Body mass index and all-cause mortality in HUNT and UK biobank studies: revised non-linear Mendelian randomisation analyses.

OBJECTIVES: To estimate the shape of the causal relationship between body mass index (BMI) and mortality risk in a Mendelian randomisation framework. DESIGN: Mendelian randomisation analyses of two prospective population-based cohorts. SETTING: Individuals of European ancestries living in Norway or the UK. PARTICIPANTS: 56 150 participants from the Trøndelag Health Study (HUNT) in Norway and 366 385 participants from UK Biobank recruited by postal invitation. OUTCOMES: All-cause mortality and cause-specific mortality (cardiovascular, cancer, non-cardiovascular non-cancer). RESULTS: A previously published non-linear Mendelian randomisation analysis of these data using the residual stratification method suggested a J-shaped association between genetically predicted BMI and mortality outcomes with the lowest mortality risk at a BMI of around 25 kg/m2. However, the 'constant genetic effect' assumption required by this method is violated. The reanalysis of these data using the more reliable doubly-ranked stratification method provided some indication of a J-shaped relationship, but with much less certainty as there was less precision in estimates at the lower end of the BMI distribution. Evidence for a harmful effect of reducing BMI at low BMI levels was only present in some analyses, and where present, only below 20 kg/m2. A harmful effect of increasing BMI for all-cause mortality was evident above 25 kg/m2, for cardiovascular mortality above 24 kg/m2, for cancer mortality above 30 kg/m2 and for non-cardiovascular non-cancer mortality above 26 kg/m2. In UK Biobank, the association between genetically predicted BMI and mortality at high BMI levels was stronger in women than in men. CONCLUSION: This research challenges findings from previous conventional observational epidemiology and Mendelian randomisation investigations that the lowest level of mortality risk is at a BMI level of around 25 kg/m2. Our results provide some evidence that reductions in BMI will increase mortality risk for a small proportion of the population, and clear evidence that increases in BMI will increase mortality risk for those with BMI above 25 kg/m2.

Sex hormones and risk of lung and colorectal cancers in women: a Mendelian randomization study.

The roles of sex hormones such as estradiol, testosterone, and sex hormone-binding globulin (SHBG) in the etiology of lung and colorectal cancers in women, among the most common cancers after breast cancer, are unclear. This Mendelian randomization (MR) study evaluated such potential causal associations in women of European ancestry. We used summary statistics data from genome-wide association studies (GWASs) on sex hormones and from the Trøndelag Health (HUNT) Study and large consortia on cancers. There was suggestive evidence of genetically predicted 1-standard deviation increase in total testosterone levels being associated with a lower risk of lung non-adenocarcinoma (hazard ratio (HR) 0.60, 95% CI 0.37-0.98) in the HUNT Study. However, this was not confirmed by using data from a larger consortium. In general, we did not find convincing evidence to support a causal role of sex hormones on risk of lung and colorectal cancers in women of European ancestry.

Increased expression of individual genes in whole blood is associated with late-stage lung cancer at and close to diagnosis.

Lung cancer (LC) mortality rates are still increasing globally. As survival is linked to stage, there is a need to identify markers for earlier LC diagnosis and individualized treatment. The whole blood transcriptome of LC patients represents a source of potential LC biomarkers. We compared expression of > 60,000 genes in whole blood specimens taken from LC cases at diagnosis (n = 128) and controls (n = 62) using genome-wide RNA sequencing, and identified 14 candidate genes associated with LC. High expression of ANXA3, ARG1 and HP was strongly associated with lower survival in late-stage LC cases (hazard ratios (HRs) = 2.81, 2.16 and 2.54, respectively). We validated these markers in two independent population-based studies with pre-diagnostic whole blood specimens taken up to eight years prior to LC diagnosis (n = 163 cases, 184 matched controls). ANXA3 and ARG1 expression was strongly associated with LC in these specimens, especially with late-stage LC within two years of diagnosis (odds ratios (ORs) = 3.47 and 5.00, respectively). Additionally, blood CD4 T cells, NK cells and neutrophils were associated with LC at diagnosis and improved LC discriminative ability beyond candidate genes. Our results indicate that in whole blood, increased expression levels of ANXA3, ARG1 and HP are diagnostic and prognostic markers of late-stage LC.

Age at Menarche, age at Natural Menopause, and Risk of Lung and Colorectal Cancers: A Mendelian Randomization Study.

Background: The roles of age at menarche and age at menopause in the etiology of lung and colorectal cancers are unclear. Objective: We aimed to investigate potential causal associations between age at menarche, age at natural menopause, and risk of lung and colorectal cancers using a Mendelian randomization (MR) approach. Methods: From the Trøndelag Health Study in Norway, we defined two cohorts of 35 477 and 17 118 women to study the effects of age at menarche and age at natural menopause, respectively. We ran univariable MR to evaluate the potential causal associations. We performed multivariable MR adjusting for genetic variants of adult body mass index (BMI) to estimate the direct effect of age at menarche. Results: Genetically predicted 1-year increase in age at menarche was associated with a lower risk of lung cancer overall (hazard ratio [HR, 0.64; 95% CI, 0.48-0.86), lung adenocarcinoma (HR, 0.61; 95% CI, 0.38-0.99), and lung non-adenocarcinoma (HR, 0.66; 95% CI, 0.45-0.95). After adjusting for adult BMI using a multivariable MR model, the direct effect estimates reduced to HR 0.72 (95% CI, 0.54-0.95) for lung cancer overall, HR 0.67 (95% CI, 0.43-1.03) for lung adenocarcinoma, and HR 0.77 (95% CI, 0.54-1.09) for lung non-adenocarcinoma. Age at menarche was not associated with colorectal cancer. Moreover, genetically predicted age at natural menopause was not associated with lung and colorectal cancers. Conclusion: Our MR study suggested that later age at menarche was causally associated with a decreased risk of lung cancer overall and its subtypes, and adult BMI might be a mediator.

Estimated cardiorespiratory fitness in relation to overall, breast and prostate cancer incidence: the Norwegian HUNT study.

PURPOSE: To investigate the relationships between the estimated cardiorespiratory fitness (eCRF) and the incidence of overall, breast, and prostate cancer in a large prospective cohort study. METHODS: We included 46,968 cancer-free adults who participated in the second survey of the Trøndelag Health Study in Norway. Sex-specific non-exercise algorithms were used to estimate CRF. eCRF was classified into sex and age-specific tertiles, that is, into low, medium and high levels. Cox proportional hazards models were used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs). RESULTS: Over a median of 22.1 years' follow-up, there were 7752 overall, 858 breast and 1376 prostate cancer cases. Medium and high levels of eCRF were associated with a reduced incidence of overall cancer in a dose-response manner in all participants (HR 0.96; 95% CI, 0.90-1.01 and HR 0.85; 95% CI, 0.79-0.91, respectively, and P-value for trend <.001). No association was observed between eCRF and breast cancer incidence in women. Only the high level of eCRF seemed to be associated with a reduced incidence of prostate cancer in men (HR 0.85; 95% CI, 0.72-1.02). CONCLUSIONS: eCRF may be a practical and cost-effective means of investigating the association between the CRF and cancer incidence.

Reproductive Factors in Relation to Incidence of Lung and Colorectal Cancers in a Cohort of Norwegian Women: The HUNT Study.

Context: The roles of reproductive factors in the etiology of lung and colorectal cancers, among the most common cancers in women, are unclear. Objective: We aimed to explore whether female reproductive factors were associated with the incidence of lung and colorectal cancers. Methods: We followed up 33 314 cancer-free women who participated in the HUNT Study in Norway from 1995-1997 to 2018. A large panel of reproductive factors were self-reported at baseline. Incident lung and colorectal cancer cases were ascertained from the Cancer Registry of Norway. Cox regression models were used to estimate hazard ratios (HRs) with 95% CIs after adjustment for important confounders. Results: During a median follow-up interval of 22.2 years, 467 women developed lung cancer (including 169 lung adenocarcinoma), 660 developed colon cancer, and 211 had rectal cancer. Early menarche (≤12 years) was associated with an increased incidence of lung adenocarcinoma (HR 1.43; 95% CI, 1.02-2.03). Women with one or no child had an increased colon cancer incidence (HR 1.26; 95% CI, 1.03-1.54). Hormone therapy appeared to be associated with a decreased incidence of rectal cancer (HR 0.68; 95% CI, 0.44-1.04). Results in the subgroup of postmenopausal women were similar or strengthened. Other reproductive factors were not related to the risk of lung, colon, and rectal cancers. Conclusion: Certain reproductive factors might play a role in the etiology of lung and colorectal cancers. Further investigations are warranted to study if they are causal associations.

Body mass index and incidence of lung cancer in the HUNT study: using observational and Mendelian randomization approaches.

BACKGROUND: Traditional observational studies have shown an inverse association between body mass index (BMI) and lung cancer risk. Mendelian randomization (MR) analysis using genetic variants as instruments for BMI may clarify the nature of the association. AIMS: We studied the causal association between BMI and lung cancer incidence using observational and MR approaches. METHODS: We followed up 62,453 cancer-free Norwegian adults from 1995-97 (HUNT2) until 2017. BMI at baseline in HUNT2 was classified as < 25.0, 25.0-29.9 and ≥ 30.0 kg/m2. BMI change over ten years between HUNT1 (1984-86) and HUNT2 was calculated and classified into quartiles. Seventy-five genetic variants were included as instruments for BMI (among which 14 also associated with smoking behavior). Incident lung cancer cases were ascertained from the Cancer Registry of Norway. Cox regression models were used to estimate hazard ratios (HRs) with 95% confidence intervals (CIs). Multivariable MR was used to examine the effect of BMI after genetically controlling for smoking. RESULTS: During a median follow-up of 21.1 years, 1009 participants developed lung cancer including 327 with lung adenocarcinoma. The HRs and 95% CIs for incidence of adenocarcinoma were 0.73 (0.58-0.92) for BMI 25.0-29.9 kg/m2 and 0.53 (0.37-0.76) for BMI ≥ 30 kg/m2 compared with BMI < 25.0 kg/m2 in HUNT2 (P for trend < 0.001). However, there was little evidence of a dose-response relationship between the BMI change from HUNT1 to HUNT2 in quartiles and the incidence of adenocarcinoma (P for trend = 0.08). Furthermore, multivariable MR approach suggested a positive association between genetically determined 1 kg/m2 increase in BMI and the incidence of adenocarcinoma (HR 1.25, 95% CI 1.02-1.53). No associations were found with other lung cancer histologic types. CONCLUSIONS: Our study suggests that the inverse association between baseline BMI and lung adenocarcinoma in observational analysis may not be causal. More MR studies are needed to confirm our finding of a positive association between BMI and lung adenocarcinoma.

The validity of self-reported number of teeth and edentulousness among Norwegian older adults, the HUNT Study.

BACKGROUND: Number of teeth is an established indicator of oral health and is commonly self-reported in epidemiological studies due to the costly and labor-intensive nature of clinical examinations. Although previous studies have found self-reported number of teeth to be a reasonably accurate measure, its accuracy among older adults ≥ 70 years is less explored. The aim of this study was to assess the validity of self-reported number of teeth and edentulousness in older adults and to investigate factors that may affect the accuracy of self-reports. METHODS: This study included two different samples of older adults ≥ 70 years drawn from the fourth wave of the Trøndelag Health Study (the HUNT Study), Norway. Sample 1 (n = 586) was used to evaluate the validity of self-reported number of teeth and sample 2 (n = 518) was used to evaluate self-reported edentulousness. Information on number of teeth and background variables (education, smoking, cognitive function, and self-perceived general and oral health) were self-reported in questionnaires, while clinical oral health examinations assessed number of teeth, number of teeth restored or replaced by fixed prosthodontics and edentulousness. Spearman and Pearson correlation coefficients, Bland-Altman plot, chi-square test and kappa statistics were used to assess the agreement between self-reported and clinically recorded number of teeth. RESULTS: The mean difference between self-reported and clinically recorded number of teeth was low (- 0.22 teeth), and more than 70% of the participants reported their number of teeth within an error of two teeth. Correlations between self-reports and clinical examinations were high for the total sample (0.86 (Spearman) and 0.91 (Pearson)). However, a lower correlation was found among participants with dementia (0.74 (Spearman) and 0.85 (Pearson)), participants having ≥ 20 teeth (0.76 (Spearman) and 0.67 (Pearson)), and participants with ≥ 5 teeth restored or replaced by fixed prosthodontics (0.75 (Spearman) and 0.77 (Pearson)). Self-reports of having teeth or being edentulous were correct in 96.3% of the cases (kappa value 0.93, p value < 0.001). CONCLUSIONS: Among older Norwegian adults, self-reported number of teeth agreed closely with clinical tooth counts and nearly all the edentulous participants correctly reported having no teeth.

Assessing the role of genome-wide DNA methylation between smoking and risk of lung cancer using repeated measurements: the HUNT study.

BACKGROUND: It is unclear if smoking-related DNA methylation represents a causal pathway between smoking and risk of lung cancer. We sought to identify novel smoking-related DNA methylation sites in blood, with repeated measurements, and to appraise the putative role of DNA methylation in the pathway between smoking and lung cancer development. METHODS: We derived a nested case-control study from the Trøndelag Health Study (HUNT), including 140 incident patients who developed lung cancer during 2009-13 and 140 controls. We profiled 850 K DNA methylation sites (Illumina Infinium EPIC array) in DNA extracted from blood that was collected in HUNT2 (1995-97) and HUNT3 (2006-08) for the same individuals. Epigenome-wide association studies (EWAS) were performed for a detailed smoking phenotype and for lung cancer. Two-step Mendelian randomization (MR) analyses were performed to assess the potential causal effect of smoking on DNA methylation as well as of DNA methylation (13 sites as putative mediators) on risk of lung cancer. RESULTS: The EWAS for smoking in HUNT2 identified associations at 76 DNA methylation sites (P < 5 × 10-8), including 16 novel sites. Smoking was associated with DNA hypomethylation in a dose-response relationship among 83% of the 76 sites, which was confirmed by analyses using repeated measurements from blood that was collected at 11 years apart for the same individuals. Two-step MR analyses showed evidence for a causal effect of smoking on DNA methylation but no evidence for a causal link between DNA methylation and the risk of lung cancer. CONCLUSIONS: DNA methylation modifications in blood did not seem to represent a causal pathway linking smoking and the lung cancer risk.

Asthma and asthma symptom control in relation to incidence of lung cancer in the HUNT study.

Large prospective studies on asthma, especially asthma symptom control, as a potential risk factor for lung cancer are limited. We followed up 62,791 cancer-free Norwegian adults from 1995-1997 to 2017. Self-reported doctor-diagnosed asthma was categorized into active and non-active asthma. Levels of asthma symptom control were classified into controlled and partially controlled (including partly controlled and uncontrolled) according to the Global Initiative for Asthma guidelines. Incident lung cancer cases were ascertained from the Cancer Registry of Norway. Cox regression models were used to estimate hazard ratios (HRs) with 95% confidence intervals (CIs) for possible associations. Totally, 984 participants developed lung cancer during a median follow-up of 21.1 years. After adjustment for smoking and other potential confounders, an increased incidence of lung cancer was found for adults with partially controlled asthma (HR 1.39, 95% CI 1.00-1.92) compared with those without asthma at baseline. Adults with active asthma had a tendency of increased lung cancer incidence (HR 1.29, 95% CI 0.95-1.75). Sensitivity analyses indicated that the observed associations were less likely resulted from reverse causation or residual confounding by smoking. Our findings suggested that proper control of asthma symptoms might contribute to a reduced incidence of lung cancer.

Hours lying down per day, as a proxy for sedentary behaviour and risk of diabetes in young and middle-aged adults in Norway: an 11-year follow-up of the HUNT study.

OBJECTIVE: We aimed to examine relationship between hours lying down per day, as a proxy for sedentary behaviour and risk of diabetes in young and middle-aged adults, and to assess if leisure-time physical activity and body mass index (BMI) modified this relationship. DESIGN: A population-based prospective cohort study. SETTING: Nord-Trøndelag, Norway. PARTICIPANTS: The cohort included 17 058 diabetes-free adults, at an age of 20-55 years in 1995-1997, who were followed-up to 2006-2008. PRIMARY OUTCOME MEASURES: Incident diabetes was defined by self-report of diabetes or non-fasting glucose levels greater than 11 mmol/L at the follow-up. METHODS: Multivariable logistic regression models were used to obtain OR with 95% CI for risk of diabetes by the categories of hours lying down (≤7, 8 and ≥9 hours/day). RESULTS: 362 individuals (2.1%) developed diabetes during an average of 11-year follow-up. Individuals who reported lying down ≥9 hours/day had an adjusted OR of 1.35 (95% CI 1.01 to 1.80) for incident diabetes compared with those lying down 8 hours/day. Lying down ≤7 hours/day was not associated with the risk of diabetes. In analysis stratified by physical activity, the ORs associated with lying down ≥9 hours/day were 1.41 (95% CI 1.05 to 1.90) and 0.90 (95% CI 0.23 to 3.55), respectively, among the less active and highly active individuals (pinteraction=0.048). There was little evidence that the association differed by BMI status (pinteraction=0.62). CONCLUSIONS: Prolonged hours lying down per day was associated with an increased risk of diabetes in young and middle-aged adults. The positive association appeared to be modified by physical activity but not by BMI.

Body mass index and all cause mortality in HUNT and UK Biobank studies: linear and non-linear mendelian randomisation analyses.

OBJECTIVE: To investigate the shape of the causal relation between body mass index (BMI) and mortality. DESIGN: Linear and non-linear mendelian randomisation analyses. SETTING: Nord-Trøndelag Health (HUNT) Study (Norway) and UK Biobank (United Kingdom). PARTICIPANTS: Middle to early late aged participants of European descent: 56 150 from the HUNT Study and 366 385 from UK Biobank. MAIN OUTCOME MEASURES: All cause and cause specific (cardiovascular, cancer, and non-cardiovascular non-cancer) mortality. RESULTS: 12 015 and 10 344 participants died during a median of 18.5 and 7.0 years of follow-up in the HUNT Study and UK Biobank, respectively. Linear mendelian randomisation analyses indicated an overall positive association between genetically predicted BMI and the risk of all cause mortality. An increase of 1 unit in genetically predicted BMI led to a 5% (95% confidence interval 1% to 8%) higher risk of mortality in overweight participants (BMI 25.0-29.9) and a 9% (4% to 14%) higher risk of mortality in obese participants (BMI ≥30.0) but a 34% (16% to 48%) lower risk in underweight (BMI <18.5) and a 14% (-1% to 27%) lower risk in low normal weight participants (BMI 18.5-19.9). Non-linear mendelian randomisation indicated a J shaped relation between genetically predicted BMI and the risk of all cause mortality, with the lowest risk at a BMI of around 22-25 for the overall sample. Subgroup analyses by smoking status, however, suggested an always-increasing relation of BMI with mortality in never smokers and a J shaped relation in ever smokers. CONCLUSIONS: The previously observed J shaped relation between BMI and risk of all cause mortality appears to have a causal basis, but subgroup analyses by smoking status revealed that the BMI-mortality relation is likely comprised of at least two distinct curves, rather than one J shaped relation. An increased risk of mortality for being underweight was only evident in ever smokers.

Prolonged Sitting, Its Combination With Physical Inactivity and Incidence of Lung Cancer: Prospective Data From the HUNT Study.

Background: Prolonged sitting as a major sedentary behavior potentially contributes to illness, but its relation with lung cancer risk is unclear. Prolonged sitting can be presented in physically active or inactive individuals. Those who are extendedly seated and also physically inactive may represent the most sedentary people. We therefore aimed to prospectively examine if total sitting time daily itself or in combination with physical activity is associated with lung cancer incidence overall and histologic types. Methods: We included 45,810 cancer-free adults who participated in the second survey of HUNT Study in Norway (1995-97), with a median follow-up of 18.3 years. Total sitting time daily and physical activity were self-reported at baseline. Lung cancer cases were ascertained from the Cancer Registry of Norway. Cox regression was used to estimate hazard ratios (HRs) with 95% confidence intervals (CIs). Results: In total, 549 participants developed lung cancer during the follow-up. Total sitting time daily was not associated with the incidence of lung cancer overall and histologic subtypes. Compared with participants sitting < 8 h daily and being physically active, those sitting ≥8 h daily (prolonged sitting) and being physically inactive had an increased incidence of lung cancer (overall: adjusted HR = 1.44, 95% CI: 1.07-1.94; small cell lung cancer: adjusted HR = 2.58, 95% CI: 1.23-5.41). Prolonged sitting only or physical inactivity only was not associated with the incidence of lung cancer. Conclusions: Our study suggested that prolonged sitting was not independently associated with lung cancer incidence. The combination of prolonged sitting and physical inactivity might increase the risk of lung cancer. However, residual confounding by smoking cannot be excluded completely even though smoking was adjusted for with detailed information.

Serum 25-hydroxyvitamin D levels and risk of lung cancer and histologic types: a Mendelian randomisation analysis of the HUNT study.

We aimed to investigate potential causal associations between serum 25-hydroxyvitamin D (25(OH)D) levels and incidence of lung cancer overall and histologic types.We performed a Mendelian randomisation analysis using a prospective cohort study in Norway, including 54 580 individuals and 676 incident lung cancer cases. A 25(OH)D allele score was generated based on the vitamin D-increasing alleles rs2282679, rs12785878 and rs10741657. Hazard ratios with 95% confidence intervals for incidence of lung cancer and histologic types were estimated in relation to the allele score. The inverse-variance weighted method using summarised data of individual single nucleotide polymorphisms was applied to calculate the Mendelian randomisation estimates.The allele score accounted for 3.4% of the variation in serum 25(OH)D levels. There was no association between the allele score and lung cancer incidence overall, with HR 0.99 (95% CI 0.93-1.06) per allele score. A 25 nmol·L-1 increase in genetically determined 25(OH)D level was not associated with the incidence of lung cancer overall (Mendelian randomisation estimate HR 0.96, 95% CI 0.54-1.69) or any histologic type.Mendelian randomisation analysis did not suggest a causal association between 25(OH)D levels and risk of lung cancer overall or histologic types in this population-based cohort study.

Associations of serum 25-hydroxyvitamin D level with incidence of lung cancer and histologic types in Norwegian adults: a case-cohort analysis of the HUNT study.

Previous prospective studies have shown inconsistent associations between serum 25-hydroxyvitamin D [25(OH)D] level and lung cancer incidence. The aim of the present study was to explore the associations of serum 25(OH)D levels with incidence of lung cancer overall and different histologic types. We performed a population-based prospective case-cohort study including 696 incident lung cancer cases and 5804 individuals in a subcohort who participated in the second survey of the Nord-Trøndelag Health Study in Norway. Cox proportional hazards regression models counting for the case-cohort design were used to estimate hazard ratios (HRs) with 95% confidence interval (CIs) for lung cancer overall or histologic types in relation to serum 25(OH)D levels. Compared with the fourth season-specific quartile of 25(OH)D (median 68.0 nmol/L), lower 25(OH)D levels were not associated with the incidence of overall, small or squamous cell lung cancer. However, the risk of adenocarcinoma was lower in the second and third quartiles (median 39.9 and 51.5 nmol/L) compared with the fourth quartile, with HRs of 0.63 (95% CI 0.41-0.98) and 0.58 (0.38-0.88), respectively. The associations of lower levels of 25(OH)D with a reduced risk of adenocarcinoma were only observed in the overweight/obese subjects [HRs for second and third quartiles: 0.40 (0.22-0.72) and 0.50 (0.27-0.92)] but not in the normal weight subjects [HRs: 0.95 (0.52-1.75) and 0.60 (0.32-1.10)]. Serum 25(OH)D levels were not associated with the risk of lung cancer in general. The observation that lower 25(OH)D levels were associated with a lower risk of adenocarcinoma should be interpreted with caution.

Mertk gene expression and photoreceptor outer segment phagocytosis by cultured rat bone marrow mesenchymal stem cells.

BACKGROUND: Bone marrow mesenchymal stem cells (BM-MSCs) are multipotential stem cells that have been used for a broad spectrum of indications. Several investigations have used BM-MSCs to promote photoreceptor survival and suggested that BM-MSCs are a potential source of cell replacement therapy for some forms of retinal degeneration. PURPOSE: To investigate the expression of the MER proto-oncogene, tyrosine kinase (Mertk), involved in the disruption of RPE phagocytosis and the onset of autosomal recessive retinitis pigmentosa in rat BM-MSCs and to compare phagocytosis of the photoreceptor outer segment (POS) by BM-MSCs and RPE cells in vitro. METHODS: MSCs were isolated from the bone marrow of Brown Norway rats. Reverse transcription-PCR (RT-PCR) and western blot analyses were used to examine the expression of Mertk. The phagocytized POS was detected with double fluorescent labeling, transmission electron microscopy, and scanning electron microscopy. RESULTS: Mertk expression did not differ among the first three passages of BM-MSCs. Mertk gene expression was greater in the BM-MSCs than the RPE cells. Mertk protein expression in the BM-MSCs was similar to that in the RPE cells in the primary passage and was greater than that in the RPE cells in the other two passages. BM-MSCs at the first three passages phagocytized the POS more strongly than the RPE cells. The process of BM-MSC phagocytosis was similar to that of the RPE cells. CONCLUSIONS: BM-MSCs may be an effective cell source for treating retinal degeneration in terms of phagocytosis of the POS.

Concurrent genotyping of Helicobacter pylori virulence genes and human cytokine SNP sites using whole genome amplified DNA derived from minute amounts of gastric biopsy specimen DNA.

BACKGROUND: Bacterial and cellular genotyping is becoming increasingly important in the diagnosis of infectious diseases. However, difficulties in obtaining sufficient amount of bacterial and cellular DNA extracted from the same human biopsy specimens is often a limiting factor. In this study, total DNA (host and bacterial DNA) was isolated from minute amounts of gastric biopsy specimens and amplified by means of whole genome amplification using the multiple displacement amplification (MDA) technique. Subsequently, MDA-DNA was used for concurrent Helicobacter pylori and human host cellular DNA genotyping analysis using PCR-based methods. RESULTS: Total DNA was isolated from gastric biopsy specimens of 12 subjects with gastritis and 16 control subjects having a normal mucosa. The DNA was amplified using a multiple displacement amplification (MDA) kit. Next, concurrent genotyping was performed using H. pylori-specific virulence gene PCR amplification assays, pyrosequencing of bacterial 16S rDNA and PCR characterisation of various host genes. This includes Interleukin 1-beta (IL1B) and Interferon-gamma receptor (IFNGR1) SNP analysis, and Interleukin-1 receptor antagonist (IL1RN) variable tandem repeats (VNTR) in intron 2. Finally, regions of the vacA-gene were PCR amplified using M13-sequence tagged primers which allowed for direct DNA sequencing, omitting cloning of PCR amplicons. H. pylori specific multiplex PCR assays revealed the presence of H. pylori cagA and vacA genotypic variations in 11 of 12 gastritis biopsy specimens. Using pyrosequencing, 16S rDNA variable V3 region signatures of H. pylori were found in 11 of 12 individuals with gastritis, but in none of the control subjects. Similarly, IL1B and IFNGR1-SNP and IL1RN-VNTR patterns could be established in all individuals. Furthermore, sequencing of M13-sequence tagged vacA-PCR amplicons revealed the presence of highly diverse H. pylori vacA-s/i/m regions. CONCLUSION: The PCR-based molecular typing methods applied, using MDA-amplified DNA derived from small amounts of gastric biopsy specimens, enabled a rapid and concurrent molecular analysis of bacterial and host genes in the same biopsy specimen. The principles and technologies used in this study could also be applied to any situation in which human host and microbial genes of interest in microbial-host interactions would need to be sequenced.

Multiple strand displacement amplification of DNA isolated from human archival plasma/serum: identification of cytokine polymorphism by pyrosequencing analysis.

BACKGROUND: DNA isolation from formalin-fixed paraffin-embedded tissue appears to be problematic due to degradation caused by fixative. Our aim was to investigate if the isolated genomic DNA from archival plasma/serum, combined with multiple strand displacement amplification (MDA) can be used for genotyping. METHODS: Nine archival plasma/serum samples and freshly frozen gastric biopsies from the same nine H. pylori-infected subjects were used for DNA isolation. Subsequently, MDA-DNA derived from the plasma/serum samples and DNA isolated from the antrum biopsies were analyzed by PCR amplification and pyrosequencing for the presence of interleukin-1beta gene (IL-1B) single nucleotide polymorphism (SNP). In addition, Southern blot and pyrosequencing analysis of H. pylori-specific PCR amplicons were performed. RESULTS: IL-1B SNP profiles obtained from the plasma/serum MDA-DNA and antrum biopsy DNA were identical. A C/C genotype was observed in 7 of 9 samples, and 2 of 9 revealed a C/T genotype for IL-1B -511. Similarly, 7 of 9 had a T/T, and 2 of 9 had a C/T genotype for IL-1B -31; 4 of 9 had a C/C, 4 of 9 had a C/T, and 1 of 9 had a T/T genotype, respectively, for IL-1B +3954. Moreover, pyrosequencing analysis revealed the presence of H. pylori 26695 and J99-like 16S rDNA variable V3 region sequence motifs in the antrum biopsies but not in the plasma/serum samples. CONCLUSIONS: We conclude that MDA combined with pyrosequencing enables a rapid and accurate molecular typing of cytokine single nucleotide polymorphisms from archival plasma/serum samples.

Effects of chronic stress on the immune response to oral human serum albumin-conjugated starch microparticles in rats.

Uptake of antigens and bacteria over the follicle-associated epithelium (FAE) is increased after chronic psychological stress. We investigated whether stress affects the immune response to particle-conjugated antigens taken up via the FAE. Rats were submitted to two 10-day periods of water avoidance stress and orally immunized during these periods. Stressed immunized rats displayed altered cell populations and a Th1-skewed immune response within the lymphoid follicles, together with enhanced delayed-type hypersensitivity. We conclude that chronic stress affects the cell-mediated immune response after oral immunization, which may have implications for the understanding of allergic and autoimmune diseases and development of oral vaccines.