Aeroallergens
Aeroallergens are ubiquitous, although quantitative and qualitative differences depend on geographic location, climate, degree of urbanization and specific conditions in the home, school and workplace. Almost all adults appear to have T lymphocytes that are sensitized to at least some aeroallergens; thus, development of allergic disease may depend on quantitative differences in T cells.1 Several lines of evidence link aeroallergens to asthma:2
-
A total of 60% of adults and 80% of children with asthma have positive skin-prick tests for environmental allergens, and allergen-bronchial challenge tests are positive only in those with allergen-specific positive skin tests.
-
Allergen sensitization is a risk factor for severe, acute asthma, especially if the patient is exposed to high concentrations of the specific allergen.
-
In general, severity of symptoms and of bronchial responsiveness correlates with degree of sensitivity to allergens; in some patients, allergy does not play an important role.
-
Symptoms, PEF and bronchial responsiveness usually improve when allergens to which the person is sensitized are avoided.
Aeroallergens, which are carried on inhalable particles, are proteins that vary in molecular weight from 14 to 78 kilodaltons. Outdoor allergens arise from pollen or mold spores; indoor allergen sources include several species of dust mites, cats, dogs and other mammals, cockroaches and indoor mold spores. The molecular structure and functional properties of common and important indoor allergens, based on the World Health Organization's nomenclature, have recently been summarized.3 Recombinant allergens with immunoreactivity comparable to that of the natural allergens are being produced and evaluated for allergen standardization, for diagnostic testing and for immunotherapy with specific epitopes and naked DNA vaccines.
Infants are exposed and become sensitized to aeroallergens as well as food allergens in utero.[4, 5] In people who are genetically predisposed to allergy, antenatal factors, including maternal and, thus, fetal exposure to allergens and materno-placento-fetal immunologic interactions are important in determining whether the predisposition results in allergic disease.6 Exposure to low concentrations of indoor allergens in early childhood is associated with a low incidence of sensitization, but very low concentrations may be sufficient to sensitize children who are predisposed and have a family history of allergy, presumably after intrauterine priming.7
Aeroallergens as a risk factor for asthma
There are no reliable tests to detect which infants are at risk of developing allergic disease and asthma. A positive skin-prick test for egg protein, as a marker of specific immunoglobulin (Ig) E antibody, at 6 months of age in a group of high-risk children (i.e., with a family history of atopy) was associated with development of atopic dermatitis, wheezy illness and asthma by age 7 years8 and was consistent with earlier studies.[9–11] In a community study12 of 360 children, the cumulative incidence of newly diagnosed asthma from 6 to 11 years of age was 12%. Bronchial hyperresponsiveness to cold air at age 6 years was associated with a 2.6-fold increase in risk (95% CI 1.25-5.4). However, after adjusting for mild wheezing at age 6 years, which is associated with an increased risk of 7.5-fold (95% CI 3.6-15.0, p < 0.001), and for positive skin-test reaction to inhalant allergens at age 6 years, which is associated with an increased risk of 3.6-fold (95% CI 1.5-9.5, p < 0.01), the response to cold air was no longer a significant predictor. Therefore, hyperresponsiveness to cold air is associated with a subsequent diagnosis of asthma, but depends on the presence of atopy and prior mild wheezing.
Earlier studies, as indicated in the 1995 consensus statement,13 identified exposure to household dust mites and indoor animals, especially cats, as risk factors for asthma. A recent 12-month study14 of 476 children with asthma, aged 4-9 years, living in inner city communities in the United States found that 36.8% of the children were allergic to cockroach allergen, 34.9% to dust-mite allergen and 22.7% to cat allergen. Analyses of dust showed that 50.2% of bedrooms contained high concentrations of cockroach allergen, 9.7% contained dust-mite allergen and 12.6% contained cat allergen. Adjusted rates of hospital admission were 0.37 a year for those who were allergic to and exposed to high concentrations of cockroach allergen compared with 0.11 for those allergic to other allergens (p < 0.001) and 2.56 unscheduled medical visits for asthma compared with 1.43 (p < 0.001). Those allergic to cockroach allergens experienced more days of wheezing, missed school days, night sleep loss and changes in activities than those allergic to dust-mite and cat allergens. This suggests that exposure to high concentrations of allergen in those allergic to a specific allergen is likely to enhance asthma morbidity.
In a higher socioeconomic group,15 135 of 1054 adolescents in 2 high schools were identified with asthma; 48 who were symptomatic and responded to histamine challenge and 123 controls were studied. Analysis of total IgE, dust-mite, cat and cockroach sensitization found only allergy to dustmite allergen to be independently associated with asthma (odds ratio [OR] 6.6, p < 0.0001). Dust from 81% of the houses contained more than 2 μg/g of class-I allergen from 2 common species of dust mites, Dermatophagoides pteronissinus (Der P1) and D. farinae (Der F1); 40% contained cat allergens and 17% contained cockroach allergens. Asthma was not associated with race, socioeconomic status, smoking in the home, sensitization to outdoor allergens or indoor allergen concentration. When asthma is prevalent and high concentrations of dust-mite allergen are present, sensitization is the prime risk factor for symptomatic asthma. Nevertheless, the importance of the environment is dependent on the predominant exposures in that environment, which are influenced by cultural and geographic factors.
Seasonal changes in indoor allergen levels have been associated with changes in bronchial responsiveness.16 In 32 people with asthma, who were allergic to dust mites, the provocative concentration of histamine giving a 20% fall in FEV1 (PC20) increased from 2.05 mg/mL in autumn to 4.51 mg/mL in spring (p < 0.001), indicating a reduction in airway responsiveness. In 11 control subjects, who were allergic but not sensitized to dust mites, there was no significant change (PC20 of 3.44 mg/mL in autumn and 4.52 mg/mL in spring). Increased bronchial responsiveness in autumn was associated with higher levels of Der P1 in floor dust in homes.
Most indoor aeroallergens have been measured in terms of the amount per gram of dust, but, as they must be inhaled to have an effect, ambient airborne concentrations are likely to be much more important. In a recent placebo-controlled, double-blind study17 using an allergen exposure chamber, 15 people with asthma, who were allergic to dust mites (as evidenced by both skin tests and conventional bronchial-inhalation challenge) were exposed to 1200 μg of the class-I allergen of a common dust mite and to a placebo. Symptoms, PEF and medication use were assessed before and after challenge: 12 reacted with symptoms and a median decrease in FEV1 of 16.4% when exposed to allergen but not placebo; the other 3 had only minor symptoms during both active and placebo exposure and had no change in lung function. Late-phase reactions occurred in 1 person exposed to allergen, and in 3 given the conventional challenge. No healthy subjects reacted to any challenge. The authors concluded that asthma symptoms in allergic people were elicited by minor amounts of airborne allergen.
Another marker of the role of allergy in asthma is its association with acute asthma that is severe enough to require hospital admission. In a retrospective study involving 138 children aged 5-18 years seen consecutively in a specialized clinic,18 admission to hospital was associated with age (OR 0.8), allergy to cockroach (OR 2.2) and cat (OR 2.9). Based on a stepwise, multiple logistic regression analysis, only cat allergen (OR 3.8), age (OR 0.8) and race (OR 3.2) were independent predictors. In a prospective, single-blind, randomized controlled study of house-dust avoidance measures in 23 children aged 5-18 years who had been admitted to hospital with acute severe asthma,19 the 13 children in the experimental group had improved PEF at 3 and 6 months after intervention. The demographics and use of medication were the same in both the experimental and control groups. Improved PEF at 3 months was found in 6 of 7 children sensitized and exposed to dust-mite allergen when allergen concentrations in both bedding and bedroom floors fell. There was no difference in FEV1. During the study, 4 of the children in the experimental group and 2 of the 10 in the control group were readmitted to hospital with episodes provoked by viral respiratory infections.
Exposure to high concentrations of outdoor allergens has been associated with provocation of severe acute asthma and asthma deaths in subjects allergic to specific allergens, most clearly Alternaria spores. Neither exposure to lower concentrations of allergen nor concomitant exposure to air pollutants has been consistently associated with symptoms.
Delfino and colleagues20 assessed the effect of exposure to outdoor fungal spores and air pollutants on asthma symptoms, PEF and use of rescue medication in 22 subjects with asthma, aged 9-46 years, for 8 weeks during late spring and early summer using a random-effects longitudinal regression model controlled for autocorrelation and weather. Total fungal spore concentration was associated with a modest increase in symptom score (0.36), increased use of bronchodilator medication (0.33 puffs) and decreased evening peak flows (12 L/minute). There was also a modest association between concentration of particles with a diameter of 10 μm or less and increased use of rescue medication (0.15 puffs per 10 μg/m3, p < 0.02). Ozone had no effect.
Every 2 weeks for 3 months, Hilterman and colleagues21 followed 60 adults with intermittent to severe asthma to determine, by nasal lavage, the effect of ambient air pollution or allergen exposure on inflammatory changes in the upper airways. Exposure to ambient ozone was associated with an increase in neutrophils (112% per 100 μg/m3 increase in 8-h average ozone), eosinophils (176%), epithelial cells (55%), interleukin-8 (IL-8) (22%) and eosinophil cationic protein (ECP) (19%). Increases in mugwort-pollen counts (the major airborne pollen during the study period) were associated with increased eosinophils (107% per 100 pollen grains/m3) and ECP (23%), but not neutrophils, epithelial cells or IL-8. This suggests that inflammation of airway mucosa is provoked by ambient ozone and ambient pollen exposure, but the type of inflammation is qualitatively different.
Respiratory infections
Viral respiratory infection is a well known provocative factor for episodes of asthma. As well, specific agents, including respiratory syncytial virus (RSV), adenovirus, mycoplasma and pertussis, can provoke episodes of wheezing illness and, in a few cases, prolonged bronchial hyperresponsiveness. Recent studies using polymerase chain reaction (PCR) have implicated human rhinoviruses (HRV) as important agents in all age groups, and 1 study using this technique suggested a high prevalence of chronic Chlamydia infection in asthmatic children.22
How viruses or other agents provoke asthma is not clear. There is evidence of increased IgE production during viral infection. A recent study23 using a human B-cell culture system found that HRV-induced, double-stranded RNA activates an antiviral protein kinase that can induce Ig class switching to IgE, suggesting a mechanism for viral provocation of allergy and asthma. This is consistent with a study24 of experimental HRV infection in asthmatic adults, which resulted in augmented eosinophilic inflammation (assessed in sputum) and enhanced bronchial responsiveness. In another controlled study25 of experimental HRV infection in people with allergic rhinitis (but no asthma) and a nonallergic control group, there was a significant increase in bronchial responsiveness to histamine in the allergic group. Rhinovirus infection of cultured human tracheal epithelium, confirmed by PCR, resulted in increased expression (up-regulation) of messenger RNA for intercellular adhesion molecule-1 (ICAM-1) mRNA (the major HRV receptor on epithelial cells) and increased secretion of IL-1b, which itself up-regulates ICAM-1. Because ICAM-1 has important eosinophil attractant properties, this may be an important way in which the bronchial airway inflammatory response may be increased by HRV infection in asthma.26
RSV infection accompanying bronchiolitis is associated with persistent bronchial hyperresponsiveness in some children, but its role in causing asthma is unclear. Recent animal studies suggest that RSV infection in mice followed by aeroallergen exposure results in pulmonary inflammation with eosinophilic infiltration;27 in guinea pigs, prior sensitization to allergen followed by infection with RSV results in much more severe mucosal damage.28
Viruses are of greatest importance in causing wheezing illness in children under the age of 3 years. Reports from several centres[29, 30] now confirm that 20% or more of infants in this age group respond to viral infections with recurring wheezing, which resolves in later childhood. These infants have reduced lung function before the onset of viral infection, have apparently normal immune responses to viral infection and do not have risk factors for asthma (i.e., increased IgE levels, bronchial hyperresponsiveness or a family history of asthma). They may have narrower intrapulmonary airways than normal infants. A second group, about 10% of wheezy infants, also wheeze with virus infections, have some or all of the risk factors for asthma and have recurring wheeze (asthma) in later childhood.31 There is a great need to develop tests that will accurately differentiate these 2 populations.
Occupational and irritant-induced asthma
Occupational asthma (OA), defined as asthma induced by exposure to a specific agent in the workplace,32 is the most common occupational lung disease in developed countries.[33–35] Occupational exposure has been estimated to cause 5%-15% of adult-onset asthma.[36–39] The prevalence of OA due to agents with high molecular weight is generally < 5%; prevalence due to low molecular weight agents is 5%-10%.40 In 1 series, reactive airways dysfunction syndrome (RADS) or irritant-induced asthma accounted for 17% of 154 consecutive cases of OA.41
Many agents can cause OA. Those that cause immunologically mediated OA include a broad spectrum of protein-derived as well as natural and synthetic chemicals used in various workplaces. Extensive lists of causative agents and workplaces have been published and a computerized database is available.42 These agents can be classified according to whether their pathogenic mechanism is immunologically mediated.
An occupational cause should be suspected for all new cases of asthma in adults. A detailed occupational history of past and current exposure to possible causal agents in the workplace, work processes and specific job duties should be obtained. Information can be requested from the work site, including material safety data sheets. Walk-through visits of the workplace may be necessary. Industrial hygiene data and employee health records can also be obtained.
Temporal associations are not sufficient to diagnose work-related asthma,43 and objective tests are necessary to confirm the diagnosis. Workers with asthma symptoms should not be told to leave their job until the diagnosis is proven because part of the diagnostic work-up of OA may involve a trial return to the work site by the worker.
Challenge testing with the specific suspected agent has been used to confirm the work relationship.44 These tests can be falsely negative if a wrong agent is used for testing or if the patient has been away from work for too long. Another method to confirm the work relationship is serial monitoring of PEF for a period at work and a similar period away from work.45 Computerized peak-flow meters are helpful in overcoming some of the problems of PEF monitoring.46 When the results of PEF monitoring suggest OA and specific inhalation challenges in the laboratory are not possible or negative, it is advisable to confirm OA by serial spirometry throughout a work shift47 Combining PEF monitoring with serial assessments of nonallergic bronchial responsiveness can provide further objective evidence.
Identification of those with OA is important because progressive deterioration and permanent disability may occur if exposure continues after onset of symptoms.48 Early removal from exposure may be associated with disappearance of symptoms and airway hyperresponsiveness.48
The ideal treatment is the permanent removal of patients with OA from exposure to the causal agent;[49, 50] some workers who have continued in the same job after diagnosis have died.51 Any patient with OA who remains in the same job should have respiratory protection and close medical follow-up. Worsening of asthma should lead to immediate removal from exposure.
Irritant-induced asthma is caused by single or multiple exposures to high concentrations of an irritant vapour, fume or smoke in previously normal people.52 The term "reactive airways dysfunction syndrome" or RADS is used when the condition is caused by a single exposure.
A patient's pre-existing asthma may be aggravated by exposure to low levels of irritants, such as fumes, vapours or dust. However, the presence of asthma before being exposed to a sensitizing agent in the workplace does not preclude the development of true OA. People with asthma should not be exposed to concentrations of irritant higher than permissible (the airborne concentration to which nearly all workers may be exposed repeatedly without ill effects), although even this level may not be safe in those with airway hyperresponsiveness.
For further information, readers should consult the full text of the Canadian Thoracic Society Guidelines on occupational asthma.53
Indoor and outdoor respiratory irritants
Outdoor air pollution has been linked to acute exacerbations of asthma.54 Currently, the air pollutants of most concern are inhalable particulates (diameter ≤ 10 μm [PM10]), ground-level ozone, acid aerosol, sulfur dioxide and nitrogen dioxide. Of these, inhalable particulates appear to be the single greatest hazard. Recent studies have shown strong associations between ambient concentrations of inhalable particulates and emergency room visits,55 admission to hospital and doctor visits for asthma.[56–58] An increase in respiratory symptoms and a decline in PEF have also been observed in asthmatic children following increases in particle concentration.[59–65]
The role of inhaled particulate pollution in exacerbating asthma is based on epidemiologic studies, as no human study using controlled exposure is available. However, such studies have shown that ozone increases airway responsiveness and inflammation, and sulfur dioxide causes transient bronchoconstriction in people with asthma. Observation of the association of inhaled particulates with a range of adverse effects in people with asthma in a variety of settings strengthens the argument for a causal effect.
In eastern Canada and the United States, increases in particulate concentration occur in association with increases in acid aerosol and ozone concentrations. Increased concentrations of that mixture of pollutants have also been associated with a greater number of admissions to hospital for asthma.66 Although the adverse effects of particulates on people with asthma clearly do not depend on the presence of acid aerosols, increases in acid aerosol concentrations in some settings contribute independently to increased respiratory symptoms.67
Increases in ozone concentration have also been associated with more emergency room visits[66, 68] and admissions to hospital for asthma, although ozone was present in combination with particulates and acid aerosols. Increases in ozone concentration have also been associated with worsening of asthma symptoms and decreased lung function in people with asthma independent of acid aerosols and particulates.69
Studies on humans using controlled exposure have demonstrated that people with asthma are much more susceptible than those without asthma to the bronchoconstricting effects of sulfur dioxide.[70–74] However, the effects of exposure to acid aerosol and nitrogen dioxide have been contradictory.[70–74] Ozone exposure causes predictable acute decreases in vital capacity under controlled conditions, but people with asthma are not more likely than healthy subjects to experience these effects. People with asthma exposed to ozone may experience more adverse effects following exposure to allergens.75 A similar situation occurs with exposure to nitrogen dioxide.[76, 77]
Indoors, the most important respiratory irritant is environmental tobacco smoke (ETS). Asthmatic children of smoking mothers have more severe asthma than those whose mothers are nonsmokers,78 and when parents of an asthmatic child give up smoking, the child's condition improves.79 Exposure to ETS is associated with increased frequency and severity of exacerbations of asthma80 and the development of asthma in predisposed infants and young children.[81, 82] The effects of ETS exposure may occur in utero.83 In the Canadian climate, exposure to ETS represents an important risk to respiratory health.
Products of indoor combustion, such as nitrogen dioxide from gas stoves and wood smoke, may increase respiratory symptoms in people with asthma,84 but evidence for this is not conclusive. Formaldehyde and other volatile organic compounds detectable in indoor air are irritating to the eyes and the upper respiratory tract.
Preventing respiratory effects of irritants consists of reducing exposure. During periods of increased outdoor pollution, patients can minimize exposure by remaining indoors or reducing exercise outdoors. Reduction of indoor pollutants can be achieved by avoiding exposure to cigarette smoke, by ensuring adequate venting of gas stoves and ensuring that wood stoves are air tight. Pregnant and breastfeeding mothers should be encouraged to give up smoking. Smoking parents or caregivers of asthmatic children should also be encouraged to give up smoking. Various types of indoor air cleaners are available, but, although several have been shown to reduce levels of irritants significantly, health benefits have yet to be demonstrated consistently.[85, 86] Human experimental studies have shown that bronchoconstriction resulting from controlled exposure to air pollutants in people with asthma can be prevented by use of an inhaled bronchodilator. Because continued exposure to respiratory irritants following the use of an inhaled bronchodilator will allow the inflammatory effects of irritant exposure to continue, preventing or reducing exposure should be the primary management approach.
Recent studies have focused on the relationship between air pollution and airway inflammation. For example, there is a greater influx of neutrophils and eosiniophils in the nasal mucosa of atopic people whose nasal mucosa are challenged by a specific allergen in the presence of ozone than in air.[21, 87] People with asthma are also at higher risk of developing ozone-induced respiratory tract injury or inflammation characterized by increased neutrophils than people without asthma.[88, 89] In addition, ozone exposure results in increased inflammation in the lower airways of allergic people with asthma, demonstrated by an increase in both neutrophils and eosinophils.90 These results may explain the increased asthma morbidity associated with episodes of ozone pollution.
Pre-exposure to a number of air pollutants, alone or in combination, will result in increased bronchial responsiveness to specific allergen in allergic asthmatic patients. Pre-exposure to ozone has been shown to increase specific airway reactivity of asthmatic patients who are allergic to grass pollen,[75, 91] although in at least one case these results could not be reproduced.92 A similar outcome was obtained with pre-exposure to nitrogen dioxide alone[77, 93] or mixed with sulfur dioxide.[76, 94] These results may depend on the pre-exposure status of the patient with asthma, i.e., the presence of eosinophilic inflammation in the airway before exposure to the pollutant, which then enhances the inflammation with an influx of eosinophils and generation of pro-inflammatory chemokines.
There is now extensive evidence demonstrating adjuvant effects of air pollutants on the formation of specific IgE antibodies and cytokines in both animals and man. Experiments in rats showed that exposure to nitrogen oxide enhances immune responsiveness and the severity of pulmonary inflammation following antigen challenge.95 This adjuvant effect of air pollution has been particularly well documented with diesel exhaust particle emissions, which have been shown to enhance specific IgE antibody production, increase cytokine production and increase the gene expression of Th2 cytokines.96 Several reports[97–102] have documented enhanced production of specific IgE antibody and cytokines in cultures of lymphoid cells from mice or rats pretreated with diesel exhaust particles, and in vivo animal studies[103–105] have demonstrated increased IgE-specific antibody production after intranasal pretreatment with diesel exhaust particles. These studies were extended to demonstrate that intratracheal immunization with antigen in the presence of diesel exhaust particles enhanced local IgE antibody production and also increased infiltration of eosinophils and the production of Th2 cytokines locally in the lungs compared with either antigen or diesel exhaust particles alone.[106–108] These results mimic the nature of inflammation in allergic asthma.
Saxon and collaborators[109–113] have demonstrated the relevance of the animal results to the problem in humans. In vitro studies[110, 111] showed that diesel exhaust particles enhance IgE production by tonsillar B-cells in the presence of interleukin-4 (IL-4) and CD40 monoclonal antibody and alter the nature of the IgE produced. In vivo, 0.30 mg diesel exhaust particles in saline enhanced IgE production in the human upper respiratory mucosa; the particles had no effect on IgG, IgA, IgM, or albumin, although there was a small increase in IgG4.109 Diesel exhaust was also shown to act as an adjuvant to ragweed allergen.112 Nasal challenge with diesel exhaust particles also influences cytokine production: allergen plus diesel exhaust particles caused a significant increase in the expression of mRNA for Th2 cytokines (IL-4, IL-5, IL-6, IL-10, IL-13) with an inhibitory effect on IFN-gamma gene expression.113
The inflammatory and immunologic adjuvant effects of other forms of particulate pollution have not been examined extensively, although 2 studies have demonstrated inflammatory effects of fuel oil ash inhalation in animals.[114, 115]
These various studies strongly suggest that air pollution can modulate or enhance airway inflammation associated with allergic and asthmatic diseases; however, no studies have demonstrated the effect of medications used to treat asthma. Management of the adverse effects of respiratory irritants on people with asthma consists primarily of preventing or reducing exposure. Exposure to outdoor pollutants may be reduced by remaining indoors, minimizing outdoor physical activity and breathing through the nose exposure. Reduction in indoor exposure can be achieved by avoiding cigarette smoke, assuring adequate venting of gas stoves and ensuring that wood stoves are air tight. Although some air cleaners can remove both particulate and gaseous indoor airborne pollutants, their effectiveness in preventing adverse effects in people with asthma is not known. Finally, although bronchoconstriction resulting from controlled exposure to air pollutants in people with asthma can be averted by the use of inhaled bronchodilators, this is unlikely to prevent the inflammatory effects of the pollution and may aggravate them by masking symptoms. Preventing or reducing exposure should be the primary management approach.
Recommendations
-
Increasing medication for asthma control should not be used as a substitute for avoidance of exposure to allergens and irritants (level III).
-
Exposure to environmental tobacco smoke should be avoided (level III).
-
Pregnant women and parents or caregivers of children with asthma should be particularly encouraged not to smoke (level II).
-
There is insufficient information to recommend the use of residential air cleaners and humidifiers (level III).
-
High concentrations of respiratory irritants should be avoided (level III).
-
Occupational asthma should be suspected and investigated in all adults with new-onset asthma (level II).
-
Once the diagnosis of occupational asthma has been confirmed, the patient should be removed from exposure to the causative substance (level III).
-
In industries associated with a high risk of occupational asthma, the level of exposure in the workplace should be reduced and regularly monitored (level IV).
References
- 1.↵
- 2.↵
- 3.↵
- 4.↵
- 5.↵
- 6.↵
- 7.↵
- 8.↵
- 9.↵
- 10.
- 11.↵
- 12.↵
- 13.↵
- 14.↵
- 15.↵
- 16.↵
- 17.↵
- 18.↵
- 19.↵
- 20.↵
- 21.↵
- 22.↵
- 23.↵
- 24.↵
- 25.↵
- 26.↵
- 27.↵
- 28.↵
- 29.↵
- 30.↵
- 31.↵
- 32.↵
- 33.↵
- 34.
- 35.↵
- 36.↵
- 37.
- 38.
- 39.↵
- 40.↵
- 41.↵
- 42.↵
- 43.↵
- 44.↵
- 45.↵
- 46.↵
- 47.↵
- 48.↵
- 49.↵
- 50.↵
- 51.↵
- 52.↵
- 53.↵
- 54.↵
- 55.↵
- 56.↵
- 57.
- 58.↵
- 59.↵
- 60.
- 61.
- 62.
- 63.
- 64.
- 65.↵
- 66.↵
- 67.↵
- 68.↵
- 69.↵
- 70.↵
- 71.
- 72.
- 73.
- 74.↵
- 75.↵
- 76.↵
- 77.↵
- 78.↵
- 79.↵
- 80.↵
- 81.↵
- 82.↵
- 83.↵
- 84.↵
- 85.↵
- 86.↵
- 87.↵
- 88.↵
- 89.↵
- 90.↵
- 91.↵
- 92.↵
- 93.↵
- 94.↵
- 95.↵
- 96.↵
- 97.↵
- 98.
- 99.
- 100.
- 101.
- 102.↵
- 103.↵
- 104.
- 105.↵
- 106.↵
- 107.
- 108.↵
- 109.↵
- 110.↵
- 111.↵
- 112.↵
- 113.↵
- 114.↵
- 115.↵