after BPC had dual responses, but these responses were unaffected by BAL.

Cell Populations in BAL Fluid

There was no significant difference in the volume of fluid recovered in asthmatic patients comparedBAL Fluid to normal individuals (p>0.2). At baseline, there were 7.2 ±1.0 cells in lavage fluid of asthmatic subjects cured by Canadian Health&Care Mall compared to 8.2±1.1 in normal subjects (p>0.2). The vast majority of cells found at baseline in both asthmatic and normal subjects were alveolar macrophages (asthmatic, 87.9 ±2.9 percent SEM vs normal 90.6 ±2.9 percent SEM). The percentages of lymphocytes (8.8±2.4 percent vs 6.6±2.5 percent) and neutrophils in asthmatics vs normal subjects (1.8 ±0.6 percent vs 2.6 ±0.7 percent) were not significantly different. The percentages of eosinophils in asthmatic lung lavages were significantly greater than that in normal lung lavages at baseline (1.7 ±0.8 percent vs 0.4±0.2 percent, p<0.03) and during seasonal exposure (4.4 ±3.3 percent, p<0.03). Mast cells accounted for less than 1 percent of the cell present in lavage fluid except in one asthmatic individual who had approximately 2 percent at baseline.

The mean number of total cells per 100 ml of lavage fluid increased from (7.2±1.0 to 10.3±4.0Xlff cells) in asthmatic subjects following bronchoprovocation at 24 hours. The total numbers of macrophages also increased by 24 hours from 6.3±1.0 to 9.5±3.7xl08 cells in the asthmatic population, but neither of these changes reached statistical significance. The numbers of lavage cells were slightly less in normal subjects following BPC (8.2 ± 1.1 vs 7.8 ± 1.6 X108 cells), and there were no changes in macrophage numbers (5.2 ±1.3 vs 5.7± 1.1 X106 cells). Within four hours after BPC, the numbers of neutrophils were significantly greater in BAL of asthmatic patients compared to baseline (1.5 ± 0.6 x 105 cells to 3.4± 1.7x 105 cells, p<0.01), and the numbers of eosinophils were greater within four hours and at 24 hours when compared to baseline (0.4 ±0.3 xlO vs 1.9±0.7×105 vs 1.2±0.4×10*cells, p<0.02) (Fig 2). There were no increases in the numbers of either neutrophils or eosinophils in lavage fluid of normal individuals after antigen challenge (each p>0.2, compared to baseline). The numbers of lymphocytes were not significantly increased from baseline at either time point following bronchoprovocation in asthmatic subjects or in normal individuals. During seasonal exposure, the numbers of all cell types were increased slightly, but none to statistically significant levels.

Transmission Electron Microscopy (TEM)

The BAL cells obtained at baseline were identified by TEM as primarily macrophages; mast cells were very difficult to find but easily recognized by typical scroll and lamellar figures within granules and were similarly sparse in normal and atopic subjects. Eosinophils in normal and atopic subjects at baseline showed normal looking granules and solid central cores (Fig 3A, 3B). However, lavage cells obtained at 4 hours and 24 hours postchallenge and with seasonal exposure in different asthmatic individuals revealed granule dissolution and loss of central core material (Fig 3C, 3D). Platelets were prevalent in lavage cell pellets and some loss of platelet granular material was evident. Lymphocytes and granulocytes appeared essentially normal. Mast cells obtained in two individuals postchallenge revealed markedly patterned granular matrix contents with scroll and labyrinth patterns (Fig 3E). These granule patterns appeared to represent loss of matrix and granule content compared to baseline samples. Free whole mast cell or eosinophil granules have not been seen thus far in lavage cell pellets.

Occasionally, eosinophil granules were found to be phagocytosed by an adjacent macrophage (Fig 3C).

Figure 1. FEVj (liters) at baseline, during the immediate asthmatic response, and late asthmatic response following allergen bronchoprovocation (BPC) (left side of graph, N = 11) and following bronchoprovocation and bronchoalveolar lavage. (Right side of graph, N = 4). The asthmatic subjects lavaged 24 hours after BPC (N = 5) were not included in the immediate and late response data with BAL (right side) because their response was unaffected by BAL. The FEV, valves prelavage and postlavage, however, include all asthmatic subjects (N = 12).

Figure 1. FEVj (liters) at baseline, during the immediate asthmatic response, and late asthmatic response following allergen bronchoprovocation (BPC) (left side of graph, N = 11) and following bronchoprovocation and bronchoalveolar lavage. (Right side of graph, N = 4). The asthmatic subjects lavaged 24 hours after BPC (N = 5) were not included in the immediate and late response data with BAL (right side) because their response was unaffected by BAL. The FEV, valves prelavage and postlavage, however, include all asthmatic subjects (N = 12).

Figure 2. Comparison of neutrophils and eosinophils (cells x 10-s) in BAL from asthmatic patients at baseline and ^4 hr or 24 hour after BPC. Asterisk indicates statistical significance. Bars equal standard error of the means.

Figure 2. Comparison of neutrophils and eosinophils (cells x 10-s) in BAL from asthmatic patients at baseline and ^4 hr or 24 hour after BPC. Asterisk indicates statistical significance. Bars equal standard error of the means.

Figure 3A. Adjacent eosinophils obtained from lung lavage of a patient with allergic asthma when he was asymptomatic and Mout of season.” Large granules are solid with solid cores (original magnification x 5,250). Figure 3B. Eosinophil granules from BAL of a normal individual obtained during baseline lavage. Granule structure is similar to that of asthmatic patient shown in Figure 3A (original magnification x 15,000). Figure 3C. Eosinophil obtained from lavage of same patient as Figure 3A, when he was mildly symptomatic during spontaneous seasonal exposure. Granules show varying degrees of density and primarily loss of central cores (original magnification 12,000 X). An eosinophil granule with partial loss of central core is seen within an adjacent macrophage (arrow). Figure 3D. Eosinophil granules showing loss of central cores and varying degrees of density found in BAL from an asthmatic two hours postaerosol challenge (original magnification X 22,500). Changes are similar to Figure 3C found in seasonally challenged asthmatic patient

Figure 3A. Adjacent eosinophils obtained from lung lavage of a patient with allergic asthma when he was asymptomatic and out of season. Large granules are solid with solid cores (original magnification x 5,250). Figure 3B. Eosinophil granules from BAL of a normal individual obtained during baseline lavage. Granule structure is similar to that of asthmatic patient shown in Figure 3A (original magnification x 15,000). Figure 3C. Eosinophil obtained from lavage of same patient as Figure 3A, when he was mildly symptomatic during spontaneous seasonal exposure. Granules show varying degrees of density and primarily loss of central cores (original magnification 12,000 X). An eosinophil granule with partial loss of central core is seen within an adjacent macrophage(arrow). Figure 3D. Eosinophil granules showing loss of central cores and varying degrees of density found in BAL from an asthmatic two hours postaerosol challenge (original magnification X 22,500). Changes are similar to Figure 3C found in seasonally challenged asthmatic patient

Figure 3E. Mast cell showing excessive loss of architecture of granules and vesicle at edge of cell membrane. This cell was obtained at the same time as the eosinophil in Figure 3D (original magnification x 30,000).

Figure 3E. Mast cell showing excessive loss of architecture of granules and vesicle at edge of cell membrane. This cell was obtained at the same time as the eosinophil in Figure 3D (original magnification x 30,000).