Foodborne Yersinia : Identification and Molecular Epidemiology of Isolates from Human Infections

Yersinia enterocolitica and Yersinia pseudotuberculosis are among the major enteropathogenic bacteria causing infections in humans in many industrialized countries. In Finland, Y. pseudotuberculosis has caused 10 outbreaks among humans during 1997-2008. Some of these outbreaks have been very extensive involving over 400 cases; mainly children attending schools and day-care. Y. enterocolitica, on the contrary, has caused mainly a large number of sporadic human infections in Finland. Y. pseudotuberculosis is widespread in nature, causing infections in a variety of domestic and wild animals. Foodborne transmission of human infections has long been suspected, however, attempts to trace the pathogen have been unsuccessful before this study that epidemiologically linked Y. pseudotuberculosis to a specific food item. Furthermore, due to modern food distribution systems, foodborne outbreaks usually involve many geographically separate infection clusters difficult to identify as part of the same outbreak. Among pathogenic Y. enterocolitica, the global predominance of one genetically homogeneous type (bioserotype 4/O:3) is a challenge to the development of genetic typing methods discriminatory enough for epidemiological purposes, for example, for tracing back to the sources of infections. Furthermore, the diagnostics of Y. enterocolitica infections is hampered because clinical laboratories easily misidentify some other members of the Yersinia species (Y. enterocolitica–like species) as Y. enterocolitica. This results in misleading information on the prevalence and clinical significance of various Yersinia isolates. The aim of this study was to develop and optimize molecular typing methods to be used in epidemiological investigations of Y. enterocolitica and Y. pseudotuberculosis, particularly in active surveillance and outbreak investigations of Y. pseudotuberculosis isolates. The aim was also to develop a simplified set of phenotypic tests that could be used in routine diagnostic laboratories for the correct identification of Y. enterocolitica and Y. enterocolitica –like species. A PFGE method designed here for typing of Y. pseudotuberculosis was efficient in linking the geographically dispersed and apparently unrelated Y. pseudotuberculosis infections as parts of the same outbreak. It proved to be useful in active laboratory-based surveillance of Y. pseudotuberculosis outbreaks. Throughout the study period, information about the diversity of genotypes among outbreak and non-outbreak related strains of human origin was obtained. Also, to our knowledge, this was the first study to epidemiologically link a Y. pseudotuberculosis outbreak of human illnesses to a specific food item, iceberg lettuce. A novel epidemiological typing method based on the use of a repeated genomic region (YeO:3RS) as a probe was developed for the detection and differentiation between strains of Y. enterocolitica subspecies palearctica. This method was able to increase the discrimination in a set of 106 previously PFGE typed Finnish Y. enterocolitica bioserotype 4/O:3 strains among which two main PFGE genotypes had prevailed. The developed simplified method was a more reliable tool than the commercially available biochemical test kits for differentiation between Y. enterocolitica and Y. enterocolitica –like species. In Finland, the methods developed for Y. enterocolitica and Y. pseudotuberculosis have been used to improve the identification protocols and in subsequent outbreak investigations.

Modern techniques in detection, identification and quantification of bacteria and peptides from foods

Standards have been placed to regulate the microbial and preservative contents to assure that foods are safe to the consumer. In a case of a food-related disease outbreak, it is crucial to be able to detect and identify quickly and accurately the cause of the disease. In addition, for every day control of food microbial and preservative contents, the detection methods must be easily performed for numerous food samples. In this present study, quicker alternative methods were studied for identification of bacteria by DNA fingerprinting. A flow cytometry method was developed as an alternative to pulsed-field gel electrophoresis, the golden method . DNA fragment sizing by an ultrasensitive flow cytometer was able to discriminate species and strains in a reproducible and comparable manner to pulsed-field gel electrophoresis. This new method was hundreds times faster and 200,000 times more sensitive. Additionally, another DNA fingerprinting identification method was developed based on single-enzyme amplified fragment length polymorphism (SE-AFLP). This method allowed the differentiation of genera, species, and strains of pathogenic bacteria of Bacilli, Staphylococci, Yersinia, and Escherichia coli. These fingerprinting patterns obtained by SE-AFLP were simpler and easier to analyze than those by the traditional amplified fragment length polymorphism by double enzyme digestion. Nisin (E234) is added as a preservative to different types of foods, especially dairy products, around the world. Various detection methods exist for nisin, but they lack in sensitivity, speed or specificity. In this present study, a sensitive nisin-induced green fluorescent protein (GFPuv) bioassay was developed using the Lactococcus lactis two-component signal system NisRK and the nisin-inducible nisA promoter. The bioassay was extremely sensitive with detection limit of 10 pg/ml in culture supernatant. In addition, it was compatible for quantification from various food matrices, such as milk, salad dressings, processed cheese, liquid eggs, and canned tomatoes. Wine has good antimicrobial properties due to its alcohol concentration, low pH, and organic content and therefore often assumed to be microbially safe to consume. Another aim of this thesis was to study the microbiota of wines returned by customers complaining of food-poisoning symptoms. By partial 16S rRNA gene sequence analysis, ribotyping, and boar spermatozoa motility assay, it was identified that one of the wines contained a Bacillus simplex BAC91, which produced a heat-stable substance toxic to the mitochondria of sperm cells. The antibacterial activity of wine was tested on the vegetative cells and spores of B. simplex BAC91, B. cereus type strain ATCC 14579 and cereulide-producing B. cereus F4810/72. Although the vegetative cells and spores of B. simplex BAC91 were sensitive to the antimicrobial effects of wine, the spores of B. cereus strains ATCC 14579 and F4810/72 stayed viable for at least 4 months. According to these results, Bacillus spp., more specifically spores, can be a possible risk to the wine consumer.

Assessment and control of Bacillus cereus emetic toxin in food

Despite of improving levels of hygiene, the incidence of registered food borne disease has been at the same level for many years: there were 40 to 90 epidemics in which 1000-9000 persons contracted food poisoning through food or drinking water in Finland. Until the year 2004 salmonella and campylobacter were the most common bacterial causes of food borne diseases, but in years 2005-2006 Bacillus cereus was the most common. Similar developement has been published i.e. in Germany already in the 1990´s. One reason for this can be Bacillus cereus and its emetic toxin, cereulide. Bacillus cereus is a common environmental bacterium that contaminates raw materials of food. Otherwise than salmonella and campylobacter, Bacillus cereus is a heat resistant bacterium, capable of surviving most cooking procedures due to the production of highly thermo resistant spores. The food involved has usually been heat treated and surviving spores are the source of the food poisoning. The heat treatment induces germination of the spore and the vegetative cells then produce toxins. This doctoral thesis research focuses on developing methods for assessing and eliminating risks to food safety by cereulide producing Bacillus cereus. The biochemistry and physiology of cereulide production was investigated and the results were targeted to offer tools for minimizing toxin risk in food during the production. I developed methods for the extraction and quantitative analysis of cereulide directly from food. A prerequisite for that is knowledge of the chemical and physical properties of the toxin. Because cereulide is practically insoluble in water, I used organic solvents; methanol, ethanol and pentane for the extraction. For extraction of bakery products I used high temperature (100C) and pressure (103.4 bars). Alternaties for effective extraction is to flood the plain food with ethanol, followed by stationary equilibration at room temperature. I used this protocol for extracting cereulide from potato puree and penne. Using this extraction method it is also possible also extract cereulide from liquid food, like milk. These extraction methods are important improvement steps for studying of Bacillus cereus emetic food poisonings. Prior my work, cereulide extraction was done using water. As the result, the yield was poor and variable. To investigate suspected food poisonings, it is important to show actual toxicity of the incriminated food. Many toxins, but not cereulide, inactivate during food processing like heating. The next step is to identify toxin by chemical methods. I developed with my colleague Maria Andesson a rapid assay for the detection of cereulide toxicity, within 5 to 15 minutes. By applying this test it is possible to rapidly detect which food was causing the food poisoning. The chemical identification of cereulide was achieved using mass spectrometry. I used cereulide specific molecular ions, m/z (+/-0.3) 1153.8 (M+H+), 1171.0 (M+NH4+), 1176.0 (M+Na+) and 1191.7 (M+K+) for reliable identification. I investigated foods to find out their amenability to accumulate cereulide. Cereulide was formed high amounts (0.3 to 5.5 microg/g wet wt) when of cereulide producing B. cereus strains were present in beans, rice, rice-pastry and meat-pastry, if stored at non refrigerated temperatures (21-23C). Rice and meat pastries are frequently consumed under conditions where no cooled storage is available e.g. picnics and outdoor events. Bacillus cereus is a ubiquitous spore former and is therefore difficult to eliminate from foods. It is therefore important to know which conditions will affect the formation of cereulide in foods. My research showed that the cereulide content was strongly (10 to 1000 fold differences in toxin content) affected by the growth environment of the bacterium. Storage of foods under nitrogen atmosphere (> 99.5 %) prevented the production of cereulide. But when also carbon dioxide was present, minimizing the oxygen contant (< 1%) did not protect the food from formation of cereulide in preliminary experiments. Also food supplements affected cereulide production at least in the laboratory. Adding free amino acids, leucine and valine, stimulated cereulide production 10 to 20 fold. In peptide bonded form these amino acids are natural constituents in all proteins. Interestingly, adding peptide bonded leucine and valine had no significant effect on cereulide production. Free amino acids leucine and valine are approved food supplements and widely used as flawour modifiers in food technology. My research showed that these food supplements may increase food poisoning risk even though they are not toxic themselves.

Pheromone of the banana-spotting bug, Amblypelta lutescens lutescens Distant (Heteroptera: Coreidae): Identification, synthesis, and field bioassay

The banana-spotting bug, Amblypelta lutescens lutescens Distant (Heteroptera: Coreidae), is one of the principal pests of tree fruits and nuts across northern and eastern Australia. Apart from visual damage assessment, there are currently no reliable methods for monitoring bug activity to aid management decisions. An attractant pheromone for this species that could be used as a trap lure could potentially fill this void. Earlier, two male-specific compounds were identified in airborne extracts from A. lutescens lutescens, (E,E)-α-farnesene and (R,E)-nerolidol; an unknown compound with a molecular weight 220 was also detected. We now report the identification of this hitherto unknown compound as (R,E,E)-α-farnesene-10,11-oxide. Synthesis of this epoxide was conducted using a regioselective asymmetric dihydroxylation of a sulfolene. A blend mimicking the natural proportions of (E,E)-α-farnesene, (R,E)-nerolidol, and (R,E,E)-α-farnesene-10,11- oxide attracted male and female A. lutescens lutescens as well as nymphs in the field, verifying that the aggregation pheromone comprises or is contained within this group of compounds. Copyright © 2012 Ashot Khrimian et al.

Identification of a class of non-linear systems

This paper considers the on-line identification of a non-linear system in terms of a Hammerstein model, with a zero-memory non-linear gain followed by a linear system. The linear part is represented by a Laguerre expansion of its impulse response and the non-linear part by a polynomial. The identification procedure involves determination of the coefficients of the Laguerre expansion of correlation functions and an iterative adjustment of the parameters of the non-linear gain by gradient methods. The method is applicable to situations involving a wide class of input signals. Even in the presence of additive correlated noise, satisfactory performance is achieved with the variance of the error converging to a value close to the variance of the noise. Digital computer simulation establishes the practicability of the scheme in different situations.

Novel fluorinated surfactants tentatively identified in firefighters using liquid chromatography quadrupole time-of flight tandem mass spectrometry and a case-control approach

Fluorinated surfactant-based aqueous film-forming foams (AFFFs) are made up of per- and polyfluorinated alkyl substances (PFAS) and are used to extinguish fires involving highly flammable liquids. The use of perfluorooctanesulfonic acid (PFOS) and other perfluoroalkyl acids (PFAAs) in some AFFF formulations has been linked to substantial environmental contamination. Recent studies have identified a large number of novel and infrequently reported fluorinated surfactants in different AFFF formulations. In this study, a strategy based on a case-control approach using quadrupole time-of-flight tandem mass spectrometry (QTOF-MS/MS) and advanced statistical methods has been used to extract and identify known and unknown PFAS in human serum associated with AFFF-exposed firefighters. Two target sulfonic acids [PFOS and perfluorohexanesulfonic acid (PFHxS)], three non-target acids [perfluoropentanesulfonic acid (PFPeS), perfluoroheptanesulfonic acid (PFHpS), and perfluorononanesulfonic acid (PFNS)], and four unknown sulfonic acids (Cl-PFOS, ketone-PFOS, ether-PFHxS, and Cl-PFHxS) were exclusively or significantly more frequently detected at higher levels in firefighters compared to controls. The application of this strategy has allowed for identification of previously unreported fluorinated chemicals in a timely and cost-efficient way.

QTL analysis in multiple sorghum populations facilitates the dissection of the genetic and physiological control of tillering

Tillering in sorghum can be associated with either the carbon supply–demand (S/D) balance of the plant or an intrinsic propensity to tiller (PTT). Knowledge of the genetic control of tillering could assist breeders in selecting germplasm with tillering characteristics appropriate for their target environments. The aims of this study were to identify QTL for tillering and component traits associated with the S/D balance or PTT, to develop a framework model for the genetic control of tillering in sorghum. Four mapping populations were grown in a number of experiments in south east Queensland, Australia. The QTL analysis suggested that the contribution of traits associated with either the S/D balance or PTT to the genotypic differences in tillering differed among populations. Thirty-four tillering QTL were identified across the populations, of which 15 were novel to this study. Additionally, half of the tillering QTL co-located with QTL for component traits. A comparison of tillering QTL and candidate gene locations identified numerous coincident QTL and gene locations across populations, including the identification of common non-synonymous SNPs in the parental genotypes of two mapping populations in a sorghum homologue of MAX1, a gene involved in the control of tiller bud outgrowth through the production of strigolactones. Combined with a framework for crop physiological processes that underpin genotypic differences in tillering, the co-location of QTL for tillering and component traits and candidate genes allowed the development of a framework QTL model for the genetic control of tillering in sorghum.

The critical period for weed control in dry-seeded rice

The critical crop-weed competition period in a dry-seeded rice system is an important consideration in formulating weed management strategies. Field experiments were conducted in the summer seasons of 2012 and 2013 at the Punjab Agricultural University, Ludhiana, India, to determine the extent of yield loss in two different rice cultivars (PR 114 and PR 115) with different periods of weed interference. Twelve weed control timings were used to identify critical periods of weed competition in dry-seeded rice. PR 114, a long-duration rice cultivar (145 d) having slower initial growth than PR 115 (125 d), was more prone to yield losses. In both years, 100% yield loss was observed where weeds were not controlled throughout the season. In weed-free plots, the grain yield of PR 114 was 6.39-6.80 t ha-1, for PR 115, it was 6.49-6.87 t ha-1. Gompertz and logistic equations fitted to yield data in response to increasing periods of weed control and weed interference showed that, PR 114 had longer critical periods than PR 115. Critical weed-free periods to achieve 95% of weed-free yield for PR 114 was longer than for PR 115 by 31 days in 2012 and 26 days in 2013. Weed infestation also influenced the duration of critical periods. Higher weed pressure in 2012 than in 2013 increased the duration of the critical period of crop-weed competition in that year. The identification of critical crop-weed competition periods for different cultivars will facilitate improved decision-making regarding the timing of weed control and the adoption of cultivars having high weed-suppressing abilities. This will also contribute to the development of integrated weed management in dry-seeded rice systems.

Top-predator control-induced trophic cascades: an alternative hypothesis to the conclusion of Colman et al.

Recently argued that observed positive relationships between dingoes and small mammals were a result of top-down processes whereby lethal dingo control reduced dingoes and increased mesopredators and herbivores, which then suppressed small mammals. Here, I show that the prerequisite negative effects of dingo control on dingoes were not shown, and that the same positive relationships observed may simply represent well-known bottom-up processes whereby more generalist predators are found in places with more of their preferred prey. Identification of top-predator controlinduced trophic cascades first requires demonstration of some actual effect of control on predators, typically possible only through manipulative experiments with the ability to identify cause and effect.

Genomic approach to organ-specific growth control

Growth is a fundamental aspect of life cycle of all organisms. Body size varies highly in most animal groups, such as mammals. Moreover, growth of a multicellular organism is not uniform enlargement of size, but different body parts and organs grow to their characteristic sizes at different times. Currently very little is known about the molecular mechanisms governing this organ-specific growth. The genome sequencing projects have provided complete genomic DNA sequences of several species over the past decade. The amount of genomic sequence information, including sequence variants within species, is constantly increasing. Based on the universal genetic code, we can make sense of this sequence information as far as it codes proteins. However, less is known about the molecular mechanisms that control expression of genes, and about the variations in gene expression that underlie many pathological states in humans. This is caused in part by lack of information about the second genetic code that consists of the binding specificities of transcription factors and the combinatorial code by which transcription factor binding sites are assembled to form tissue-specific and/or ligand-regulated enhancer elements. This thesis presents a high-throughput assay for identification of transcription factor binding specificities, which were then used to measure the DNA binding profiles of transcription factors involved in growth control. We developed ‘enhancer element locator’, a computational tool, which can be used to predict functional enhancer elements. A genome-wide prediction of human and mouse enhancer elements generated a large database of enhancer elements. This database can be used to identify target genes of signaling pathways, and to predict activated transcription factors based on changes in gene expression. Predictions validated in transgenic mouse embryos revealed the presence of multiple tissue-specific enhancers in mouse c- and N-Myc genes, which has implications to organ specific growth control and tumor type specificity of oncogenes. Furthermore, we were able to locate a variation in a single nucleotide, which carries a susceptibility to colorectal cancer, to an enhancer element and propose a mechanism by which this SNP might be involved in generation of colorectal cancer.

Thyroid Hormone Control of Cardiac Substrate Metabolism

Thyroid hormone (TH) plays an important role in maintaining a homeostasis in all the cells of our body. It also has significant cardiovascular effects, and abnormalities of its concentration can cause cardiovascular disease and even morbidity. Especially development of heart failure has been connected to low levels of thyroid hormone. A decrease in TH levels or TH-receptor binding adversely effects cardiac function. Although, this occurs in part through alterations in excitation-contraction and transport proteins, recent data from our laboratory indicate that TH also mediates changes in myocardial energy metabolism. Thyroid dysfunction may limit the heart s ability to shift substrate pathways and provide adequate energy supply during stress responses. Our goals of these studies were to determine substrate oxidation pattern in systemic and cardiac specific hypothyroidism at rest and at higher rates of oxygen demand. Additionally we investigated the TH mediated mechanisms in myocardial substrate selection and established the metabolic phenotype caused by a thyroid receptor dysfunction. We measured cardiac metabolism in an isolated heart model using 13Carbon isotopomer analyses with MR spectroscopy to determine function, oxygen consumption, fluxes and fractional contribution of acetyl-CoA to the citric acid cycle (CAC). Molecular pathways for changes in cardiac function and substrate shifts occurring during stress through thyroid receptor abnormalities were determined by protein analyses. Our results show that TH modifies substrate selection through nuclear-mediated and rapid posttranscriptional mechanisms. It modifies substrate selection differentially at rest and at higher rates of oxygen demand. Chronic TH deficiency depresses total CAC flux and selectively fatty acid flux, whereas acute TH supplementation decreases lactate oxidation. Insertion of a dominant negative thyroid receptor (Δ337T) alters metabolic phenotype and contractive efficiency in heart. The capability of the Δ337T heart to increase carbohydrate oxidation in response to stress seems to be limited. These studies provided a clearer understanding of the TH role in heart disease and shed light to identification of the molecular mechanisms that will facilitate in finding targets for heart failure prevention and treatment.

Assessment of motivation to control alcohol use: The motivational thought frequency and state motivation scales for alcohol control

Introduction The Elaborated Intrusion Theory of Desire holds that desires for functional and dysfunctional goals share a common form. Both are embodied cognitive events, characterised by affective intensity and frequency. Accordingly, we developed scales to measure motivational cognitions for functional goals (Motivational Thought Frequency, MTF; State Motivation, SM), based on the existing Craving Experience Questionnaire (CEQ). When applied to increasing exercise, MTF and SM showed the same three-factor structure as the CEQ (Intensity, Imagery, Availability). The current study tested the internal structure and concurrent validity of the MTF and SM Scales when applied to control of alcohol consumption (MTF-A; SM-A). Methods Participants (N = 417) were adult tertiary students, staff or community members who had recently engaged in high-risk drinking or were currently trying to control alcohol consumption. They completed an online survey comprising the MTF-A, SM-A, Alcohol Use Disorders Identification Test (AUDIT), Readiness to Change Questionnaire (RCQ) and demographics. Results Confirmatory Factor Analysis gave acceptable fit for the MTF-A, but required the loss of one SM-A item, and was improved by intercorrelations of error terms. Higher scores were associated with more severe problems on the AUDIT and with higher Contemplation and Action scores on the RCQ. Conclusions The MTF-A and SM-A show potential as measures of motivation to control drinking. Future research will examine their predictive validity and sensitivity to change. The scales' application to both increasing functional and decreasing dysfunctional behaviours is consistent with EI Theory's contention that both goal types operate in similar ways.

Identification of loci for migraine with aura

Common migraine, i.e. migraine with (MA) or without aura (MO), is a chronic neurological disorder affecting about 10% of the Caucasian population. In MA, migraine headache is preceded by visual, sensoric and/or dysphasic reversible aura symptoms. Twin and family studies have suggested a multifactorial mode of inheritance for common migraine, and a stronger genetic component for MA than for MO. Since there is no biological or genetic marker to identify common migraine, aura symptoms provide a distinctive character to identify those suspected of suffering from migraine. The aim of this study was to identify MA susceptibility loci in well-phenotyped migraine samples with familial predisposition using different gene mapping methods. Genes coding for endothelin1 and its receptors EDNRA and ENDRB are potential candidate genes for cortical spreading depression (CSD), which is considered to be the underlying mechanism of migraine aura. The role of these genes in MA was studied in 850 Finnish migraine cases and 890 control individuals. Rare homozygous EDNRA SNPs showed nominal association with MA and with the age of onset trait (20 years). This result was also detected in the pooled analysis on 648 German MA cases and 651 control individuals when the test was adjusted for gender and sample origin. Evaluation of SNP genotyping reactions with two different DNA polymerase enzymes ensured that the genotype quality was high, and thus the discovered associations are considered reliable. The role of the 19p13 region was studied in a linkage analysis of 72 Finnish MA families. This region contains two migraine-associated genes: CACNA1A, which is associated with a predisposition to a rare Mendelian form of MA, familial hemiplegic migraine (FHM), and the insulin receptor gene (INSR) that is associated with common migraine. No evidence of linkage between the 19p13 and MA was detected. A novel visual aura locus was mapped to chromosome 9q21-q22 with significant evidence of linkage using a genome-wide linkage approach in 36 Finnish MA families. Five additional, potential loci were also detected. The 9q21-q22 region has previously been linked to occipitotemporal lobe epilepsy and MA, both of which involve prominent visual symptoms. Our result further supports a shared background for these episodic disorders.

Tumour necrosis factor receptor-associated periodic syndrome (TRAPS) : Identification of novel TNFRSF1A mutations and intracellular signalling defects

The systemic autoinflammatory disorders are a group of rare diseases characterized by periodically recurring episodes of acute inflammation and a rise in serum acute phase proteins, but with no signs of autoimmunity. At present eight hereditary syndromes are categorized as autoinflammatory, although the definition has also occasionally been extended to other inflammatory disorders, such as Crohn s disease. One of the autoinflammatory disorders is the autosomally dominantly inherited tumour necrosis factor receptor-associated periodic syndrome (TRAPS), which is caused by mutations in the gene encoding the tumour necrosis factor type 1 receptor (TNFRSF1A). In patients of Nordic descent, cases of TRAPS and of three other hereditary fevers, hyperimmunoglobulinemia D with periodic fever syndrome (HIDS), chronic infantile neurologic, cutaneous and articular syndrome (CINCA) and familial cold autoinflammatory syndrome (FCAS), have been reported, TRAPS being the most common of the four. Clinical characteristics of TRAPS are recurrent attacks of high spiking fever, associated with inflammation of serosal membranes and joints, myalgia, migratory rash and conjunctivitis or periorbital cellulitis. Systemic AA amyloidosis may occur as a sequel of the systemic inflammation. The aim of this study was to investigate the genetic background of hereditary periodically occurring fever syndromes in Finnish patients, to explore the reliability of determining serum concentrations of soluble TNFRSF1A and metalloproteinase-induced TNFRSF1A shedding as helpful tools in differential diagnostics, as well as to study intracellular NF-κB signalling in an attempt to widen the knowledge of the pathomechanisms underlying TRAPS. Genomic sequencing revealed two novel TNFRSF1A mutations, F112I and C73R, in two Finnish families. F112I was the first TNFRSF1A mutation to be reported in the third extracellular cysteine-rich domain of the gene and C73R was the third novel mutation to be reported in a Finnish family, with only one other TNFRSF1A mutation having been reported in the Nordic countries. We also presented a differential diagnostic problem in a TRAPS patient, emphasizing for the clinician the importance of differential diagnostic vigiliance in dealing with rare hereditary disorders. The underlying genetic disease of the patient both served as a misleading factor, which possibly postponed arrival at the correct diagnosis, but may also have predisposed to the pathologic condition, which led to a critical state of the patient. Using a method of flow cytometric analysis modified for the use on fresh whole blood, we studied intracellular signalling pathways in three Finnish TRAPS families with the F112I, C73R and the previously reported C88Y mutations. Evaluation of TNF-induced phosphorylation of NF-κB and p38, revealed low phosphorylation profiles in nine out of ten TRAPS patients in comparison to healthy control subjects. This study shows that TRAPS is a diagnostic possibility in patients of Nordic descent, with symptoms of periodically recurring fever and inflammation of the serosa and joints. In particular in the case of a family history of febrile episodes, the possibility of TRAPS should be considered, if an etiology of autoimmune or infectious nature is excluded. The discovery of three different mutations in a population as small as the Finnish, reinforces the notion that the extracellular domain of TNFRSF1A is prone to be mutated at the entire stretch of its cysteine-rich domains and not only at a limited number of sites, suggesting the absence of a founder effect in TRAPS. This study also demonstrates the challenges of clinical work in differentiating the symptoms of rare genetic disorders from those of other pathologic conditions and presents the possibility of an autoinflammatory disorder as being the underlying cause of severe clinical complications. Furthermore, functional studies of fresh blood leukocytes show that TRAPS is often associated with a low NF-κB and p38 phosphorylation profile, although low phosphorylation levels are not a requirement for the development of TRAPS. The aberrant signalling would suggest that the hyperinflammatory phenotype of TRAPS is the result of compensatory NF-κB-mediated regulatory mechanisms triggered by a deficiency of the innate immune response.