Acute Asthma in Children

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					Acute Asthma in Children
Relationships among CD14 and CC16 Genotypes, Plasma Levels, and Severity
Andrew C. Martin, Ingrid A. Laing, Siew-Kim Khoo, Guicheng Zhang, Kristina Rueter, Laurel Teoh,
Shahir Taheri†, Catherine M. Hayden, Gary C. Geelhoed, Jack Goldblatt, and Peter N. LeSouef
School of Paediatrics and Child Health, University of Western Australia, Perth, Western Australia, Australia

Rationale: The majority of previous studies investigating asthma                          extensively studied in adults and children (9–12). Differences in
genetics have focused on cohorts with stable disease and have not                         gene expression have been identified among individuals with
defined mechanisms important during acute asthma. CD14 and                                stable asthma, yet dysregulation of pro- or antiinflammatory
CC16 each play a key role in biologically important inflammatory                          processes is likely to have the most critical influence during the
pathways and the gene of each has a functional promoter-region                            early stages of an acute asthma attack, when the loss of control
polymorphism.                                                                             of inflammation could be expected to be maximal. CD14 is a
Objectives: This study was designed to determine the influence of                         key component of the innate immune system and its gene is
these polymorphisms on plasma levels of their products and clinical                       located on chromosome 5q31.1. It is expressed on monocytes
disease during acute asthma. We hypothesized that genotype-
                                                                                          and macrophages, functions as a receptor for LPS (13), and
related differences in CD14 and CC16 production would be more
                                                                                          exists in a membrane-bound form and a soluble form (sCD14).
marked during acute asthma and related to disease severity.
                                                                                          CD14 plays a critical role in determining the balance of Th1:Th2
Methods: We studied 148 children on presentation with acute
asthma and again in convalescence. CD14 C-159T and CC16 A38G                              cytokines, with activation promoting the release of interleukin
genotypes were determined, and plasma levels of soluble CD14                              12 (IL-12) and deviation of immune responses toward an antivi-
(sCD14) and CC16 were measured at both times.                                             ral T-helper type 1 (Th1) response (14). Levels of sCD14 have
Measurements and Main Results: During acute asthma, plasma sCD14                          been noted to be higher in individuals with asthma than in those
levels were higher for the whole group (p      0.003), but increases                      without asthma and also higher during acute asthma exacerba-
were only in subjects with CD14 159TT (p         0.003) and 159CT                         tions as compared with convalescence (15). A promoter poly-
(p 0.004), and not in those with 159CC. Plasma CC16 levels were                           morphism (C-159T) was identified, and the 159C allele associ-
also elevated acutely for the whole group (p      0.013), but only in                     ated with lower levels of sCD14 in an unselected population of
those with CC16 38GG (p 0.043) and 38AG (p 0.014), and not                                children (11), but the role of this polymorphism during acute
in those with CC16 38AA. Subjects with CD14 159CC and CC16                                asthma is unknown. Clara cell secretory protein (CC16) is pro-
38AA were more likely to have moderate or severe acute asthma.                            duced by bronchiolar, nonciliated epithelial cells (5) and is the
Conclusions: Plasma levels of sCD14 and CC16 were higher during acute                     most abundant protein secreted into the airway (16), functioning
asthma in the subjects. Those with CD14 159CC and CC16 38AA                               as an immunosuppressive and antiinflammatory agent (5, 17).
had no change in sCD14 and CC16 levels and more severe asthma.                            Although CC16 is produced in the airway, serum levels mirror
                                                                                          those in the lung and individuals with asthma have lower circulat-
Keywords: asthma; children; single nucleotide polymorphism
                                                                                          ing levels than those without asthma (18). The gene for CC16
Acute asthma is the most common diagnosis in children admitted                            is located on chromosome 11q13 and has a functional promoter-
to hospitals in Western society and is characterized by acute                             region polymorphism (A38G) (8, 9). The 38G allele was associ-
episodes of obstruction related to loss of control of airway in-                          ated with a decreased risk of developing childhood asthma (9)
flammation mostly in response to a viral respiratory-tract infec-                          and increased gene expression levels in vitro, but the role of this
tion (1). Many genetic variations associated with asthma and                              allele in acute asthma is unknown.
asthma phenotypes have been reported (2–4). However, these                                    Given the rapid onset of inflammation in an acute asthma
investigations have focused on asthma phenotypes in cohorts                               attack, we hypothesized that genotype-related differences in pro-
with stable disease and no studies have systematically evaluated                          duction of CD14 and CC16 would be more marked during acute
the influence of genetic differences in acute asthma.                                      asthma than convalescence and that these differences would be
   CD14 and CC16 are biologically important in clearly defined                             associated with altered phenotypes.
immunologic and inflammatory pathways (5, 6). Single nucleo-                                   Some of the results of this study have been previously re-
tide polymorphisms (SNPs) in the promoter regions of their                                ported in the form of abstracts (19, 20).
genes alter the amount of expressed protein (7, 8) and have been
(Received in original form September 3, 2005; accepted in final form December 27, 2005)   A total of 148 children, aged 2 to 16 yr, presenting with acute asthma to
                                                                                          the Emergency Department at Princess Margaret Hospital for Children,
Supported by grants from the National Health and Medical Research Council of              Perth, Western Australia, were recruited between July 2002 and
Australia, Asthma Foundation of Western Australia, and the West Australian Insti-
                                                                                          September 2004. An acute asthma attack was diagnosed by the emer-
tute of Medical Research.
                                                                                          gency department physician based on the presence of wheezing with
    Dr. Shahir Taheri is deceased.                                                        increased difficulty of breathing. Parents of all participants gave
Correspondence and requests for reprints should be addressed to Andrew Martin,            informed consent and the Princess Margaret Hospital Ethics Committee
M.B.B.S., M.R.C.P., Princess Margaret Hospital for Children, Roberts Road, Subiaco,       approved the study.
Western Australia 6008, Australia. E-mail:                     The severity of the acute asthma attack at presentation was deter-
This article has an online supplement, which is accessible from this issue’s table        mined using a previously validated scoring system with a possible score
of contents at                                                        in the range 5 to 15 determined for each subject: 5 to 7, mild; 8 to 11,
Am J Respir Crit Care Med Vol 173. pp 617–622, 2006
                                                                                          moderate; 12 to 15, severe (21). Additional details of the scoring system
Originally Published in Press as DOI: 10.1164/rccm.200509-1367OC on December 30, 2005     are provided in the online supplement. All children received inhaled
Internet address:                                                     salbutamol and ipratropium bromide at 20-min intervals for the first
618                                                          AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 173 2006

hour, prednisolone 1 mg/kg orally, and if saturations were less than or              The C allele frequency for CD14 C-159T was 45.3% and A
equal to 94%, supplemental oxygen was administered. The pattern                  allele frequency for CC16 A38G was 31.9%, both similar to
of asthma severity was also assessed according to National Asthma                frequencies from an unselected population in the same city (25,
Council of Australia guidelines to determine whether children suffered
from infrequent episodic, frequent episodic, or persistent asthma
                                                                                 26). The genotype distributions did not deviate from Hardy-
exacerbations (22).                                                              Weinberg equilibrium. Table 2 shows the genotype frequencies.

Samples                                                                          sCD14 and CC16: Acute versus Convalescent Plasma Levels
Peripheral blood samples were obtained as soon as possible after the             Paired acute and convalescent plasma levels were available for 94
first dose of prednisolone and always within 24 h of presentation to              children who were representative of the cohort. During the acute
the hospital. A further blood sample was obtained from those who                 period, GM levels of both sCD14 and CC16 were higher than during
were clinically well and able to return at least 6 wk after the acute            convalescence: CD14, 3.03 g/ml (95% CI, 2.80–3.28 g/ml)
attack. Buffy coat and plasma were separated and stored at 80 C. At
                                                                                 versus 2.61 g/ml (95% CI, 2.42–2.82 g/ml), p 0.003; CC16,
presentation, a per-nasal aspirate was obtained for detection of common
viral respiratory pathogens and a skin-prick test for 11 common aller-           2.23 ng/ml (95% CI, 1.89–2.64 ng/ml) versus 1.78 ng/ml (95%
gens was done. A positive reaction was defined as a wheal size that               CI, 1.48–2.14 ng/ml), p     0.013 (Figure 1).
was larger than the negative control and greater than or equal to 3 mm
in diameter. Atopy was defined as at least one positive skin-prick test.          sCD14 and CC16 versus the Corresponding Genotypes
                                                                                 During the acute episodes of asthma (n          148), plasma levels
                                                                                 of sCD14 and CC16 were significantly related to genotype for
Genomic DNA was extracted by standard techniques (23) and the CD14               CD14 C-159T and CC16 A38G, respectively. Mean plasma
C-159T and CC16 A38G genotypes were determined by restriction
                                                                                 sCD14 levels were highest in subjects with CD14 159TT (GM,
digestion of polymerase chain reaction products using the restriction
enzymes AvaII and Sau96I (Promega, Madison, WI), respectively (9, 11).           3.6 g/ml; 95% CI, 3.3–4.0 g/ml), followed by heterozygotes
                                                                                 (GM, 3.2 g/ml; 95% CI, 2.9–3.5 g/ml), and lowest for those
Plasma Assays                                                                    with CD14 159CC (GM, 2.5 g/ml; 95% CI, 2.1–2.9 g/ml),
Plasma levels of sCD14 and CC16 were determined using commercially               p 0.001. When levels during the acute attacks were compared
available ELISA kits (R&D Systems, Minneapolis, MN; and Bio-                     with levels during convalescence (n 94), higher plasma levels
Vendor, Brno, Czech Republic; respectively).                                     of sCD14 were present during the acute attack in those with
                                                                                   159CT and 159TT (p 0.004 and p               0.003, respectively),
Statistical Analysis
                                                                                 but no difference was present in those with CD14 159CC
Because plasma levels of sCD14 and CC16 were both positively skewed,             (Figure 2). Similarly, during the acute attack, mean plasma CC16
geometric means (GM) and 95% confidence intervals (CI) were calcu-                level was highest in those with 38GG (GM, 2.4 ng/ml; 95% CI,
lated after applying a logarithmic transformation that resulted in a
normal distribution. As the distribution of asthma severity scores was
                                                                                 2.0–2.9), intermediate in heterozygotes (GM, 2.0 ng/ml; 95% CI,
approximately normal, parametric statistics were employed. The differ-           1.7–2.5), and lowest in those with 38AA (GM, 1.2 ng/ml; 95%
ences in plasma levels of sCD14 and CC16 between acute asthma and                CI, 0.8–1.8 ng/ml), p 0.025. When levels during acute attacks
convalescence were compared by paired-sample t tests. Analysis of                were compared with levels during convalescence (n 94), higher
variance was used to compare the difference in plasma levels of sCD14            plasma levels of CC16 were present during the acute attack in
and CC16 and asthma severity scores between the genotypes, with a                those with 38GG and 38AG (p 0.014 and p 0.043, respec-
polynomial linear analysis for trend. Bivariate Pearson correlation was
employed to explore the association between asthma severity scores
                                                                                 tively), whereas in 38AA homozygotes, CC16 levels were actu-
and plasma sCD14 and CC16. To estimate the odds ratios (OR) for                  ally lower during the acute attacks than during convalescence,
moderate or severe acute asthma attacks in children with CD14 -159CC             although this difference was not statistically significant (p
and CC16 38AA, logistic regression models were applied. All statistics           0.088; Figure 3). For CD14 C-159T and CC16 A38G, there was
were analyzed using SPSS (SPSS for Windows, Release 11; SPSS,                    a significant linear effect for each additional C or A allele, respec-
Chicago, IL) (24).                                                               tively, on plasma sCD14 and CC16 levels (p           0.001 and p
                                                                                 0.007, respectively). During convalescence, no significant differ-
RESULTS                                                                          ence in plasma levels of sCD14 and CC16 was found between
Characteristics of the children recruited for the study are shown                the genotype groups for the CD14 C-159T and CC16 A38G
in Table 1.                                                                      polymorphisms. We also explored the impact of viral infection
                                                                                 and atopy on plasma sCD14 and CC16 levels and found that
                                                                                 neither had a significant effect. (Comparing subjects with and
TABLE 1. CHARACTERISTICS OF THE STUDY POPULATION                                 without viral infection, p      0.89 and p     0.59 for sCD14 and
                                                                                 CC16 levels, respectively; and for atopy, p 0.58 and p 0.80,
Characteristic                                              Subjects (n   148)   respectively.)
Mean age, yr SD                                                  6.6 3.6
Males/females                                                     89/59          Asthma Severity in Relation to CD14 C-159T and CC16 A38G
Ethnicity, % white                                                 92.3          Genotypes and Plasma Levels
Preceding symptoms of URTI, n (%)                               120 (81.1)
                                                                                 In the 148 children assessed during acute attacks, children with
Virus isolated, n* (%)                                           89 (75.4)
Oxygen saturation on arrival, % SD                              93.8 3.6         the CD14 159CC and CC16 38AA genotypes had higher mean
Mean asthma severity score SD                                    9.5 2.4         asthma severity scores compared with the other genotypes
Mild asthma, n (%)                                               32 (21.6)
Moderate asthma, n (%)                                           86 (58.1)
Severe asthma, n (%)                                             30 (20.3)
Infrequent episodic asthma, n (%)                                98 (66.2)       TABLE 2. CD14 AND CC16 GENOTYPE FREQUENCIES
Frequent episodic asthma, n (%)                                  23 (15.5)
Persistent asthma, n (%)                                         27 (18.3)       CD14 C-159T              CC                 CT                 TT
Atopy, n* (%)                                                    94 (79.3)        n, %                 36 (24.3)          62 (41.9)          50 (33.8)
                                                                                 CC16 A38G                AA                 AG                 GG
  Definition of abbreviation: URTI upper respiratory tract infection.
                                                                                  n, %                 13 (8.8)           68 (45.9)          67 (45.3)
  * Test completed in only 118 subjects.
Martin, Laing, Zhang, et al.: Genetics and Acute Asthma in Children                                                                  619

                                                                                  Figure 1. Paired plasma levels of soluble CD14 (sCD14)
                                                                                  and CC16: acute versus convalescence (n     94).

(CD14 C-159T, CC 10.1 vs. CT 9.0 vs. TT 9.7, p           0.06; and    clude a larger panel of genes, examining both individual SNPs
CC16 A38G, AA 9.9 vs. AG 9.6 vs. GG 9.3, p 0.76). Logistic            and haplotype patterns.
regression analysis was undertaken to further investigate the             As previously noted, CD14 is known to play an important
association between asthma severity and these genotypes. After        role in the immune system and plasma levels were increased
adjustment for age and sex, children with CD14 159CC were             with acute asthma. However, the precise physiologic role of
more likely to have an asthma severity score indicating a moder-      sCD14 is still unclear, as it may be beneficial as a scavenger to
ate or severe attack (odds ratio [OR], 3.7; 95% CI, 1.04–13.2;        neutralize circulating LPS, or its absence on endothelial cells
p     0.043) compared with those with 159CT and 159TT.                that do not express membrane-bound CD14 may allow LPS to
An inverse correlation (coefficient, 0.17; p 0.05) was found           produce an aggressive and harmful proinflammatory cytokine
between asthma severity scores and the log value of sCD14             response. Increased serum levels of sCD14 have been reported
during acute asthma attacks. Subjects with CC16 38AA also             in severe acute systemic conditions, such as gram-negative septi-
tended to have an asthma severity score in the moderate or            cemia, polytrauma, burns, and acute respiratory distress syn-
severe range, but the difference from those with 38AG and             drome and have been associated with increased morbidity and
38GG were not significant (OR, 3.7; 95% CI, 0.45–30; p 0.231).         mortality (27–29). Studies of IC14, a CD14-specific monoclonal
This same pattern for both CD14 and CC16 was found when               antibody, showed the potential of this treatment to limit the
only the subjects with paired samples (n 94) were analyzed.           excessive systemic inflammatory response in subjects with severe
   We also investigated the possibility of an additive or syner-      sepsis (30). Consistent with these reports, the current study found
gistic effect of the CD14 159CC and CC16 38AA genotypes               that during acute asthma attacks in children, plasma sCD14
on the severity of an acute asthma attack, but found no significant    levels were inversely correlated with severity, suggesting a pro-
gene–gene interaction.                                                tective role for sCD14. Plasma sCD14 levels have been directly
                                                                      correlated with IFN- and inversely correlated with IL-4 (11),
DISCUSSION                                                            suggesting that in children with acute asthma, the beneficial
The present study is the first to our knowledge to use genetics        effect of greater CD14 activity may result from increased Th1
to investigate the mechanisms of asthma in children during acute      and decreased Th2 responses. A relative predominance of Th1
exacerbation. This approach clearly has the potential to elucidate    over Th2 cytokines assists in the elimination of viral infections
mechanisms underlying acute asthma, as transcription of genes         (31), thus lower levels of sCD14 may allow ongoing viral replica-
involved in asthmatic inflammation is likely to be maximal during      tion and inflammation.
the acute exacerbation, and therefore, allele-specific differences         Acute asthma attacks are associated with a marked increase
should be most evident.                                               in airway inflammation and this was reflected by the finding of
    In the present study, we have investigated two key agents         mean CC16 plasma levels being 95.9% higher during the acute
involved in inflammation and demonstrated clear patterns relat-        attacks compared with convalescence. Plasma levels of CC16
ing their genotypes to both asthma severity and plasma levels.        are reported to reflect CC16 levels in bronchoalveolar lavage in
These patterns were present during the acute attacks and not          healthy adults (16). This study is the first to demonstrate altered
detectable on recovery. Specifically, we found that levels of both     circulating CC16 levels in subjects during an acute attack. The
CD14 and CC16 were increased during the acute episode, but            only other study comparing acute and convalescent plasma CC16
that the increases were seen with one but not the other allele        levels did not find a difference, but compared paired plasma
of each of the polymorphisms. These findings were plausible            CC16 levels in only 10 adults with asthma (18).
and linked to clinical status, as, for both CD14 and CC16, the            This study demonstrated that genotype-specific differences
allele for which there was no increase in plasma level was associ-    in plasma levels of sCD14 and CC16 during acute exacerbation
ated with more severe acute asthma. Because these findings             were even more pronounced than during convalescence. For
could not be made studying individuals with stable asthma, they       CD14, subjects with 159TT and 159CT genotypes had 42
make an important contribution to current knowledge of the            and 31% higher plasma sCD14 levels, respectively, compared
mechanisms of asthma. The two genes we studied were specifi-           with 10% lower levels in those with 159CC. The finding that
cally selected because of their involvement in inflammatory path-      during acute asthma 159TT subjects had the highest sCD14
ways, but studies on similar cohorts should be extended to in-        levels, heterozygotes intermediate, and 159CC the lowest
620                                                AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 173 2006

                                                                                              Figure 2. Relationship between plasma lev-
                                                                                              els of sCD14 and genotypes of CD14 C-159T.

levels was consistent in direction with those from a normal popu-     greatly exceeds production during acute asthma. Subjects with
lation of American school children (11). However, in contrast to      asthma with the 38A allele may be unable to increase levels of
the study by Baldini and colleagues, which suggested a dominant       CC16 in the airway at the time of viral infections when protection
model, we found a linear effect, with each additional T allele        from excessive airway inflammation becomes most critical. This
having an additive effect on plasma sCD14 levels. Interestingly,      may result in 38AA subjects experiencing more severe acute
this linear effect was only present during the acute asthma exac-     asthma due to lower levels of this endogenous antiinflammatory
erbation, suggesting that gene expression at this time is likely      agent.
to be exaggerated. Levels of sCD14 were similar for the different        Those subjects who were either CD14 159CC or CC16
genotypes in convalescence, further demonstrating that this           38AA were over three times more likely to have moderate or
pathogenetic process could only have been identified by studying       severe attacks of acute asthma compared with the other geno-
children during acute attacks. Our findings were also consistent       types. A potential mechanism in determining severity of an acute
with functional studies that showed that the T allele produced        attack is impaired ability to increase sCD14 and CC16. The
32% greater activity than the C allele (7).                           effect of CD14 C-159T on asthma severity was partly dependent
    For CC16, subjects with 38GG and 38AG genotypes had               on plasma sCD14 levels, with higher levels associated with a
134% and 80% higher plasma CC16 levels, respectively, com-            milder asthma attack, possibly because of increased Th1 cytokine
pared with 38AA subjects, who paradoxically had plasma CC16           release. Although differences for CC16 did not reach statistical
levels that were 33% lower. These genotype-specific differences        significance, mildly affected subjects tended to have the highest
in plasma levels were also consistent in direction with the findings   CC16 levels and severely affected the lowest. However, as CC16
from a case control study of Australian children (25). However,       levels in plasma are approximately 10,000 times lower than in
in addition to having the lowest levels of CC16 during an acute       bronchoalveolar lavage (32), any changes seen in the plasma are
asthma attack, subjects with 38AA appeared to have had a de-          likely to be substantially more pronounced in the airway.
crease in plasma CC16 levels when the degree of airway inflam-            Although this study population was relatively large compared
mation was maximal. This difference may reflect decreased gene         with other studies of children with acute asthma, the number of
expression by the A allele or that the consumption of CC16            subjects with some genotypes, such as CC16 38AA, was relatively
Martin, Laing, Zhang, et al.: Genetics and Acute Asthma in Children                                                                 621

                                                                                              Figure 3. Relationship between plasma lev-
                                                                                              els of CC16 and genotypes of CC16 A38G.

small compared with other genetic association studies. However,       effect on the polymorphisms studied. Furthermore, the highly
this study differed from previous studies in that the stimulation     significant findings of genotype-specific variation in acute plasma
of inflammatory pathways was likely to have been near maximal          levels of sCD14 and CC16 suggested that, even if steroids influ-
and, for the genes assessed, the genes’ own product was an            enced the absolute level of gene product, genotype differences
important primary outcome variable. Also, genotype-specific            still play a major role in determining altered levels of gene
plasma levels of sCD14 and CC16 were consistent with previous         product between individuals and asthma severity. Ethnicity is
studies, as well as being internally consistent between the geno-     known to influence the genotype frequency for both CD14
types, and these consistencies strongly support the validity of       C-159T and CC16 A38G (35). However, as more than 90% of
the findings. Thus, the striking and consistent associations found,    our population were white Australians, of families originally
despite the relatively small numbers of subjects, vindicate this      from Europe, the low frequency of other ethnic origins meant
novel approach of studying genetic associations at a time when        that we were unable to investigate the effect of ethnicity in this
the loss of control of inflammation could be expected to be            study. In studying genetic associations, consideration must be
maximal. Such an approach has the potential to allow the identi-      also given to the presence of multiple SNPs per gene and to
fication of important genetic associations in relatively small         functional haplotypes. In the present study, only one SNP was
cohorts.                                                              examined for each gene, but CD14 C-159T and CC16 A38G are
    Other potential limitations should be considered. Although        considered the most important CD14 and CC16 SNPs, as they
children were studied early in the course of the acute attack, in     are the only ones shown to have functional significance.
all cases blood samples were obtained after the first dose of              In summary, this study of children during acute asthma at-
prednisolone. Exogenous steroids can promote down-regulation          tacks identified significant relationships between two common
of sCD14 and up-regulation of CC16 (33, 34), but there is no          promoter SNPs in the CD14 and CC16 genes and genotype-
evidence to suggest that steroids would have an allele-specific        specific differences in their gene products’ plasma levels, which
622                                                            AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 173 2006

in turn reflected the severity of the acute asthma attacks. These                     15. Garty BZ, Monselise Y, Nitzan M. Soluble CD14 in children with status
results suggest that in acute asthma production of both CD14                               asthmaticus. Isr Med Assoc J 2000;2:104–107.
                                                                                     16. Bernard A, Marchandise FX, Depelchin S, Lauwerys R, Sibille Y. Clara
and CC16 is increased in an attempt to control airway inflamma-                             cell protein in serum and bronchoalveolar lavage. Eur Respir J 1992;
tion, and for subjects whose genotype limits or prevents these                             5:1231–1238.
increases, the ability to control airway inflammation is impaired,                    17. Chen LC, Zhang Z, Myers AC, Huang SK. Cutting edge: altered pulmo-
resulting in more severe asthma. Studies of this nature have the                           nary eosinophilic inflammation in mice deficient for Clara cell secre-
potential to provide new insight into mechanisms that control                              tory 10-kDa protein. J Immunol 2001;167:3025–3028.
                                                                                     18. Shijubo N, Itoh Y, Yamaguchi T, Sugaya F, Hirasawa M, Yamada T,
airway inflammation, and to facilitate the development of indi-                             Kawai T, Abe S. Serum levels of Clara cell 10-kDa protein are de-
vidually tailored treatments.                                                              creased in patients with asthma. Lung 1999;177:45–52.
                                                                                     19. Martin AC, Taheri S, Rueter K, Khoo S, Zhang G, Laing IA, Goldblatt
Conflict of Interest Statement : None of the authors have a financial relationship
                                                                                           J, Le Souef PN. Relationship between CD14C–159T and soluble CD14
with a commercial entity that has an interest in the subject of this manuscript.
                                                                                           levels in acute asthma. Am J Respir Crit Care Med 2004;169:A584.
Acknowledgment : The authors thank the children and families who participated              (abstract).
in this study and acknowledge the technical assistance of Jenny Tizard.              20. Martin AC, Laing IA, Taheri S, Teoh L, Rueter K, Zhang G, Khoo S,
                                                                                           Hayden CM, Goldblatt J, Le Souef PN. Relationship between CC16
                                                                                           A38G, plasma CC16 levels and severity of acute asthma exacerbations
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