Adiponectin receptor genes : mutation screening in syndromes of insulin resistance and association studies for type 2 diabetes and metabolic traits in UK populations Aims/hypothesis Adiponectin is an adipokine with insulin-sensitising and anti-atherogenic properties . Several reports suggest that genetic variants in the adiponectin gene are associated with circulating levels of adiponectin , insulin sensitivity and type 2 diabetes risk . Recently two receptors for adiponectin have been cloned . Genetic studies have yielded conflicting results on the role of these genes and type 2 diabetes predisposition . In this study we aimed to evaluate the potential role of genetic variation in these genes in syndromes of severe insulin resistance , type 2 diabetes and in related metabolic traits in UK Europid populations . Introduction Adiponectin , encoded by the gene ADIPOQ -LRB- also known as 30-kDa adipocyte complement-related protein , Acrp30 , APM-1 , APM1 , ACDC , and gelatin-binding protein-28 or GBP28 -RRB- , is an adipokine with insulin-sensitising -LSB- 1 , 2 -RSB- and anti-atherogenic actions -LSB- 3 -RSB- . Its levels correlate strongly with insulin sensitivity in humans and animal models , and increasing levels of plasma adiponectin produce a sensitising effect to the biological action of insulin -LSB- 4 -RSB- . Several genetic reports have detected association between adiponectin gene variants and obesity , insulin resistance , type 2 diabetes , and adiponectin levels -LSB- 5 -- 9 -RSB- . Recently , two adiponectin receptors were identified , adiponectin receptor 1 -LRB- ADIPOR1 -RRB- , cloned from a human skeletal muscle expression library , and adiponectin receptor 2 -LRB- ADIPOR2 -RRB- , identified using computational tools by Yamauchi et al. -LSB- 10 -RSB- . In mice , Adipor1 is expressed ubiquitously , with higher levels in skeletal muscle , and has a higher affinity for the globular form of adiponectin . Adipor2 , on the other hand , is most abundant in the liver and preferentially binds the full-length form of adiponectin -LSB- 10 -RSB- . In contrast , both human ADIPOR1 -LRB- 375aa -RRB- and ADIPOR2 -LRB- 311aa -RRB- were predominantly expressed in skeletal muscle -LSB- 10 , 11 -RSB- . In Mexican Americans , glucose-tolerant individuals with a family history of type 2 diabetes were reported to exhibit significantly lower levels of mRNA for ADIPOR1 and ADIPOR2 in skeletal muscle than subjects without a family history of diabetes . mRNA levels of both receptors were also reported to positively correlate with glucose disposal -LSB- 11 -RSB- . It is possible therefore that lower expression or altered function of the receptors would predispose to increased insulin resistance and type 2 diabetes . In fact , common variants in the ADIPOR1 gene were recently tested for association in a case -- control study with white and African American individuals , but no association was reported -LSB- 12 -RSB- . Two additional studies have evaluated the role of adiponectin receptor variants and risk of type 2 diabetes . In the Old Order Amish population , two intronic variants in ADIPOR1 were reported to associate with risk of type 2 diabetes , while in ADIPOR2 an extended haplotype block was associated with increased risk of disease -LSB- 13 -RSB- . In contrast , in a Japanese population no associations between polymorphisms in adiponectin receptor genes and risk of type 2 diabetes were detected -LSB- 14 -RSB- . More recently , studies in French and Finnish populations reported no association between ADIPOR1 single nucleotide polymorphisms -LRB- SNPs -RRB- and type 2 diabetes -LSB- 15 , 16 -RSB- , although evidence for association between rs767870 in ADIPOR2 and type 2 diabetes in a French population has been suggested -LSB- 15 -RSB- . In light of these studies , and the potential role of these receptors in insulin action and diabetes , we sought to identify and investigate the effects of genetic variants in these genes in UK populations . Subjects and methods Participants Severe insulin resistance cohort A cohort of human patients with severe insulin resistance -LRB- SIR cohort -RRB- was collected at the University of Cambridge , UK . The inclusion criteria for this cohort were : -LRB- 1 -RRB- fasting insulin > 150 pmol/l or exogenous insulin requirement > 200 U/day ; -LRB- 2 -RRB- acanthosis nigricans ; and -LRB- 3 -RRB- BMI < 33 kg/m2 . In the present study , 129 patients from this cohort were screened for mutations in exons and splice junctions of ADIPOR1 and ADIPOR2 genes . Cambridge Local Research Ethics Committee approval was obtained , and informed consent was received from all individuals before participation . Cambridgeshire Case -- Control Study The Cambridgeshire Case -- Control Study has been described previously -LSB- 17 -RSB- . Briefly , this population-based case -- control study consists of 552 type 2 diabetes patients and matched control subjects . DNA was available from 516 cases and control subjects for this study . The cases were a random sample of Europid men and women with type 2 diabetes , aged 47 to 75 years , from a population-based diabetes register in a geographically defined region in Cambridgeshire , UK . The presence of type 2 diabetes in these participants was defined as onset of diabetes after the age of 30 years without use of insulin therapy in the first year after diagnosis . The control participants were individually matched to each of the diabetic subjects by age , sex and geographical location , but not by BMI . Potential control subjects with HbA1c levels greater than 6 % were excluded , as this subgroup could have contained a higher proportion of individuals with previously undiagnosed diabetes . Ethical approval for the study was granted by the Cambridge Local Research Ethics Committee . EPIC-Norfolk participants This is a nested case -- control study within the EPIC-Norfolk prospective cohort study ; both the case -- control and full cohort study -LSB- 18 , 19 -RSB- have been described in detail previously . Briefly , the case -- control study consists of 417 incident type 2 diabetes cases and two sets of 417 control subjects , matched on age , sex , time in study and family physician , with the second set additionally matched for BMI . A case was defined by a physician 's diagnosis of type 2 diabetes , with no insulin prescribed within the first year after diagnosis , and/or HbA1c > 7 % at baseline or the follow-up health check . Controls were selected from those in the cohort who had not reported diabetes , cancer , stroke or myocardial infarction at baseline , and who had not developed diabetes by the time of selection . Potential control subjects with measured HbA1c levels > 6 % were excluded . DNA was available for this analysis from 354 cases and 741 control subjects . Ethical approval for the study was granted by the Norwich Local Research Ethics Committee . Ely Study This is a population-based cohort study of the aetiology and pathogenesis of type 2 diabetes and related metabolic disorders in the UK -LSB- 20 -RSB- . It uses an ethnically homogeneous Europid population , in which phenotypic data were recorded at the outset and after 4.5 years . The cohort was recruited from a population sampling frame with a high response rate -LRB- 74 % -RRB- , making it representative of the general population for this area in Eastern England . This analysis included 1,721 men and women , aged 35 -- 79 years and without diagnosed diabetes , who attended the study clinic for a health check between 2000 and 2004 . Of these , 1,005 were attending a follow-up health check , while the remaining 716 were newly recruited in 2000 from the original population sampling frame . Participants attending the health check underwent standard anthropometric measurements and a 75-g oral glucose tolerance test . Plasma glucose was measured using the hexokinase method . Plasma insulin was measured by two-site immunometric assays with either 125I or alkaline phosphatase labels . Cross-reactivity with intact proinsulin was less than 0.2 % and interassay CVs were less than 7 % . Ethical approval for the study was granted by the Cambridge Local Research Ethics Committee . PCR and sequencing Genomic DNA from patients was randomly preamplified in a GenomiPhi reaction -LRB- GE Healthcare UK , Chalfont St. Giles , UK -RRB- prior to amplification with gene-specific primers . Primers were designed using Primer3 software -LSB- 21 -RSB- to cover all coding exons and splice junctions . PCR primers and expected product sizes are described in Electronic supplementary material -LRB- ESM -RRB- Table 1 . Following PCR , performed using standard conditions , products were purified using exonuclease I and shrimp alkaline phosphatase -LRB- USB Corporation , Cleveland , OH , USA -RRB- , and bi-directional sequencing was performed using a DNA sequencing kit -LRB- Big Dye Terminator 3.1 ; Applied Biosystems , Foster City , CA , USA -RRB- . Sequencing reactions were run on ABI3700 capillary machines -LRB- Applied Biosystems -RRB- and sequences were analysed using Mutation Surveyor version .2.20 -LRB- SoftGenetics LLC , State College , PA , USA -RRB- . Genotyping SNP selection All SNPs with a minor allele frequency greater than 2 % in our SIR cohort were selected for genotyping . To increase coverage in areas not re-sequenced , a number of dbSNPs were selected in an attempt to eliminate gaps , between genotyped SNPs , of greater than 2.5 -- 3 kb on average -LRB- additional SNPs were selected prior to HapMap phase I data release -RRB- . SNP choice was based on the following criteria : -LRB- 1 -RRB- all putative non-synonymous SNPs in dbSNP were selected irrespective of whether or not there was frequency or validation information for the SNP ; -LRB- 2 -RRB- SNPs with frequency information were selected if their minor allele frequency was greater than or equal to 5 % ; -LRB- 3 -RRB- for SNPs with no frequency information the choice was based on whether the SNP was a double-hit SNP , had been validated by-cluster or by-submitter ; and finally -LRB- 4 -RRB- some SNPs with no validation information were included to try and eliminate gaps of greater than 2.5 -- 3 kb between SNPs selected for genotyping . Genotyping and quality control Samples were arrayed on 96-well plates with three replicates and one water control per plate . For the case -- control populations , cases and control samples were randomly distributed across each 96-well plate , with approximately the same number of cases and controls per plate . Genotyping of samples was performed in 384-well plates at the Wellcome Trust Sanger Institute , Cambridge , using an adaptation of the homogenous MassExtend protocol for the MassArray system -LRB- Sequenom , San Diego , CA , USA -RRB- -LSB- 22 -RSB- . Assay results for case -- control are described in ESM Table 2 . The following criteria were used to pass assays resulting from genotyping : -LRB- 1 -RRB- call rates had to be greater than or equal to 90 % -LRB- in one case a call rate of 88 % was accepted -RRB- ; -LRB- 2 -RRB- concordance rates between duplicate samples had to be greater than or equal to 98 % ; -LRB- 3 -RRB- minor allele frequency had to be greater than or equal to 1 % in the genotyped populations ; -LRB- 4 -RRB- agreement with Hardy -- Weinberg equilibrium was tested separately in cases and controls using a χ2 goodness-of-fit test , and if p < 0.01 in controls , the assay was failed , while if p < 0.01 in cases , the assay was flagged but included in primary analysis . Assays that failed quality control were excluded from further analysis . In total we analysed results from nine SNPs in ADIPOR1 and 15 SNPs from ADIPOR2 . Statistical analysis All analyses used SAS 8.02 -LRB- SAS Institute , Cary , NC , USA -RRB- or Stata 7.0 -LRB- Stata Corporation , College Station , TX , USA -RRB- statistical programs , unless otherwise stated . All used genotypes were in Hardy -- Weinberg equilibrium . The pair-wise linkage disequilibrium -LRB- LD -RRB- coefficient for the controls -LRB- r2 -RRB- was calculated for genotyped SNPs and is represented in Fig. 1 . For each SNP , two primary models were used to assess association with diabetes and quantitative traits , the linear trend -LRB- additive model -RRB- on 1df and the general model on 2df . Since the results from these analyses were not materially different , we present only the results from the linear trend test . Tests for association were performed by logistic regression combined in the two case -- control populations adjusting for age , sex and population . Between-study heterogeneity was tested by log-likelihood ratio tests . Quantitative trait analysis was undertaken in the Ely Study population . Association between fasting plasma insulin , fasting and 2-h post-load plasma glucose , 30-min insulin increment -LRB- 30-min insulin minus fasting insulin over 30-min glucose in an OGTT -RRB- , BMI and genotype was tested in a multiple regression model , adjusted for age and sex . Fig. 1Genomic structure and pair-wise marker LD in ADIPOR1 -LRB- a -RRB- and ADIPOR2 -LRB- b -RRB- . The location of SNPs identified in this study and/or genotyped is represented along the gene -LRB- bold type : SNPs selected for genotyping , and failed or monomorphic SNPs -RRB- . Exons are represented in boxes -LRB- black for coding and open for untranslated -RRB- . Introns and flanking sequences appear as lines . The pair-wise marker LD measured by r2 statistics is shown below the genomic structures and indicated by the shade of grey blocks -LRB- white to black -RRB- and the r2 value . a , b , isoforms a and b in ADIPOR2 Results To evaluate whether genetic variation in ADIPOR1 and ADIPOR2 contributed to severe insulin resistance in humans , we sequenced exons and splice junctions in both genes in 129 individuals from our SIR cohort . These individuals were unrelated and had a variety of syndromes of severe insulin resistance -LSB- 23 -RSB- . We identified 13 and 29 polymorphisms in ADIPOR1 and ADIPOR2 respectively , none of which altered the protein sequence of either gene -LRB- ESM Table 3 -RRB- . This suggests that no functional mutations were identified in these genes . We next tested whether common variants at these genes impacted on type 2 diabetes predisposition or related quantitative traits in UK Europid populations . We selected all variants with a minor allele frequency greater than 2 % in the SIR cohort and supplemented our SNP selection with additional variants from the dbSNP database plus SNPs with significant association results in other published studies . Thirteen polymorphisms from ADIPOR1 and 23 polymorphisms from ADIPOR2 were selected for genotyping in two type 2 diabetes case -- control studies -LRB- n = 2,127 -RRB- and one metabolic quantitative trait study -LRB- n = 1,721 -RRB- -LRB- Fig. 1 -RRB- . Those SNPs that passed our genotyping quality control criteria -LRB- described in methods -RRB- were used to investigate the degree of LD in control individuals across ADIPOR1 and ADIPOR2 . In total nine SNPs in ADIPOR1 and 15 SNPs in ADIPOR2 were included in the analysis . LD was measured by r2 statistic and is depicted in Fig. 1 . Under both general and linear trend models no evidence was found for statistically significant associations between SNPs and disease risk -LRB- Table 1 -RRB- . In ADIPOR1 , SNPs rs2275738 , rs2275735 and rs10581 were removed from the quantitative trait analysis as they were out of Hardy -- Weinberg equilibrium -LRB- p < 0.01 -RRB- . For the remaining SNPs there was also no evidence for association of the SNPs tested with BMI , fasting and 2-h glucose levels , fasting insulin or 30-min insulin incremental response -LRB- ESM Table 4 -RRB- . In ADIPOR2 a few SNPs showed nominally significant association with BMI and 2-h glucose levels . However , these results are likely to be chance findings given the number of statistical tests performed -LRB- ESM Table 4 -RRB- . Table 1ADIPOR1 and ADIPOR2 SNP association results with type 2 diabetesGene SNPAlleleCasesControlsOdds ratiop value trendp value homogeneity1/211 -LRB- % -RRB- 12 -LRB- % -RRB- 22 -LRB- % -RRB- 11 -LRB- % -RRB- 12 -LRB- % -RRB- 22 -LRB- % -RRB- -LRB- 95 % CI -RRB- ADIPOR1rs6666089G/A378 -LRB- 47.6 -RRB- 340 -LRB- 42.8 -RRB- 76 -LRB- 9.6 -RRB- 536 -LRB- 46.2 -RRB- 525 -LRB- 45.2 -RRB- 100 -LRB- 8.6 -RRB- 1.00 -LRB- 0.86,1.15 -RRB- 0.96010.750 rs1539355A/G374 -LRB- 46.6 -RRB- 346 -LRB- 43.1 -RRB- 82 -LRB- 10.2 -RRB- 558 -LRB- 45.9 -RRB- 547 -LRB- 45.0 -RRB- 111 -LRB- 9.1 -RRB- 1.02 -LRB- 0.89,1.18 -RRB- 0.73500.649 rs2275738C/T277 -LRB- 34.4 -RRB- 379 -LRB- 47.1 -RRB- 149 -LRB- 18.5 -RRB- 395 -LRB- 34.0 -RRB- 551 -LRB- 47.4 -RRB- 216 -LRB- 18.6 -RRB- 0.97 -LRB- 0.85,1.1 -RRB- 0.61090.496 IVS3 +85 C/G707 -LRB- 92.1 -RRB- 61 -LRB- 7.9 -RRB- 0 -LRB- 0 -RRB- 1046 -LRB- 92.6 -RRB- 82 -LRB- 7.3 -RRB- 2 -LRB- 0.2 -RRB- 1.03 -LRB- 0.73,1.45 -RRB- 0.87210.654 rs2275735C/T763 -LRB- 92.0 -RRB- 66 -LRB- 8.0 -RRB- 0 -LRB- 0 -RRB- 1152 -LRB- 93.5 -RRB- 79 -LRB- 6.4 -RRB- 1 -LRB- 0.1 -RRB- 1.24 -LRB- 0.88,1.75 -RRB- 0.22390.228 rs1342387C/T249 -LRB- 30.4 -RRB- 414 -LRB- 50.5 -RRB- 156 -LRB- 19.0 -RRB- 362 -LRB- 29.5 -RRB- 634 -LRB- 51.7 -RRB- 231 -LRB- 18.8 -RRB- 0.96 -LRB- 0.85,1.1 -RRB- 0.59090.603 rs10581G/A741 -LRB- 93.8 -RRB- 46 -LRB- 5.8 -RRB- 3 -LRB- 0.4 -RRB- 1079 -LRB- 95.7 -RRB- 46 -LRB- 4.1 -RRB- 3 -LRB- 0.3 -RRB- 1.35 -LRB- 0.92,1.97 -RRB- 0.12810.466 rs7539542C/G373 -LRB- 47.4 -RRB- 336 -LRB- 42.7 -RRB- 78 -LRB- 9.9 -RRB- 554 -LRB- 46.9 -RRB- 510 -LRB- 43.2 -RRB- 117 -LRB- 9.9 -RRB- 1.00 -LRB- 0.87,1.15 -RRB- 0.95660.193 rs2185781C/T532 -LRB- 63.6 -RRB- 256 -LRB- 30.6 -RRB- 49 -LRB- 5.9 -RRB- 789 -LRB- 63.8 -RRB- 398 -LRB- 32.2 -RRB- 50 -LRB- 4.0 -RRB- 1.08 -LRB- 0.92,1.26 -RRB- 0.34250.495 ADIPOR2rs1029629A/C353 -LRB- 44.6 -RRB- 372 -LRB- 47.0 -RRB- 67 -LRB- 8.5 -RRB- 537 -LRB- 44.9 -RRB- 528 -LRB- 44.1 -RRB- 132 -LRB- 11.0 -RRB- 0.94 -LRB- 0.81,1.08 -RRB- 0.34970.265 rs11061971A/T228 -LRB- 28.1 -RRB- 409 -LRB- 50.4 -RRB- 174 -LRB- 21.5 -RRB- 349 -LRB- 29.5 -RRB- 589 -LRB- 49.7 -RRB- 246 -LRB- 20.8 -RRB- 1.05 -LRB- 0.93,1.20 -RRB- 0.43940.092 rs4766415A/T207 -LRB- 25.6 -RRB- 411 -LRB- 50.7 -RRB- 192 -LRB- 23.7 -RRB- 323 -LRB- 27.1 -RRB- 586 -LRB- 49.2 -RRB- 282 -LRB- 23.7 -RRB- 1.04 -LRB- 0.92,1.19 -RRB- 0.51590.074 rs767870A/G575 -LRB- 71.3 -RRB- 214 -LRB- 26.5 -RRB- 18 -LRB- 2.2 -RRB- 857 -LRB- 72.3 -RRB- 310 -LRB- 26.1 -RRB- 19 -LRB- 1.6 -RRB- 1.08 -LRB- 0.9,1.3 -RRB- 0.38430.095 rs2286384G/C214 -LRB- 25.7 -RRB- 429 -LRB- 51.5 -RRB- 190 -LRB- 22.8 -RRB- 329 -LRB- 26.8 -RRB- 618 -LRB- 50.3 -RRB- 282 -LRB- 22.9 -RRB- 1.03 -LRB- 0.9,1.17 -RRB- 0.68370.037 * rs2286383C/T237 -LRB- 28.6 -RRB- 418 -LRB- 50.4 -RRB- 174 -LRB- 21.0 -RRB- 368 -LRB- 30.1 -RRB- 602 -LRB- 49.2 -RRB- 253 -LRB- 20.7 -RRB- 1.05 -LRB- 0.92,1.19 -RRB- 0.45980.068 I290C/A618 -LRB- 75.2 -RRB- 193 -LRB- 23.5 -RRB- 11 -LRB- 1.3 -RRB- 940 -LRB- 77.9 -RRB- 252 -LRB- 21.0 -RRB- 14 -LRB- 1.2 -RRB- 1.18 -LRB- 0.97,1.44 -RRB- 0.09850.118 rs9805042C/T603 -LRB- 75.8 -RRB- 180 -LRB- 22.6 -RRB- 12 -LRB- 1.5 -RRB- 900 -LRB- 79.0 -RRB- 225 -LRB- 19.8 -RRB- 14 -LRB- 1.2 -RRB- 1.17 -LRB- 0.96,1.43 -RRB- 0.12820.051 rs2286382G/A791 -LRB- 94.5 -RRB- 45 -LRB- 5.4 -RRB- 1 -LRB- 0.1 -RRB- 1159 -LRB- 94.2 -RRB- 71 -LRB- 5.8 -RRB- 0 -LRB- 0 -RRB- 0.98 -LRB- 0.67,1.43 -RRB- 0.90540.664 rs12342C/T370 -LRB- 44.6 -RRB- 376 -LRB- 45.3 -RRB- 84 -LRB- 10.1 -RRB- 543 -LRB- 44.3 -RRB- 540 -LRB- 44.1 -RRB- 142 -LRB- 11.6 -RRB- 0.96 -LRB- 0.84,1.1 -RRB- 0.54610.398 rs1044471C/T227 -LRB- 27.8 -RRB- 420 -LRB- 51.5 -RRB- 169 -LRB- 20.7 -RRB- 334 -LRB- 27.6 -RRB- 592 -LRB- 49.0 -RRB- 282 -LRB- 23.3 -RRB- 0.93 -LRB- 0.82,1.05 -RRB- 0.25000.115 rs2286380A/T621 -LRB- 77.0 -RRB- 176 -LRB- 21.8 -RRB- 10 -LRB- 1.2 -RRB- 947 -LRB- 79.0 -RRB- 238 -LRB- 19.8 -RRB- 15 -LRB- 0.0125 -RRB- 1.12 -LRB- 0.91,1.37 -RRB- 0.27620.155 rs13219T/C251 -LRB- 30.2 -RRB- 403 -LRB- 48.4 -RRB- 178 -LRB- 21.4 -RRB- 369 -LRB- 30.1 -RRB- 618 -LRB- 50.4 -RRB- 238 -LRB- 19.4 -RRB- 1.04 -LRB- 0.91,1.18 -RRB- 0.56100.276 rs2286379T/C259 -LRB- 31.0 -RRB- 399 -LRB- 47.7 -RRB- 178 -LRB- 21.3 -RRB- 376 -LRB- 30.6 -RRB- 614 -LRB- 50.0 -RRB- 239 -LRB- 19.4 -RRB- 1.03 -LRB- 0.91,1.17 -RRB- 0.64270.269 rs3815325G/A490 -LRB- 60.6 -RRB- 273 -LRB- 33.7 -RRB- 46 -LRB- 5.7 -RRB- 712 -LRB- 58.7 -RRB- 437 -LRB- 36.0 -RRB- 64 -LRB- 5.3 -RRB- 0.96 -LRB- 0.82,1.11 -RRB- 0.57850.26311 , homozygous for allele 1 , 12 , heterozygous , 22 , homozygous for allele 2 . Genotype counts are shown with frequency -LRB- % -RRB- , odds ratios are shown per allele 2 with significance calculated for the linear trend -LRB- p value trend -RRB- . Homogeneity in results from association between EPIC and Cambridge Case -- Control populations was tested -LRB- p value homogeneity -RRB- * p values ≤ 0.05 Discussion The current study , including sequencing of 129 patients with syndromes of severe insulin resistance and genotyping of both population-based type 2 diabetes case -- control studies -LRB- n = 2,127 -RRB- and a metabolic quantitative trait study -LRB- n = 1,721 -RRB- , suggests that ADIPOR1 and ADIPOR2 genetic variants are unlikely to be major risk factors for type 2 diabetes and insulin resistance in UK Europid populations . Although sequencing of ADIPOR1 and ADIPOR2 genes in a cohort of patients with syndromes of severe insulin resistance -LRB- n = 129 -RRB- led to the identification of 42 polymorphisms , including 21 novel rare variants , none altered the protein sequence . Given that this group of patients comprises a heterogeneous cohort representative of a variety of syndromes of extreme insulin resistance , the lack of variants affecting the protein sequence suggests that functional mutations in the genes ADIPOR1 and ADIPOR2 are not major causes of extreme insulin resistance in humans . For ADIPOR1 , our data in case -- control studies are consistent with previous reports showing that SNPs in this gene are not associated with type 2 diabetes risk in Europid -LSB- 12 , 15 , 16 -RSB- , African -LSB- 12 -RSB- or Japanese populations -LSB- 14 -RSB- . This is in contrast to evidence from the Old Order Amish , where an association of rs2275738 -LRB- and rs2275737 which is in perfect LD with it -RRB- and rs1342387 with type 2 diabetes risk has been reported -LSB- 13 -RSB- . Since the Amish represent an isolated population , it is possible that variants in ADIPOR1 play a role in type 2 diabetes predisposition among them , which is not apparent in more heterogeneous populations . However , given that both reported associated SNPs are present in all populations , it is unlikely that either is the true causal variant , although they could be detecting , through LD , the effect of an untested SNP . Alternatively , given the relative small sample sizes used and lack of adjustment for multiple testing , the authors may have reported a false -- positive association . For ADIPOR2 the data are less consistent . While no evidence of association between ADIPOR2 SNPs and type 2 diabetes was present in a Japanese population -LSB- 14 -RSB- , an association has been suggested between SNP rs767870 and type 2 diabetes risk in French populations -LSB- 15 -RSB- . Our data , and those from the Old Order Amish -LSB- 13 -RSB- , do not support this finding . Notably , in the French population , meta-analysis of rs767870 , including 1,380 individuals with type 2 diabetes and 1,496 controls , demonstrated allelic association of nominal significance only -LRB- p = 0.02 -RRB- , while the most significant result was under a recessive model -LRB- p = 0.0018 -RRB- . Our study has only 23 % power to detect such small recessive effects -LRB- odds ratio 1.3 -RRB- with an allele frequency of 0.15 , and this could explain our discrepant result . Further large scale studies of this SNP in additional populations will be required to elucidate its role in conferring risk of disease . A haplotype was also reported to increase risk of diabetes in the Old Order Amish -LSB- 13 -RSB- . However , when we performed haplotype analyses in both ADIPOR1 and ADIPOR2 , we were unable to detect any significant associations with diabetes risk -LRB- data not shown -RRB- , and this discrepancy might also be accounted for by differences in the populations studied . With regard to quantitative metabolic traits , to date two studies have reported nominally significant results with insulin sensitivity and body size -LSB- 16 , 24 -RSB- . The first study showed nominally significant associations between two tightly linked SNPs -LRB- rs6666089 and an intronic -- 1927 SNP -RRB- in ADIPOR1 and decreased insulin sensitivity and increased HbA1c levels -LSB- 24 -RSB- . We did not test association with HbA1c , but have not replicated any association with insulin sensitivity -LRB- including with SNP rs6666089 -RRB- as assessed by fasting insulin measurements . Recently Kantartzis and colleagues reported that the association between rs6666089 and insulin sensitivity is observed only in more obese , but not in lean individuals -LSB- 25 -RSB- . This dependence on the degree of adiposity could explain some of the discrepant results observed for this SNP , if there are substantial differences in mean BMI between populations tested . To explore this hypothesis , we performed SNP × BMI interaction tests -LRB- using BMI both as a continuous trait and splitting the population above and below the median -RRB- on all ADIPOR1 SNPs tested . Our data provided no evidence for such an interaction -LRB- data not shown -RRB- . We also specifically tested for association between rs6666089 , and measures of insulin and glucose in participants with BMI above and below 25 . Again we found no statistically significant difference between the two groups -LRB- p > 0.3 -RRB- . Therefore , it is unlikely that BMI differences between populations are at the root of our discrepant results . Of note , our data can not exclude possible small effects of this SNP on insulin sensitivity , in particular if this effect is only observed in subjects with higher BMI . The second study suggested there was evidence of association between two markers -LRB- rs10920534 and rs2275738 -RRB- and BMI , but this evidence came from men only . Furthermore , three other markers -LRB- rs10920534 , rs12045862 and rs7539542 -RRB- were reported to associate with fasting and 2-h insulin levels , particularly in men at baseline -LSB- 16 -RSB- . We did not test rs10920534 , but did test rs6666089 -LRB- D ′ = r2 = 1 with rs10920534 -RRB- and found no evidence for SNP × sex interaction on any of the quantitative traits we analysed . Our data also did not replicate the sex effect of SNP rs7539542 on insulin measurements . Although we did not test SNP rs12045862 , previously published data was conflicting . Thus while the C allele was suggested to be associated with higher 2-h insulin levels in men -LRB- p = 0.027 -RRB- , in women the T allele was associated with higher levels -LRB- p = 0.029 -RRB- -LSB- 16 -RSB- . Given that neither we , nor others -LSB- 24 -RSB- , have found evidence for sex × SNP interaction effects on measures of insulin sensitivity , we suggest that further confirmatory studies are required to test this hypothesis . Until recently , remaining uncertainty regarding the identity of the true physiological receptors for adiponectin -LSB- 26 -RSB- had hampered interpretation of the functional relevance of polymorphisms in ADIPOR1 and ADIPOR2 with respect to adiponectin 's insulin sensitising effects . However , a recent yeast two-hybrid screen identified an ADIPOR1 interacting molecule , APPL1 , thought to mediate many of the effects of adiponectin -LSB- 27 -RSB- . This molecule was shown to interact with both ADIPOR1 and ADIPOR2 in an adiponectin-sensitive manner , and was shown to mediate many of adiponectin 's insulin-sensitising effects . This suggests that ADIPOR1 and ADIPOR2 could be therapeutic targets for drug development and should renew interest in association studies , such as those we present here , testing polymorphisms in ADIPOR1 and ADIPOR2 for effects on type 2 diabetes risk and metabolic traits . In summary , sequencing of ADIPOR1 and ADIPOR2 genes in a cohort of patients with syndromes of severe insulin resistance -LRB- n = 129 -RRB- suggests that functional mutations in these genes are not a major cause of extreme insulin resistance in humans . Furthermore , testing of common genetic variants -LRB- n = 24 -RRB- did not find evidence for association of these genes with type 2 diabetes risk -LRB- n = 2,127 -RRB- or with five additional quantitative metabolic traits -LRB- n = 1,721 -RRB- . These data suggest that ADIPOR1 and ADIPOR2 variants are unlikely to be major risk factors for type 2 diabetes and insulin resistance in UK Europid populations , although more detailed analyses of gene variants may be required to exclude a potential minor role of these genes in insulin resistance and glucose homeostasis . Electronic supplementary material Below is the link to the electronic supplementary material . Table 1 Primer sequences and PCR product sizes used for sequencing ADIPOR1 and ADIPOR2 -LRB- DOC 46 kb -RRB- Table 2 Genotyping assay results for case-control studies -LRB- DOC 61 kb -RRB- Table 3 SNPs detected by sequencing ADIPOR1 and ADIPOR2 in severely insulin-resistant patients -LRB- DOC 78 kb -RRB- Table 4 ADIPOR1 and ADIPOR2 SNP association results with quantitative traits -LRB- DOC 215 kb -RRB-

O O O O O O O O O B I O B I O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O B I O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B I I O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B I I O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O B O O O O O O O B I I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B I I O O O O O O O O O O O O O O O O O O O B O O B O O O O B I I O O O O O O O O O O O O O B O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O B O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O B O B O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O B I I O B I O O O O O O O O B O B O O O O O O O O O O B I O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B O B O O O O O O B I O O O O B O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O B O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O B I I O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O B I O O O O O O O O O B O B O B O O O B I I O O O O O O O O O O B O B O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O B I I O B I O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O B O B O O O O O O O O O O O

adipor1
adipor2
association studies
insulin resistance
polymorphisms
type 2 diabetes


No evidence for circulating HuD-specific CD8 + T cells in patients with paraneoplastic neurological syndromes and Hu antibodies Aim In paraneoplastic neurological syndromes -LRB- PNS -RRB- associated with small cell lung cancer -LRB- SCLC -RRB- and Hu antibodies -LRB- Hu-PNS -RRB- , Hu antigens expressed by the tumour hypothetically trigger an immune response that also reacts with Hu antigens in the nervous system , resulting in tumour suppression and neuronal damage . To gain more insight into the hypothesized CD8 + T cell-mediated immune pathogenesis of these syndromes , we searched for circulating HuD-specific CD8 + T cells in a large cohort of Hu-PNS patients and controls . Introduction Paraneoplastic neurological syndromes -LRB- PNS -RRB- are considered as naturally occurring , successful anti-tumour immune responses in humans -LSB- 6 -RSB- . However , this tumour immunity goes along with autoaggression against the nervous system , resulting in severe neurological dysfunction -LSB- 9 -RSB- . The mechanisms responsible for the anti-tumour response and neuronal damage are poorly understood . Antigens expressed by the tumour that are normally restricted to neurons -LRB- so-called onconeuronal antigens -RRB- hypothetically trigger an immune response that cross-reacts with the same antigens in the nervous system -LSB- 7 -RSB- . One of the most frequently involved tumours is small cell lung cancer -LRB- SCLC -RRB- and approximately 50 % of patients with PNS and SCLC have high-titre Hu antibodies -LRB- Hu-PNS -RRB- . Hu antibodies are directed against a family of neuron specific , mRNA binding proteins , of which HuD is the best-documented member . Consistent expression of HuD in all SCLC suggests that HuD plays a central role in triggering the immune response -LSB- 15 , 17 -RSB- . High titres of Hu antibodies in serum and cerebrospinal fluid suggested a pathogenic role for these antibodies , that could , however , never be proven in animal models -LSB- 3 , 22 -RSB- . Furthermore , expression of Hu antigens is exclusively intracellular , and it is therefore difficult to understand how such antibodies could target tumours or neurons -LSB- 5 -RSB- . In addition , pathological examination of PNS neuronal tissue demonstrated localized inflammatory cell infiltrates , containing B cells , CD4 + and CD8 + T cells , in the proximity of overt neuronal cell damage -LSB- 2 , 13 , 18 , 25 -RSB- . The presence of an oligoclonal CD8 + T cell infiltrate in nervous tissues and tumours of Hu-PNS patients further suggests an immunopathogenic role for such cells -LSB- 18 , 25 -RSB- . Some authors report the presence of HuD peptide-specific CD8 + T cells in the blood of these patients , but also in that of apparently healthy controls -LRB- HC -RRB- -LSB- 19 , 20 , 24 -RSB- . In addition , the presence of circulating HuD-specific CD4 + T cells has been suggested -LSB- 1 -RSB- . Here , we have investigated the presence of circulating HuD-specific T cells in a large cohort of Hu-PNS patients and controls . Despite a multifaceted approach mainly geared towards the detection of HuD-reactive CD8 + T cells and , to a somewhat lesser extent , CD4 + T cells , no such cells were detected in the blood of Hu-PNS patients and controls . Materials and methods Patients Forty-three patients with high-titred Hu antibodies and a definite clinical diagnosis of PNS -LSB- 12 -RSB- , 31 Hu antibody negative SCLC patients without neurological symptoms or signs -LRB- SCLC -RRB- and 54 apparently HC were tested . The Erasmus MC Institutional Review Board approved the study and all individuals provided written informed consent . The individuals ' class I HLA alleles were typed by standard diagnostic PCR at the two-digit resolution level . Patient characteristics are shown in the Table 1 . Anti-Hu IgG titres were determined as described previously -LSB- 21 -RSB- and in Hu-PNS the dependence in activities of daily living was scored using the modified Rankin scale -LSB- 21 -RSB- . Twenty-five HC were male and 29 female , their median age was 46 years -LRB- range 17 -- 89 -RRB- and all were Hu antibody negative . No HC had received previous chemotherapy or immunosuppressive treatment and 30 HC -LRB- 56 % -RRB- were CMV-seropositive . Table 1Patient characteristics at the time of study entryHu-PNS SCLC N4331Age -LRB- median , range -RRB- 64 -LRB- 4 -- 81 -RRB- a61 -LRB- 40 -- 83 -RRB- Gender -LRB- M/F -RRB- 15/2821/10Hu-Ab titre -LRB- median , range -RRB- 12,800 -LRB- 400 -- 204,800 -RRB- NegativeCMV serostatus -LRB- pos/neg -RRB- 29/1417/14Paraneoplastic neurological syndromeNA PSN27 PEM5 PCD4 PLE / BE3 Pseudo-obstruction2 Motor neuron disease2Tumour No tumour8b0 SCLC3131 Limited2817 Extended314 NSCLC20 Prostate10 Neuroblastoma10Prior treatment None2727 Chemotherapy ± immunosuppression164Neurological symptomsNA Interval onset symptoms -LRB- study entry -RRB- 5 months -LRB- 2 -- 15 -RRB- c Interval onset symptoms -LRB- diagnosis -RRB- 4 months -LRB- 1 -- 12 -RRB- c Progressive at study entryd34 -LRB- 79 % -RRB- Modified Rankin scoreNA MRS = 27 MRS = 322 MRS = 410 MRS = 54Hu-Ab Hu antibody , CMV cytomegalovirus , pos positive , neg negative , NA not applicable , PSN paraneoplastic sensory neuronopathy , PEM paraneoplastic encephalomyelitis , PCD paraneoplastic cerebellar degeneration , PLE paraneoplastic limbic encephalitis , BE brainstem encephalitis , SCLC small cell lung cancer , NSCLC non-small cell lung cancer , MRS modified Rankin scorea One Hu-PNS patient was a 4-year-old boy with an underlying neuroblastoma . The remaining Hu-PNS patients were aged between 49 and 81 yearsb No tumour mass visible on CT-scan or FDG-PET scanc Median -LRB- ranges -RRB- of intervals are shownd Progression of neurological symptoms was defined by the increase of at least one point on the modified Rankin scale during 2 months prior to study entry Reagents Ninety-three HuD protein-spanning synthetic peptides , 15-mers with 11 amino acids overlap , were pooled to constitute the HuD peptide mix -LRB- HuDmix -RRB- and smaller peptide pools -LSB- Jerini Peptide Technologies -LRB- JPT -RRB- , Berlin , Germany -RSB- . For interferon-γ enzyme-linked immunosorbent spot-forming -LRB- IFN-γ ELISPOT -RRB- assays and the construction of HLA class-I multimers , HuD-derived 9 - and 10-mers were selected based on previous studies -LSB- 19 , 20 -RSB- . The phycoerythrin -LRB- PE -RRB- - labeled multimers and corresponding peptides used were : HLA-A * 0101-147ELEQLFSQY155 , HLA-A * 0101-245RLDNLLNMAY254 , HLA-A * 0201-86SLGYGFVNYI95 , HLA-A * 0201-248NLLNMAYGV256 , HLA-A * 0201-315QLFGPFGAV323 , HLA-A * 0201-362RLGDRVLQV370 , and HLA-A * 2402-154QYGRIITSRI163 -LRB- ProImmune , Oxford , UK -RRB- . As positive and negative controls , HLA-A * 0201-495NLVPMVATV503 -LSB- CMV phosphoprotein-65 -LRB- CMV-pp65 -RRB- ; Beckman Coulter , San Diego , CA -RSB- and HLA-A * 0201 presenting an irrelevant peptide -LRB- ProImmune -RRB- , respectively , were included . To measure general T-cell responsiveness , we used phorbol myristate acetate -LRB- PMA -RRB- plus ionomycin , or phytohemagglutinin -LRB- PHA -RRB- . A peptide pool containing 15-mers spanning CMV-pp65 -LRB- JPT -RRB- was used as positive - and negative antigen-specific control in CMV seropositive and seronegative individuals , respectively . Cytokine flow cytometry Peripheral blood mononuclear cells -LRB- PBMC -RRB- were isolated within 12 h after venipuncture and stimulated in duplicate as described elsewhere -LSB- 14 -RSB- . Briefly , 2 × 106 PBMC were incubated at 37 °C in a CO2 incubator for 18 h with 1 μg/ml HuDmix , 1 μg/ml CMV-pp65 , 1 μg/ml ionomycin plus 25 ng/ml PMA , or without antigen . After 2 h of stimulation , brefeldin A was added to one of the duplicate tubes allowing for intracellular accumulation of cytokines in activated T cells . Brefeldin A was not added to the second tube to allow detection of secreted cytokines in the supernatant . In some individuals , additional stimulation was performed in the presence of co-stimulatory monoclonal antibodies -LRB- mAb -RRB- directed against CD28 and CD49d -LRB- BD Biosciences , San Jose , CA -RRB- . Stimulated PBMC were stained and analysed using anti-CD3 conjugated with peridinyl chlorophyllin -LRB- PerCP -RRB- , anti-CD8 conjugated with allophycocyanin -LRB- APC -RRB- , anti-interferon -LRB- IFN -RRB- - γ conjugated with fluorescein isothiocyanate -LRB- FITC -RRB- , anti-tumour necrosis factor -LRB- TNF -RRB- - α conjugated with PE , or appropriate isotype control mAb -LRB- all from BD Biosciences -RRB- -LSB- 14 -RSB- . CD4 + T cells were defined as CD3 + , 8 − . Positive responses were defined by -LRB- 1 -RRB- percentage of cytokine-positive CD4 + or CD8 + T cells > 2 times the negative control -LRB- i.e. , no antigen -RRB- and -LRB- 2 -RRB- ≥ 0.1 % of the total number of CD4 + or CD8 + T cells , each after subtraction of isotype control results . Detection of secreted cytokines The secretion levels of IFN-γ , TNF-α , interleukin -LRB- IL -RRB- -2 , IL-4 , IL-5 and IL-10 were measured in supernatants using a cytometric bead array -LRB- BD Biosciences -RRB- . Based on CMV data -LRB- not shown -RRB- , a positive result was defined as cytokine concentration > 2 times the background -LRB- no antigen -RRB- and a minimum level of 50 pg/ml . IFN-γ ELISPOT PBMC -LRB- 2 × 105/well -RRB- were pre-stimulated in duplicate in 96-well plates with culture medium containing 3 μg/ml HuD 9-mers , HuDmix , CMV-pp65 , PHA , or no antigen for 1.5 h at 37 °C and 5 % CO2 -LSB- 10 -RSB- . The PBMC were subsequently transferred to anti-IFN-γ-coated ELISPOT plates -LRB- Nalge Nunc , Rochester , NY -RRB- for a further 18-h incubation . The ELISPOT assay was performed using standard protocols and automated reading -LRB- AELVIS GmbH , Hanover , Germany -RRB- -LSB- 10 -RSB- . The mean number of spot-forming cells -LRB- SFC -RRB- in duplicate wells was used as assay outcome . Positive results were defined by numbers of SFC/well > 3 times background -LRB- no antigen -RRB- and a minimum of 15 SFC/well . Detection of HuD-specific CD8 + T cells using Class-I HLA multimers Thawed PBMC were stained as described previously -LSB- 11 -RSB- . Following acquisition of 1 × 105 viable -LRB- i.e. , 7-amino-actinomycin-D -LSB- 7AAD -RSB- negative -RRB- CD8 + T cells , a positive result required a percentage of ≥ 0.1 % of viable CD8 + T cells binding the HuD multimer and a brightly staining HuD multimer-binding CD8 + T-cell population that did not overlap with the dimly staining irrelevant multimer-binding T-cell population . Results Cytokine flow cytometry of PBMC after stimulation with HuD-derived peptides Stimulation with PMA and ionomycin induced IFN-γ production in both CD4 + and CD8 + T cells of all individuals -LRB- not shown -RRB- . Whilst all CMV-seropositive individuals specifically responded to CMV-pp65 , no HuD-specific T-cell reactivity was observed in any of the Hu-PNS patients , SCLC patients or HC -LRB- Fig. 1a , b -RRB- . Similar results were obtained when intracellular TNF-α -LRB- not shown -RRB- or secreted cytokines -LRB- Fig. 1c -RRB- were measured . The use of co-stimulatory antibodies in combination with HuDmix did not result in the detection of HuD-specific T-cell reactivity -LRB- not shown -RRB- . Fig. 1Cytokine production in response to HuDmix and HLA-matched HuD 9-mers . Proportions of CD8 + -LRB- panel a -RRB- and CD4 + -LRB- panel b -RRB- T cells expressing intracellular IFN-γ after stimulation with HuDmix in Hu-PNS patients , SCLC and healthy controls . Each dot represents the result observed in a single individual -LRB- panel c -RRB- . After stimulation of PBMC with HuDmix and control antigens , the indicated cytokines were measured in assay supernatants . The results are shown for Hu-PNS patients only . Horizontal lines indicate median values of each group -LRB- panel d -RRB- . ELISPOT assay showing the number of IFN-γ SFC after stimulation of 2 × 105 PBMC with HuDmix or HuD 9-mers . Each dot represents the mean result of duplicates for each stimulus in each individual -LRB- panels a -- d -RRB- . Responses to CMV antigens are shown for CMV seropositive individuals only , as they were consistently negative in CMV seronegative individuals -LRB- not shown -RRB- . The numbers of individuals tested are given in between brackets . NEG negative control -LRB- incubation without antigen -RRB- , HuDmix HuD protein-spanning peptide pool , PP65 CMV pp-65 protein-spanning peptide pool , IL interleukin TNF tumour necrosis factor , IFN interferon , SFC spot-forming cell , PHA phytohemagglutinin . ELE , RLD , SLG , NLL , QLF , RLG and QYG designate individual HuD-based peptides IFNγ-ELISPOT assay on PBMC stimulated with HuD-derived 9-mers All individuals responded to PHA as determined by IFNγ-ELISPOT assay . In addition , all CMV-seropositive individuals responded to CMV-pp65 . However , no T cell reactivity towards the previously described class-I HLA-binding HuD peptides -LSB- 20 -RSB- was detected in individuals with the appropriate HLA types in any of the study groups -LRB- Fig. 1d -RRB- . Analysis of HuD-specific CD8 + T cells using Class-I HLA multimers Finally , we investigated the presence of HuD-specific CD8 + T cells in PBMC using Class-I HLA multimers containing HuD-derived peptides . Whilst CMV-seropositive individuals with the appropriate HLA types showed distinct populations of pp65 multimer-binding CD8 + T cells , no HuD-specific CD8 + T cells were observed in individuals with the appropriate HLA types in any of the study groups -LRB- Fig. 2 -RRB- . Fig. 2Analysis of HuD peptide-loaded , Class-I HLA multimer-binding CD8 + T cells . PBMC from a CMV-seropositive Hu-PNS patient were stained with HLA-A * 0201 multimers loaded with the HuD peptide SLGYGFVNYI -LRB- panel a -RRB- , an irrelevant peptide -LRB- panel b -RRB- or with the CMV-pp65 peptide NLVPMVATV -LRB- panel c -RRB- . All data shown are obtained after selection of T cells -LRB- i.e. , CD3 + , low sideward scatter signals -RRB- -LSB- 12 -RSB- . Binding of Class-I HLA multimers -LRB- panels a -- c , horizontal axes -RRB- was analysed in relation to CD8 expression . In this example , the proportion of SLGYGFVNYI multimer-binding CD8 + T cells -LRB- 0.36 % -RRB- was similar to that of irrelevant multimer-binding CD8 + T cells -LRB- 0.46 % -RRB- ; binding resulted in low-intensity fluorescence signals only . In contrast , 2.46 % of CD8 + T cells bound the NLVPMVATV multimer resulting in high-intensity fluorescence signals from most CD8 + T cells Discussion We set out to detect HuD-specific T cells in the blood of Hu-PNS patients as they are postulated to play a pivotal role in the immunopathology of this disease . Although we applied three different approaches we could not detect circulating HuD-specific T cells . First , to induce cytokine responses in T cells , we used 15-mer protein-spanning peptide pools that have the advantage of covering the full protein sequence and of eliciting both CD8 + and CD4 + T-cell responses -LSB- 14 -RSB- , as demonstrated by the CD8 + and CD4 + CMV-pp65-specific T-cell responses . However , no CD8 + or CD4 + HuD-specific T-cell responses were observed . These results are at variance with the detection of HuD-specific CD4 + T-cell proliferative responses in PBMC of Hu-PNS patients by Benyahia et al. -LSB- 1 -RSB- . This discrepancy may be explained by differences in read-out -LRB- i.e. , 3-day lymphocyte proliferation in Benyahia 's study -LSB- 1 -RSB- vs. overnight cytokine production in ours -RRB- and the use of recombinant HuD protein -LSB- 1 -RSB- versus a protein-spanning 15-mer peptide pool -LRB- this manuscript -RRB- . We then studied responses to HLA class-I binding HuD peptides that were previously selected -LSB- 20 -RSB- . Using the same experimental setup and HuD 9-mer peptides , Rousseau et al. -LSB- 20 -RSB- reported HuD-specific T-cell reactivity in 7/10 Hu-PNS patients and in 3/10 HC -LSB- 20 -RSB- . In that small study a positive response was defined as an experimental value ≥ 2 times above background . With that criterion , 3 PNS , 2 SCLC and 4 HC would have been classified as HuD T-cell responders in our study . However , using that cut-off we would also have detected T-cell reactivity in individuals whose Class-I HLA molecules did not have the appropriate binding motifs -LRB- data not shown -RRB- . Therefore , we used more stringent cut-off levels resulting in a negative outcome . As most patients in both studies had progressive neurological disease and were tested shortly after start of symptoms and prior to therapy , differences between the study populations do not explain this discrepancy . Finally , we could not detect HuD-specific circulating CD8 + T cells using Class-I HLA multimers with the same fine specificities as defined by Rousseau et al. -LSB- 20 -RSB- . The absence of detectable circulating HuD-specific CD8 + T cells may not be surprising . In a PCR-based study , Plonquet et al. -LSB- 18 -RSB- detected the same T-cell clone in neoplastic and nervous tissues , but not in blood . This finding suggests that T cells involved in the pathogenesis of Hu-PNS circulate in concentrations below detection level . An immune response taking place in the central nervous system parenchyma may deplete the circulating pool of CD8 + T cells with that specificity -LSB- 23 -RSB- . Furthermore , vaccination studies in melanoma patients demonstrate that clinically effective anti-tumour immune responses may occur despite low levels of melanoma-specific cytotoxic T cells , i.e. , below the detection limit of multimer-based assays -LSB- 4 , 16 -RSB- . In conclusion , we were unable to detect HuD-specific T cells in a large cohort of Hu-PNS patients and controls . However , two of our three assays were designed for the detection of CD8 + T cells only . The IgG1 isotype predominance of serum Hu antibodies in Hu-PNS indicates a T-helper response to the Hu antigen -LSB- 13 -RSB- . Therefore , further studies are warranted that focus on the detection of circulating HuD-specific CD4 + T cells . In this context , regulatory CD4 + T cells -- which down regulate immune responses towards auto-antigens and tumour-antigens -- are of interest . Although the numbers of regulatory T cells are increased in cancer patients -LSB- 26 -RSB- and in PNS patients -LSB- 8 -RSB- -LRB- de Beukelaar et al. unpublished data -RRB- their -LRB- possibly impaired -RRB- function in PNS remains to be studied . Finally , examination of the antigen-specificity of T cells in affected tissues may shed further light on the role of HuD-specific T cells in the pathogenesis of Hu-PNS .

O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O B O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

anti-tumour immunity
t lymphocyte
hla class i multimer


Clinical review : The implications of experimental and clinical studies of recruitment maneuvers in acute lung injury Mechanical ventilation can cause and perpetuate lung injury if alveolar overdistension , cyclic collapse , and reopening of alveolar units occur . The use of low tidal volume and limited airway pressure has improved survival in patients with acute lung injury or acute respiratory distress syndrome . The use of recruitment maneuvers has been proposed as an adjunct to mechanical ventilation to re-expand collapsed lung tissue . Many investigators have studied the benefits of recruitment maneuvers in healthy anesthetized patients and in patients ventilated with low positive end-expiratory pressure . However , it is unclear whether recruitment maneuvers are useful when patients with acute lung injury or acute respiratory distress syndrome are ventilated with high positive end-expiratory pressure , and in the presence of lung fibrosis or a stiff chest wall . Moreover , it is unclear whether the use of high airway pressures during recruitment maneuvers can cause bacterial translocation . This article reviews the intrinsic mechanisms of mechanical stress , the controversy regarding clinical use of recruitment maneuvers , and the interactions between lung infection and application of high intrathoracic pressures . Introduction Mechanical ventilation -LRB- MV -RRB- is a supportive and life saving therapy in patients with acute lung injury -LRB- ALI -RRB- and/or acute respiratory distress syndrome -LRB- ARDS -RRB- . Despite advances in critical care , mortality in these patients remains over 40 % -LSB- 1 -RSB- . During the past decade the possibility that MV can produce morphologic and physiologic alterations in the lung has been recognized -LSB- 2 -RSB- . On histopathologic examination , findings in ventilator-induced lung injury -LRB- VILI -RRB- do not differ from those in ARDS -LSB- 2 -RSB- . To minimize this damage , lung protective strategies to avoid overdistension and cyclic collapse and reopening of alveoli have successfully been used in patients with ARDS receiving MV -LSB- 3,4 -RSB- . Recruitment maneuvers -LRB- RMs -RRB- consisting of sustained inflation to open the collapsed alveolar units have been proposed as an adjunct to MV in anesthesia and ARDS -LSB- 5,6 -RSB- . In most ARDS patients , however , lung recruitment and overdistension occur simultaneously at higher intrathoracic pressure -LSB- 7 -RSB- . Whether RMs can initiate cellular mechanisms of injury in healthy parts of the lung is unknown . In the present review of the literature we describe the intrinsic mechanisms that explain how MV inflicts alveolar damage and the controversy regarding the use of RMs as an adjunct to MV . Finally , we discuss the interactions between lung infection and periodic application of high intrathoracic pressure , both in experimental models of ALI and in patients with ARDS . Method To identify the most relevant English language publications , the Medline database was searched using the following keywords : mechanotransduction , acute lung injury , acute respiratory distress syndrome , mechanical ventilation , ventilator-induced lung injury , overdistension , recruitment maneuvers , and bacterial translocation . Many different methods of RM delivery have been proposed in the literature -LRB- Table 1 -RRB- . Several investigators have demonstrated that RMs can increase oxygenation and lung volume in collapsed prone lungs . However , the benefits of RMs in terms of oxygenation and lung recruitment in ARDS patients and in experimental models with alveolar flooding or consolidation are unclear . Intrinsic mechanism of ventilator-induced lung injury The mechanical stresses produced by MV at high pressure or volumes , and the forces generated by repeated opening and collapse lead to upregulation of an inflammatory response , with release of cytokines and chemokines and activation of neutrophils and macrophages that produce lung damage -LSB- 2 -RSB- . Injurious MV can lead to end-organ dysfunction , and the inflammatory cascade also plays a pivotal role in the systemic inflammatory response syndrome and in multiple organ system failure -LSB- 8-10 -RSB- . Like all adherent cells , alveolar epithelial cells interact with extracellular matrix through transmembrane adhesion receptors such as integrins . These receptors transmit forces from the surrounding matrix to the cytoskeleton via the focal adhesion complex -LSB- 11 -RSB- . When the basement membrane is strained , adherent epithelial cells must change shape and the ratio of their surface -LRB- plasma membrane -RRB- to their volume must increase . If the plasma membrane is disrupted the intracellular lipid stores are utilized to repair the cell surface . Most breaks are repaired within seconds , usually via a calcium-dependent response -LSB- 12 -RSB- . This dynamic remodeling process is the most important determinant of cell wounding -LSB- 13 -RSB- . Mechanotransduction is the conversion of mechanical stimuli , such as cell deformation , into biochemical and biomolecular alterations . How mechanical forces can be sensed by cells and converted into intracellular signals is still unclear , but in various experiments it was observed that mechanical stimuli activate the nuclear factor-κB -- a critical transcription factor that is required for maximal expression of many cytokines involved in the pathogenesis of VILI -LSB- 14 -RSB- . It is unknown whether a single stimulus such as RMs applied during MV can trigger the above-mentioned pathways of lung injury , and the long-term benefits and safety of RMs will depend on the extent of this effect . Experimental evidence on recruitment maneuvers In saline lavaged rabbit lungs , Bond and coworkers -LSB- 15 -RSB- found an improvement in respiratory system compliance and oxygenation during high frequency oscillatory ventilation after RMs . In a similar model , Rimensberger and coworkers -LSB- 16 -RSB- showed that a single RM resulted in better oxygenation without augmenting histologic injury at positive end-expiratory pressure -LRB- PEEP -RRB- below the lower inflection point of the respiratory system pressure -- volume curve , as compared with the group with PEEP set above the lower inflection point without RM . Furthermore , those investigators showed that a single sustained inflation to 30 cmH2O boosted the ventilatory cycle onto the deflation limb of the pressure -- volume curve -LRB- Fig. 1 -RRB- . In other words , a RM applied in a recruitable lung increases the amount of recruited tissue at end expiration , favoring tidal ventilation . Some data suggest that RMs have different effects depending on the type of lung insult and on the use of various combinations of tidal volume and PEEP . Whether RMs are necessary to prevent alveolar collapse when optimal PEEP is used remains controversial . Van der Kloot and colleagues -LSB- 17 -RSB- studied the effects of RMs on gas exchange and lung volumes in three experimental models of ALI : saline lavage , oleic acid , and pneumonia . After application of RMs , oxygenation improved only in the surfactant depletion group when low PEEP was used . At high PEEP in any model , RMs had no effect . Similar effects were observed in the study conducted by Bond and coworkers -LSB- 15 -RSB- . Takeuchi and colleagues -LSB- 18 -RSB- highlighted the difficulties in maintaining tidal ventilation at high lung volumes . Those investigators showed that , after RMs , PEEP set at 2 cmH2O above the lower inflection point was more effective in maintaining gas exchange and minimizing inflammation and lung injury than was PEEP set at the maximum curvature of the deflation pressure -- volume curve . When recruitment is achieved with posture , Cakar and coworkers -LSB- 19 -RSB- showed better oxygenation after RMs in the prone than in the supine position , and importantly the benefit was sustained at lower PEEP . In other studies other adjuncts to MV were necessary to keep the lung open after RMs -LSB- 20 -RSB- . Lu and coworkers -LSB- 21 -RSB- demonstrated that RMs completely reversed the atelectasis , bronchoconstriction , and decrease in arterial oxygen saturation observed after endotracheal suctioning in an anesthetized sheep model . In summary , the beneficial effects of RMs have been demonstrated in animal models of alveolar collapse induced by surfactant depletion . However , the pathobiology of ARDS is more complex and includes an altered vascular barrier function and alveolar flooding or consolidation . Indeed , in animal models other than that involving surfactant depletion , the effect of RMs on lung function is less evident . Role of recruitment maneuvers in anesthetized patients Formation of atelectasis and airway closure are mechanisms of impaired gas exchange in anesthetized patients with healthy lungs -LSB- 22 -RSB- . RMs have successfully been used to reverse collapsed dependent areas in these patients . Rothen and coworkers -LSB- 22 -RSB- found that a pressure of 40 cmH2O maintained for 7 -- 8 s entirely re-expanded the collapsed lung tissue in anesthetized humans , although the net effect on gas exchange might be rather small if low ventilation/perfusion areas still coexist at the time of decrease in intrapulmonary shunt . Long-term effects of RMs in anesthetized patients depend on gas composition . Re-expanded lung tissue remained inflated for at least 40 min at low oxygen concentration -LSB- 5 -RSB- , whereas lung collapse reappears within minutes with pure oxygen -LSB- 23 -RSB- . Finally , RMs followed by moderate PEEP may produce physiologic benefits in patients undergoing upper abdominal , thoracic , or laparoscopic surgery -LSB- 24,25 -RSB- , and in patients prone to develop a moderate degree of lung injury after surgical procedures -LSB- 26,27 -RSB- . Recruitment maneuvers in patients with acute respiratory distress syndrome Since the publication of the reports from Amato and coworkers -LSB- 3 -RSB- and the Consensus Conference on ARDS -LSB- 28 -RSB- , the application of periodic RMs in patients with ARDS has gained acceptance among clinicians , although controversy remains . Among the earliest reports providing evidence that RMs improve lung function was that from Pelosi and coworkers -LSB- 29 -RSB- , who demonstrated that sighs at 45 cmH2O plateau pressure in patients ventilated with PEEP at 14 ± 2.2 cmH2O significantly improved oxygenation , intrapulmonary shunt , and lung mechanics . Foti and colleagues -LSB- 6 -RSB- observed that RMs were effective in improving oxygenation and alveolar recruitment only during MV at low PEEP , suggesting that high PEEP better stabilized alveoli and prevented loss of lung volume . In the same line , Lapinsky and coworkers -LSB- 30 -RSB- reported beneficial effects on oxygenation , but this effect was sustained only if PEEP was increased after RMs . Lim and colleagues -LSB- 31 -RSB- found an improvement in oxygenation that persisted for 1 hour after an ` extended sigh ' ; this effect was partially lost soon after ventilatory support returned to the baseline PEEP level . Other studies have shown a modest and variable effect of RMs on oxygenation when ARDS patients are ventilated with high PEEP . Richard and coworkers -LSB- 32 -RSB- demonstrated decreased oxygenation when tidal volume was switched from 10 to 6 ml/kg with PEEP set above the lower inflection point of the pressure -- volume curve . However , increasing PEEP and RMs prevented alveolar derecruitment , and RMs performed in patients already ventilated with high PEEP had minimal effects on requirements for oxygenation support . Similarly , Villagrá and colleagues -LSB- 33 -RSB- , studying the effect of RMs superimposed on a lung protective strategy -LRB- tidal volume < 8 ml/kg and PEEP 3 -- 4 cmH2O higher than the lower inflection point on the pressure -- volume curve -RRB- , found no effect on oxygenation regardless of the stage of ARDS , and in some patients venous admixture increased during RMs -LRB- Fig. 2 -RRB- . This deleterious effect suggested that the RMs increased lung volume by overdistending the more compliant already-opened and aerated alveolar units , favoring blood flow redistribution from overdistended to collapsed lung regions . Furthermore , a negative correlation was found between recruited lung volume induced by PEEP before RMs and RM-induced changes in oxygenation , suggesting that RMs are less effective when the lungs have been near optimally recruited by PEEP and tidal volume . Recently Hubmayr -LSB- 34 -RSB- suggested that alveolar flooding is probably the main mechanism of end-expiratory loss of lung aeration in human ARDS . This may explain , at least in part , why RMs are less beneficial in patients with ARDS . Nevertheless , when sudden lung derecruitment occurs in conditions of adequate PEEP ventilation , such as the loss in lung volume produced during secretion aspiration , Maggiore and coworkers -LSB- 35 -RSB- observed that suctioning-induced lung derecruitment can be prevented by performing RMs . The cause of ARDS may also influence the response to RMs . In a majority of trauma patients developing ARDS , Johannigman and coworkers -LSB- 36 -RSB- found an improvement in oxygenation after RMs in patients receiving MV with low tidal volume and high PEEP . However , when Bein and colleagues -LSB- 37 -RSB- analyzed the impact of RMs on intracranial pressure and cerebral metabolism in patients with acute cerebral injury and respiratory failure , they observed an increase in intracranial pressure at the end of RMs and a subsequent reduction in mean arterial pressure resulting in a decrease in cerebral perfusion pressure . Both normalized 10 min after RMs . Grasso and coworkers -LSB- 38 -RSB- found that RMs significantly improved arterial oxygenation and lung volume in patients with early ARDS without impaired chest wall mechanics -LRB- i.e. with large recruitment potential -RRB- . Nevertheless , in the group with low chest wall compliance , RM-induced lung overdistension reduced blood pressure and cardiac output , making RMs ineffective and potentially harmful -LRB- Fig. 3 -RRB- . RMs can also be applied during assisted breathing in non-sedated patients . Patroniti and coworkers -LSB- 39 -RSB- applied one sigh per minute to baseline pressure support ventilation in patients with early ARDS . They observed a significant improvement in arterial oxygenation associated with an increase in end-expiratory lung volume and respiratory system compliance during the sigh , suggesting that sighs promote alveolar recruitment . These changes returned to baseline after the sighs were discontinued . Other studies emphasize the importance of body posture -LRB- supine or prone -RRB- on regional distribution of intrapulmonary ventilation and perfusion , and the beneficial effects of prone position in limiting VILI in experimental animals -LSB- 40 -RSB- . Lim and coworkers -LSB- 31 -RSB- found that the benefit was significantly greater when patients were in the supine position as compared with those in the prone position , suggesting that patients in the prone position have less collapsed lung . These findings were recently confirmed by Pelosi and colleagues -LSB- 41 -RSB- , who demonstrated that adding cyclical sighs during ventilation in the prone position provided optimal lung recruitment in the early stage of human ARDS . Finally , two randomized physiologic pilot studies of RMs superimposed on low tidal volume ventilation and moderate to high PEEP conducted in approximately 100 patients with ALI showed no clear benefits in terms of oxygenation -LSB- 42,43 -RSB- . Moreover , RMs were potentially harmful because some patients developed hemodynamic instability , ventilator dysynchrony , and pneumothorax after RM . In summary , RMs can be useful in improving oxygenation in patients receiving MV with low PEEP and low tidal volume . However , in patients with ARDS receiving MV with high PEEP levels , the beneficial effects of RMs disappear . RMs may restore lung volume and oxygenation in endotracheal suctioning-induced lung derecruitment in mechanically ventilated patients diagnosed with ALI/ARDS . RMs should be avoided in patients with suspected or documented intracranial hypertension , in patients with a stiff chest wall , and in patients in the late stage of ARDS . Lung infection and mechanical ventilation Recent studies suggest that the detrimental effect of MV may be aggravated when lungs are infected or primed with endotoxin . In ex vivo rat lungs , Ricard and coworkers -LSB- 44 -RSB- showed that ventilation that severely injures lungs does not lead to release of significant amounts of inflammatory cytokines by the lung in the absence of lipopolysaccharide challenge . Likewise , in experimental studies other investigators have shown that MV predisposes to development of pneumonia -LSB- 45 -RSB- and that coexisting MV and infection have a strong impact on the lung because they appear to act synergistically in causing alveolar damage -LSB- 46 -RSB- . Finally , when bacteria were injected in animals with previous severe ALI , MV produced a clinical picture closely resembling that of hyperdynamic sepsis in humans -LSB- 47 -RSB- . These experimental studies taken together suggest that in the presence of lung infection MV -LRB- cyclic positive intrathoracic pressure -RRB- predisposes to greater bacterial burden and bacterial translocation from the lung into the systemic circulation than would occur without MV . These effects are particularly important when using ventilatory strategies that apply large transpulmonary pressures -LRB- high tidal volume and/or high alveolar pressures without PEEP -RRB- -LSB- 48 -RSB- and are partially attenuated when protective ventilatory strategies are used -LSB- 49 -RSB- . RMs can be applied as sighs or as periodic sustained inflations that can damage or transiently alter the integrity of the alveolar -- capillary barrier -LSB- 50,51 -RSB- . Whether such strategies to improve lung function can result in failure of the alveolar -- capillary barrier and promote transient bacterial translocation in humans remains unknown . The amount of recruitable lung parenchyma in patients with ALI/ARDS receiving MV is a matter of debate , and controversy exists on the use of RMs in such patients for two main reasons . First , consolidation -LRB- non-recruitable lung parenchyma -RRB- and sticky atelectasis -LRB- potentially recruitable -RRB- coexist in different amounts in ALI/ARDS , and can not be distinguished and quantified at the bedside to inform a decision regarding a recruitment strategy . Second , the amount of lung tissue to be recruited in some ARDS patients is sparse -LSB- 52 -RSB- . Therefore , RMs can exert little effect on consolidated lung areas but can cause overdistension in some lung regions where bacteria are compartmentalized at the site of infection or colonization . Because spillover of lung cytokines into the systemic circulation is observed in lung inflammation and is potentiated with MV -LSB- 53 -RSB- , a similar phenomenon is likely to occur when the concentration of bacteria in the lungs is high enough . In a recent study -LSB- 54 -RSB- it was found that high pressure ventilation promoted early translocation of bacteria ; however , intermittent RMs applied as a sustained inflation superimposed on low-pressure ventilation without PEEP did not cause translocation of intratracheally inoculated Pseudomonas aeruginosa in rats with previously healthy lungs . However , we do not yet know whether the lung injury model used is valid for human ARDS or the degree of reproducibility of short-term experimental studies in patients receiving MV for days or weeks -LSB- 55 -RSB- . Conclusion On the basis of our review of the literature on experimental and clinical studies , considerable uncertainty remains regarding the use of RMs in humans with ARDS . RMs may have a role to play in patients with early ARDS and normal chest wall mechanics because there is great potential for alveolar recruitment , and after disconnections from the ventilator , when sudden loss of lung volume promotes alveolar instability and derecruitment . Recommendations to use RMs as adjuncts during lung protection ventilatory strategies seem unnecessary because sustained improvements in lung function have not been found when the strategies are combined . The presence of lung infection must be considered a major limitation for aggressive RMs because translocation of bacteria and the occurrence of systemic sepsis have been demonstrated in animal models . Finally , large randomized studies do not support the use of RMs in patients with ARDS . In conclusion , the use of RMs can not be recommended in the light of current knowledge , and if RMs are used they should be restricted to an individualized clinical decision or to a last resort to improve oxygenation and lung mechanics in a severely hypoxemic ARDS patient . Competing interests None declared . Abbreviations ALI = acute lung injury ; ARDS = acute respiratory distress syndrome ; MV = mechanical ventilation ; PEEP = positive end-expiratory pressure ; RM = recruitment maneuver ; VILI = ventilator-induced lung injury .

O O O O O O O O O O O O O O B I I B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O B I O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O

acute lung injury
lung collapse
lung infection
mechanical stress
mechanical ventilation