Helminth exposure and its association with allergies and lung function in Northern European cohorts

Bakgrunn: De siste tiårene har forekomsten av astma og allergi økt over hele verden. I land hvor parasittinfeksjoner er endemiske, er forekomsten av astma og allergi lavere enn i land med lavere forekomst av parasitter. En hypotese er at parasittinfeksjoner kan beskytte mot allergiske sykdommer. Studier som har undersøkt dette er fremdeles kontroversielle, de fleste studier er fra endemiske områder og studiene gjelder oftest barn og ikke voksne. Rundormen Ascaris lumbricoides forårsaker en av de vanligste parasittinfeksjonene på verdensbasis med om lag 800 millioner smittetilfeller årlig. I løpet av livssyklusen, passerer Ascaris lungene. I tillegg til direkte skade på vev, kan infeksjonen føre til vedvarende immunologiske forandringer. I europeiske land antar man at denne typen infeksjoner begrenses av gode sanitære forhold, men forekomsten av parasitter som helminter (rundorm) og hvordan disse påvirker astma og allergi er lite studert i høyinntektsland. Formålet med studien er å: 1) Beskrive forekomst av Ascaris og Toxocara seropositivitet og eventuell sammenheng med allergiske sykdommer i to generasjoner i Norge; 2) Undersøke sammenhengen mellom helminteksponering og lungefunksjon, astma og DNA-metylering i tre kohorter fra Nord-Europa; 3) Undersøke om humant serum med Ascaris IgG4 binder seg til Ascaris-larver, og om husstøvmidd (HDM) sensibilisering bidrar til anti-Ascaris IgE- og IgG4-responser i humant serum. Material og metode: 1) Konsentrasjon av serum IgG4 mot Ascaris spp. og Toxocara spp. ble målt med endepunkttitrering i to kohorter: Foreldre fra ECRHS III-studien født 1945-1972 (n=171), og barna til disse deltagerne født 1969-2003 (n=264) (RHINESSA- studien). Data om allergisk sykdom og symptomer og andre faktorer ble samlet inn gjennom intervju og kliniske undersøkelser, bl.a. med måling av IgE i serum og hudprikktest for allergisk sensibilisering. Multivariat logistisk regresjon ble benyttet for å analysere sammenheng mellom seropositivitet og allergiske utfall. 2) IgG-antistoffer mot Ascaris i serum ble kvantifisert med ImmunoCAP (ThermoFisher) hos 671 voksne i alderen 18-47 år (46% kvinner) fra RHINESSA sentre i Aarhus, Bergen og Tartu. Seropositivitet ble definert som IgG over 90 persentilen. Multivariat logistisk/lineær regresjon ble anvendt for å analysere sammenhengen mellom Ascaris seropositivitet og astma/lungefunksjon. Alle modeller ble justert for alder, høyde og røyking, og vektet i forhold til studiesenter. DNA-metylering ble målt med Human-MethylationEPIC-chip (Illumina, USA), som dekker 850 CpG sites. I tillegg ble IgG-antistoffer mot fem helminter målt med kommersielt tilgjengelig ELISA kits (NovaTec Immunodiagnostica, Tyskland) for deltakere fra Bergen RHINESSA. 3) ELISA med fire forskjellige Ascaris-antigener for IgG4 og IgE binding ble benyttet for å bekrefte Ascaris-eksponering, hos mannlige RHINESSA deltagere fra Bergen senter kategorisert på bakgrunn av HDM sensibilisering og Ascaris IgG4 serum positivitet. Kruskal-Wallis test og Dunns Multiple sammenligningstest ble bruk for å identifisere signifikante forskjeller (p<0.05) mellom gruppene. Fluorescensmikroskopi ble benyttet for å undersøke IgG i serum og IgE binding til Ascaris-larvene. Resultater: 1) Anti-Ascaris spp. IgG4 påvist i serum hos 29% av deltakerne fra den eldre generasjonen (foreldre) og hos 10% av deres «barn», den yngre generasjonen. Anti-Toxocara spp. IgG4 ble påvist hos 18% av foreldrene og hos 8% av barna. Hos den yngre generasjonen var Toxocara seropositivitet assosiert med å ha hatt kjæledyr før 15 års alder (odds ratio (OR) = 6.15; 95%CI = 1.37-27.5) og økende BMI (1.16 [1.06-1.25] per kg/m2). Toxocara positivitet var assosiert med piping i brystet (justert OR 2.97 [1.45- 7.76]), høysnue (4.03[1.63-9.95]), eksem (2.89 [1.08-7.76]) og sensibilisering mot katt (5.65 [1.92-16.6]) for deltakere i den yngste generasjonen, men ikke for den eldre generasjonen. Selv når kjæledyrhold ble tatt med i betraktningen endret ikke dette sammenhengen mellom Toxocara seropositivitet og allergi. Toxocara spp. Seropositivitet i foreldre-generasjonen var assosiert med høyere forekomst av allergi hos deres barn (kjønns-spesifikke mønster); det bemerkes at eksponeringen ble målt flere år etter at barna var født. 2) Av de fem helmintene som ble målt med kommersielt ELISA kit, var positivt utslag på Ascaris mest hyppig. Ascaris seropositivitet var assosiert med lav FEV1 (-247 mL [-460, -34]) og høyere odds for noen gang å ha hatt astma (aOR 5.84 [1.67, 20.37]); dette gjaldt kun for menn. Funnene endret seg ikke etter å ha tatt høyde for HDM sensibilisering, og til stede for alle studiesentre (Aarhus, Bergen og Tartu). Epigenetikk analysene viste at Ascaris eksponering var forbundet med endret DNA metylering for 23 CpG lokasjoner hos menn og tre hos kvinner. Vi identifiserte hypermetylering i MYBPC1 gener, som er involvert i regulering av luftveiskontraksjon, samt endret metylering av gener som påvirker astma patogenese, slik som CRHR1 og GRK1, og av PRSS22 genet som er assosiert med nematode infeksjon. De epigenetiske analysene understøttet dermed helmint eksponeringsmålingene i populasjonene våre, sammenheng med lungehelseutfall samt kjønnsforskjell i sammenhengene. 3) Serum IgG4 binding mot Ascaris var konsistent, selv når vi benyttet forskjellige antigener. Vi påviste IgG4 binding til Ascaris larver kun for serum med forhøyet nivå av Ascaris spesifikk IgG4 (målt med ELISA), mens sensibilisering mot husstøvmidd alene (uten Ascaris IgG4 seropositivitet) ikke førte til IgG4 eller IgE binding mot Ascaris larver. Ingen av prøvene hadde påvisbare nivåer av IgE mot Ascaris og det ble heller ikke observert spesifikk eller uspesifikk IgE binding mot larvene. Konklusjon: 1) I Norge var eksponering for parasittene Ascaris og Toxocara mindre vanlig hos yngre enn hos eldre generasjoner. Imidlertid var seropositivitet mot disse assosiert med økt risiko for allergisk sykdom og symptomer hos den yngre generasjonen, men ikke hos den eldre generasjonen. Våre funn tyder på at parasitt eksponering per i dag påvirker immunsystemet på en slik måte at det kan gi økt risiko for allergi også i høyinntektsland. 2) Ascaris eksponering var assosiert med lavere lungefunksjon og økt risiko for astma hos menn. Deltagere som var Ascaris seropositive hadde endringer i DNA-metylering, forskjellig for menn og kvinner. Dette kan tyde på at Ascaris eksponering kan føre til kjønnsspesifikke epigenetiske endringer i DNA metylering som kan påvirke lungepatologi. 3) Resultatene tyder på at sensibilisering mot husstøvmidd ikke gir IgG4 eller IgE kryss-sensibilisering mot Ascaris, men at IgG4 mot Ascaris også kan binde mot allergen fra husstøvmidd. Oppsummert er dette en indikasjon på at IgG4 mot Ascaris og HDM reflekterer Ascaris eksponering og ikke HDM sensibilisering. Implikasjoner: Resultatene fra dette PhD-prosjektet tyder på at parasitteksponering i Nord-Europa kan være en risikofaktor for astma, allergi og nedsatt lungefunksjon som har vært oversett. I et folkehelseperspektiv bør det forskes mer på kilder til og eksponeringsveier for parasitter. Vi har vist i dette prosjektet at det er viktig å undersøke hvordan parasitt eksponering påvirker lungehelse i et globalt perspektiv, det gjelder både høy- og middelinntektsland, og ikke minst lavinntektsland hvor parasittinfeksjoner er svært vanlig.Background: The prevalence of asthma and allergies have increased all over the world during the past few decades. In countries where helminth infections are endemic, the prevalence of asthma and allergies appears to be lower. Therefore, it has been hypothesized that helminth infections might confer protection against allergic diseases. Results from studies linking helminth infections and allergies are still controversial with most studies addressing allergy in children in endemic regions. The round worm, Ascaris lumbricoides, is a common helminth infection worldwide with an estimated prevalence of 800 million people. During its life cycle, Ascaris migrates through the lungs causing direct damage. The infection is also likely to cause long-term altered immune responses. Data addressing the prevalence of helminths infections and their association with allergies and asthma in Europe is scarce. High sanitary standards are believed to have broken the life cycle of many helminthiases. Objectives: 1) Describe the prevalence of Ascaris and Toxocara seropositivity and test for association with allergies in two generations in Norway. 2) Investigate the association of lung function, asthma, and DNA methylation with helminth exposure in three Northern European cohorts. 3) Explore if serum anti-Ascaris IgG4 from European cohorts binds to Ascaris larvae, and test if house dust mite (HDM) sensitization leads to anti-Ascaris IgE and IgG4 responses in humans. Materials and Methods: 1) Serum levels of IgG4 against Ascaris spp. and Toxocara spp. were established by end point titration ELISA in two cohorts: parents born 1945-1972 (n = 171) from the ECRHS III study and their offspring born 1969-2003 (n = 264) from the RHINESSA study. Serum samples were first pre-incubated on plates covered with Ascaris proteins before measuring antibody levels towards Toxocara, in order to reduce cross-reactivity with Toxocara spp. Allergic outcomes and covariates were recorded through interviews and clinical examinations including measurement of serum IgE levels and skin prick tests. Multivariate logistic regression models were used to measure the association between seropositivity and allergic outcomes. 2) Serum IgG antibodies against Ascaris were measured with ImmunoCAP (ThermoFisher) in 671 adults aged 18-47 years (46% women) from Aarhus, Bergen, and Tartu RHINESSA study centres. Seropositivity was defined as IgG above the 90th percentile. Multivariate linear and logistic regression models were used to analyse Ascaris seropositivity as associated with lung function measured via spirometry, and asthma. All models were adjusted for age, height, smoking, and clustered by study centre. DNA methylation was measured from peripheral blood using Human- MethylationEPIC (Illumina, USA) chip, which measures 850k 5′-C-phosphate-G-3′ (CpG) sites. Additionally, in the Bergen study centre, IgG antibodies against five helminths were measured with commercially available ELISA kits (NovaTec Immunodiagnostica, Germany). 3) Men from the RHINESSA Bergen study centre were divided into groups, based on their HDM sensitization and Ascaris IgG4 seropositivity. Four different Ascaris antigens were used as coating antigens for the IgG4 and IgE ELISAs to confirm Ascaris exposure. Kruskal-Wallis test followed by Dunn’s Multiple comparison test was used to identify significant differences (p<0.05) between the groups. The presence of serum IgG4 and IgE binding to the larvae was imaged using fluorescence microscopy. Results: 1) Anti-Ascaris spp. IgG4 was detected in 29% of the parent generation and in 10% of the offspring, anti-Toxocara spp. IgG4 in 18% and 8% of parents and offspring, respectively. Among offspring, Toxocara seropositivity was associated with pet keeping before age the of 15 (OR = 6.15; 95%CI = 1.37-27.5) and increasing BMI (1.16 [1.06-1.25] per kg/m2). Toxocara seropositivity was associated with wheezing (adjusted odds ratio 2.97 [1.45- 7.76]), hay fever (4.03[1.63-9.95]), eczema (2.89 [1.08- 7.76]) and sensitization towards cat allergens (5.65 [1.92-16.6]) among offspring, but was not associated with allergic outcomes among parents. Adjustment for childhood or current pet keeping did not alter associations with allergies. Parental Toxocara spp. seropositivity was associated with increased offspring allergies following a sex-specific pattern, although the exposure was measured years after the birth of the offspring. 2) Among the five helminths measured, Ascaris exposure was the most common. Ascaris seropositivity was associated with lower FEV1 (-247 mL [-460, -34]) and higher odds for ever reporting asthma (aOR 5.84 [1.67, 20.37]) among men but not women, also after further adjusting for HDM sensitization. These results were consistent across study centres. At a genome-wide level, Ascaris exposure was associated with 23 differentially methylated DNA sites in men and three in women. We identified hypermethylation of the MYBPC1 gene, which is involved with regulation of airway muscle contraction. We also identified genes linked to asthma pathogenesis such as CRHR1 and GRK1, as well as a differentially methylated region in the PRSS22 gene, previously linked to nematode infection, suggesting that we are measuring true helminth exposure. 3) Serum IgG4 levels against Ascaris were consistent using the different antigens. We detected IgG4 binding to Ascaris larvae only from sera that had raised Ascaris specific IgG4 levels measured with the ELISAs. HDM sensitization only (without concurrent Ascaris IgG4 seropositivity) did not facilitate serum IgG4 or IgE binding to Ascaris larva. Furthermore, no Ascaris specific IgE levels were detected in any of the samples, and no specific or non-specific IgE binding to larvae was detected. Conclusion: 1) Helminth exposure in Norway was less frequent in younger than older adults. However, seropositivity was associated with increased risk of allergic manifestations in the younger generation, but not among their older parents. Potential changes in response to helminth exposure may provide insights into the increase in allergy incidence in affluent countries. 2) Ascaris exposure was associated with lower lung function and increased asthma risk among men. Seropositive participants had sex- specific differences in DNA methylation compared to the unexposed, thus, suggesting that Ascaris exposure may lead to sex-specific epigenetic changes that might contribute to effects on lung pathology. 3) Our results suggest that HDM sensitization does not lead to IgG4 or IgE cross-sensitization towards Ascaris, but that anti-Ascaris IgG4 appears to also bind to HDM allergens. Thus, IgG4 against Ascaris and HDM allergens appear to reflect Ascaris exposure and not HDM sensitization. Implications: Results from this PhD project suggest that exposure to helminths might be an overlooked risk factor in the development of allergies, asthma and low lung function in Northern Europe. From a public health perspective, future research should focus on pinpointing the source of this neglected exposure. Overall, our findings identify a need to investigate the role of helminths on long-term lung health globally, including in high- and middle-income countries, as well as in low-income countries, where helminth exposure is highly prevalent.Doktorgradsavhandlin

Tomographic ventilation-perfusion lung scintigraphy in cardiopulmonary disease

Respiration relies, among other things, on the balance between regional ventilation and perfusion in the lungs. There are many cardiopulmonary diseases, such as pulmonary embolism (PE), chronic obstructive pulmonary disease (COPD) and left heart failure (LHF), which can affect respiration negatively. The diagnosis of PE, COPD and LHF follows separate diagnostic pathways. However, the symptoms that cause the patient to seek medical care are overlapping. This results in a diagnostic dilemma that is complicated by the fact that cardiopulmonary diseases often coexist. Ventilation and perfusion can be imaged with lung scintigraphy. Lung scintigraphy is primarily used to diagnose PE. The introduction of 3-dimensional tomographic lung scintigraphy (V/P SPECT) has resulted in an improved accuracy in the diagnosis of PE. Follow-up with V/P SPECT may lead to better individualization of PE treatment, but has not yet been evaluated. Changes in ventilation and perfusion are also found in COPD and LHF. V/P SPECT may have a clinical role in the diagnosis and characterization of COPD and LHF, but this has been insufficiently studied. Therefore, this thesis focuses on the potential role for V/P SPECT in the follow-up of PE and in the diagnosis and classification of LHF and COPD. In study I, we found that V/P SPECT can be used to diagnose LHF with a high positive predictive value. We developed an algorithm to objectively calculate perfusion gradients and found that an inverted gravitational gradient in the lungs is indicative of LHF. It was also shown that LHF was common among patients with suspected PE. In study II we compared ventilation studies performed with 99mTc-DTPA and 99mTc-Technegas, in patients with and without COPD. This study showed that 99mTc-Technegas, due to a more homogeneous distribution with less focal deposition and better peripheral penetration, should be regarded as the preferred radioaerosol in V/P SPECT studies. Study III indicated an additional value of V/P SPECT in the diagnosis of COPD. V/P SPECT could also be used to characterize the severity of COPD. In study IV, we found that restoration of regional perfusion after acute PE occurred during the first 3 months of treatment, but not thereafter. Follow-up after an episode of PE, using V/P SPECT, seems important since about 20% of the patients in the study had remaining perfusion defects at 3 months after diagnosis, although all were free from symptoms

Reduced administered activity, reduced acquisition time, and preserved image quality for the new CZT camera.

BACKGROUND: For a 1-day myocardial perfusion SPECT (MPS) the recommendations for administered activity stated in the EANM guidelines results in an effective dose of up to 16 mSv per patient. Recently, a gamma camera system, based on cadmium zinc telluride (CZT) technology, was introduced. This technique has the potential to reduce the effective dose and scan time compared to the conventional NaI gamma camera. The aim of this study was to investigate if the effective dose can be reduced with a preserved image quality using CZT technology in MPS. METHODS: In total, 150 patients were included in the study. All underwent a 1-day (99m)Tc-tetrofosmin stress-rest protocol and were divided into three subgroups (n = 50 in each group) with 4, 3, and 2.5 MBq/kg body weight of administered activity in the stress examination, respectively. The acquisition time was increased in proportion to the decrease in administered activity. All examinations were analyzed for image quality by visual grading on a 4-point scale (1 = poor, 2 = adequate, 3 = good, 4 = excellent), by two expert readers. RESULTS: The total effective dose (stress + rest) decreased from 9.3 to 5.8 mSv comparing 4 to 2.5 MBq/kg body weight. For the patients undergoing stress examination only (35%) the effective dose, administrating 2.5 MBq/kg, was 1.4 mSv. The image acquisition times for 2.5 MBq/kg body weight were 475 and 300 seconds (stress and rest) compared to 900 seconds for each when using conventional MPS. The average image quality was 3.7 ± 0.5, 3.8 ± 0.5, and 3.8 ± 0.4 for the stress images and 3.5 ± 0.6, 3.6 ± 0.6, and 3.5 ± 0.6 for the rest images and showed no statistically significant difference (P = .62) among the 4, 3, and 2.5 MBq/kg groups. CONCLUSIONS: The new CZT technology can be used to considerably decrease the effective dose and acquisition time for MPS with preserved high image quality

Weighted Fixed Points in Self-Similar Analysis of Time Series

The self-similar analysis of time series is generalized by introducing the notion of scenario probabilities. This makes it possible to give a complete statistical description for the forecast spectrum by defining the average forecast as a weighted fixed point and by calculating the corresponding a priori standard deviation and variance coefficient. Several examples of stock-market time series illustrate the method.Comment: two additional references are include

Cancer cell differentiation heterogeneity and aggressive behavior in solid tumors

The differentiation stage of tumors is a central aspect in the histopathological classification of solid malignancies. The differentiation stage is strongly associated with tumor behavior, and generally an immature tumor is more aggressive than the more differentiated counterpart. While this is common knowledge in surgical pathology, the contribution of differentiation-related gene expression and functions to tumor behavior is often overlooked in the experimental, tumor biological setting. The mechanisms by which tumor cell differentiation stages are perturbed or affected are poorly explored but have recently come into focus with the introduction.of the tumor stem cell concept. While developmental biologists view the differentiation as a unidirectional event, pathologists and tumor biologists have introduced the concept of dedifferentiation to explain phenotypic changes occurring in solid tumors. In this review we discuss the impact of the tumor cell differentiation stage as used in surgical pathology. We further discuss knowledge gained from exploring the molecular basis of the differentiation and dedifferentiation processes in neuroblastoma and breast cancer, two tumor forms where the tumor cell differentiation concept is used in the clinical diagnostic work and where the tumor stem cell theory has been applied

A prospective study of asthma incidence and its predictors: the RHINE study.

To access publisher full text version of this article. Please click on the hyperlink in Additional LinkThe objective of this longitudinal study was to estimate the incidence rate of asthma, and to compare the incidence between subjects with or without baseline reporting of certain respiratory symptoms. A follow-up of the random population samples in the European Respiratory Health Survey (ECRHS) in Sweden, Norway, Denmark, Iceland and Estonia was conducted in 1999-2001, in a population aged 30-54 yrs at follow-up (n=14,731). Asthma was defined as reporting either asthma or physician-diagnosed asthma, and a reported year when asthma symptoms were first noticed. Incidence rates, incidence rate ratios and hazard ratios were calculated with 95% confidence intervals. The incidence rate of asthma was 2.2 cases per 1,000 person-yrs. The incidence was higher among females (2.9 cases.1,000 person-yrs(-1)) than among males (1.5 cases.1,000 person-yrs(-1)). When subjects with baseline reporting of wheezing were excluded, the incidence rate decreased to 1.7 cases.1,000 person-yrs(-1), with a further decrease to 1.5 cases.1,000 person-yrs(-1) after exclusion of subjects with wheezing, nocturnal dyspnoea, chest tightness and cough. There was a strong association between onset of asthma and wheezing at baseline. In this prospective, population-based study, the incidence rate of asthma in the whole population sample ranged 1.5-2.2.1,000 person-yrs(-1), with a higher incidence range among females. The incidence was dependent on the extent to which subjects with respiratory symptoms were excluded from follow-up. Hence, for comparability between studies, the exclusion criteria in the follow-up population must be stated