2006 AAAAI/CIS Hypersensitivity/Allergic Diseases School
Diogenes Ferreira, M.D. Department of Pathology University of Sao Paulo Medical School, Brazil
Objectives
1. Demographic and clinical characterization of 73 autopsy-proven fatal asthmatics 2. Pilot study - Compare the gene expression of myosin light chain kinase (contractile phenotype) and connective tissue growth factor (synthetic phenotype) in airway smooth muscle cells of fatal asthma patients
Characterization of 73 autopsyproven cases of fatal asthma in Sao Paulo, Brazil
Introduction
• Asthma accounts for 1 in every 250 deaths worldwide (GINA 2004) • In Brazil the prevalence of asthma is high (11.4%) • The overall mortality rate per 100,000 inhabitants is 2.04 (2000 deaths/year) • Many of these deaths are probably preventable, but there is little information about fatal asthma patients in Brazil
Methods
• 73 autopsy-proven fatal asthma patients (1996-2004) • Autopsies performed at the Death Verification Service, University of Sao Paulo • All patients had a history of asthma and died during an acute exacerbation • Macro and microscopic examination confirmed the diagnosis of fatal asthma
Methods
• A questionnaire to investigate socioeconomic factors and the history and treatment of asthma was applied to the next of kin after written informed consent • Information was obtained from medical institutions when available
Macro and microscopic examination
Dep. Patologia-USP
Simões SM et al., 2005
Demographics
Age group (yr) Number
0-5
5-34
34-70
Total
2 (2.7%)
15 (20.6%)
56 (76.7%)
73 (100%)
Gender Race
F: 42 (57.5%) M: 31 (42.5%) White: 46 (63%) African: 24 (32.9%) Asian: 3 (4.1%) Current smoker: 22 (30.1%) Non-smoker: 37 (50.7%) Ex-smoker: 14 (19.2%) Illiterate: 11 (16.7%) <8yr : 40 (60.6%) >8yr : 15 (22.7%) NA = 5 1.6 (0.26 - 8.86) 23.5 kg/m2
Smoking status Education
Per capita income (times minimum wage) BMI
Asthma characterization
• Age at onset of asthma <12 yrs → 33/68 (48.5%) >12 yrs → 35/68 (51.5%) Parental history of asthma 16/44 (36.4%) Rhinitis 28/73 (38.4%)
• •
Treatment of asthma
• 25 (34.2%) patients regularly followed by a doctor • 12 (16.4%) seen by a respiratory specialist MEDICATIONS 9 (12.3%) inhaled steroids 20 (27.4%) oral steroids 35 (47.9%) methylxanthines 30 (41.1%) no controllers 71 (97.3%) short-acting bronchodilators
• • • • •
Estimation of severity
• 35 (47.9%) patients:
last year - previous ICU admission - FEV1<60% or - high dose ICS or OS
- hospitalized
Fatal crisis
• Duration of symptoms before death < 2h → 11/66 (16.7%) 2h-1day → 17/66 (25.7%) >1day → 38/66 (57.6%) • 55 (75.3%) patients died outside a hospital
Conclusions
These 73 autopsy-proven fatal asthmatics are characterized by: • • • • 47.9% moderate-severe asthma Low socio-economic level Lack of regular medical care Lack of steroid use
Contractile and synthetic phenotype gene expression of airway smooth muscle in fatal asthma
Introduction
• Airway smooth muscle (ASM) is the main effector of AHR and also secretes inflammatory mediators and ECM proteins • Phenotypic plasticity of ASM cells in vitro: contractile and synthetic phenotype (Halayko et al., 1996; Ma et al., 1998)
Myosin light chain kinase (MLCK)
• MLCK is a key regulatory enzyme of ASM contraction • Ma et al. (2002) – correlated ↑ contractile properties of asthmatic ASM cells with ↑ expression of MLCK mRNA
• Benayoun et al. (2003) – ↑ expression of MLCK in severe asthma (IHC) • Woodruff et al. (2004) – no difference in MLCK mRNA in ASM from mildmoderate asthmatics and controls
Connective tissue growth factor (CTGF)
• Overexpression of CTGF linked to various fibrotic diseases (Grotendorst, 1997) • CTGF may represent a downstream effector molecule of the profibrotic activities of TGF-β (Leask et al., 2002)
• Burgess et al. (2003) – ASM cells from asthmatic individuals release CTGF in ↑ amount following TGF-β stimulation compared with controls • Johnson et al. (2006) – asthmatic and non-asthmatic ASM cells produce fibronectin and collagen I in response to CTGF
Hypothesis
ASM cells of fatal asthma patients show altered expression of both contractile (MLCK) and synthetic (CTGF) phenotype genes
Methods
• 20 fatal asthma patients • 10 patients that have died due to nonpulmonary causes and without any lung disease (controls)
5 μm sections of frozen lung tissue on membrane-coated slides, H&E stained 10 fields containing ASM in each slide obtained by laser microdissection (PALM Microbeam IP Z System, Carl Zeiss)
Joubert and Hamid, 2005
Catapulted fragments kept into an inverted microcentrifuge tube cap Total RNA extraction with the RNeasy Micro Kit (Qiagen) Quantification of the genes MLCK and CTGF by real-time RTPCR
• Measurement of airway area, basement membrane thickness and ASM area • The expression of MLCK and CTGF will be compared between the fatal asthma group and control group and among the fatal asthma individuals • Expression of the genes will be correlated with airway wall area, basement membrane thickness and ASM area
Acknowledgements
• Thomas A.E. Platts-Mills, MD PhD (AAAAI) • Alkis Togias, MD (CIS) • James Gern, MD (Summer School tutor) • Thais Mauad, MD PhD (PhD mentor)