Tag Archives: von Mutius

Schutz vor Allergie auf dem Bauernhof: Ist das zentrale Paradigma falsch?

Keine Frage, die Lebensbedingungen auf Bauernhöfen sind anders. Mehr Tiere, mehr Dreck, mehr frische Luft, vieles ist anders als in derr Großstadt. Dass es hier auch weniger Allergien gibt, wird wohl auf Selektionsbedingungen zurückzuführen sein, und ist gut zu sehen bereits an einer der ersten Studien vor 30 Jahren.

Clin Exp Allergy. 1999 Jan;29(1):28-34. https://doi.org/10.1016/S0140-6736(01)06252-3 Hier nicht relevante Daten ausgegraut

Auch die Eltern hatten hierweniger Allergien. Der “protektive” Effekt kann also einfach dadurch erklärt werden, dass mit weniger Eltern als Risikoträger auch weniger Kinder mit Allergien haben (nennt sich popoulation attributable fraction oder PAF).

Nehmen wir an, alle grau markierten Personen haben keine Allergie, nur die rot markierten. Dunkel sind alle Kinder mit Risikofaktor Genetik markiert, hell alle ohne. Das genetische Risiko ist gleich hoch, aber auf Bauernhöfen hat nur die Hälfte der Kinder eine Allergie. Man kann für den nicht existierenden Risikofaktor auch nicht bereinigen, wenn man eine Studie nur auf Bauernhöfen bzw der Landbevölkerung macht.

Wenn man genau hinschaut, dann haben alle Bauernhofstudien immer wieder dieselbe Argumentationsstruktur: weil die Bedingung X dort so ist, dann kann die Folge Y auch auf die Bedingung X zurückgeführt werden. Allerdings machen immer mehr Beschreibungen von X die Story nicht glaubwürdiger.  Keine der jemals beschriebenen Bedingungen X, ist aus der Bauernhofsituation auf eine allgemeine Situation übertragbar gewesen, von einem einzigen verunglückten Versuch einmal abgesehen.

Natürlich kann ein hoher Endotoxin Spiegel auf den Bauernhöfen eine bestimmte Wirkung haben –  zumindest bei einigen Menschen und bei einigen Mäusen – aber ist das nicht nur ein Effekt modifizierender Faktor?

Da Lebensbedingungen auf dem Bauernhof angeblich protektiv sind, müsste es eigentlich auch ein Kind geben, das eine Allergie haben müssten (zB mit doppelter Familienanamnese über die letzten zwei Generation) aber nun doch keine Allergien bekommen hat. Aber solche Kinder gib es nicht, weil auch schon die Eltern keine Allergien hatten.

Farming and allergy prevention could be caused by Berkson’s fallacy

Lets look at the Wikipedia definition first

Berkson’s fallacy is a result in conditional probability and statistics which is often found to be counterintuitive, and hence a veridical paradox. It is a complicating factor arising in statistical tests of proportions. Specifically, it arises when there is an ascertainment bias inherent in a study design … The most common example of Berkson’s paradox is a false observation of a negative correlation between two positive traits, i.e., that members of a population which have some positive trait tend to lack a second.

The original example is developed using the example of an hospital based group of patients. The only thing to know is that diabetes is a risk for cholecystitis in the general population.

Any given hospital in-patient without diabetes must have another disease (otherwise he would not be there), for example cholecystitis. And by definition this will be cholecystitis without diabetes caused by some other risk facors (female, fat, forty…) So in this group of in-patients there maybe a spurious negative association between cholecystitis and diabetes.

My example here is with families who are living on farms. Since around 1960  [Leynaert 2001] there is this interesting observation that farming families have less allergy, an effect that I found back in 1989 and that is most likely a healthy farmer effect.
This selected farm population has a lower allergy prevalence and of course their children will also have less allergy. All the negative correlations (that are interpreted as protection) with endotoxin, microbiome, etc could be caused by Berkson’s fallacy (Linneberg 2005). The observation will also be even replicated as the same selection criteria are also present in the replication sample.

It seems that I am not the first to

Many more cognitive biases could also be involved: anchoring, availability cascade, confirmation and expectation bias and of course: law of the instrument.

What is wrong with the 2011 NEJM paper?

N Engl J Med 2011;364:701-9 is another paper with 1000+ citations  that had a lasting impression on some but not all people.

First, I can’t remember of any study with such an enormous selection bias  where >94% of individuals have been lost.

Second, we should not forget that farm is not protective per se – farmers may just avoid a known allergy risk factor.  PARSIFAL participants in this study included Steiner schools — anthroposophic medicine mostly avoids vitamin D (ref). This is of course a major issue for any cross-sectional study that doesn’t take into account the temporality of events.

Third, in PARSIFAL dust from children’s mattresses were collected by vacuuming — it is not very likely that many helminthic eggs were transported  from stable to bedroom. In GABRIELA, only airborne dust samples  were collected which again may miss helminth eggs although being certainly present in stable dust.

Fourth, more  microbial exposure and more fungal taxa on farms are a trivial finding.

The inverse associations of the diversity scores with asthma were not confounded by status with respect to living on a farm because adjustment did not change the respective point estimates for asthma (Table 2), although the associations became nonsignificant.

Small sample size, borderline p-values even after a long fishing expedition?

What do these strange “probability” plots  really show – the probability of asthma or the probability to live on a farm?

N Engl J Med 2011;364:701-9 Figure 3 Does it refute any general effect of diversity?

The plots are misleading if adjustment for farm living does not change the parameter estimates for bacterial/fungal diversity.

Sixth – even many years later, the main findings of this study have not been independently replicated. There is not any single study that shows listeriosis (Listeria) or diphtheria (Corynebacterium)  to be protective.

Allergy protection on farms – why also studies in mice could have failed

There are  many immunological differences in humans and mice (follow my link) that are never discussed — not even in Science 2015;349/6252:1106.

Ignoring the long-standing paradox that endotoxin is also acting as a natural adjuvant to atopic inflammation, the credibility of the Science paper is further reduced.

IMHO it is also a  strange experimental condition to have all animals on a standard vitamin D diet – a known co-sensitizers – and looking then for A20 which is co-regulated by vitamin D ??

Does a healthy worker effect explain the “allergy protection” at Bavarian farms?

Unfortunately most studies in the farming environment did not report the prevalence of parental history nor did they report the effect size of parental risk in the farming population. This is, however, a critical issue as the so called healthy worker effect (HEW) may be a rather trivial explanation of the results.

Specifically, it is a sampling bias: the kind of subjects that voluntarily enroll in a clinical trial and actually follow the experimental regimen are not representative of the general population. They can be expected, on average, to be healthier as they are concerned for their health [or as ill people already dropped out]

At least Braun-Fahrländer 1999 reported that allergic parents were seen much less at farms: allergic rhinitis 12.7% versus 29.4% (P=0.001). A history of allergy at farms is no more a risk factor as it is otherwise in the general population. How that? Risk genes or risk behaviour (supplementation) has been eliminated.

Are there any studies in adults? I know of three studies (and one review Le Moual N 2008).

Leynaert 2001 showed only a slightly reduced prevalence of “allergy” (39.1% vs 41.5%, NS) while her table 4 is most interesting. The association started only after year 1960 which points towards misclassification as far as the analysis is not stratified by year of birth.

Remes 2002 showed a dose dependent effect decline between farming (36.2%) and controls (31.6%, P=0.075),

Perkin 2006 also found some significant lower prevalence in farmers 47.3% versus 57.7%, P<0.001.

A HWE is therefore likely.

There are six studies (Thelin 1994, Braback 2006, Chenard 2007, Thaon 2011, Elholm 2013 and Spierenburg 2015) that examined in detail a possible relationship of HWE, allergy and farming. Unfortunately the examination period in five of these studies is too short for any conclusion while Braback 2006 seems to be the only reliable study.

Source: Braback 2006

Also from this study, we can safely conclude, that there is a significant HWE.

Addendum 22 Nov 2019

It seems that I missed some papers on HWE and farming. For asthma it is important to discriminate atopic and non-atopic asthma.

Timm 2019: a hard to understand 3 generation study of unclear asthma  type. Point estimates of parental asthma on farm upbringing are not really a measure of HWE – shuffling exposure and outcome distorts temporality. Neither protection by farm nor HWE found but also selection bias (approx. 50% dropout) and non-differential bias for residency.

Eduard 2015: 1,964 farming students. Current asthma in farmers was 3.0% compared to 6.3% in farmers who had retired early. No HWE found at entry level but during farm work.

Farmers who had changed the type of production had an increased risk of current asthma (OR 9.8), compared with those who had not changed production, indicating a strong selection effect.

Vogelzang 1999: 400 pig farmers, X-sectional point estimates, not a  real HWE study, although HWE offered as explanation.

Health-based selection of nonasthmatics for pig farming, which tends to mask a work-related hazard for asthma, is offered as an explanation for these results.

Taken together – more data but not more knowledge.

Claim to fame of the hygiene hypothesis

The recent encyclopedia article about the hygiene hypothesis seems to be well written. At least on the first instance … in reality it is more a novel than a scientific review.

For many years already, the hygiene hypothesis has been called an outdated concept; various times it was revised and transformed, and finally it gave birth to novel hypotheses.

In other words, the hypothesis has been rejected for being wrong . Even many revisions did not change that. There seem to be only one proven fact – the obsession of some authors with hygiene and nouvel Rousseauism.

Anyway, the hygiene hypothesis has promoted radical rethinking of infections, microbiota, and coevolution of mankind and microbes.

There is nothing radical in backward thinking. We still carry tons of microbes, freezer and antibiotics only did some qualitative but not so much quantitative changes,

With the advent of novel high-throughput sequencing technologies the human microbiome, which is sometimes called the ‘forgotten organ,’ has attracted much attention and is currently being implemented in a wider concept of self-foreign relationship, which may even include recognition of the nonmicrobial nonself as a vital stimulus to a well-developing immune system.

  1. So the interest is technology and not science driven.
  2. The microbiome is not an organ.
  3. The hype is already over.
  4. The Self is not defined by any bacterium.
  5. Most bacteria are excreted and not vital stimulus.

Given the many molecule classes regulating immune functions across individuals such as short RNAs, the hygiene hypothesis may eventually come back as a surprising explanation of the phenomena evoked by crowding, day care, sibship size, orofecally transmitted diseases, and respiratory infections.

Why that?
A comeback of the hygiene hypothesis by short RNA?
The listed phenomena are not intrinsically related, but are occuring only at the same time scale.

Even the old birth order effect might be rediscovered as epigenetic programming someday. Admittedly, these notions are entirely hypothetical, but without hypotheses, proven or not, science hardly advances.

So if David Strachan’s birth order effect would be really caused by  epigenetic programming – why would that be related to hygiene at all?

Science is is not so much about proven or unproven but about reasonable and non reasonable hypotheses.

 

The endotoxin phantom – discrepancies in the NEJM 2002 farm paper

N Engl J Med 2002; 347:869-877 made a strong point that the farm effect is mediated by endotoxin but could show only a 1,7 fold higher endotoxin exposure at farms.

N Engl J Med 2002; 347:869-877 table 1 shows environmental exposure between farm and non-farming households

LPS therefore will not explain the negative farm association as endotoxin effects are so much similar between farming and non-farming environment. BTW why is only the result of the total sample given here and not just the farm result?

N Engl J Med 2011; 364:701-709 table 2 endotoxin effect is not stronger at farms – more some unspecific immunosuppressive effect?

So, what is the reason for the lower IL10 capacity in Figure 2D after LPS exposure – exhaustion, adaptation? And even more important: What is the reason for the lower allergy rate at farms?

If we now go back to table 1 there is a 5,0 fold Der p1 excess in farm – much higher than the 1,7 fold increase of endotoxin. As nearly all allergens have helminth homologues –  the question is what did the authors really measure? Da Costa Santiago 2015 has a nice table that could give an answer.

J Immunol. 2015 Jan 1;194(1):93-100 shows Der p1 homologs

Der p1 is a cysteine protease of 25kDa that has homologues for example in Loa loa. Unfortuneately Loa loa is not an explanation at Bavarian farms – as it is more common in tropical Africa. Cysteine proteases are nevertheless a big tool for helminths –  in Schistosoma japonicum a cathepsin B2 cysteine protease is considered the main penetration tool.

Usually cysteine proteases are not allergenic, but the excessive (and rather isolated) rise combined with a reduced Der  p1 sensitization in the children,  is definitely an unusual finding. The Dermatophagoides pteronyssinus habitat is cosmopolitan, house dust,  also influenced by altitude, but there is no known reasons for this excess in farms. Is the Der p1 value just a false positive and has it helminthic and not mite origin?

I am therefore blasting Der p1  sequence (P08176) against all known cow endoparasite genomes. Fasciola has a strong hit with CL6, a papain family cysteine protease with e+48.

Wormbase blastp results – e+ values are somewhat lower than my local blast result at the same locus

With identity values between 40% and 70% there could well be a confusion of mite Der p1 and  helminth (Fasciola?) CL6, which should be tested for cross-reactivity.

The story even gets more interesting when reading more about parasites

Helminth parasites have complicated life cycles … at the same time as skewing the immune system toward a Th2-driven response, they have a general suppressive effect on the host immune system that prevents their elimination and reduces immune-mediated tissue damage. It has been suggested that cytokines of the anti-inflammatory network, particularly IL-10 and transforming growth factor- (TGF-), that are produced in response to continual stimulation of the immune system by parasite antigens, are pivotal to regulating the damage they cause and that, coincidentally, these have a bystander protective affect against allergic reactions.

This is exactly what N Engl J Med 2002; 347:869-877 showed: increasingly exhausted IL10 capacity. May the x-axis label should be CL6 instead of LPS?

N Engl J Med 2002; 347:869-877 Figure 2. Smoothed Plots of the Log-Transformed Capacity of Peripheral-Blood Leukocytes (PBL) to Produce Interleukin-10 (Panel D) after Stimulation with Lipopolysaccharide (LPS) or Staphylococcal Enterotoxin B (SEB) in Relation to the Log-Transformed Endotoxin-Load Values.

 

 

The Amish paradox in NEJM 2016 explained

A recent study in the NEJM found remarkable differences in the asthma prevalence between Amish and Hutterite populations. The lifestyle of both communities is similar but their farming practice is distinct as the Amish follow a more traditional style of outdoor grazing whereas the Hutterities use industrialized farming practices. Gene expression data in the Amish children have been interpreted as „intense exposure to microbes“ because protection of experimental asthma by Amish derived house dust was nearly abrogated in mice deficient for MyD88.

Any helminth exposure has been excluded due to low IgE and eosinophil counts in the children while I still think that this could be an explanation in particular as the attempt to show an effect of bacterial exposure was unsuccessful since the discovery of the farming effect.

One difference between conventional stable (Hutterites) and outdoor grazing (Amish) is the higher helminthic infection rate on pasture, mainly with Fasciola, Ostertagia, Eimeria, Cooperia, Dictyocaulus and Trichostrongylos species.

Infected cattle rarely demonstrate clinical disease, while it is known that Fasciola (as for example Schistosome) has numerous immunosuppressive functions in the host. IgE is not always raised as Fasciola can degrade human immunoglobulin or even induce eosinophil apoptosis.

Re-analysis of Gene Expression Network using string-db.org (String Consortium 2019). The gene expression network in Amish children {Stein et al., 2016, #73074} in the upper area has similarities with the network observed in sheep after Fasciola infection {Fu et al., 2017, #6751} module #1 and #3, in the lower plot.

FaRMI

I will add now a special collection of farming studies here as many of them are just candidates for the Ig nobel prize.

The most recent study introduces FaRMI, a “bacterial relative abundance farm home microbiota index”, probably introduced as the authors couldn’t find anything else. It reminds me very much to the polygenic risk score that rescues your study if you could not find the gene.

Asthma prevalence has increased in epidemic proportions with urbanization

Already the first sentence is wrong if we look at the following plot.

Urbanization happened in the late späten 19th century and not after 1950. Source of plot: The prevalence of asthma in children: a reversing trend ( ERJ 2005 )

Unfortunately, the difference between farm and non farm children is never explained in the Kirjavainen et al. paper.

What is for example the average distance of a non farm house to a farm house? Are there any joint school or sports activities of children from farms and non farms (allergens travel in the classroom)? And why is there such a strong conclusion in the title?

Farm-like indoor microbiota in non-farm homes protects children from asthma development

A lower risk score is not equivalent to protection.

And did somebody of the authors or reviewers ever look at the plots?

I do not understand Figure 3c. It even makes only sense when I cross out the top labels. But even then it menas: GABRIELA does not show a significant replication.

or tables?

What should these values tell us? 5 times p<0.05 in a grid of 6×11=66 tests? Quantile regression that has been “adjusted” by the data?

And isn’t that  just an association that may have a rather simple explanation?

As FaRMI is weakly associated with muramic acid concentration in dust, the authors make Gram-positive bacteria responsible for the effect. The rhizosphere of soil is extremly rich of bacteria. The world’s first soil atlas showed  hundreds of taxa but never differentiated between water resistant, gram positive and less water resistant gram-negative taxa. Maybe Gram positive Streptococcaceae are ubiquitous and depend on where you draw your samples?

FaRMI is found in non farm / rural children by bacterial/archaeal operational taxonomic units (OTUs) of soil origin which basically confirms my initial assumption: There was the same contamination of soil both in farm and non-farm homes if we look at supplement table 6 where walking indoors with outdoor shoes results in significant higher FaRMI values…

In conclusion, while the asthma-protective effect of farming is intriguing, it has little practical relevance unless the protective effect can be functionally transferred to non-farming environments.

I do not find this data derived score intriguing. Maybe the microbiome hype is already over.

Our results warrant translational studies to confirm the causal relationship through indoor microbial exposure-modifying intervention that may also form a novel strategy for primary asthma prevention.

Good luck with your translational studies, as we are now somewhere in the nowhere.

 

BTW – The scripts at Github are useless references to shell and Python scripts that will never run due to “—” characters. And what about that baby girl code?

writerow <- paste("Eigenvalue min / max: ", min.eigen, " / ", max.eigen, sep="")
write(writerow, file=eigenfile, append=F)
writerow <- paste("Sum of all eigenvalues: ", round(neg.eigensum, digits=6), sep="")
write(writerow, file=eigenfile, append=T)
writerow <- paste("Sum of all eigenvalues (negatives as 0): ", round(nonneg.eigensum, digits=6), sep="")
write(writerow, file=eigenfile, append=T)
writerow <- "Eigenvalues (pos & neg): "
write(writerow, file=eigenfile, append=T)
writerow <- paste(pcoa$value$Eigenvalues, collapse="\t")
write(writerow, file=eigenfile, append=T)
writerow <- "Percents (Negatives as negatives): "
write(writerow, file=eigenfile, append=T)
writerow <- paste(paste(neg.percent, " %", sep=""), collapse="\t")
write(writerow, file=eigenfile, append=T)
writerow <- "Percents (Negatives as 0): "
write(writerow, file=eigenfile, append=T)
writerow <- paste(paste(nonneg.percent, " %", sep=""), collapse="\t")
write(writerow, file=eigenfile, append=T)

Using R heredoc syntax I can rewrite 18 unreadable to 10 readable lines. And 9x disc access to 1x just doing

tmp <- 'Eigenvalue min / max: min.eigen / max.eigen
Sum of all eigenvalues: neg.eigensum
Sum of all eigenvalues (negatives as 0): nonneg.eigensum
Eigenvalues (pos & neg): pcoa
Percents (Negatives as negatives):  neg.percent %
Percents (Negatives as 0): nonneg.percent %'
for (i in c("min.eigen","max.eigen","neg.eigensum","nonneg.eigensum","pcoa$value$Eigenvalues","neg.percent","nonneg.percent") ) { tmp <- gsub(i,get(i),tmp) }
write(tmp, file=eigenfile)

And why moving to SAS for a simple logistic regression? Is there anyone else in the academic world who pays $8,700 annually for a basic SAS Windows Analytics package?

The end of the hygiene hypothesis

The authors put a question mark at the end of the above statement while I would not hesitate to put an exclamation mark there. Writing this as a comment to a new study in the IJE they summarize the evidence that the “epidemics” of asthma in Western countries has begun to decline – as hygiene standards are not declining this might indicate the end of the hygiene hypothesis. Continue reading The end of the hygiene hypothesis

Dung hill counting

Wikipedia writes about Imre Lakatos the famous Hungarian mathematician and philosopher who graduated 1961 in Cambridge with “Essays in the Logic of Mathematical Discovery”

He showed that in some cases one research programme can be described as progressive while its rivals are degenerative. A progressive research programme is marked by its growth, along with the discovery of stunning novel facts, development of new experimental techniques, more precise predictions, etc. A degenerative research program is marked by lack of growth, or growth of the protective belt that does not lead to novel facts.

 

One of these degenerate research program relates to the hypothesis that farming protects you from allergy

E 2006:

There is increasing evidence that environmental exposures determining childhood illnesses operate early in life. Prenatal exposure to a farming environment through the mother might also play an important role … Both atopic sensitization … and the gene expression of receptors of innate immunity were strongly determined by maternal exposure to stables during pregnancy, whereas current exposures had much weaker or no effects … Each additional farm animal species increased the expression of TLR2, TLR4, and CD14 by a factor of 1.16

Keep in mind – it’s the farm animal.

K 2008:

Several epidemiological studies have shown that the farm environment impacts allergy protection mechanisms in children … In investigating the link between farming lifestyle and prevention of childhood allergy, we examined the prevalence of Listeria spp. in dust specimens from the environment of rural children … The dominant species found by culturing methods were L. innocua (n=12) and L. monocytogenes (n=8).

Sorry – it’s listeria.

K 2006:

There is increasing evidence that the farming environment has a protective effect as regards allergic diseases. Exposure to animal parasites, particularly helminth infections, is common in the farming environment. Exposure to nematodes, as determined by the levels of antibody to A. lumbricoides, was more frequent among farmers’ children than non-farmers’ children… This positive serology was found to be significantly associated with high total IgE levels … and eosinophilia.

Sorry again – it’s ascaris.


E 2007
:

In recent years, studies have shown a protective effect of being raised in a farm environment on the development of hay fever and atopic sensitization…Inverse relations with a diagnosis of asthma were found for pig keeping …, farm milk consumption …, frequent stay in animal sheds …, child’s involvement in haying …, and use of silage … Protective factors were related with higher expression levels of genes of the innate immunity.

Sorry, it’s everything: the pig, the milk, haying and silage.

W 2007:

Some studies in rural environments claimed an inverse association between consumption of farm-produced dairy products … Farm milk consumption ever in life showed a statistically significant inverse association with asthma… rhinoconjunctivitis … and sensitization to pollen and the food mix fx5 …, and sensitization to horse dander.

Hey, milky ways ahead something new: the horse!

K 2007:

There is still uncertainty about the determinants of atopic eczema … In multivariate analyses, helping with haying was the only variable related to a farming environment having a consistent inverse association with both current symptoms and a doctor’s diagnosis of AE.

Yes,  haying makes sense with hayfever.

W 2005:

An increasing number of studies report pet exposure to be associated with lower risk of asthma and allergies … Current contact with dogs was inversely associated with diagnosed hay fever (OR 0.26, 95% CI 0.11-0.57), diagnosed asthma (OR 0.29, 95% CI 0.12-0.71), sensitization…

Oh no, the dog.

V 2008:

Numerous epidemiologic studies have demonstrated an allergy-protective effect of farm life early in childhood …In vitro, B. licheniformis spores activated a T(H)1 cytokine expression profile. In vivo application of these spores resulted in less spore-specific but long-lasting immune activation preventing eosinophilia and goblet cell hyperplasia; however, they provoked an influx of neutrophils in lung tissue of asthmatic mice.

What about bacillus spores?

vM 2008

Contact with farm animals, at least in childhood, likely confers protection; other factors have not been completely identified. Also, the consumption of milk directly from the farm during childhood has been shown to be beneficial with respect to childhood asthma and allergies.

Ok, it is milk. Are you still readings here?

This week I am back with the most exciting research

Previous cross-sectional surveys have suggested that maternal exposure to animal sheds during pregnancy exerted a protective effect on atopic sensitization in children lasting until school age … Different sensitization patterns in cord blood of farm and nonfarm children were observed. In multivariable analysis consumption of boiled, but not unboiled, farm milk during pregnancy was positively associated with specific IgE to cow’s milk independently from maternal IgE.

This paper counts dung hills The authors even invent a new classification (sorry, not dung hill height but “50 m distance between dung hill and house”).

And did you also wonder why paternal history is no more a risk in thesel studies? There are only a few allergic parent due to healthy worker effect…
No adjustment for multiple testing “because it will lead to fewer errors of interpretation when the data under evaluation are not random numbers but actual observations on nature” That is one of the most stupid sentences I have ever read.

The overall response rate in this study is 32% and the strongest risk for cord blood IgE is maternal IgE. Is there any statistical model that can account for poor data by contamination of newborn cord blood with maternal IgE? And uhh, 32% response is that really a representative sample?

Did you notice that being a farm child now suddenly becomes a risk for seasonal sensitization (OR=1.18, NS) and food allergy as well (OR=1.25, NS)? And that farm milk consumption is suddenly a risk! for IgE to cow’s milk (OR=3.64, p=0.01)?

The mantra at the beginning at each of the abstract above is certainly necessary to let us believe in the rest of these papers.

Addendum 8/8/2008
Poster E3269: Prenatal exposure to a farm environment affects atopic sensitization at birth at ERS Berlin Tuesday, October 7, 2008.

Furthermore, inverse associations of CB IgE to seasonal allergens with positive maternal records for Toxoplasma (T.) gondii (adjusted odds ratio = 0.37 [0.17-0.81]) and rubella virus (adjusted odds ratio = 0.35 [0.13-0.96]) were found.

gotcha – Toxoplasma + Rubella.

Addendum 11/12/2009
a new paper & a new cowshed derived bacterium: Acinetobacter

Using the cowshed-derived bacterium Acinetobacter lwoffii F78 together with a mouse model of experimental allergic airway inflammation, this study investigated the hygiene hypothesis.

Addendum 28/2/2011
a new press release Eurotium

Mikrobielle Vielfalt allein reicht vermutlich allerdings nicht aus, um Asthma zu verhindern. Wahrscheinlich ist es eine Kombination spezifischer Arten, die eine Schutzwirkung entfalten kann. „Im gesamten untersuchten Spektrum fanden sich einige Keime, die besonders interessant sein könnten”, berichtet Ege, „dazugehören außer bestimmten Bazillen und Staphylokokken – etwa die Art Staphylococcus sciuri – auch Schimmelpilze der Gattung Eurotium.“

Addendum 1/1/2018
The research above has now lead to the highest German Science Prize, an honorary doctorate, an ERC advanced grant, a Leopoldina and Bavarian Academy membership.

Is there an association of CARD15 variants with allergy?

An association of functional CARD 15 polymorphism and allergy has been described recently in this journal [1]. This paper has been widely cited [2] and is promoted as one of the main outcomes of the German National Genome Research Network I (http://www.ngfn.de/22_190.htm). In this study three SNPs previously found to be associated with Crohn ́s disease are examined for association with allergy. These are SNP8 (akin 2104C/T, 2023C/T, Arg675Trp, Arg702Trp, R702W, rs2066844), SP12 (akin 2722G/C, 2641G/C, Gly881Arg, Gly908Arg, G908R, rs2066845) and SNP13 (akin 3020-/C deletion, 2936 C insertion, L1007fsinsC, 980/981 frameshift) [3], [4]. While genotyping several CARD15 SNPs in our own family sample we noted several errors, inconsistencies and omissions in the primary report [1].

Laboratory Methods. There is no information available about DNA extraction procedures, re-identification of samples, quality control, pre-amplification, details of PCR reaction, size of restriction fragments and scoring of genotypes.

The SNP8 assay seems to include a MspI restriction site at the forward primer which could interfere with cutting the amplified fragment. The SNP12 assay has identical forward and backward primer and would not lead to any amplification. The assay for SNP13 has a poor design as it does not include any control site to ensure enzyme activity.

Neither results of duplicate sample testing nor results of Hardy- Weinberg equilibrium are being reported. There is also no rationale given why only half of the Dresden samples are being genotyped. According to table I and II only 81% of genotyping attempts were successful while it is known that high genotyping failure rates is leading to false positive associations [5]. Even if the noted errors in the primer sequence may be attributed to simple typing errors, this still raises doubts about the validity of the laboratory procedures.

Analysis. A control group created by the absence of any genetic variant seems to inflate risks if during the following step the presence of a particular genetic variant is being tested against this control group. It may even be argued that the calculation of relative risks would be more appropriate than that of odds ratios (resulting in considerable lower effect estimates). Without any detailed information of phenotypic and genotypic details of the control group, effects are hard to understand and might be also a reason why this definition of controls has been abandoned in further analysis of this population [6]. The 1:2 matching of “supernormal” controls in the consecutive analysis [6] is also questionable as selecting individuals with certain homogenous levels of confounders imposes restrictions of the analysis. With the absence of “normal” controls even a more severe bias may be introduced.

No information is given on the prevalence of Crohn ́s disease in this sample although this would have been a unique opportunity to verify the initial findings [7] in an independent patient based cohort.

Results. The method section details the estimation of haplotypes but results are being omitted. Instead of haplotypes associations, the risk of co-occurrence of any two SNPs is reported which increase from 3.16 to 4.64. This result is considerably lower from what is being reported in the Crohn ́s literature [8]. Neither results of separate allelic and genotypic analysis are being reported and -as only significant risk estimates are given in table III- it is impossible to judge about any possible dose-response effect.

The risks are also not adjusted for strong confounders like season and there is no assessment of goodness-of-fit of the models used [9] which further undermines the validity of this study.

The numbers in table III are confusing: The legend in table III refers to a total sample of 1873 children while introduction and methods reports 1872; row numbers in table III do not exceed 1805 and column numbers do not exceed 1765 individuals. Table I, II and III data do not match: For example 8,6% of 1161 genotyped children in table I have atopic rhinitis (may be rounded to N=100) plus 9,2% of 711 genotyped children in table II (may be rounded to N=65) which does not add to N=154 genotyped children with atopic rhinitis (table III). Similar restrictions apply to all other traits tested.

A sample of 1872 children is reported in another paper to originate also from Leipzig and not only from Munich and Dresden [6]. Other reportsonthesamecollectionreportmorethan3,000samples [10], a number in the same range as here [11], but also less than half of the sample size [12], [13], [14], [15], [16], [17].

The results section of the current paper report that “In Dresden, [….] polymorphism T2104 was also associated with atopic rhinitis to a lesser degree [than in Munich] (16,9% vs 7.6%; OR 2,43; 95% CI 1,24 to 4,78; P<.05)”. Results for Munich are not given, but the overall result for C2104T and atopic rhinitis in table III may indicate that the above sentence should read to a higher degree.

In the results section the allele frequency of SNP8 is reported to be 5,6%, SNP12 of 2,0% and SNP13 of 3,8%. In contrast percentages calculated from column 1 of table III give 11,2% for SNP8, 4,0% for SNP12 and 7,3% for SNP13.

No significant linkage disequilibrium was observed between the examined SNPs. It is unclear why only homozygous subjects were included for this procedure. As linkage disequilibrium results seems to also unlikely in another paper from the same group [18] this might refer to outstanding problems in the genetics module of SAS version 8.2 used by the authors (SAS notes SN-011039 and SN-008611). Linkage disequilibrium is not reported correctly if at least one haplotype frequency for a marker pair is estimated to be greater than the frequency of either allele. Another another error may be introduced if there are individuals with all missing alleles as already noted above.

The moderate increase of total IgE probably does not indicate a “higher severity of atopy” [1]. The difference between mean 186,6 IU/ml and 312,1 IU/ml reflect less than the transition between 70thand 80th percentile as may be assumed from another paper [6]. IgE is a laboratory value that may be influenced also by other reasons and is otherwise not used as severity marker of atopy.

Sources of bias. There is no information if the difference observed between the study centres is caused by population stratification (which could have been tested with anonymous marker and been completely avoided by using family-based samples). This is expected to be a particular problem as the authors reported a much lower prevalence of allergy of the Turkish minority in the Munich study center [19] that have a considerable different genetic background (unpublished own observation during the Genetic Analysis Workshop 11/2000). Was ancestry defined by passport or by self-reported affiliation? How were probands with mixed ancestry treated? Mild stratification might exist also in less admixed populations when looking for alleles with modest disease effect [5]. Which steps have been taken to ensure that controls are non-cases [9]?

References. There are numerous referencing errors and misunderstandings: Fig. 1 locates SNP12 in the 6th LRR and SNP13 in the 9th LRR. According to Ogura [7] SNP12 resides in the 7th LRR and SNP13 in the 10th LRR. Table I and II footnotes refers to reference 8 which belongs to another topic. Reference 10 cited in the methods is misleading as it relates to a different skin prick test device. Reference 14 is used to show that impaired LPS recognition by NOD2 polymorphism reduces the capability to interact with bacteria and to develop a Treg reservoir. Unfortunately this is not the content of their reference 14: “NOD” denotes “non-obese diabetic mice” and the review discusses helminth (but not bacterial) effects on T cells. CARD15 gene is also not located in the pericentromeric region of chromosome 16 (which would be q11.1) but on the cytogenetic band q12.1. There exists also a 12 exon isoform of CARD15 [20] (and not only the reported 11 exon form).

Even more important is the omission, that at the time of the study 67 (and not only the reported 13) polymorphism have been known [20]. Linkage disequilibrium with untyped SNPs could therefore confound the current analysis.

Interpretation. It is hard to understand why SNP8 and SNP12 impose the highest risk for atopic rhinitis while the risk for Crohn ́s disease comes mainly with SNP13 [8]. Why is the excess risk for atopic rhinitis not found with the underlying biological traits? Although not being discussed in the paper, this could point towards chance effects introduced by multiple testing [21].

The overall number of tests performed is not given in the paper. If we assume, however, 6 traits (total IgE, number of skin prick tests, atopy, atopic dermatitis, atopic rhinitis, asthma), tested in 3 groups (total and 2 subgroups), 4 series (as single SNP plus all combinations of 2 SNPs) and assume their “best” p value from table III to be 0,001 (an exact estimate is not given), the Bonferoni corrected p value would be pcorr= 1-(1-0,001)6*3*4 = 0,0695 which is above conventional standards.

There seem to be misunderstandings on the role and function of CARD15. In my opinion the main result of CARD15 activation is not so much apoptosis but infection control by activating the adaptive immune system [22]; its function is also not so much sensing of endotoxin (LPS) from gram negative bacteria but peptidoglycan (PGN) of practically all bacteria [23], [22]. The authors discuss only in part the apparent paradox at the time of submission that lacking the entire LRR region resulted in enhanced NF-κB activity whereas the frameshift mutation by SNP13 resulted in low NF-κB levels [24] (for an updated discussion see [25]). Protein truncation of the most terminal C- terminal LRRs of CARD15 lead to an unresponsiveness to bacterial components but leaves CARD15 still able to activate NF-κB at a level comparable to that of the wild-type protein [26]. An antagonistic effect of these SNPs is therefore possible [23]. It is difficult, however, to follow any further discussion [1] as the authors even mix up the amino acid and genomic positions of SNP8 and SNP12 (which was otherwise correctly denoted in their figure) and assume that SNP8 leads to reduced NF-κB activity.

In a more general view, it does not seem to be adequate to make any conclusions about causal interference from the statistical association in one study as the authors repeatedly do [27]. There are many known fallacies with such an approach [28], [29] and imposes a particular problem in a field where most studies are never reproduced [30]. The main factors accounted so far for non replication are inadequate statistical power, biased analysis and selective reporting [9]. Further criteria for meaningful associations modified after [31] are: (a) functional importance of the tested protein with the trait of interest (b) functional importance of the mutation (c) genetic background and interaction with other genes (d) time of onset of functional change and interaction with relevant pathway (e) interaction with the environment and (f) the existence of alternative pathways. None of these points are being examined in the current study.

The authors conclude in the last sentence of their abstract that “The shared genetic background between Crohn’s disease and atopy may indicate that an impaired recognition of microbial exposures results in an insufficient downregulation of excessive immune responses, giving rise to either TH2 dominated allergies or TH1 related Crohn’s disease.” This seems to be unwarranted: The authors have not examined the genetic background (genomewide association studies are still out of reach) but association of a few gene variants. They have neither examined any patient with Crohn’s disease nor the process how microbes are recognized. Even if we follow their conclusion, how can a shared mutation give rise to either TH2 dominated allergies or TH1 related Crohn’s disease?

History. A first report of this study published as a poster at the American Thoracic Society Meeting in Atlanta May, 2002 included 528 children and found that there is “No association between polymorphisms in the NOD2 gene and atopic phenotypes”. The results changed with the target sample of 1872 children at the NGFN meeting in Berlin, November 2002, where the authors reported “allele C2722 had a more than 3-fold risk to develop allergic rhinitis (p<0.0001) and an almost 2-fold risk for atopic dermatitis (p<0.01)”. The current paper was submitted in Sept 2002 and report a 10-fold higher p-value, e.g. p < 0.001 for allergic rhinitis and 5-fold higher p-value, e.g. p<0.05 for atopic dermatitis. A third abstract submitted three months after the current paper to the European Respiratory Society in Vienna, Oct 2003, reported again the lower p-values of p<0.0001 and p<0.01, respectively. Although there was never an association of CARD15 variants with asthma, this study is now being cited by the same group that “mutations in the related gene NOD2 have been shown to predispose to Crohn ́s disease (…) as well as to asthma (…)” [6] or again with “asthma, atopy, total Ig E, atopic rhinitis and asthma” [27]. This association now even changes to be “associated with the development and severity of atopic diseases and airway hyper- reactivity” [32] where neither development, nor severity of atopic diseases nor hyperreactivity was tested here.

Omissions. Why do the authors ignore all genomewide linkage studies conducted over the past decade? It would also be interesting to know why the authors omitted the existence of the comprehensive Innate Immunity Net genotyping results of CARD 15 published in the Internet on May 6, 2002 before the submission of their own article (http://innateimmunity.net/IIPGA/IIPGASNPs/IIPGA2/PGAs/InnateIm munity/CARD15/ADsas) and known to the authors [6]. There are no acknowledgments and the list of authors does not match the list of principal investigators (http://medweb.uni- muenster.de/institute/epi/forschung/index.php). Funding sources are also incomplete as the NGFN funding did not start before 2001 (http://www.ngfn.de/15_102.htm).

Editorial problems. There are numerous meaningless and inaccurate statements (“putative amino acid exchange”). Editing errors like double author names in the references and typing errors disrupt the text. The commercial IgE assay “Insulite” which is the main laboratory outcome of this study should probably read “Immulite”. This error is particular interesting as it allows to trace this text block to several other papers. The lengthy description of children that never participated in this study is superfluous as well as the discussion of functional properties of CARD 15 that have not been examined here.

Ethics. It is an open question how study methods in 2002 could have been reviewed by an ethics committee more than 7 years before (a paper on CARD 4 variants in the same population reports different ethics committees consulted for this study [6]). As the authors describe variants with a 17-fold risk for Crohn’s disease , it would be interesting to know if and how children and parents have been informed on these results.

Post Scriptum. Is there an association of CARD15 with allergy? I don ́t know. Unfortunately any criticism of a published paper affects all collaborators and the scientific network (who is loosing credibility), department heads (who do not establish proper control mechanisms), participating subjects (who would not have consented to such a study), funding agencies (who are loosing their money), journal editors and reviewer (who do not follow accepted standards), colleagues that cite this paper (as it shows that they have not read it) and finally to the whistle blower (who experiences moral pressures). Also others raised doubts about results from this laboratory [18] but I think this is more a general problem of current biomedical research that is centered on impact points [9],[33]. Non-reproducibility of studies is a fundamental problem in this field where genetic association is becoming a dirty word [21]. W hile in the beginning researchers have directly been accused of having falsified data [34] genetic heterogeneity is now assumed to be responsible for non- reproducibility. I am more inclined to think of a complex mélange where improper study design and poor methods result in unwarranted conclusions. Although there are several checklists available to ensure minimum quality [9],[35] there seems to be an increasing number of reports where the peer-review failed.

References

1. M Kabesch, W Peters, D Carr, W Leupold, SK Weiland, E von Mutius: Association between polymorphisms in caspase recruitment domain containing protein 15 and allergy in two German populations. J Allergy Clin Immunol 2003, 111:813-7.

2. W Cookson: The immunogenetics of asthma and eczema: a new focus on the epithelium. Nat Rev Immunol 2004, 4:978-88.

3. J Hampe, A Cuthbert, PJ Croucher, MM Mirza, S Mascheretti, S Fisher, H Frenzel, K King, A Hasselmeyer, AJ MacPherson, et al:

IS THERE REALLY AN ASSOCIATION OF CARD15 WITH ALLERGY P 11/14

Association between insertion mutation in NOD2 gene and Crohn’s disease in German and British populations. Lancet 2001, 357:1925-8.

4. J Hampe, J Grebe, S Nikolaus, C Solberg, PJ Croucher, S Mascheretti, J Jahnsen, B Moum, B Klump, M Krawczak, et al:Association of NOD2 (CARD 15) genotype with clinical course of Crohn’s disease: a cohort study. Lancet 2002, 359:1661-5.

5. JN Hirschhorn, MJ Daly: Genome-wide association studies for common diseases and complex traits. Nat Rev Genet 2005, 6:95-108.

6. P Hysi, M Kabesch, MF Moffatt, M Schedel, D Carr, Y Zhang, B Boardman, E von Mutius, SK Weiland, W Leupold, et al: NOD1 variation, Immunoglobulin E, and asthma. Hum Mol Genet; online preview Feb 17, 2005 2005.

7. Y Ogura, DK Bonen, N Inohara, DL Nicolae, FF Chen, R Ramos, H Britton, T Moran, R Karaliuskas, RH Duerr, et al: A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease.Nature 2001, 411:603-6.

8. M Economou, TA Trikalinos, KT Loizou, EV Tsianos, JP Ioannidis:

Differential effects of NOD2 variants on Crohn’s disease risk and phenotype in diverse populations: a metaanalysis. Am J Gastroenterol 2004, 99:2393-404.

9. J Little, L Bradley, MS Bray, M Clyne, J Dorman, DL Ellsworth, J Hanson, M Khoury, J Lau, TR O’Brien, et al: Reporting, appraising, and integrating data on genotype prevalence and gene-disease associations. Am J Epidemiol 2002, 156:300-10.

10. M Kabesch, C Hoefler, D Carr, W Leupold, SK Weiland, E von Mutius: Glutathione S transferase deficiency and passive smoking increase childhood asthma. Thorax 2004, 59:569-73.

11. M Kabesch, K Hasemann, V Schickinger, I Tzotcheva, A Bohnert, D Carr, M Baldini, H Hackstein, W Leupold, SK Weiland, et al: A promoter polymorphism in the CD14 gene is associated with elevated levels of soluble CD14 but not with IgE or atopic diseases.Allergy 2004, 59:520-5.

12. M Kabesch, D Carr, SK Weiland, E von Mutius: Association between polymorphisms in serine protease inhibitor, kazal type 5 and asthma phenotypes in a large German population sample. Clin Exp Allergy 2004, 34:340-5.

13. PE Graves, M Kabesch, M Halonen, CJ Holberg, M Baldini, C

IS THERE REALLY AN ASSOCIATION OF CARD15 WITH ALLERGY P 12/14

Fritzsch, SK Weiland, RP Erickson, E von Mutius, FD Martinez: A cluster of seven tightly linked polymorphisms in the IL-13 gene is associated with total serum IgE levels in three populations of white children. J Allergy Clin Immunol 2000, 105:506-13.

14. M Schedel, D Carr, N Klopp, B Woitsch, T Illig, D Stachel, I Schmid, C Fritzsch, SK Weiland, E von Mutius, et al: A signal transducer and activator of transcription 6 haplotype influences the regulation of serum IgE levels. J Allergy Clin Immunol 2004,114:1100-5.

15. B Woitsch, D Carr, D Stachel, I Schmid, SK Weiland, C Fritzsch, E von Mutius, M Kabesch: A comprehensive analysis of interleukin-4 receptor polymorphisms and their association with atopy and IgE regulation in childhood. Int Arch Allergy Immunol 2004, 135:319-24.

16. M Kabesch, I Tzotcheva, D Carr, C Hofler, SK Weiland, C Fritzsch, E von Mutius, FD Martinez: A complete screening of the IL4 gene: novel polymorphisms and their association w ith asthma and IgE in childhood. J Allergy Clin Immunol 2003, 112:893-8.

17. M Allen, A Heinzmann, E Noguchi, G Abecasis, J Broxholme, CP Ponting, S Bhattacharyya, J Tinsley, Y Zhang, R Holt, et al: Positional cloning of a novel gene influencing asthma fro m chromosome 2q14.Nat Genet 2003, 35:258-63.

18. MJ Basehore, TD Howard, LA Lange, WC Moore, GA Hawkins, PL Marshik, MS Harkins, DA Meyers, ER Bleecker: A comprehensive evaluation of IL4 variants in ethnically diverse populations: association of total serum IgE levels and asthma in white subjects. J Allergy Clin Immunol 2004, 114:80-7.

19. M Kabesch, W Schaal, T Nicolai, E von Mutius: Lower prevalence of asthma and atopy in Turkish children living in Germany. Eur Respir J 1999, 13:577-82.

20. S Lesage, H Zouali, JP Cezard, JF Colombel, J Belaiche, S Almer, C Tysk, C O’Morain, M Gassull, V Binder, et al: CARD15/NOD2 mutational analysis and genotype-phenotype correlation in 612 patients with inflammatory bowel disease. Am J Hum Genet 2002,70:845-57.

21. Editorial: In search of genetic precision. The Lancet 2003,361:357.

22. KS Kobayashi, M Chamaillard, Y Ogura, O Henegariu, N Inohara, G Nunez, RA Flavell: Nod2-dependent regulation of innate and

IS THERE REALLY AN ASSOCIATION OF CARD15 WITH ALLERGY P 13/14

adaptive immunity in the intestinal tract. Science 2005, 307:731-4.

23. M Chamaillard, D Philpott, SE Girardin, H Zouali, S Lesage, F Chareyre, TH Bui, M Giovannini, U Zaehringer, V Penard-Lacronique, et al: Gene-environment interaction modulated by allelic heterogeneity in inflammatory diseases. Proc Natl Acad Sci U S A2003, 100:3455-60.

24. DP McGovern, DA van Heel, T Ahmad, DP Jewell: NOD2 (CARD15), the first susceptibility gene for Crohn’s disease. Gut2001, 49:752-4.

25. S Maeda, LC Hsu, H Liu, LA Bankston, M Iimura, MF Kagnoff, L Eckmann, M Karin: Nod2 mutation in Crohn’s disease potentiates NF- kappaB activity and IL-1beta processing. Science 2005, 307:734-8.

26. T Tanabe, M Chamaillard, Y Ogura, L Zhu, S Qiu, J Masumoto, P Ghosh, A Moran, MM Predergast, G Tromp, et al: Regulatory regions and critical residues of NOD2 involved in muramyl dipeptide recognition. Embo J 2004, 23:1587-1597.

27. M Kabesch: Bald Gentests für (potentielle) Allergiker? MMW Fortschr Med 2004, 146:1017-1020.

28. P Skrabanek, McCormick, J: Follies and Fallacies in Medicine.Prometheus, Buffalo 1990.

29. S Milloy: Science without sense. Cato Institute, Washington1995.

30. JN Hirschhorn, K Lohmueller, E Byrne, K Hirschhorn: A comprehensive review of genetic association studies. Genet Med2002, 4:45-61.

31. P Vineis, P Schulte, AJ McMichael: Misconceptions about the use of genetic tests in populations. Lancet 2001, 357:709-12.

32. M Kabesch, RP Lauener: Why Old McDonald had a farm but no allergies: genes, environments, and the hygiene hypothesis. J Leukoc Biol 2004, 75:383-7.

33. E Garcia-Berthou, C Alcaraz: Incongruence between test statistics and P values in medical papers. BMC Med Res Methodol2004, 4:13.

34. W Cookson: Die Jagd nach den Genen. VCH Wiley 2000.

IS THERE REALLY AN ASSOCIATION OF CARD15 WITH ALLERGY P 14/14

35. ST Weiss: Association studies in asthma genetics. Am J Respir Crit Care Med 2001, 164:2014-5.

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