Bacillus cereus spores and cereulide in food-borne illness

B. cereus is a gram-positive bacterium that possesses two different forms of life:the large, rod-shaped cells (ca. 0.002 mm by 0.004 mm) that are able to propagate and the small (0.001 mm), oval shaped spores. The spores can survive in almost any environment for up to centuries without nourishment or water. They are insensitive towards most agents that normally kill bacteria: heating up to several hours at 90 ºC, radiation, disinfectants and extreme alkaline (≥ pH 13) and acid (≤ pH 1) environment. The spores are highly hydrophobic and therefore make them tend to stick to all kinds of surfaces, steel, plastics and live cells. In favorable conditions the spores of B. cereus may germinate into vegetative cells capable of producing food poisoning toxins. The toxins can be heat-labile protein formed after ingestion of the contaminated food, inside the gastrointestinal tract (diarrhoeal toxins), or heat stable peptides formed in the food (emesis causing toxin, cereulide). Cereulide cannot be inactivated in foods by cooking or any other procedure applicable on food. Cereulide in consumed food causes serious illness in human, even fatalities. In this thesis, B. cereus strains originating from different kinds of foods and environments and 8 different countries were inspected for their capability of forming cereulide. Of the 1041 isolates from soil, animal feed, water, air, used bedding, grass, dung and equipment only 1.2 % were capable of producing cereulide, whereas of the 144 isolates originating from foods 24 % were cereulide producers. Cereulide was detected by two methods: by its toxicity towards mammalian cells (sperm assay) and by its peculiar chemical structure using liquid-chromatograph-mass spectrometry equipment. B. cereus is known as one of the most frequent bacteria occurring in food. Most foods contain more than one kind of B. cereus. When randomly selected 100 isolates of B. cereus from commercial infant foods (dry formulas) were tested, 11% of these produced cereulide. Considering a frequent content of 103 to 104 cfu (colony forming units) of B. cereus per gram of infant food formula (dry), it appears likely that most servings (200 ml, 30 g of the powder reconstituted with water) may contain cereulide producers. When a reconstituted infant formula was inoculated with >105 cfu of cereulide producing B. cereus per ml and left at room temperature, cereulide accumulated to food poisoning levels (> 0.1 mg of cereulide per serving) within 24 hours. Paradoxically, the amount of cereulide (per g of food) increased 10 to 50 fold when the food was diluted 4 - 15 fold with water. The amount of the produced cereulide strongly depended on the composition of the formula: most toxin was formed in formulas with cereals mixed with milk, and least toxin in formulas based on milk only. In spite of the aggressive cleaning practices executed by the modern dairy industry, certain genotypes of B. cereus appear to colonise the silos tanks. In this thesis four strategies to explain their survival of their spores in dairy silos were identified. First, high survival (log 15 min kill ≤ 1.5) in the hot alkaline (pH >13) wash liquid, used at the dairies for cleaning-in-place. Second, efficient adherence of the spores to stainless steel from cold water. Third, a cereulide producing group with spores characterized by slow germination in rich medium and well preserved viability when exposed to heating at 90 ºC. Fourth, spores capable of germinating at 8 ºC and possessing the psychrotolerance gene, cspA. There were indications that spores highly resistant to hot 1% sodium hydroxide may be effectively inactivated by hot 0.9% nitric acid. Eight out of the 14 dairy silo tank isolates possessing hot alkali resistant spores were capable of germinating and forming biofilm in whole milk, not previously reported for B. cereus. In this thesis it was shown that cereulide producing B. cereus was capable of inhibiting the growth of cereulide non-producing B. cereus occurring in the same food. This phenomenon, called antagonism, has long been known to exist between B. cereus and other microbial species, e.g. various species of Bacillus, gram-negative bacteria and plant pathogenic fungi. In this thesis intra-species antagonism of B. cereus was shown for the first time. This brother-killing did not depend on the cereulide molecule, also some of the cereulide non-producers were potent antagonists. Interestingly, the antagonistic clades were most frequently found in isolates from food implicated with human illness. The antagonistic property was therefore proposed in this thesis as a novel virulence factor that increases the human morbidity of the species B. cereus, in particular of the cereulide producers.

Äidinmaito - terveysjuomaa ja normaalibakteereita

The chemical composition of breast milk has been studied in detail in the past decades. Hundreds of new antibacterial and antiviral components have been found. Several molecules have been found to promote the proper function of neonatal intestine. However, microbiological studies of breast milk have been, until recently, focused mainly on detecting harmful and pathogenic bacteria and viruses. Natural microbial diversity of human milk has not been widely studied before the work reported in this thesis. This is mainly because breast milk has traditionally been thought to be sterile - even if a certain amount of commensal bacteria have usually been detected in milk samples. The first part of this licentiate thesis contains a short literature review about the anatomy and physiology of breast feeding, human milk chemical and microbiological composition, mastitis, intestinal flora and bacteriocins. The second part reports on the experiments of the licentiate work, concentrating on the microbial diversity in the milk of healthy breast-feeding mothers, and the ability of these bacteria to produce antibacterial substances against pathogenic bacteria. The results indicate that human milk is a source of commensal bacteria for infant intestine. 509 random isolates from 40 breast milk samples were isolated and identified by 16S rRNA sequencing. Median bacterial count was about 600 colony forming units per milliliter. Over half of the isolates were staphylococci, and almost one third streptococci. The most common species were skin bacteria Staphylococcus epidermidis and oral bacteria Streptococcus salivarius and Streptococcus mitis. Lactic acid bacteria, identified as members of Lactobacillus-, Lactococcus- and Leuconostoc -genera, were found in five milk samples. Enterococci were found in three samples. A novel finding in this study is the capability of these commensal bacteria to inhibit the growth of pathogens. In 90 precent of the milk samples commensal bacteria inhibiting the growth of Staphylococcus aureus were found. In 40 precent of samples the colonies could block the growth completely. One fifth of the isolated Staph. epidermidis strains, half of Str. salivarius strains, and all lactic acid bacteria and enterococci could inhibit or block the growth of Staph. aureus. In further study also Listeria innocua- and Micrococcus luteus active isolates were found in 33 and 11 precent of milk samples (out of 140). Furthermore, two Lactococcus lactis isolates from the breast milk were shown to produce bacteriocin nisin, which is an antimicrobial molecule used as a food preservative. The importance of these human milk commensal bacteria in the development of newborn intestinal flora and immune system, as well as in preventing maternal breast infections, should be further explored.

Chemical and source characterisation of size-segregated urban air particulate matter

Recent epidemiological studies have shown a consistent association of the mass concentration of urban air thoracic (PM10) and fine (PM2.5) particles with mortality and morbidity among cardiorespiratory patients. However, the chemical characteristics of different particulate size ranges and the biological mechanisms responsible for these adverse health effects are not well known. The principal aims of this thesis were to validate a high volume cascade impactor (HVCI) for the collection of particulate matter for physicochemical and toxicological studies, and to make an in-depth chemical and source characterisation of samples collected during different pollution situations. The particulate samples were collected with the HVCI, virtual impactors and a Berner low pressure impactor in six European cities: Helsinki, Duisburg, Prague, Amsterdam, Barcelona and Athens. The samples were analysed for particle mass, common ions, total and water-soluble elements as well as elemental and organic carbon. Laboratory calibration and field comparisons indicated that the HVCI can provide a unique large capacity, high efficiency sampling of size-segregated aerosol particles. The cutoff sizes of the recommended HVCI configuration were 2.4, 0.9 and 0.2 μm. The HVCI mass concentrations were in a good agreement with the reference methods, but the chemical composition of especially the fine particulate samples showed some differences. This implies that the chemical characterization of the exposure variable in toxicological studies needs to be done from the same HVCI samples as used in cell and animal studies. The data from parallel, low volume reference samplers provide valuable additional information for chemical mass closure and source assessment. The major components of PM2.5 in the virtual impactor samples were carbonaceous compounds, secondary inorganic ions and sea salt, whereas those of coarse particles (PM2.5-10) were soil-derived compounds, carbonaceous compounds, sea salt and nitrate. The major and minor components together accounted for 77-106% and 77-96% of the gravimetrically-measured masses of fine and coarse particles, respectively. Relatively large differences between sampling campaigns were observed in the organic carbon content of the PM2.5 samples as well as the mineral composition of the PM2.5-10 samples. A source assessment based on chemical tracers suggested clear differences in the dominant sources (e.g. traffic, residential heating with solid fuels, metal industry plants, regional or long-range transport) between the sampling campaigns. In summary, the field campaigns exhibited different profiles with regard to particulate sources, size distribution and chemical composition, thus, providing a highly useful setup for toxicological studies on the size-segregated HVCI samples.

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.

Characterization of genomic diversity in extraintestinal pathogenic Escherichia coli (ExPEC) and development of a diagnostic DNA microarray for the differentiation of ExPEC isolates causing urinary tract infections

Extraintestinal pathogenic Escherichia coli (ExPEC) represent a diverse group of strains of E. coli, which infect extraintestinal sites, such as the urinary tract, the bloodstream, the meninges, the peritoneal cavity, and the lungs. Urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC), the major subgroup of ExPEC, are among the most prevalent microbial diseases world wide and a substantial burden for public health care systems. UTIs are responsible for serious morbidity and mortality in the elderly, in young children, and in immune-compromised and hospitalized patients. ExPEC strains are different, both from genetic and clinical perspectives, from commensal E. coli strains belonging to the normal intestinal flora and from intestinal pathogenic E. coli strains causing diarrhea. ExPEC strains are characterized by a broad range of alternate virulence factors, such as adhesins, toxins, and iron accumulation systems. Unlike diarrheagenic E. coli, whose distinctive virulence determinants evoke characteristic diarrheagenic symptoms and signs, ExPEC strains are exceedingly heterogeneous and are known to possess no specific virulence factors or a set of factors, which are obligatory for the infection of a certain extraintestinal site (e. g. the urinary tract). The ExPEC genomes are highly diverse mosaic structures in permanent flux. These strains have obtained a significant amount of DNA (predictably up to 25% of the genomes) through acquisition of foreign DNA from diverse related or non-related donor species by lateral transfer of mobile genetic elements, including pathogenicity islands (PAIs), plasmids, phages, transposons, and insertion elements. The ability of ExPEC strains to cause disease is mainly derived from this horizontally acquired gene pool; the extragenous DNA facilitates rapid adaptation of the pathogen to changing conditions and hence the extent of the spectrum of sites that can be infected. However, neither the amount of unique DNA in different ExPEC strains (or UPEC strains) nor the mechanisms lying behind the observed genomic mobility are known. Due to this extreme heterogeneity of the UPEC and ExPEC populations in general, the routine surveillance of ExPEC is exceedingly difficult. In this project, we presented a novel virulence gene algorithm (VGA) for the estimation of the extraintestinal virulence potential (VP, pathogenicity risk) of clinically relevant ExPECs and fecal E. coli isolates. The VGA was based on a DNA microarray specific for the ExPEC phenotype (ExPEC pathoarray). This array contained 77 DNA probes homologous with known (e.g. adhesion factors, iron accumulation systems, and toxins) and putative (e.g. genes predictably involved in adhesion, iron uptake, or in metabolic functions) ExPEC virulence determinants. In total, 25 of DNA probes homologous with known virulence factors and 36 of DNA probes representing putative extraintestinal virulence determinants were found at significantly higher frequency in virulent ExPEC isolates than in commensal E. coli strains. We showed that the ExPEC pathoarray and the VGA could be readily used for the differentiation of highly virulent ExPECs both from less virulent ExPEC clones and from commensal E. coli strains as well. Implementing the VGA in a group of unknown ExPECs (n=53) and fecal E. coli isolates (n=37), 83% of strains were correctly identified as extraintestinal virulent or commensal E. coli. Conversely, 15% of clinical ExPECs and 19% of fecal E. coli strains failed to raster into their respective pathogenic and non-pathogenic groups. Clinical data and virulence gene profiles of these strains warranted the estimated VPs; UPEC strains with atypically low risk-ratios were largely isolated from patients with certain medical history, including diabetes mellitus or catheterization, or from elderly patients. In addition, fecal E. coli strains with VPs characteristic for ExPEC were shown to represent the diagnostically important fraction of resident strains of the gut flora with a high potential of causing extraintestinal infections. Interestingly, a large fraction of DNA probes associated with the ExPEC phenotype corresponded to novel DNA sequences without any known function in UTIs and thus represented new genetic markers for the extraintestinal virulence. These DNA probes included unknown DNA sequences originating from the genomic subtractions of four clinical ExPEC isolates as well as from five novel cosmid sequences identified in the UPEC strains HE300 and JS299. The characterized cosmid sequences (pJS332, pJS448, pJS666, pJS700, and pJS706) revealed complex modular DNA structures with known and unknown DNA fragments arranged in a puzzle-like manner and integrated into the common E. coli genomic backbone. Furthermore, cosmid pJS332 of the UPEC strain HE300, which carried a chromosomal virulence gene cluster (iroBCDEN) encoding the salmochelin siderophore system, was shown to be part of a transmissible plasmid of Salmonella enterica. Taken together, the results of this project pointed towards the assumptions that first, (i) homologous recombination, even within coding genes, contributes to the observed mosaicism of ExPEC genomes and secondly, (ii) besides en block transfer of large DNA regions (e.g. chromosomal PAIs) also rearrangements of small DNA modules provide a means of genomic plasticity. The data presented in this project supplemented previous whole genome sequencing projects of E. coli and indicated that each E. coli genome displays a unique assemblage of individual mosaic structures, which enable these strains to successfully colonize and infect different anatomical sites.

Differentiation of neural stem cells in fragile X syndrome

Multipotent stem cells can self-renew and give rise to multiple cell types. One type of mammalian multipotent stem cells are neural stem cells (NSC)s, which can generate neurons, astrocytes and oligodendrocytes. NSCs are likely involved in learning and memory, but their exact role in cognitive function in the developing and adult brain is unclear. We have studied properties of NSCs in fragile X syndrome (FXS), which is the most common form of inherited mental retardation. FXS is caused by the lack of functional fragile X mental retardation protein (FMRP). FMRP is involved in the regulation of postsynaptic protein synthesis in a group I metabotropic glutamate receptor 5 (mGluR5)-dependent manner. In the absence of functional FMRP, the formation of functional synapses is impaired in the forebrain which results in alterations in synaptic plasticity. In our studies, we found that FMRP-deficient NSCs generated more neurons and less glia than control NSCs. The newborn neurons derived from FMRP-deficient NSCs showed an abnormally immature morphology. Furthermore, FMRP-deficient NSCs exhibited aberrant oscillatory Ca2+ responses to glutamate, which were specifically abolished by an antagonist of the mGluR5 receptor. The data suggested alterations in glutamatergic differentiation of FMRP-deficient NSCs and were further supported by an accumulation of cells committed to glutamatergic lineage in the subventricular zone of the embryonic Fmr1-knockout (Fmr1-KO) neocortex. Postnatally, the aberrant cells likely contributed to abnormal formation of the neocortex. The findings suggested a defect in the differentiation of distinct glutamatergic mGluR5 responsive cells in the absence of functional FMRP. Furthermore, we found that in the early postnatal Fmr1-KO mouse brain, the expression of mRNA for regulator of G-protein signalling-4 (RGS4) was decreased which was in line with disturbed G-protein signalling in NSCs lacking FMRP. Brain derived neurotrophic factor (BDNF) promotes neuronal differentiation of NSCs as the absence of FMRP was shown to do. This led us to study the effect of impaired BDNF/TrkB receptor signaling on NSCs by overexpression of TrkB.T1 receptor isoform. We showed that changes in the relative expression levels of the full-length and truncated TrkB isoforms influenced the replication capacity of NSCs. After the differentiation, the overexpression of TrkB.T1 increased neuronal turnover. To summarize, FMRP and TrkB signaling are involved in normal differentiation of NSCs in the developing brain. Since NSCs might have potential for therapeutic interventions in a variety of neurological disorders, our findings may be useful in the design of pharmacological interventions in neurological disorders of learning and memory.

CMV-, EBV-, and HHV-6-DNAemia after liver transplantation

Viral infections caused by herpesviruses are common complications after organ transplantation and they are associated with substantial morbidity and even mortality. Herpesviruses remain in a latent state in a host after primary infection and may reactivate later. CMV infection is the most important viral infection after liver transplantation. Less is known about the significance of human herpesvirus-6 (HHV-6). EBV is believed to play a major role in the development of post-transplant lymphoproliferative disorders (PTLD). The aim of this study was to investigate the CMV-, EBV- and HHV-6 DNAemia after liver transplantation by frequent monitoring of adult liver transplant patients. The presence of CMV, EBV and HHV-6 DNA were demonstrated by in situ hybridization assays and by real-time PCR methods from peripheral blood specimens. CMV and HHV-6 antigens were demonstrated by antigenemia assays and compared to the viral DNAemia. The response to antiviral therapy was also investigated. CMV-DNAemia appeared earlier than CMV pp65-antigenemia after liver transplantation. CMV infections were treated with ganciclovir. However, most of the treated patients demonstrated persistence of CMV-DNA for up to several months. Continuous CMV-DNA expression of peripheral blood leukocytes showed that the virus is not eliminated by ganciclovir and recurrences can be expected during several months after liver transplantation. HHV-6 DNAemia / antigenemia was common and occurred usually within the first three months after liver transplantation together with CMV. The HHV-6 DNA expression in peripheral blood mononuclear cells correlated well with HHV-6 antigenemia. Antiviral treatment significantly decreased the number of HHV-6 DNA positive cells, demonstrating the response to ganciclovir treatment. Clinically silent EBV reactivations with low viral loads were relatively common after liver transplantation. These EBV-DNAemias usually appeared within the first three months after liver transplantation together with betaherpesviruses (CMV, HHV-6, HHV-7). One patient developed high EBV viral loads and developed PTLD. These results indicate that frequent monitoring of EBV-DNA levels can be useful to detect liver transplant patients at risk of developing PTLD.

Primary total hip arthroplasty for primary osteoarthritis in Finland : A national register based analysis

The first aim of the current study was to evaluate the survival of total hip arthroplasty (THA) in patients aged 55 years and older on a nation-wide level. The second aim was to evaluate, on a nation wide-basis, the geographical variation of the incidence of primary THA for primary OA and also to identify those variables that are possibly associated with this variation. The third aim was to evaluate the effects of hospital volume: on the length of stay, on the numbers of re-admissions and on the numbers of complications of THR on population-based level in Finland. The survival of implants was analysed based on data from the Finnish Arthroplasty Register. The incidence and hospital volume data were obtained from the Hospital Discharge Register. Cementless total hip replacements had a significantly reduced risk of revision for aseptic loosening compared with cemented hip replacements. When revision for any reason was the end point in the survival analyses, there were no significant differences found between the groups. Adjusted incidence ratios of THA varied from 1.9- to 3.0-fold during the study period. Neither the average income within a region nor the morbidity index was associated with the incidence of THA. For the four categories of volume of total hip replacements performed per hospital, the length of the surgical treatment period was shorter for the highest volume group than for the lowest volume group. The odds ratio for dislocations was significantly lower in the high volume group than in the low volume group. In patients who were 55 years of age or older, the survival of cementless total hip replacements was as good as that of the cemented replacements. However, multiple wear-related revisions of the cementless cups indicate that excessive polyethylene wear was a major clinical problem with modular cementless cups. The variation in the long-term rates of survival for different cemented stems was considerable. Cementless proximal porous-coated stems were found to be a good option for elderly patients. When hip surgery was performed on with a large repertoire, the indications to perform THAs due to primary OA were tight. Socio-economic status of the patient had no apparent effect on THA rate. Specialization of hip replacements in high volume hospitals should reduce costs by significantly shortening the length of stay, and may reduce the dislocation rate.

Placental abruption : Studies on incidence, risk factors and potential predictive biomarkers

Placental abruption, one of the most significant causes of perinatal mortality and maternal morbidity, occurs in 0.5-1% of pregnancies. Its etiology is unknown, but defective trophoblastic invasion of the spiral arteries and consequent poor vascularization may play a role. The aim of this study was to define the prepregnancy risk factors of placental abruption, to define the risk factors during the index pregnancy, and to describe the clinical presentation of placental abruption. We also wanted to find a biochemical marker for predicting placental abruption early in pregnancy. Among women delivering at the University Hospital of Helsinki in 1997-2001 (n=46,742), 198 women with placental abruption and 396 control women were identified. The overall incidence of placental abruption was 0.42%. The prepregnancy risk factors were smoking (OR 1.7; 95% CI 1.1, 2.7), uterine malformation (OR 8.1; 1.7, 40), previous cesarean section (OR 1.7; 1.1, 2.8), and history of placental abruption (OR 4.5; 1.1, 18). The risk factors during the index pregnancy were maternal (adjusted OR 1.8; 95% CI 1.1, 2.9) and paternal smoking (2.2; 1.3, 3.6), use of alcohol (2.2; 1.1, 4.4), placenta previa (5.7; 1.4, 23.1), preeclampsia (2.7; 1.3, 5.6) and chorioamnionitis (3.3; 1.0, 10.0). Vaginal bleeding (70%), abdominal pain (51%), bloody amniotic fluid (50%) and fetal heart rate abnormalities (69%) were the most common clinical manifestations of placental abruption. Retroplacental blood clot was seen by ultrasound in 15% of the cases. Neither bleeding nor pain was present in 19% of the cases. Overall, 59% went into preterm labor (OR 12.9; 95% CI 8.3, 19.8), and 91% were delivered by cesarean section (34.7; 20.0, 60.1). Of the newborns, 25% were growth restricted. The perinatal mortality rate was 9.2% (OR 10.1; 95% CI 3.4, 30.1). We then tested selected biochemical markers for prediction of placental abruption. The median of the maternal serum alpha-fetoprotein (MSAFP) multiples of median (MoM) (1.21) was significantly higher in the abruption group (n=57) than in the control group (n=108) (1.07) (p=0.004) at 15-16 gestational weeks. In multivariate analysis, elevated MSAFP remained as an independent risk factor for placental abruption, adjusting for parity ≥ 3, smoking, previous placental abruption, preeclampsia, bleeding in II or III trimester, and placenta previa. MSAFP ≥ 1.5 MoM had a sensitivity of 29% and a false positive rate of 10%. The levels of the maternal serum free beta human chorionic gonadotrophin MoM did not differ between the cases and the controls. None of the angiogenic factors (soluble endoglin, soluble fms-like tyrosine kinase 1, or placental growth factor) showed any difference between the cases (n=42) and the controls (n=50) in the second trimester. The levels of C-reactive protein (CRP) showed no difference between the cases (n=181) and the controls (n=261) (median 2.35 mg/l [interquartile range {IQR} 1.09-5.93] versus 2.28 mg/l [IQR 0.92-5.01], not significant) when tested in the first trimester (mean 10.4 gestational weeks). Chlamydia pneumoniae specific immunoglobulin G (IgG) and immunoglobulin A (IgA) as well as C. trachomatis specific IgG, IgA and chlamydial heat-shock protein 60 antibody rates were similar between the groups. In conclusion, although univariate analysis identified many prepregnancy risk factors for placental abruption, only smoking, uterine malformation, previous cesarean section and history of placental abruption remained significant by multivariate analysis. During the index pregnancy maternal alcohol consumption and smoking and smoking by the partner turned out to be the major independent risk factors for placental abruption. Smoking by both partners multiplied the risk. The liberal use of ultrasound examination contributed little to the management of women with placental abruption. Although second-trimester MSAFP levels were higher in women with subsequent placental abruption, clinical usefulness of this test is limited due to low sensitivity and high false positive rate. Similarly, angiogenic factors in early second trimester, or CRP levels, or chlamydial antibodies in the first trimester failed to predict placental abruption.

Microsurgical Aneurysm Model in Rats and Mice: Development of Endovascular Treatment and Optimization of Magnetic Resonance Imaging

The rupture of a cerebral artery aneurysm causes a devastating subarachnoid hemorrhage (SAH), with a mortality of almost 50% during the first month. Each year, 8-11/100 000 people suffer from aneurysmal SAH in Western countries, but the number is twice as high in Finland and Japan. The disease is most common among those of working age, the mean age at rupture being 50-55 years. Unruptured cerebral aneurysms are found in 2-6% of the population, but knowledge about the true risk of rupture is limited. The vast majority of aneurysms should be considered rupture-prone, and treatment for these patients is warranted. Both unruptured and ruptured aneurysms can be treated by either microsurgical clipping or endovascular embolization. In a standard microsurgical procedure, the neck of the aneurysm is closed by a metal clip, sealing off the aneurysm from the circulation. Endovascular embolization is performed by packing the aneurysm from the inside of the vessel lumen with detachable platinum coils. Coiling is associated with slightly lower morbidity and mortality than microsurgery, but the long-term results of microsurgically treated aneurysms are better. Endovascular treatment methods are constantly being developed further in order to achieve better long-term results. New coils and novel embolic agents need to be tested in a variety of animal models before they can be used in humans. In this study, we developed an experimental rat aneurysm model and showed its suitability for testing endovascular devices. We optimized noninvasive MRI sequences at 4.7 Tesla for follow-up of coiled experimental aneurysms and for volumetric measurement of aneurysm neck remnants. We used this model to compare platinum coils with polyglycolic-polylactic acid (PGLA) -coated coils, and showed the benefits of the latter in this model. The experimental aneurysm model and the imaging methods also gave insight into the mechanisms involved in aneurysm formation, and the model can be used in the development of novel imaging techniques. This model is affordable, easily reproducible, reliable, and suitable for MRI follow-up. It is also suitable for endovascular treatment, and it evades spontaneous occlusion.

Mediastinitis after cardiac surgery

Mediastinitis as a complication after cardiac surgery is rare but disastrous increasing the hospital stay, hospital costs, morbidity and mortality. It occurs in 1-3 % of patients after median sternotomy. The purpose of this study was to find out the risk factors and also to investigate new ways to prevent mediastinitis. First, we assessed operating room air contamination monitoring by comparing the bacteriological technique with continuous particle counting in low level contamination achieved by ultra clean garment options in 66 coronary artery bypass grafting operations. Second, we examined surgical glove perforations and the changes in bacterial flora of surgeons' fingertips in 116 open-heart operations. Third, the effect of gentamicin-collagen sponge on preventing surgical site infections (SSI) was studied in randomized controlled study with 557 participants. Finally, incidence, outcome, and risk factors of mediastinitis were studied in over 10,000 patients. With the alternative garment and textile system (cotton group and clean air suit group), the air counts fell from 25 to 7 colony-forming units/m3 (P<0.01). The contamination of the sternal wound was reduced by 46% and that of the leg wound by >90%. In only 17% operations both gloves were found unpunctured. Frequency of glove perforations and bacteria counts of hands were found to increase with operation time. With local gentamicin prophylaxis slightly less SSIs (4.0 vs. 5.9%) and mediastinitis (1.1 vs. 1.9%) occurred. We identified 120/10713 cases of postoperative mediastinitis (1.1%). During the study period, the patient population grew significantly older, the proportion of women and patients with ASA score >3 increased significantly. In multivariate logistic regression analysis, the only significant predictor for mediastinitis was obesity. Continuous particle monitoring is a good intraoperative method to control the air contamination related to the theatre staff behavior during individual operation. When a glove puncture is detected, both gloves are to be changed. Before donning a new pair of gloves, the renewed disinfection of hands will help to keep their bacterial counts lower even towards the end of long operation. Gentamicin-collagen sponge may have beneficial effects on the prevention of SSI, but further research is needed. Mediastinitis is not diminishing. Larger populations at risk, for example proportions of overweight patients, reinforce the importance of surveillance and pose a challenge in focusing preventive measures.

Determinants of outcome in critically ill patients

Assessment of the outcome of critical illness is complex. Severity scoring systems and organ dysfunction scores are traditional tools in mortality and morbidity prediction in intensive care. Their ability to explain risk of death is impressive for large cohorts of patients, but insufficient for an individual patient. Although events before intensive care unit (ICU) admission are prognostically important, the prediction models utilize data collected at and just after ICU admission. In addition, several biomarkers have been evaluated to predict mortality, but none has proven entirely useful in clinical practice. Therefore, new prognostic markers of critical illness are vital when evaluating the intensive care outcome. The aim of this dissertation was to investigate new measures and biological markers of critical illness and to evaluate their predictive value and association with mortality and disease severity. The impact of delay in emergency department (ED) on intensive care outcome, measured as hospital mortality and health-related quality of life (HRQoL) at 6 months, was assessed in 1537 consecutive patients admitted to medical ICU. Two new biological markers were investigated in two separate patient populations: in 231 ICU patients and 255 patients with severe sepsis or septic shock. Cell-free plasma DNA is a surrogate marker of apoptosis. Its association with disease severity and mortality rate was evaluated in ICU patients. Next, the predictive value of plasma DNA regarding mortality and its association with the degree of organ dysfunction and disease severity was evaluated in severe sepsis or septic shock. Heme oxygenase-1 (HO-1) is a potential regulator of apoptosis. Finally, HO-1 plasma concentrations and HO-1 gene polymorphisms and their association with outcome were evaluated in ICU patients. The length of ED stay was not associated with outcome of intensive care. The hospital mortality rate was significantly lower in patients admitted to the medical ICU from the ED than from the non-ED, and the HRQoL in the critically ill at 6 months was significantly lower than in the age- and sex-matched general population. In the ICU patient population, the maximum plasma DNA concentration measured during the first 96 hours in intensive care correlated significantly with disease severity and degree of organ failure and was independently associated with hospital mortality. In patients with severe sepsis or septic shock, the cell-free plasma DNA concentrations were significantly higher in ICU and hospital nonsurvivors than in survivors and showed a moderate discriminative power regarding ICU mortality. Plasma DNA was an independent predictor for ICU mortality, but not for hospital mortality. The degree of organ dysfunction correlated independently with plasma DNA concentration in severe sepsis and plasma HO-1 concentration in ICU patients. The HO-1 -413T/GT(L)/+99C haplotype was associated with HO-1 plasma levels and frequency of multiple organ dysfunction. Plasma DNA and HO-1 concentrations may support the assessment of outcome or organ failure development in critically ill patients, although their value is limited and requires further evaluation.

Long-term outcomes of esophageal atresia

Esophageal atresia (EA), a common congenital anomaly comprising interrupted esophagus with or without a tracheoesophageal fistula (TEF), affects one in 2840 newborns. Over half have associated anomalies. After EA repair in infancy, gastroesophageal reflux (GER) and esophageal dysmotility and respiratory problems are common. As there exist no previous population-based long-term follow-up-studies on EA, its long-term sequelae are unclear. The aims of this study were to assess the cancer incidence (I), esophageal morbidity and function (II), respiratory morbidity (III), and the spinal defects (IV) in adults with repaired EA. All patients treated for EA at the Hospital for Children and Adolescents, University of Helsinki, from 1947 to 1985 were identified, and those alive with their native esophagus were contacted, and the first hundred who replied made up the study group. The patients were interviewed, they filled in symptom questionnaires, and they underwent esophageal endoscopy and manometry, pulmonary function tests, and a full orthopedic evaluation was performed with radiographs of the spine. The questionnaire was also sent by mail to adults with repaired EA not attending the clinical study, and to 287 general population-derived controls matched for age, gender, and municipality of residence. Incidence of cancer among the study population was evaluated from the population-based countrywide cancer registry. 169 (72%) adults with repaired EA replied; 101 (42%) (58 male) participated in the clinical studies at a median age of 36 years (range, 22-56). Symptomatic GER occurred in 34% and dysphagia in 85% of the patients and in 8% and 2% of the controls (P<0.001 for both). The main endoscopic findings included hiatal hernia (28%), Barrett´s esophagus (11%), esophagitis (8%), and stenotic anastomosis (8%). Histology revealed esophagitis in 25 individuals, and epithelial metaplasia in another 21. At immunohistochemistry, CDX2-positive columnar epithelial metaplasia was present in all 21 individuals, and 6 of these also demonstrated goblet cells and MUC2 positivity. In all histological groups, GER and dysphagia were equally common (P=ns). Esophageal manometry demonstrated non-propagating peristalsis in most of the patients, and low ineffective pressure of the distal esophageal body in all. The changes were significantly worse in those with epithelial metaplasia (P≤0.022). Anastomotic complications (OR 8.6-24, 95%CI 1.7-260, P=0.011-0.008), age (OR 20, 95%CI 1.3-310, P=0.034), low distal esophageal body pressure (OR 2.6, 95%CI 0.7-10, P=0.002), and defective esophageal peristalsis (OR 2.2, 95%CI 0.4-11, P=0.014) all predicted development of epithelial metaplasia. Despite the high incidence of esophageal metaplasia, none of the EA patients had suffered esophageal cancer, according to the Finnish Cancer Registry. Although three had had cancer (SIR, 1.0; 95% CI, 0.20-2.8). The overall cancer incidence among adults with repaired EA did not differ from that of the general Finnish population. Current respiratory symptoms occurred in 11% of the patients and 2% of the controls (P<0.001). Of the patients, 16%, and 6% of the controls had doctor-diagnosed asthma (P<0.001). A total of 56% and 70% of the patients and 20% and 50% of the controls had a history of pneumonia and of bronchitis (P<0.001 for both). Respiratory-related impaired quality of life was observable in 11% of the patients in contrast to 6% of the controls (P<0.001). PFT revealed obstruction in 21 of the patients, restriction in 21, and both in 36. A total of 41 had bronchial hyper-responsiveness (BHR) in HCT, and 15 others had an asthma-like response. Thoracotomy-induced rib fusion (OR 3.4, 95%CI 1.3-8.7, P=0.01) and GER-associated epithelial metaplasia in adulthood (OR 3.0, 95%CI 1.0-8.9, P=0.05) were the most significant risk factors for restrictive ventilatory defect. Vertebral anomalies were evident in 45 patients, predominating in the cervical spine in 38. The most significant risk factor for the occurrence of vertebral anomalies was any additional anomaly (OR 27, 95%C I8-100). Scoliosis (over 10 degrees) was observable in 56 patients, over 20 degrees in 11, and over 45 degrees in one. In the EA patients, risk for scoliosis over 10 degrees was 13-fold (OR 13, 95%CI 8.3-21) and over 20 degrees, 38-fold (OR 38, 95%CI 14-106) when compared to that of the general population. Thoracotomy-induced rib fusion (OR 3.6, 95%CI 0.7-19) and other associated anomalies (OR 2.1, 95%CI 0.9-2.9) were the strongest predictive factors for scoliosis. Significant esophageal morbidity associated with EA extends into adulthood. No association existed between the esophageal symptoms and histological findings. Surgical complications, increasing age, and impaired esophageal motility predicted development of epithelial metaplasia after repair of EA. According to our data, the risk for esophageal cancer is less than 500-fold that of the general population. However, the overall cancer incidence among adults with repaired EA did not differ from that of the general population. Adults with repaired EA have had significantly more respiratory symptoms and infections, as well as more asthma, and allergies than does the general population. Thoracotomy-induced rib fusion and GER-associated columnar epithelial metaplasia were the most significant risk factors for the restrictive ventilatory defect that occurred in over half the patients. Over half the patients with repaired EA are likely to develop scoliosis. Risk for scoliosis is 13-fold after repair of EA in relation to that of the general population. Nearly half the patients had vertebral anomalies. Most of these deformities were diagnosed neither in infancy nor during growth. The natural history of spinal deformities seems, however, rather benign, with spinal surgery rarely indicated.

Cytomegalovirus in the Process of Chronic Allograft Nephropathy

Cytomegalovirus (CMV) is a major cause of morbidity, costs and even mortality in organ transplant recipients. CMV may also enhance the development of chronic allograft nephropathy (CAN), which is the most important cause of graft loss after kidney transplantation. The evidence for the role of CMV in chronic allograft nephropathy is somewhat limited, and controversial results have also been reported. The aim of this study was to investigate the role of CMV in the pathogenesis of CAN. Material for the purpose of this study was available from altogether 70 kidney transplant recipients who received a kidney transplant between the years 1992-2000. CMV infection was diagnosed with pp65 antigenemia test or by viral culture from blood, urine, or both. CMV proteins were demonstrated in the kidney allograft biopsies by immunohistochemisrty and CMV-DNA by in situ hybridization. Cytokines, adhesion molecules, and growth factors were demonstrated from allograft biopsies by immunohistochemistry, and from urinary samples by ELISA-methods. CMV proteins were detectable in the 6-month protocol biopsies from 18/41 recipients with evidence of CMV infection. In the histopathological analysis of the 6-month protocol biopsies, presence of CMV in the allograft together with a previous history of acute rejection episodes was associated with increased arteriosclerotic changes in small arterioles. In urinary samples collected during CMV infection, excretion of TGF-β was significantly increased. In recipients with increased urinary excretion of TGF-β, increased interstitial fibrosis was recorded in the 6- month protocol biopsies. In biopsies taken after an active CMV infection, CMV persisted in the kidney allograft in 17/48 recipients, as CMV DNA or antigens were detected in the biopsies more than 2 months after the last positive finding in blood or urine. This persistence was associated with increased expression of TGF-β, PDGF, and ICAM-1 and with increased vascular changes in the allografts. Graft survival and graft function one and two years after transplantation were reduced in recipients with persistent intragraft CMV. Persistent intragraft CMV infection was also a risk factor for reduced graft survival in Cox regression analysis, and an independent risk factor for poor graft function one and two years after transplantation in logistic regression analysis. In conclusion, these results show that persistent intragraft CMV infection is detrimental to kidney allografts, causing increased expression of growth factors and increased vascular changes, leading to reduced graft function and survival. Effective prevention, diagnosis and treatment of CMV infections may a major factor in improving the long term survival of kidney allograft.

Day Surgery in Finland : randomized and cross-sectional studies on treatment, quality, and outcome

The aims of this study were to describe Finnish day surgery practice at present and to evaluate quality of care by assessing postdischarge minor morbidity and quality indicators. Potential treatment options were approached by investigating the role of oral dexamethasone as a part of multimodal analgesia and the feasibility of day surgery in patients aged 65 years and older. Over a 2-month period, all patient cases at 14 Finnish day surgery or short-stay units were analyzed (Study I). Quality indicators included rates and reasons for overnight admission, readmission, reoperation, cancellations, and patient satisfaction. Recovery during the first postoperative week was assessed at two units (Study II). Altogether 2732 patients graded daily the intensity of predefined symptoms. To define risk factors of postdischarge symptoms, multinomial regression analysis was used. Sixty patients scheduled to undergo day surgery for hallux valgus were randomized to receive twice perioperatively dexamethasone 9 mg or placebo (Study III). Paracetamol 1 g was administered 3 times daily. Rescue medication (oxycodone) consumption during 0-3 postoperative days (POD), maximal pain scores and adverse effects were documented. Medically stable patients aged 65 years or older, scheduled for open inguinal hernia repair, were randomized to receive treatment either as day cases or inpatients (Study IV). Complications, unplanned admissions, healthcare visits, and patients’ acceptance of the type of care provided were assessed during 2 weeks postoperatively. In Study I, unplanned overnight admissions were reported in 5.9%, return hospital visits during PODs 1-28 in 3.7%, and readmissions in 0.7% of patients. Patient satisfaction was high. In Study II, pain was the most common symptom in adult patients (57%). Postdischarge symptoms were more frequent in adults aged < 40 years, children aged ≥ 7 years, females, and following a longer duration of surgery. In Study III, the total median (range) oxycodone consumption during the study period was 45 (0–165) mg in the dexamethasone group, compared with 78 (15–175) mg in the placebo group (P < 0.049). On PODs 0-1, patients in the dexamethasone group reported significantly lower pain scores. Following inguinal hernia repair, no significant differences in outcome measures were seen between the study groups. Patient satisfaction was equally high in day cases and inpatients (Study IV). Finnish day surgery units provide good-quality services. Minor postdischarge symptoms are common, and they are influenced by several patient-, surgery-, and anesthesia-related factors. Oral dexamethasone combined with paracetamol improves pain relief and reduces the need for oxycodone rescue medication following correction of hallux valgus. Day surgery for open inguinal hernia repair is safe and well accepted by patients aged 65 years or older and can be recommended as the primary choice of care for medically stable patients.