TLR2 mediates renal apoptosis in neonatal mice subjected experimentally to obstructive nephropathy.

Urinary tract obstruction during renal development leads to inflammation, tubular apoptosis, and interstitial fibrosis. Toll like receptors (TLRs) expressed on leukocytes, myofibroblasts and renal cells play a central role in acute inflammation. TLR2 is activated by endogenous danger signals in the kidney; its contribution to renal injury in early life is still a controversial topic. We analyzed TLR2 for a potential role in the neonatal mouse model of congenital obstructive nephropathy. Inborn obstructive nephropathies are a leading cause of end-stage kidney disease in children. Thus, newborn Tlr2-/- and wild type (WT) C57BL/6 mice were subjected to complete unilateral ureteral obstruction (UUO) or sham-operation on the 2nd day of life. The neonatal kidneys were harvested and analyzed at days 7 and 14 of life. Relative expression levels of TLR2, caspase-8, Bcl-2, Bax, GSDMD, GSDME, HMGB1, TNF, galectin-3, α-SMA, MMP-2, and TGF-ß proteins were quantified semi-quantitatively by immunoblot analyses. Tubular apoptosis, proliferation, macrophage- and T-cell infiltration, tubular atrophy, and interstitial fibrosis were analyzed immunohistochemically. Neonatal Tlr2-/- mice kidneys exhibited less tubular and interstitial apoptosis as compared to those of WT C57BL/6 mice after UUO. UUO induced neonatally did trigger pyroptosis in kidneys, however to similar degrees in Tlr2-/- and WT mice. Also, tubular atrophy, interstitial fibrosis, tubular proliferation, as well as macrophage and T-cell infiltration were unremarkable. We conclude that while TLR2 mediates apoptosis in the kidneys of neonatal mice subjected to UUO, leukocyte recruitment, interstitial fibrosis, and consequent neonatal obstructive nephropathy might lack a TLR2 involvement.

Oral Coenzyme Q10 supplementation leads to better preservation of kidney function in steroid-resistant nephrotic syndrome due to primary Coenzyme Q10 deficiency.

Primary Coenzyme Q10 (CoQ10) deficiency is an ultra-rare disorder caused by defects in genes involved in CoQ10 biosynthesis leading to multidrug-resistant nephrotic syndrome as the hallmark kidney manifestation. Promising early results have been reported anecdotally with oral CoQ10 supplementation. However, the long-term efficacy and optimal prescription remain to be established. In a global effort, we collected and analyzed information from 116 patients who received CoQ10 supplements for primary CoQ10 deficiency due to biallelic pathogenic variants in either the COQ2, COQ6 or COQ8B genes. Median duration of follow up on treatment was two years. The effect of treatment on proteinuria was assessed, and kidney survival was analyzed in 41 patients younger than 18 years with chronic kidney disease stage 1-4 at the start of treatment compared with that of an untreated cohort matched by genotype, age, kidney function, and proteinuria. CoQ10 supplementation was associated with a substantial and significant sustained reduction of proteinuria by 88% at 12 months. Complete remission of proteinuria was more frequently observed in COQ6 disease. CoQ10 supplementation led to significantly better preservation of kidney function (5-year kidney failure-free survival 62% vs. 19%) with an improvement in general condition and neurological manifestations. Side effects of treatment were uncommon and mild. Thus, our findings indicate that all patients diagnosed with primary CoQ10 deficiency should receive early and life-long CoQ10 supplementation to decelerate the progression of kidney disease and prevent further damage to other organs.

Variation of the clinical spectrum and genotype-phenotype associations in Coenzyme Q10 deficiency associated glomerulopathy.

Primary Coenzyme Q10 deficiency is a rare mitochondriopathy with a wide spectrum of organ involvement, including steroid-resistant nephrotic syndrome mainly associated with disease-causing variants in the genes COQ2, COQ6 or COQ8B. We performed a systematic literature review, PodoNet, mitoNET, and CCGKDD registries queries and an online survey, collecting comprehensive clinical and genetic data of 251 patients spanning 173 published (47 updated) and 78 new cases. Kidney disease was first diagnosed at median age 1.0, 1.2 and 9.8 years in individuals with disease-causing variants in COQ2, COQ6 and COQ8B, respectively. Isolated kidney involvement at diagnosis occurred in 34% of COQ2, 10.8% of COQ6 and 70.7% of COQ8B variant individuals. Classic infantile multiorgan involvement comprised 22% of the COQ2 variant cohort while 47% of them developed neurological symptoms at median age 2.7 years. The association of steroid-resistant nephrotic syndrome and sensorineural hearing loss was confirmed as the distinctive phenotype of COQ6 variants, with hearing impairment manifesting at average age three years. None of the patients with COQ8B variants, but 50% of patients with COQ2 and COQ6 variants progressed to kidney failure by age five. At adult age, kidney survival was equally poor (20-25%) across all disorders. A number of sequence variants, including putative local founder mutations, had divergent clinical presentations, in terms of onset age, kidney and non-kidney manifestations and kidney survival. Milder kidney phenotype was present in those with biallelic truncating variants within the COQ8B variant cohort. Thus, significant intra- and inter-familial phenotype variability was observed, suggesting both genetic and non-genetic modifiers of disease severity.

Pediatric idiopathic steroid-sensitive nephrotic syndrome: diagnosis and therapy -short version of the updated German best practice guideline (S2e) - AWMF register no. 166-001, 6/2020.

Idiopathic nephrotic syndrome is the most frequent glomerular disease in children in most parts of the world. Children with steroid-sensitive nephrotic syndrome (SSNS) generally have a good prognosis regarding the maintenance of normal kidney function even in the case of frequent relapses. The course of SSNS is often complicated by a high rate of relapses and the associated side effects of repeated glucocorticoid (steroid) therapy. The following recommendations for the treatment of SSNS are based on the comprehensive consideration of published evidence by a working group of the German Society for Pediatric Nephrology (GPN) based on the systematic Cochrane reviews on SSNS and the guidelines of the KDIGO working group (Kidney Disease - Improving Global Outcomes).

Refining genotype-phenotype correlations in 304 patients with autosomal recessive polycystic kidney disease and PKHD1 gene variants.

Autosomal recessive polycystic kidney disease (ARPKD) is a severe disease of early childhood that is clinically characterized by fibrocystic changes of the kidneys and the liver. The main cause of ARPKD are variants in the PKHD1 gene encoding the large transmembrane protein fibrocystin. The mechanisms underlying the observed clinical heterogeneity in ARPKD remain incompletely understood, partly due to the fact that genotype-phenotype correlations have been limited to the association of biallelic null variants in PKHD1 with the most severe phenotypes. In this observational study we analyzed a deep clinical dataset of 304 patients with ARPKD from two independent cohorts and identified novel genotype-phenotype correlations during childhood and adolescence. Biallelic null variants frequently show severe courses. Additionally, our data suggest that the affected region in PKHD1 is important in determining the phenotype. Patients with two missense variants affecting amino acids 709-1837 of fibrocystin or a missense variant in this region and a null variant less frequently developed chronic kidney failure, and patients with missense variants affecting amino acids 1838-2624 showed better hepatic outcome. Variants affecting amino acids 2625-4074 of fibrocystin were associated with poorer hepatic outcome. Thus, our data expand the understanding of genotype-phenotype correlations in pediatric ARPKD patients and can lay the foundation for more precise and personalized counselling and treatment approaches.

De novo TRIM8 variants impair its protein localization to nuclear bodies and cause developmental delay, epilepsy, and focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is the main pathology underlying steroid-resistant nephrotic syndrome (SRNS) and a leading cause of chronic kidney disease. Monogenic forms of pediatric SRNS are predominantly caused by recessive mutations, while the contribution of de novo variants (DNVs) to this trait is poorly understood. Using exome sequencing (ES) in a proband with FSGS/SRNS, developmental delay, and epilepsy, we discovered a nonsense DNV in TRIM8, which encodes the E3 ubiquitin ligase tripartite motif containing 8. To establish whether TRIM8 variants represent a cause of FSGS, we aggregated exome/genome-sequencing data for 2,501 pediatric FSGS/SRNS-affected individuals and 48,556 control subjects, detecting eight heterozygous TRIM8 truncating variants in affected subjects but none in control subjects (p = 3.28 × 10-11). In all six cases with available parental DNA, we demonstrated de novo inheritance (p = 2.21 × 10-15). Reverse phenotyping revealed neurodevelopmental disease in all eight families. We next analyzed ES from 9,067 individuals with epilepsy, yielding three additional families with truncating TRIM8 variants. Clinical review revealed FSGS in all. All TRIM8 variants cause protein truncation clustering within the last exon between residues 390 and 487 of the 551 amino acid protein, indicating a correlation between this syndrome and loss of the TRIM8 C-terminal region. Wild-type TRIM8 overexpressed in immortalized human podocytes and neuronal cells localized to nuclear bodies, while constructs harboring patient-specific variants mislocalized diffusely to the nucleoplasm. Co-localization studies demonstrated that Gemini and Cajal bodies frequently abut a TRIM8 nuclear body. Truncating TRIM8 DNVs cause a neuro-renal syndrome via aberrant TRIM8 localization, implicating nuclear bodies in FSGS and developmental brain disease.

Generation and Validation of a Limited Sampling Strategy to Monitor Mycophenolic Acid Exposure in Children With Nephrotic Syndrome.

BACKGROUND: Mycophenolate mofetil (MMF) plays an increasingly important role in the treatment of children with nephrotic syndrome, especially in steroid sparing protocols. Recent publications show the relationship of exposure to its active moiety mycophenolic acid (MPA) and clinical efficacy. Performance of full-time pharmacokinetic (PK) profiles, however, is inconvenient and laborious. Established limited sampling strategies (LSS) to estimate the area under the concentration (AUC) versus time curve of MPA (MPA-AUC) in pediatric renal transplant recipients cannot be easily transferred to children suffering from nephrotic syndrome, mainly because of the lack of concomitant immunosuppressive therapy. We therefore aimed for the generation and validation of a LSS to estimate MPA exposure to facilitate therapeutic drug monitoring in children with nephrotic syndrome. METHODS: We performed 27 complete PK profiles in 23 children in remission [mean age (±SD):12.3 ± 4.26 years] to generate and validate an LSS. Sampling time points were before administration (C0) and 0.5, 1, 1.5, 2, 4, 6, 8, and 12 hours after the administration of MMF. MPA was measured by enzyme multiplied immunoassay technique. There was no concomitant treatment with calcineurin inhibitors. RESULTS: Mean daily dose of MMF was 927 ± 209 mg/m of body surface area resulting in a mean MPA-AUC0-12 value of 59.2 ± 29.3 mg × h/L and a predose level of 3.03 ± 2.24 mg/L. Between-patient variability of dose-normalized MPA-AUC0-12 was high (coefficient of variation: 45.5%). Correlation of predose levels with the corresponding MPA-AUC0-12 was moderate (r = 0.59) in a subgroup of 18 patients (20 PK profiles, generation group). An algorithm based on 3 PK sampling time points during the first 2 hours after MMF dosing (estimated AUC0-12 = 8.7 + 4.63 × C0 + 1.90 × C1 + 1.52 × C2) was able to predict MPA-AUC with a low percentage prediction error (3.88%) and a good correlation of determination (r = 0.90). Validation of this algorithm in a randomized separate group of 6 patients (7 PK profiles, validation group) resulted in comparably good correlation (r = 0.95) and low percentage prediction error (5.57%). CONCLUSIONS: An abbreviated profile within the first 2 hours after MMF dosing gives a good estimate of MPA exposure in children with nephrotic syndrome and hence has the potential to optimize MMF therapy.

Initial treatment of steroid-sensitive idiopathic nephrotic syndrome in children with mycophenolate mofetil versus prednisone: protocol for a randomised, controlled, multicentre trial (INTENT study).

INTRODUCTION: Idiopathic nephrotic syndrome is the most common glomerular disease in childhood with an incidence of 1.8 cases per 100 000 children in Germany. The treatment of the first episode implies two aspects: induction of remission and sustainment of remission. The recent Kidney Disease Improving Global Outcomes, American Academy of Pediatrics and German guidelines for the initial treatment of the first episode of a nephrotic syndrome recommend a 12-week course of prednisone. Despite being effective, this treatment is associated with pronounced glucocorticoid-associated toxicity due to high-dose prednisone administration over a prolonged period of time. The aim of the INTENT study (Initial treatment of steroid-sensitive idiopathic nephrotic syndrom in children with mycophenolate mofetil versus prednisone: protocol for a randomised, controlled, multicentre trial) is to show that an alternative treatment regimen with mycophenolic acid is not inferior regarding sustainment of remission, but with lower toxicity compared with treatment with glucocorticoids only. METHODS AND DESIGN: The study is designed as an open, randomised, controlled, multicentre trial. 340 children with a first episode of steroid-sensitive nephrotic syndrome and who achieved remission by a standard prednisone regimen will be enrolled in the trial and randomised to one of two treatment arms. The standard care group will be treated with prednisone for a total of 12 weeks; in the experimental group the treatment is switched to mycophenolate mofetil, also for a total of 12 weeks in treatment duration. The primary endpoint is the occurrence of a treated relapse within 24 months after completion of initial treatment. ETHICS AND DISSEMINATION: Ethics approval for this trial was granted by the ethics committee of the Medical Faculty of the University of Heidelberg (AFmu-554/2014). The study results will be published in accordance with the Consolidated Standards of Reporting Trials statement and the Standard Protocol Items: Recommendations for Interventional Trials guidelines. Our findings will be submitted to major international paediatric nephrology and general paediatric conferences and submitted for publication in a peer-reviewed, open-access journal. TRIAL REGISTRATION NUMBER: DRKS0006547; EudraCT2014-001991-76; Pre-result. DATE OF REGISTRATION: 30 October 2014; 24 February 2017.

No Impact of the Analytical Method Used for Determining Cystatin C on Estimating Glomerular Filtration Rate in Children.

BACKGROUND: Measurement of inulin clearance is considered to be the gold standard for determining kidney function in children, but this method is time consuming and expensive. The glomerular filtration rate (GFR) is on the other hand easier to calculate by using various creatinine- and/or cystatin C (Cys C)-based formulas. However, for the determination of serum creatinine (Scr) and Cys C, different and non-interchangeable analytical methods exist. Given the fact that different analytical methods for the determination of creatinine and Cys C were used in order to validate existing GFR formulas, clinicians should be aware of the type used in their local laboratory. In this study, we compared GFR results calculated on the basis of different GFR formulas and either used Scr and Cys C values as determined by the analytical method originally employed for validation or values obtained by an alternative analytical method to evaluate any possible effects on the performance. METHODS: Cys C values determined by means of an immunoturbidimetric assay were used for calculating the GFR using equations in which this analytical method had originally been used for validation. Additionally, these same values were then used in other GFR formulas that had originally been validated using a nephelometric immunoassay for determining Cys C. The effect of using either the compatible or the possibly incompatible analytical method for determining Cys C in the calculation of GFR was assessed in comparison with the GFR measured by creatinine clearance (CrCl). RESULTS: Unexpectedly, using GFR equations that employed Cys C values derived from a possibly incompatible analytical method did not result in a significant difference concerning the classification of patients as having normal or reduced GFR compared to the classification obtained on the basis of CrCl. Sensitivity and specificity were adequate. On the other hand, formulas using Cys C values derived from a compatible analytical method partly showed insufficient performance when compared to CrCl. CONCLUSION: Although clinicians should be aware of applying a GFR formula that is compatible with the locally used analytical method for determining Cys C and creatinine, other factors might be more crucial for the calculation of correct GFR values.

Identification of 47 novel mutations in patients with Alport syndrome and thin basement membrane nephropathy.

BACKGROUND: Alport syndrome (ATS) is a progressive hereditary nephropathy characterized by hematuria and proteinuria. It can be associated with extrarenal manifestations. In contrast, thin basement membrane nephropathy (TBMN) is characterized by microscopic hematuria, is largely asymptomatic, and is rarely associated with proteinuria and end-stage renal disease. Mutations have been identified in the COL4A5 gene in ATS and in the COL4A3 and COL4A4 genes in ATS and TBMN. To date, more than 1000 different mutations in COL4A5, COL4A3, and COL4A4 are known. METHODS: In this study mutational analysis by exon sequencing and multiplex ligation-dependent probe amplification was performed in a large European cohort of families with ATS and TBMN. RESULTS: Molecular diagnostic testing of 216 individuals led to the detection of 47 novel mutations, thereby expanding the spectrum of known mutations causing ATS and TBMN by up to 10 and 6%, respectively, depending on the database. Remarkably, a high number of ATS patients with only single mutations in COL4A3 and COL4A4 were identified. Additionally, three ATS patients presented with synonymous sequence variants that possible affect correct mRNA splicing, as suggested by in silico analysis. CONCLUSIONS: The results of this study clearly broaden the genotypic spectrum of known mutations for ATS and TBMN, which will in turn now facilitate future studies into genotype-phenotype correlations. Further studies should also examine the significance of single heterozygous mutations in COL4A3 and COL4A4 and of synonymous sequence variants associated with ATS.

Clinical and molecular characterization of patients with heterozygous mutations in wilms tumor suppressor gene 1.

BACKGROUND AND OBJECTIVES: The Wilms tumor suppressor gene 1 (WT1) plays an essential role in urogenital and kidney development. Genotype/phenotype correlations of WT1 mutations with renal function and proteinuria have been observed in world-wide cohorts with nephrotic syndrome or Wilms tumor (WT). This study analyzed mid-European patients with known constitutional heterozygous mutations in WT1, including patients without proteinuria or WT. DESIGN, SETTING, PARTICIPANTS & MEASUREMENTS: Retrospective analysis of genotype, phenotype, and treatment of 53 patients with WT1 mutation from all pediatric nephrology centers in Germany, Austria, and Switzerland performed from 2010 to 2012. RESULTS: Median age was 12.4 (interquartile range [IQR], 6-19) years. Forty-four of 53 (83%) patients had an exon mutation (36 missense, eight truncating), and nine of 53 (17%) had an intronic lysine-threonine-serine (KTS) splice site mutation. Fifty of 53 patients (94%) had proteinuria, which occurred at an earlier age in patients with missense mutations (0.6 [IQR, 0.1-1.5] years) than in those with truncating (9.7 [IQR, 5.7-11.9]; P<0.001) and splice site (4.0 [IQR, 2.6-6.6]; P=0.004) mutations. Thirteen of 50 (26%) were treated with steroids and remained irresponsive, while three of five partially responded to cyclosporine A. Seventy-three percent of all patients required RRT, those with missense mutations significantly earlier (at 1.1 [IQR, 0.01-9.3] years) than those with truncating mutations (16.5 [IQR, 16.5-16.8]; P<0.001) and splice site mutations (12.3 [IQR, 7.9-18.2]; P=0.002). Diffuse mesangial sclerosis was restricted to patients with missense mutations, while focal segmental sclerosis occurred in all groups. WT occurred only in patients with exon mutations (n=19). Fifty of 53 (94%) patients were karyotyped: Thirty-one (62%) had XY and 19 (38%) had XX chromosomes, and 96% of male karyotypes had urogenital malformations. CONCLUSIONS: Type and location of WT1 mutations have predictive value for the development of proteinuria, renal insufficiency, and WT. XY karyotype was more frequent and associated with urogenital malformations in most cases.

Mutations in multiple PKD genes may explain early and severe polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is typically a late-onset disease caused by mutations in PKD1 or PKD2, but about 2% of patients with ADPKD show an early and severe phenotype that can be clinically indistinguishable from autosomal recessive polycystic kidney disease (ARPKD). The high recurrence risk in pedigrees with early and severe PKD strongly suggests a common familial modifying background, but the mechanisms underlying the extensive phenotypic variability observed among affected family members remain unknown. Here, we describe severely affected patients with PKD who carry, in addition to their expected familial germ-line defect, additional mutations in PKD genes, including HNF-1ß, which likely aggravate the phenotype. Our findings are consistent with a common pathogenesis and dosage theory for PKD and may propose a general concept for the modification of disease expression in other so-called monogenic disorders.

Hyperintense lesions in brain MRI after exposure to a mercuric chloride-containing skin whitening cream.

Exposure to inorganic mercury (Hg) is a serious problem presenting with a combination of neurological and psychiatric symptoms along with weight loss, pruritus, erythema, arterial hypertension, tachycardia, and renal tubular dysfunction. We report a 4-year-old girl with chronic intoxication of inorganic mercury secondary to the accidental use of an Hg2Cl2- and HgCl2-containing skin whitening cream (urine level of Hg, 41.1 µg/l; reference level, < 25 µg/l). Under treatment with dimercapto-1-propansulficacid, Hg level in the urine raised to 1,175.5 µg/l, neurological deterioration occurred, and brain magnetic resonance imaging (MRI) showed on fluid attenuated inversion recovery sequences new hyperintense lesions in the subcortical white matter. After 4 months, clinical signs and symptoms and brain MRI findings resolved. This is a first case of inorganic mercury poisoning showing hyperintense lesions in brain MRI and confirms earlier cases showing transient deterioration during chelation therapy. Although urinary excretion could be enhanced during chelation therapy, signs and symptoms of intoxication could be worsened.

Reduced methotrexate clearance and renal impairment in a boy with osteosarcoma and earlier undetected autosomal dominant polycystic kidney disease (ADPKD).

We report a 12-year-old boy with osteoblastic osteosarcoma of the right femur. He was started on chemotherapy according to the EURAMOS/COSS 1 protocol. Chemotherapy with doxorubicin/cisplatin resulted in reversible acute renal failure and methotrexate levels were repeatedly elevated. Family history suggested an autosomal dominant polycystic kidney disease. Genetic testing revealed a novel mutation c.10707_10712del (p.Val3569_3570del) in exon 36 of the PKD1 gene. Patients with autosomal dominant polycystic kidney disease may be at risk for acute renal failure during chemotherapy without signs of renal impairment. A careful family history is important to exclude risk factors for renal impairment before introducing high-dose chemotherapy.

Novel heterozygous COL4A3 mutation in a family with late-onset ESRD.

Thin basement membrane nephropathy (TBMN) and Alport syndrome (ATS) are genetically heterogeneous conditions characterized by structural abnormalities in the glomerular basement membrane (GBM). TBMN presents with hematuria, minimal proteinuria, and normal renal function. Although TBMN is an autosomal dominant disease (COL4A3 and COL4A4), ATS can be inherited X-linked (COL4A5), autosomal recessive, or autosomal dominant (both COL4A3 and COL4A4). The clinical course of TBMN is usually benign, whereas ATS typically results in end-stage renal disease (ESRD). Nevertheless, there is a broad spectrum of clinical phenotypes caused by mutations in COL4A3 or COL4A4. We report an Italian family who presented with hematuria and mild proteinuria. Mutational analysis showed a novel heterozygous mutation p.G291E in exon 15 of the COL4A3 gene. Many different mutations in COL4A3 and COL4A4 that cause TBMN have already been identified, but most genetic variability in these genes has been found to cause autosomal ATS. A valid genotype-phenotype correlation for TBMN or ATS is not yet known. Therefore, it is important to identify new mutations by direct sequencing to clarify their clinical importance, to assess the prognosis of the disease, and to avoid renal biopsy.

A novel mutation in AVPR2 causing congenital nephrogenic diabetes insipidus with complete resistance to antidiuretic hormone.

A 6-month-old male infant presented with failure to thrive. Hypernatraemia and elevated serum osmolality in the presence of low urine sodium and osmolality led to the diagnosis of diabetes insipidus. Administration of 1-deamino-8-D-arginine vasopressin (dDAVP) neither decreased urine volume nor increased urine osmolality indicating congenital nephrogenic diabetes insipidus. Molecular analysis in the arginine-vasopressin receptor-2 gene (AVPR2) located on chromosome Xq28 demonstrated a novel 5-base pair deletion (c.962-966delACCCC; g.1429-1433delACCCC) leading to a shift of the reading frame (p.Asn321fs) and a premature termination codon implying an absent or non-functional protein. Treatment with hydrochlorothiazide, amiloride and indomethacin led to a favourable clinical course.

Not all farming environments protect against the development of asthma and wheeze in children.

BACKGROUND: 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. Inconsistent data on the relation of farming to asthma and wheeze have raised some doubt about a true protective effect. OBJECTIVE: We sought to study the differential effects of farm-associated exposures on specific asthma-related health outcomes. METHODS: The cross-sectional Prevention of Allergy Risk Factors for Sensitization in Children Related to Farming and Anthroposophic Lifestyle study included 8263 school-age children from rural areas in 5 European countries. Information on farm-related exposures and health outcomes was obtained by using questionnaires. In subsamples allergen-specific IgE and RNA expression of CD14 and Toll-like receptor genes were measured, and dust from children's mattresses was evaluated for microbial components. RESULTS: Inverse relations with a diagnosis of asthma were found for pig keeping (odds ratio [OR], 0.57; 95% CI, 0.38-0.86), farm milk consumption (OR, 0.77; 95% CI, 0.60-0.99), frequent stay in animal sheds (OR, 0.71; 95% CI, 0.54-0.95), child's involvement in haying (OR, 0.56; 95% CI, 0.38-0.81), and use of silage (OR, 0.55; 95% CI, 0.31-0.98; for nonatopic asthma) and in Germany for agriculture (OR, 0.34; 95% CI, 0.22-0.53). Protective factors were related with higher expression levels of genes of the innate immunity. Potential risk factors for asthma and wheeze were also identified in the farm milieu. Levels of endotoxin and extracellular polysaccharides were related to the health outcomes independently of the farm exposures. CONCLUSIONS: The protective effect of being raised in a farm environment was ascribed to distinct exposures. CLINICAL IMPLICATIONS: The development of atopic sensitization and atopic and nonatopic asthma is most likely determined by different environmental factors, possibly reflecting distinct pathomechanisms.