Gene Polymorphisms of Micrornas in Helicobacter pylori-Induced High Risk Atrophic Gastritis and Gastric Cancer

Background and aims

MicroRNAs (miRNAs) are known for their function as translational regulators of tumor suppressor or oncogenes. Single nucleotide polymorphisms (SNPs) in miRNAs related genes have been shown to affect the regulatory capacity of miRNAs and were linked with gastric cancer (GC) and premalignant gastric conditions. The purpose of this study was to evaluate potential associations between miRNA-related gene polymorphisms (miR-27a, miR-146a, miR-196a-2, miR-492 and miR-608) and the presence of GC or high risk atrophic gastritis (HRAG) in European population.

Methods

Gene polymorphisms were analyzed in 995 subjects (controls: n = 351; GC: n = 363; HRAG: n = 281) of European descent. MiR-27a T>C (rs895819), miR-146a G>C (rs2910164), miR-196a-2 C>T (rs11614913), miR-492 G>C (rs2289030) and miR-608 C>G (rs4919510) SNPs were genotyped by RT-PCR.

Results

Overall, SNPs of miRNAs were not associated with the presence of GC or HRAG. We observed a tendency for miR-196a-2 CT genotype to be associated with higher risk of GC when compared to CC genotype, however, the difference did not reach the adjusted P-value (odds ratio (OR) - 1.46, 95% confidence interval (CI) 1.03-2.07, P = 0.032). MiR-608 GG genotype was more frequent in GC when compared to controls (OR −2.34, 95% CI 1.08–5.04), but significance remained marginal (P = 0.029). A similar tendency was observed in a recessive model for miR-608, where CC + CG vs GG genotype comparison showed a tendency for increased risk of GC with OR of 2.44 (95% CI 1.14–5.22, P = 0.021). The genotypes and alleles of miR-27a, miR-146a, miR-196a-2, miR-492 and miR-608 SNPs had similar distribution between histological subtypes of GC and were not linked with the presence of diffuse or intestinal-type GC.

Conclusions

Gene polymorphisms of miR-27a, miR-146a, miR-196a-2, miR-492, miR-492a and miR-608 were not associated with the presence of HRAG, GC or different histological subtypes of GC in European subjects.     

Identification and Validation of Oncologic miRNA Biomarkers for Luminal A-like Breast Cancer

Introduction

Breast cancer is a common disease with distinct tumor subtypes phenotypically characterized by ER and HER2/neu receptor status. MiRNAs play regulatory roles in tumor initiation and progression, and altered miRNA expression has been demonstrated in a variety of cancer states presenting the potential for exploitation as cancer biomarkers. Blood provides an excellent medium for biomarker discovery. This study investigated systemic miRNAs differentially expressed in Luminal A-like (ER+PR+HER2/neu-) breast cancer and their effectiveness as oncologic biomarkers in the clinical setting.

Methods

Blood samples were prospectively collected from patients with Luminal A-like breast cancer (n = 54) and controls (n = 56). RNA was extracted, reverse transcribed and subjected to microarray analysis (n = 10 Luminal A-like; n = 10 Control). Differentially expressed miRNAs were identified by artificial neural network (ANN) data-mining algorithms. Expression of specific miRNAs was validated by RQ-PCR (n = 44 Luminal A; n = 46 Control) and potential relationships between circulating miRNA levels and clinicopathological features of breast cancer were investigated.

Results

Microarray analysis identified 76 differentially expressed miRNAs. ANN revealed 10 miRNAs for further analysis (miR-19b, miR-29a, miR-93, miR-181a, miR-182, miR-223, miR-301a, miR-423-5p, miR-486-5 and miR-652). The biomarker potential of 4 miRNAs (miR-29a, miR-181a, miR-223 and miR-652) was confirmed by RQ-PCR, with significantly reduced expression in blood of women with Luminal A-like breast tumors compared to healthy controls (p = 0.001, 0.004, 0.009 and 0.004 respectively). Binary logistic regression confirmed that combination of 3 of these miRNAs (miR-29a, miR-181a and miR-652) could reliably differentiate between cancers and controls with an AUC of 0.80.

Conclusion

This study provides insight into the underlying molecular portrait of Luminal A-like breast cancer subtype. From an initial 76 miRNAs, 4 were validated with altered expression in the blood of women with Luminal A-like breast cancer. The expression profiles of these 3 miRNAs, in combination with mammography, has potential to facilitate accurate subtype-specific breast tumor detection.     

Tissue MicroRNAs as Predictors of Outcome in Patients with Metastatic Colorectal Cancer Treated with First Line Capecitabine and Oxaliplatin with or without Bevacizumab

Purpose

We tested the hypothesis that expression of microRNAs (miRNAs) in cancer tissue can predict effectiveness of bevacizumab added to capecitabine and oxaliplatin (CAPEOX) in patients with metastatic colorectal cancer (mCRC).

Experimental Design

Patients with mCRC treated with first line CAPEOX and bevacizumab (CAPEOXBEV): screening (n = 212) and validation (n = 121) cohorts, or CAPEOX alone: control cohort (n = 127), were identified retrospectively and archival primary tumor samples were collected. Expression of 754 miRNAs was analyzed in the screening cohort using polymerase chain reaction (PCR) arrays and expression levels were related to time to disease progression (TTP) and overall survival (OS). Significant miRNAs from the screening study were analyzed in all three cohorts using custom PCR arrays. In situ hybridization (ISH) was done for selected miRNAs.

Results

In the screening study, 26 miRNAs were significantly correlated with outcome in multivariate analyses. Twenty-two miRNAs were selected for further study. Higher miR-664-3p expression and lower miR-455-5p expression were predictive of improved outcome in the CAPEOXBEV cohorts and showed a significant interaction with bevacizumab effectiveness. The effects were strongest for OS. Both miRNAs showed high expression in stromal cells. Higher expression of miR-196b-5p and miR-592 predicted improved outcome regardless of bevacizumab treatment, with similar effect estimates in all three cohorts.

Conclusions

We have identified potentially predictive miRNAs for bevacizumab effectiveness and additional miRNAs that could be related to chemotherapy effectiveness or prognosis in patients with mCRC. Our findings need further validation in large cohorts, preferably from completed randomized trials.     

Circulating Methylated XAF1 DNA Indicates Poor Prognosis for Gastric Cancer

Background

Methylated DNA in fluids may be a suitable biomarker for cancer patients. XAF1 has been shown to be frequently down-regulated in human gastric cancer (GC). Here, we investigated if XAF1 methylation in GC could be a useful biomarker.

Methods

Real-time RT-PCR was used to detect XAF1 mRNA expression; immunohistochemistry and western blot were used to examine XAF1 protein expression in GC tissues (n = 202) and their corresponding para-cancerous histological normal tissues (PCHNTs). Real-time methylation specific-PCR was used to investigate XAF1 promoter methylation in the same panel of GC tissues, their PCHNTs and sera.

Results

We confirmed frequent XAF1 down-regulation in both mRNA and protein levels in GC tissues as compared to normal controls and PCHNTs. XAF1 hypermethylation was evidenced in 83.2% (168/202) of GC tissues and 27.2% (55/202) of PCHNTs, while no methylation was detected in the 88 normal controls. The methylation level in GC tissues was significantly higher than that in PCHNTs (p<0.05). The hypermethylation of XAF1 significantly correlated with the down-regulation of XAF1 in GC tissues in both mRNA and protein levels (p<0.001 each). Moreover, we detected high frequency of XAF1 methylation (69.8%, 141 out of 202) in the sera DNAs from the same patients, while the sera DNAs from 88 non-tumor controls were negative for XAF1 methylation. The XAF1 methylation in both GC tissues and in the sera could be a good biomarker for diagnosis of GC (AUC = 0.85 for tissue and AUC = 0.91 for sera) and significantly correlated with poorer prognosis (p<0.001). In addition, after-surgery negative-to-positive transition of XAF1 methylation in sera strongly associated with tumor recurrence.

Conclusions

1) Dysfunction of XAF1 is frequent and is regulated through XAF1 promoter hypermethylation; 2) Detection of circulating methylated XAF1 DNAs in the serum may be a useful biomarker in diagnosis, evaluating patient’s outcome (prognosis and recurrence) for GC patients.     

A High Degree of LINE-1 Hypomethylation Is a Unique Feature of Early-Onset Colorectal Cancer

Objective

Early-onset colorectal cancer (CRC) represents a clinically distinct form of CRC that is often associated with a poor prognosis. Methylation levels of genomic repeats such as LINE-1 elements have been recognized as independent factors for increased cancer-related mortality. The methylation status of LINE-1 elements in early-onset CRC has not been analyzed previously.

Design

We analyzed 343 CRC tissues and 32 normal colonic mucosa samples, including 2 independent cohorts of CRC diagnosed ≤50 years old (n = 188), a group of sporadic CRC >50 years (MSS n = 89; MSI n = 46), and a group of Lynch syndrome CRCs (n = 20). Tumor mismatch repair protein expression, microsatellite instability status, LINE-1 and MLH1 methylation, somatic BRAF V600E mutation, and germline MUTYH mutations were evaluated.

Results

Mean LINE-1 methylation levels (±SD) in the five study groups were early-onset CRC, 56.6% (8.6); sporadic MSI, 67.1% (5.5); sporadic MSS, 65.1% (6.3); Lynch syndrome, 66.3% (4.5) and normal mucosa, 76.5% (1.5). Early-onset CRC had significantly lower LINE-1 methylation than any other group (p<0.0001). Compared to patients with <65% LINE-1 methylation in tumors, those with ≥65% LINE-1 methylation had significantly better overall survival (p = 0.026, log rank test).

Conclusions

LINE-1 hypomethylation constitutes a potentially important feature of early-onset CRC, and suggests a distinct molecular subtype. Further studies are needed to assess the potential of LINE-1 methylation status as a prognostic biomarker for young people with CRC.     

First Trimester Screening of Circulating C19MC microRNAs Can Predict Subsequent Onset of Gestational Hypertension

Objective

The objective of the study was to evaluate risk assessment for gestational hypertension based on the profile of circulating placental specific C19MC microRNAs in early pregnancy.

Study design

The prospective longitudinal cohort study of women enrolled at first trimester screening at 10 to 13 weeks was carried out (n = 267). Relative quantification of placental specific C19MC microRNAs (miR-516-5p, miR-517*, miR-518b, miR-520a*, miR-520h, miR-525 and miR-526a) was determined in 28 normal pregnancies and 18 pregnancies which developed gestational hypertension using real-time PCR and a comparative Ct method relative to synthetic C. elegans microRNA (cel-miR-39).

Results

Increased extracellular C19MC microRNA plasmatic levels (miR-516-5p, p<0.001; miR-517*, p = 0.007; miR-520h, p<0.001; miR-518b, p = 0.002) were detected in patients destined to develop gestational hypertension. MiR-520h had the best predictive performance with a PPV of 84.6% at a 7.1% false positive rate. The combination of miR-520h and miR-518b was able to predict 82.6% of women at the same false positive rate. The overall predictive capacity of single miR-518b (73.3% at 14.3% FPR), miR-516-5p (70.6% at 17.9% FPR) and miR-517* (57.9% at 28.6% FPR) biomarkers was lower.

Conclusion

The study brought interesting finding that the up-regulation of miR-516-5p, miR-517*, miR-520h and miR-518b is associated with a risk of later development of gestational hypertension. First trimester screening of extracellular miR-520h alone or in combination with miR-518b identified a significant proportion of women with subsequent gestational hypertension.     

Integrated miRNA-mRNA Analysis Revealing the Potential Roles of miRNAs in Chordomas

Introduction

Emerging evidence suggests that microRNAs (miRNAs) are crucially involved in tumorigenesis and that paired expression profiles of miRNAs and mRNAs can be used to identify functional miRNA-target relationships with high precision. However, no studies have applied integrated analysis to miRNA and mRNA profiles in chordomas. The purpose of this study was to provide insights into the pathogenesis of chordomas by using this integrated analysis method.

Methods

Differentially expressed miRNAs and mRNAs of chordomas (n = 3) and notochord tissues (n = 3) were analyzed by using microarrays with hierarchical clustering analysis. Subsequently, the target genes of the differentially expressed miRNAs were predicted and overlapped with the differentially expressed mRNAs. Then, GO and pathway analyses were performed for the intersecting genes.

Results

The microarray analysis indicated that 33 miRNAs and 2,791 mRNAs were significantly dysregulated between the two groups. Among the 2,791 mRNAs, 911 overlapped with putative miRNA target genes. A pathway analysis showed that the MAPK pathway was consistently enriched in the chordoma tissue and that miR-149-3p, miR-663a, miR-1908, miR-2861 and miR-3185 likely play important roles in the regulation of MAPK pathways. Furthermore, the Notch signaling pathway and the loss of the calcification or ossification capacity of the notochord may also be involved in chordoma pathogenesis.

Conclusion

This study provides an integrated dataset of the miRNA and mRNA profiles in chordomas, and the results demonstrate that not only the MAPK pathway and its related miRNAs but also the Notch pathway may be involved in chordoma development. The occurrence of chordoma may be associated with dysfunctional calcification or ossification of the notochord.     

Urinary miR-29 Correlates with Albuminuria and Carotid Intima-Media Thickness in Type 2 Diabetes Patients

Background

Cell-free microRNAs stably and abundantly exist in body fluids and emerging evidence suggests cell-free microRNAs as novel and non-invasive disease biomarker. Deregulation of miR-29 is involved in the pathogenesis of diabetic nephropathy and insulin resistance thus may be implicated in diabetic vascular complication. Therefore, we investigated the possibility of urinary miR-29 as biomarker for diabetic nephropathy and atherosclerosis in patients with type 2 diabetes.

Methods

83 patients with type 2 diabetes were enrolled in this study, miR-29a, miR-29b and miR-29c levels in urine supernatant was determined by TaqMan qRT-PCR, and a synthetic cel-miR-39 was added to the urine as a spike-in control before miRNAs extraction. Urinary albumin excretion rate and urine albumin/creatinine ratio, funduscopy and carotid ultrasound were used for evaluation of diabetic vascular complication. The laboratory parameters indicating blood glucose level, renal function and serum lipids were also collected.

Results

Patients with albuminuria (n = 42, age 60.62±12.00yrs) showed significantly higher comorbidity of diabetic retinopathy (p = 0.015) and higher levels of urinary miR-29a (p = 0.035) compared with those with normoalbuminuria (n = 41, age 58.54±14.40yrs). There was no significant difference in urinary miR-29b (p = 0.148) or miR-29c level (p = 0.321) between groups. Urinary albumin excretion rate significantly correlated with urinary miR-29a level (r = 0.286, p = 0.016), while urinary miR-29b significantly correlated with carotid intima-media thickness (cIMT) (r = 0.286, p = 0.046).

Conclusion

Urinary miR-29a correlated with albuminuria while urinary miR-29b correlated with carotid intima-media thickness (cIMT) in patients with type 2 diabetes. Therefore, they may have the potential to serve as alternative biomarker for diabetic nephropathy and atherosclerosis in type 2 diabetes.     

The Clinical Significance of MiR-148a as a Predictive Biomarker in Patients with Advanced Colorectal Cancer

Aim

Development of robust prognostic and/or predictive biomarkers in patients with colorectal cancer (CRC) is imperative for advancing treatment strategies for this disease. We aimed to determine whether expression status of certain miRNAs might have prognostic/predictive value in CRC patients treated with conventional cytotoxic chemotherapies.

Methods

We studied a cohort of 273 CRC specimens from stage II/III patients treated with 5-fluorouracil-based adjuvant chemotherapy and stage IV patients subjected to 5-fluorouracil and oxaliplatin-based chemotherapy. In a screening set (n = 44), 13 of 21 candidate miRNAs were successfully quantified by multiplex quantitative RT-PCR. In the validation set comprising of the entire patient cohort, miR-148a expression status was assessed by quantitative RT-PCR, and its promoter methylation was quantified by bisulfite pyrosequencing. Lastly, we analyzed the associations between miR-148a expression and patient survival.

Results

Among the candidate miRNAs studied, miR-148a expression was most significantly down-regulated in advanced CRC tissues. In stage III and IV CRC, low miR-148a expression was associated with significantly shorter disease free-survival (DFS), a worse therapeutic response, and poor overall survival (OS). Furthermore, miR-148a methylation status correlated inversely with its expression, and was associated with worse survival in stage IV CRC. In multivariate analysis, miR-148a expression was an independent prognostic/predictive biomarker for advanced CRC patients (DFS in stage III, low vs. high expression, HR 2.11; OS in stage IV, HR 1.93).

Discussion

MiR-148a status has a prognostic/predictive value in advanced CRC patients treated with conventional chemotherapy, which has important clinical implications in improving therapeutic strategies and personalized management of this malignancy.     

miR-485-5p Binding Site SNP rs8752 in HPGD Gene Is Associated with Breast Cancer Risk

Background

Single nucleotide polymorphisms (SNPs) that reside in microRNA target sites may play an important role in breast cancer development and progression. To reveal the association between microRNA target site SNPs and breast cancer risk, we performed a large case-control study in China.

Methods

We performed a two-stage case-control study including 2744 breast cancer cases and 3125 controls. In Stage I, we genotyped 192 SNPs within microRNA binding sites identified from the “Patrocles” database using custom Illumina GoldenGate VeraCode assays on the Illumina BeadXpress platform. In Stage II, genotyping was performed on SNPs potentially associated with breast cancer risk using the TaqMan platform in an independent replication set.

Results

In stage I, 15 SNPs were identified to be significantly associated with breast cancer risk (P<0.05). In stage II, one SNP rs8752 was replicated at P<0.05. This SNP is located in the 3’ untranslated region (UTR) of the 15-hydroxyprostaglandin dehydrogenase (HPGD) gene at 4q34-35, a miR-485-5p binding site. Compared with the GG genotype, the combined GA+AA genotypes has a significantly higher risk of breast cancer (OR = 1.18; 95% CI: 1.06-1.31, P = 0.002). Specifically, this SNP was associated with estrogen receptor (ER) positive breast cancer (P = 0.0007), but not with ER negative breast cancer (P = 0.23), though p for heterogeneity not significant.

Conclusion

Through a systematic case-control study of microRNA binding site SNPs, we identified a new breast cancer risk variant rs8752 in HPGD in Chinese women. Further studies are warranted to investigate the underling mechanism for this association.     

No Correlation between TIMP2 -418 G>C Polymorphism and Increased Risk of Cancer: Evidence from a Meta-Analysis

Aim

Tissue inhibitor of metalloproteinase (TIMP2) is involved in the regulation of matrix metalloproteinase 2 (MMP2) and shown to implicate in cancer development and progression. The results from the published studies based on the association between TIMP2 -418 G>C polymorphism and cancer risk are inconsistent. In this meta-analysis, we aimed to evaluate the potential association between TIMP2 -418 G>C polymorphism and cancer risk.

Methodology

We searched PubMed (Medline) and EMBASE web databases to cover all studies based on relationship of TIMP2 -418 G>C polymorphism and risk of cancer until October 2013. The meta-analysis was performed for selected case-control studies and pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated for all genetic models.

Results

A total of 2225 cancer cases and 2532 controls were included from ten eligible case-control studies. Results from overall pooled analysis suggested no evidence of significant risk between TIMP2 -418 G>C polymorphism and cancer risk in any of the genetic models, such as, allele (C vs. G: OR = 1.293, 95% CI = 0.882 to 1.894, p = 0.188), homozygous (CC vs. GG: OR = 0.940, 95% CI = 0.434 to 2.039, p = 0.876), heterozygous (GC vs. GG: OR = 1.397, 95% CI = 0.888 to 2.198, p = 0.148), dominant (CC+GC vs. GG: OR = 1.387, 95% CI = 0.880 to 2.187, p = 0.159) and recessive (CC vs. GG+GC: OR = 0.901, 95% CI = 0.442 to 1.838, p = 0.774) models. No evidence of publication bias was detected during the analysis.

Conclusions

The present meta-analysis suggests that the TIMP2 -418 G>C polymorphism may not be involved in predisposing risk factor for cancer in overall population. However, future larger studies with group of populations are needed to analyze the possible correlation.     

Unique Behavioral Characteristics and microRNA Signatures in a Drug Resistant Epilepsy Model

Background

Pharmacoresistance is a major issue in the treatment of epilepsy. However, the mechanism underlying pharmacoresistance to antiepileptic drugs (AEDs) is still unclear, and few animal models have been established for studying drug resistant epilepsy (DRE). In our study, spontaneous recurrent seizures (SRSs) were investigated by video-EEG monitoring during the entire procedure.

Methods/Principal Findings

In the mouse pilocarpine-induced epilepsy model, we administered levetiracetam (LEV) and valproate (VPA) in sequence. AED-responsive and AED-resistant mice were naturally selected after 7-day treatment of LEV and VPA. Behavioral tests (open field, object exploration, elevated plus maze, and light-dark transition test) and a microRNA microarray test were performed. Among the 37 epileptic mice with SRS, 23 showed significantly fewer SRSs during administration of LEV (n = 16, LEV sensitive (LS) group) or VPA (n = 7, LEV resistant/VPA sensitive (LRVS) group), while 7 epileptic mice did not show any amelioration with either of the AEDs (n = 7, multidrug resistant (MDR) group). On the behavioral assessment, MDR mice displayed distinctive behaviors in the object exploration and elevated plus maze tests, which were not observed in the LS group. Expression of miRNA was altered in LS and MDR groups, and we identified 4 miRNAs (miR-206, miR-374, miR-468, and miR-142-5p), which were differently modulated in the MDR group versus both control and LS groups.

Conclusion

This is the first study to identify a pharmacoresistant subgroup, resistant to 2 AEDs, in the pilocarpine-induced epilepsy model. We hypothesize that modulation of the identified miRNAs may play a key role in developing pharmacoresistance and behavioral alterations in the MDR group.     

Clinical Evidence for Three Distinct Gastric Cancer Subtypes: Time for a New Approach

Background

Recently, a new classification for gastric cancer (GC) has been proposed, based on Lauren''s histology and on anatomic tumour location, identifying three subtypes of disease: type 1 (proximal non diffuse GC), type 2 (diffuse GC) and type 3 (distal non diffuse GC). Aim of our analysis was to compare clinical outcome according to different GC subtypes (1,2,3) in metastatic GC patients receiving first-line chemotherapy.

Patients and Methods

Advanced GC pts treated with a first-line combination chemotherapy were included in our analysis. Pts were divided in three subgroups (type 1, type 2 and type 3) as previously defined.

Results

A total of 248 advanced GC pts were included: 45.2% belonged to type 2, 43.6% to type 3 and 11.2% to type 1. Patients received a fluoropyrimidine-based chemotherapy doublet or three drugs regimens including a platinum derivate and a fluoropyrimidine with the addition of an anthracycline, a taxane or mytomicin C. RR was higher in type 1 pts (RR = 46.1%) and type 3 (34,3%) compared to type 2 (20,4%), (p = 0.015). Type 2 presented a shorter PFS, median PFS = 4.2 months, compared to type 1, mPFS = 7.2 months, and type 3, mPFS = 5.9 months (p = 0.011) and also a shorter OS (p = 0.022).

Conclusions

Our analysis suggests that GC subtypes may be important predictors of benefit from chemotherapy in advanced GC patients. Future clinical trials should take in account these differences for a better stratification of patients.     

Determination of 14 Circulating microRNAs in Swedes and Iraqis with and without Diabetes Mellitus Type 2

Background

Recent reports suggest that immigrants from Middle Eastern countries are a high-risk group for type 2 diabetes (T2D) compared with Swedes, and that the pathogenesis of T2D may be ethnicity-specific. Deregulation of microRNA (miRNA) expression has been demonstrated to be associated with T2D but ethnic differences in miRNA have not been investigated. The aim of this study was to explore the ethnic specific expression (Swedish and Iraqi) of a panel of 14 previously identified miRNAs in patients without T2D (including those with prediabetes) and T2D.

Methods

A total of 152 individuals were included in the study (84 Iraqis and 68 Swedes). Nineteen Iraqis and 14 Swedes were diagnosed with T2D. Expression of the 14 selected miRNAs (miR-15a, miR-20, miR-21, miR-24, miR-29b, miR-126, miR-144, miR-150, miR-197, miR-223, miR-191, miR-320a, miR-486-5p, and miR-28-3p) in plasma samples was measured by real-time PCR.

Results

In the whole study population, the expression of miR-24 and miR-29b was significantly different between T2D patients and controls after adjustment for age, sex, waist circumference, family history of T2D, and a sedentary lifestyle. Interestingly, when stratifying the study population according to country of birth, we found that higher expression of miR-144 was significantly associated with T2D in Swedes (OR = 2.43, p = 0.035), but not in Iraqis (OR = 0.54, p = 0.169). The interaction test was significant (p = 0.017).

Conclusion

This study suggests that the association between plasma miR-144 expression and T2D differs between Swedes and Iraqis.