Lääketieteellinen tiedekunta

 

Recent Submissions

  • Hyvärinen, Satu (Helsingin yliopisto, 2016)
    The complement system is a network of over 30 plasma proteins that act in, for example, protecting the body against invading microbes and removing damaged self cells. Complement activation proceeds in a cascade-like fashion, resulting in deposition of certain protein fragments on cell surfaces and liberation of others to the fluid nearby. Due to the destructive nature of complement attacks, it is imperative that this system be stringently controlled. When regulation fails, deleterious complement activation on self cells follows. This is the case for example in atypical hemolytic uremic syndrome (aHUS), a rare thrombotic microangiopathy characterized by hemolysis, thrombocytopenia and renal impairment. In roughly two thirds of aHUS patients, abnormalities in complement genes can be detected. A frequent observation is a mutation in the regulator factor H (FH). The majority of FH mutations in aHUS cluster in the two C-terminal domains of the protein, i.e. FH19-20. These are critical for directing FH onto cell surfaces under complement attack. Several important aspects of the syndrome have been uncovered, but the mechanisms underlying aHUS pathogenesis remain incompletely understood. The general aim of this work was to better explain the molecular events leading to aHUS. First it was asked, whether disturbances in the recently described interactions between FH19-20 and malondialdehyde (MDA) adducts could be involved in aHUS. To study this, binding assays employing MDA-modified proteins and wt and mutant FH19-20 fragments were performed. FH19-20 binding was observed only if a high density of MDA adducts on the surface was present. Since such extensive MDA modification of cell surface proteins is improbable in vivo, it was concluded that disturbed recognition of MDA adducts on cells by FH is unlikely to be relevant in aHUS. Next, the hypothesis of plasminogen to regulate propagation of the complement cascade on cell surfaces was tested. Serum complement was activated on erythrocytes, endothelial cells, and platelets with or without added plasminogen, and complement activation determined as cell lysis or deposition of C5b-9 or C3b. Plasminogen was observed to cause only a very minor inhibition of complement activation on platelets. It was concluded that reduced complement control by plasminogen does not properly explain the recent association of plasminogen deficiency with aHUS. The final aim of this work was to determine the role of sialic acid in FH-mediated regulation on not only erythrocytes, but also on endothelial cells and platelets. To this end, the abilities of several FH19-20 fragments carrying aHUS-associated mutations to antagonize FH function on different types of cells was compared. Flow cytometry detection of FH binding and C3b deposition revealed identical patterns of FH19-20 function on all types of cells. Removal of sialic acid from cells impaired FH function on them. With the help of recent structural data, it was concluded that mutations impair simultaneous binding of the C-terminus of FH to surface sialic acid and C3b, providing a unifying explanation for association between the C-terminus mutations of FH and the clinical disease aHUS. In conclusion, the work presented in this thesis improves our understanding of the pathogenic mechanisms involved in aHUS. Most importantly, the results show that in aHUS, the underlying common defect of various FH C-terminus mutations is the inability to simultaneously bind sialic acid and C3b on cells under complement attack.
  • Barreto, Goncalo (Hansaprint Oy, 2016)
    Osteoarthritis (OA), the most common form of arthritis, is estimated to be in the top 5 leading causes of disability worldwide. Yet OA incidence is estimated to keep growing partly due to the overall worldwide trend of increased obesity and ageing population. Cartilage erosion, a hallmark of OA, has its onset in the traumatic events caused by incorrect biomechanical loading of the joint and the consequent biological response. Currently we still poorly comprehend the molecular pathophysiology of preclinical and clinical symptomatic OA, which consequently results in no current available therapy to prevent OA progression. We hypothesize that innate immunity and its receptor, in particularly toll-like receptors (TLRs), could be major drivers of OA disease progression and onset. The process could be initiated as a proinflammatory reaction against extracellular matrix (ECM)-derived damage-associated molecular patterns (DAMPs). DAMPs accumulate in avascular articular cartilage as a result of traumatization and degeneration, leading directly at their source to a reactive chondrocyte-mediated and TLR-dependent production of proinflammatory and algogenic secondary mediators, which then cause a secondary synovitis with consequent joint pain. For this propose, we collected cartilage and isolated primary chondrocytes from a total of 27 OA patients. Synovial fluid was obtained from knee meniscectomy, total knee arthroplasty (TKA) due to OA, and rheumatoid arthritis (RA) patients generating a total of 30 patient samples. HEK (human embryonic kidney)-blue TLR4 reporter cell line, primary OA chondrocytes, and cartilage explants were used for functional studies. Our results confirmed that TLR1, TLR2 and TLR9 expression is present in healthy primary chondrocytes isolated from articular cartilage, and derived from chondroprogenitors. During our chondrogenesis differentiation studies initial high expression of TLR1, TLR2 and TLR9 was significantly reduced to baseline levels. We demonstrated that proinflammatory cytokine tumour necrosis factor alpha (TNF-α) is able to increase the expression of TLR2 in both healthy primary chondrocytes and mesenchymal stem cells (MSC) derived chondrocytes cultured for 21 days. TNF-α stimulation was demonstrated to induce cartilage degradation in de novo ECM matrix from pellet cultures of MSC-derived chondrocytes cultured for 21 days. This implicates TNF-α as an inducer of matrix degradation, with wide implications in the use of MSCs strategies in cartilage repair strategies for OA. Our study also added further evidence of a role for TNF-α in TLR-innate immunity in the OA synovial joint. TLRs protein expression in cartilage between knee and first carpometacarpal (CMC-I) joints from OA patients was shown to be strikingly different. Our study demonstrated for the first time all TLRs being expressed at protein levels in articular cartilage from knee OA patients. Moreover, we demonstrated that their expression is up-regulated in a cartilage zone-dependent fashion accordingto the histological progression of knee OA. TLRs expression in cartilage from CMC-I OA patients was highly heterogeneous although it followed an expression pattern according to TLRs cellular organization. This indicates that TLR-mediated innate immune response between the two joints may be significantly different. Decorin (DCN), a known small structural proteoglycan with leucine-rich repeats (SLRP) ligand able to activate TLR2 and TLR4, was discovered in knee synovial fluid from OA and RA patients. We confirmed the ability of soluble DCN (sDCN) to activate to TLR4 signaling. However, the observed low and stable concentration levels across the studied groups mean that this may not be of clinical relevance in OA pathogenesis and the associated TLR-mediated inflammatory events. Biglycan (BGN), another known SLRP ligand able to activate TLR2 and TLR4, was discovered in knee synovial fluid from OA and RA patients. Interestingly, we discovered that soluble BGN (sBGN) is upregulated in synovial fluid from OA and RA patients. sBGN ability to activate TLR-innate immunity as confirmed to be essentially activated through TLR4 signaling by studies in articular chondrocytes and human HEK-blue TLR4 reporter cell line. The sBGN stimulation lead to the upregulation and release of proinflammatory cytokines, matrix-degrading enzymes and the release of ECM degradation products. Overall, the results of this thesis demonstrate that TLRs are markedly present in articular cartilage from OA patients at different progression stages of the disease. The detection of BGN and DCN in synovial fluid, and their ability to activate TLR4-mediated proinflammatory cellular responses gives new knowledge of proinflammatory molecules present in the OA synovial joint. An enhanced molecular understanding of the triggering mechanisms by which TLRs are activated and regulated during OA progression stages may help find therapeutic options in the treatment of OA.
  • Ylösmäki, Leena (Helsingin yliopisto, 2016)
    Src homology 3 (SH3) domains are small modular protein structures that recognize and bind to short proline-rich sequence motifs in their ligand proteins. Viral proteins may also harbor such binding motifs and thereby serve as SH3 ligands in order to regulate the host cell signaling to support virus growth and replication, and to modulate virulence. The aim of this study was to examine if influenza A virus (IAV) might also use this strategy to take control of its host cells, and to characterize possible SH3 domain-containing host cell binding partners of IAV to establish their role in the cell biology of IAV infection. IAVs cause seasonal epidemics and occasional pandemics that pose a major threat to human health. The nonstructural protein 1 (NS1) is an important virulence factor of IAV. It is a multifunctional protein that suppresses the host interferon response via multiple mechanisms. Another function of NS1 is to activate phosphatidylinositol-3 kinase (PI3K) signaling in the host cell through direct binding to the p85β regulatory subunit of PI3K. The NS1-induced activation of PI3K is required for efficient replication of many IAV strains. We found that NS1 proteins from some IAV strains contain an SH3 binding site that mediates strong and selective binding to the N-terminal SH3 (nSH3) domain of Crk-family proteins, an important class of adaptor proteins involved in the coordination of cellular signal transduction. This Crk SH3 binding motif was present in the NS1 of infamous 1918 Spanish Flu pandemic virus as well as in many contemporary avian IAV strains. In contrast, it is not found in most NS1 proteins of seasonal human IAV strains. We found that the capacity of avian and Spanish Flu NS1 proteins to interact with Crk SH3 domains provided them with a greatly enhanced capacity to activate PI3K signaling. The molecular mechanism underlying this potentiation was found to be due to a reorganization of the natural PI3K-Crk complex by the SH3-binding competent NS1 protein. Of note, Crk proteins were found to indirectly (via p85β binding) contribute also to PI3K regulation by NS1 proteins of common human IAV strains that lack an SH3 binding motif and a capacity for direct Crk recruitment. Moreover, we found that the role of the NS1/Crk interaction is not limited to PI3K regulation. We observed that binding of NS1 to the Crk SH3 domain induced a robust nuclear accumulation of the predominantly cytoplasmic Crk proteins. This nuclear translocation of Crk proteins was shown to lead to a change in tyrosine phosphorylation pattern of nuclear proteins. In summary, our studies establish Crk adaptor proteins as important cellular co-factors exploited by the IAV virulence factor NS1 to manipulate host cell signaling. These results increase our understanding of the role of NS1 in IAV cell biology, and reveal possible new targets for future antiviral drug development aimed against critical host cell interactions rather than highly mutable viral proteins.
  • Muona, Mikko (Helsingin yliopisto, 2016)
    Epilepsies are a heterogeneous group of central nervous system diseases characterised by recurrent epileptic seizures. They are one of the most common neurological diseases with a lifetime prevalence of ~4%. Epileptic seizures are also a common comorbidity of various neurobiological disorders where epilepsy is not the primary diagnosis. Most epilepsies have a genetic origin, either monogenic or polygenic, however, the causal genetic variants have remained unknown in a substantial proportion of individuals with epilepsies. Over the past decade, technological advances in DNA sequencing have allowed the characterisation of the genetic basis of human disorders rapidly and efficiently. One of the most widely used methods is whole-exome sequencing (WES) where genetic variants in the protein coding regions of the genome, the exome, are captured. Even though the exome constitutes only ~1.5% of the genome, the majority of disease-causing variants underlying severe, monogenic diseases are located in the protein coding regions. Here, we aimed to decipher the molecular genetic basis of severe epilepsy syndromes by utilising WES to identify disease-causing genetic variants in patients without a genetic diagnosis. We studied patients with progressive myoclonus epilepsy (PME, n=84) or severe infantile-onset epileptic syndromes (n=30), which are one of the most devastating forms of genetic syndromes with epilepsy and characterised by frequent, pharmacoresistant seizures and poor prognosis. Given that the patients had undergone genetic testing to varying extent prior to this study, we specifically aimed to establish novel genes and molecular biological mechanisms underlying these syndromes. We made substantial progress in understanding the genetic architecture and molecular basis of the studied syndromes. For PMEs, we established a new major genetic cause and also expanded the genotypic and phenotypic spectrum of previously established disease genes. For severe infantile-onset epileptic syndromes, we identified one new, definite causal gene and one that requires identification of additional patients to confirm the causal role. The three newly identified disease genes represent three different molecular functions that together give new insight on epileptogenic mechanisms. The new PME subtype is caused by a heterozygous missense variant c.959G>A (p.Arg320His) in KCNC1 that was identified in 11 unrelated patients (13%) in the PME exome sequencing cohort. We have subsequently identified six additional patients. The gene encodes a potassium ion channel KV3.1 that has an important role in generating action potentials in the central nervous system, with the mutation disrupting the ability to transport potassium ions across the cell membrane. This mutation occurs in most families de novo, that is, it is a newly arising mutation. Based on the estimated mutation rate, the recurrent KCNC1 mutation is a worldwide cause of PME with likely hundreds of affected individuals globally. In five families with altogether nine affected siblings, we identified compound heterozygous variants in UBA5 as the cause of an infantile-onset syndrome characterised initially by irritability, followed by epilepsy, dystonic movements, moderate to severe intellectual disability, microcephaly and stagnation of development. The gene encodes an activating enzyme for UFM1, which is a small ubiquitin-like protein that is conjugated to its target proteins. The function of the highly conserved UFM1 conjugation system is still largely unknown. Functional analysis of the UBA5 mutants suggest that the identified variants cause reduced enzymatic activity of UBA5. Symptoms of the UBA5 patients and our findings in the central nervous system specific knockout mice for Ufm1 together indicate that UFM1-cascade is essential for normal development and function of the central nervous system. Finally, we identified compound heterozygous variants in ADAM22 as the likely cause of the disease in a patient with an infantile-onset rapidly progressing encephalopathy with epilepsy and cortical atrophy. The gene encodes a postsynaptic protein that functions as a receptor for LGI1, and we show that the identified variants abolish the ability of ADAM22 to bind to LGI1. The LGI1-ADAM22 complex is an antiepileptogenic factor regulating synaptic transmission throughout life. Highlighting the important role of this complex, knockout of Adam22 and Lgi1 in mice causes lethal epilepsy. Autosomal dominant LGI1 variants also cause epilepsy in humans. Identification of a patient with loss-of-function variants in ADAM22 suggest that also this gene is linked to epilepsy in humans. This connection should be confirmed through identification of additional affected individuals with ADAM22 variants. Altogether, this thesis demonstrates the power of WES in identification of causal genetic variants even in phenotypically heterogeneous patient cohorts subjected to prior genetic screenings. The findings improve diagnostics of these syndromes, increase knowledge of the underlying molecular mechanisms and potentially aid in developing new therapeutic interventions. Finally, for these families, establishment of the genetic diagnosis ends years of uncertainty and frustration of not knowing the cause of the disease and prevents need for unnecessary diagnostic testing.
  • Salo, Kati Hannele (Helsingin yliopisto, 2016)
    This work deals with the paleopathology of 555 skeletons from nine cemeteries in southern Finland dating from the 11th to the beginning of the 19th century. This is the first large-scale osteological investigation of Finnish human remains, since skeletal material in Finland is not usually preserved well. A greater number of younger individuals were identified from coastal sites than from inland sites, which may be due to real health differences, or to fertility differences between coastal towns and inland sites, or simply to taphonomy. Previous bioarchaeological studies have shown that early towns were unhealthy environments, which may also be a factor. Dental diseases were found to be more common in females, and trauma in males, as in previous bioarchaeological studies. Dental diseases, joint diseases, and trauma are age-progressive diseases, and cribra orbitalia and metabolic diseases were found more often in subadult individuals, as expected. No association between stature and any of the health-related parameters could be observed. Dental diseases are more common in more modern populations than in more ancient ones, as expected. These questions should, however, be studied further with the help of paleodietary/paleogenetic analyses. Most of the pathological lesions that were statistically significantly associated with each other were expected to be so, but some unexpected relationships also appeared. For example, skeletal trauma and entheseal changes were statistically significantly found more often in individuals with more dental calculus, whereas periapical lesions were statistically significantly associated with vertebral osteoarthritis. These unexpected co-ocurrences should be studied further, not only in bioarchaeology, but also in modern medicine, to discover whether these are just rare coincidences or whether these pathologies really do/did co-occur for some reason.
  • Arvilommi, Petri (Helsingin yliopisto, 2016)
    This study is part of a collaborative bipolar research project between the Unit of Mental Health of the National Institute for Health and Welfare, Helsinki (the former Department of Mental Health and Alcohol Research of the National Public Health Institute, Helsinki) and the Department of Psychiatry, Jorvi Hospital, Helsinki University Central Hospital (HUCH), Espoo, Finland. The Jorvi Bipolar Study (JoBS) is a prospective, naturalistic cohort study of 191 secondary-level care psychiatric in- and outpatients with a new episode of DSM-IV bipolar disorder (BD). Overall, the study involved screening 1,630 adult patients (aged 18-59 years) using the Mood Disorder Questionnaire (MDQ) for symptoms of bipolar disorder in the Department of Psychiatry, Jorvi Hospital, from January 1, 2002, to February 28, 2003, for a possible new episode of bipolar disorder. A clinical diagnosis of ICD-10 schizophrenia was an exclusion criterion for screening. The 490 consenting patients were interviewed with a semi-structured interview (SCID-I/P). Thereby, 191 patients were diagnosed with an acute phase of DSM-IV BD and included in the study. The patients participating were interviewed again 6 and 18 months after baseline. The course of the disease, with timing and durations of different phases, was examined by gathering all available data, which were then combined in the form of a graphical life chart. Observer- and self-reported scales were included at baseline and at both follow-up assessments. Also, the treatments provided were investigated at baseline and at both follow-up interviews. The aim in the first study was to investigate the adequacy of acute phase pharmacotherapy received by psychiatric in- and outpatients with a research diagnosis of BD I or BD II, including patients with and without a clinical diagnosis of BD. Information about treatments received during the index acute episode was gathered in the interview and from psychiatric records. Definitions of adequate acute-phase pharmacotherapy were based on published treatment guidelines. Only 42% of all 191 patients and 65% of those diagnosed with bipolar disorder received adequate treatment for the acute index phase. Clinical diagnosis of bipolar disorder was the factor most strongly independently associated with adequate treatment. In addition, rapid cycling, polyphasic index episode, or depressive index phase independently predicted inadequate treatment. Outpatients received adequate treatment markedly less often than inpatients. Lack of attention to the longitudinal course of the illness was another major problem area of treatment. Next, our aim was to investigate the adequacy of the maintenance-phase pharmacotherapy received during the first maintenance phase after an acute episode, following the same patients as in the first study. We defined adequate maintenance-phase pharmacotherapy based on published treatment guidelines. Of the patients with a maintenance phase in follow-up, adequate maintenance treatment was received by 75% for some time, but by only 61% throughout the maintenance phase and for 69% of the total maintenance time. Having adequate maintenance treatment throughout the maintenance phase was most strongly independently associated with having a clinical diagnosis of BD. In addition, inpatient treatment, rapid cycling, and not having a personality disorder predicted receiving adequate maintenance treatment throughout the maintenance phase. In addition, we investigated the continuity of attitudes toward and adherence to various types of psychopharmacological and psychosocial treatments among psychiatric in- and outpatients with BD I or II. During the 18-month follow-up, a quarter of the patients using mood stabilizers or atypical antipsychotics discontinued medication by their own decision, and of the medications continued, a third were not used regularly enough to provide a benefit. Overall, more than half of BD patients either discontinued pharmacotherapy or used it irregularly. The highest risk for discontinuing pharmacotherapy was present when the patients were depressed. Also, a quarter of the patients receiving psychosocial treatments did not adhere to the treatment. The main reasons patients gave for nonadherence toward pharmacological treatment were side-effects, lack of motivation, and a negative attitude toward the offered treatment; for individual/supportive psychotherapy, the reasons included practical barriers to coming to sessions and lack of motivation. Rates of nonadherence to mood stabilizers and antipsychotics did not differ, but the predictors did. Last, we investigated the prevalence and clinical factors predicting the granting of a long-term disability pension for patients with BD. We used register data to gather precise information on the pensions granted and their timing. During the 18-month follow-up after an acute episode, a quarter of the patients belonging to the labor force were granted a disability pension. Higher age, male gender, depressive index episode, comorbidity with generalized anxiety disorder (GAD) or avoidant personality disorder, and a higher number of psychiatric hospital treatments all independently predicted the granting of a disability pension. Moreover, patients subjective estimations of their vocational ability were surprisingly accurate in forecasting the granting of a future disability pension. In addition, the depression-related cumulative burden and the proportion of time spent in depression during the follow-up were important predictors. However, the predictors may vary depending on the subtype of illness, gender, and age group of the patient.
  • Paatela, Hanna (Helsingin yliopisto, 2016)
    Dehydroepiandrosterone (DHEA) and its sulfate ester DHEA sulfate (DHEAS) are the most abundant steroid hormones in the circulation. These prohormones, secreted by the adrenal glands, are important precursors of biologically active androgens and estrogens. Adipose tissue is an important site for estrogen synthesis after menopause, when all estrogens are produced from hormone precursors in peripheral tissues. In the circulation, DHEA exists also as fatty acyl esters. These lipophilic derivatives of DHEA are transported by circulating lipoprotein particles. DHEA and high-density lipoprotein (HDL) both improve endothelial function. The aims of the present thesis were to study the role of DHEA fatty acyl esters in the HDL-mediated vasodilation, to study the cellular uptake and metabolism of HDL-associated DHEA fatty acyl esters in endothelial cells, and to investigate the metabolism of DHEAS in female adipose tissue. The role of DHEA fatty acyl esters in HDL-mediated vasodilation was studied in isolated rat arterial rings. DHEA fatty acyl ester-enriched human HDL showed a stronger vasodilatory effect compared to native HDL. This relaxation was mediated by HDL receptor, scavenger receptor class B, type I (SR-BI), and was partly dependent on the function of endothelial nitric oxide synthase. The metabolism of DHEA fatty acyl esters was studied in human endothelial cells. These cells were able to internalize and slowly hydrolyze HDL-associated DHEA fatty acyl esters and to further secrete the liberated free DHEA from the cells. In abdominal subcutaneous and visceral adipose tissue obtained from pre- and postmenopausal women, steroid sulfatase activity was assessed by the conversion of DHEAS to DHEA, and mRNA expression of steroid-converting enzyme genes was quantified. Steroid sulfatase activity was higher in postmenopausal than in premenopausal women both in subcutaneous and in visceral adipose tissue. Visceral fat showed a higher sulfatase activity compared to subcutaneous fat. Three genes in the estradiol-producing pathway, aromatase, 17β-hydroxysteroid dehydrogenase type 12, and hormone-sensitive lipase, were more expressed in postmenopausal than in premenopausal adipose tissue. In conclusion, DHEA fatty acyl esters enhanced the vasodilatory effect of HDL, suggesting that DHEA esters, associated with HDL as cargos, may improve the antiatherogenic function of HDL and thus promote cardiovascular health. Endothelial cells were able to internalize and hydrolyze HDL-associated DHEA fatty acyl esters. The hydrolysis was slow and thus presumably not responsible for the rapid vasodilatory effect of DHEA ester-enriched HDL. Steroid sulfatase activity in adipose tissue was higher in postmenopausal compared to premenopausal women, suggesting that circulating DHEAS could be more efficiently hydrolyzed and utilized in postmenopausal adipose tissue for the formation of biologically active androgens and estrogens. Depot-differences in the sulfatase activity and the gene expression of steroid-converting enzymes suggest that steroid hormone metabolism may differ between subcutaneous and visceral adipose tissue.
  • Korpela, Katri (Helsingin yliopisto, 2016)
    This thesis characterises the development of the intestinal microbiota in healthy children. The influence of four common factors potentially modulating the microbiota prenatal stress, breastfeeding duration, antibiotic use, and probiotic use were investigated, as well as the association between early-life microbiota composition and the development of BMI. In addition, the microbiota in healthy children was contrasted with that that of children with inflammatory bowel disease, characterising the association between treatment response and microbiota. The bacterial composition was analysed from faecal samples using two DNA-based methods, a phylogenetic microarray, as well as sequencing of the 16S rRNA gene amplicons. In addition, real-time qPCR was conducted to measure bile-salt hydrolase genes and antibiotic resistance genes. Bacteria were cultured anaerobically for antibiotic susceptibility testing. The results showed that the microbiota in childhood are sensitive to modulating factors, and are predictive of later-life health. Maternal stress during pregnancy was associated with altered microbiota development over the first months of life. Long duration of breastfeeding was associated with slow microbiota maturation, normal BMI, and low antibiotic use in preschool age, if the microbiota were not disrupted by antibiotic use before weaning. The results indicate that some of the benefits of breastfeeding are microbiota-dependent. Early microbiota maturation was associated with fast growth in infancy and increased BMI in preschool age. Antibiotic use emerged as a central regulator of the microbiome, with potential effects on the metabolic development of the child. Lactobacillus rhamnosus GG supplementation prevented some of the penicillin-associated changes, but failed to prevent the macrolide-associated loss of bifidobacteria. The probiotic supplementation also reduced antibiotic use for at least 3 years after the intervention. In IBD patients, the microbiota composition varied along a gradient of intestinal inflammation and resembled the microbiota composition of antibiotic-treated healthy children. High microbiota similarity to non-antibiotic treated healthy controls predicted positive response to anti-TNF-α treatment in IBD patients. This work suggests that maternal wellbeing is the first step towards healthy microbiota in the child. Promoting a natural microbiota development in childhood by breastfeeding, avoiding unnecessary antibiotics, careful selection of the antibiotic when it is needed, and possibly the use of specific probiotic strains, may have long-term health benefits, particularly in terms of weight development and immune health.
  • Immonen, Tuuli (Helsingin yliopisto, 2016)
    Long-chain acyl-CoA dehydrogenase deficiency (LCHADD), a severe long-chain β-oxidation disorder which, without treatment, usually leads to death, is the most frequent β-oxidation disorder in Finland. Its typical manifestations are hypoketotic hypoglycemia, hepatopathy, failure to thrive, cardiomyopathy, and metabolic crisis during the first year of life. The long term complications are retinopathy and polyneuropathy. In recent years, diagnostics and treatment of LCHADD have produced major advantages. Many countries have implemented LCHADD in their newborn screening programs. Treatment with a low-fat, high-carbohydrate diet with avoidance of fasting is effective. Few follow-up studies, ones on the outcome of LCHADD and whether the current dietary treatment prevents the long-term complications retinopathy and peripheral neuropathy, exist. The aims of the study were to evaluate the clinical course and outcome of LCHADD patients in Finland who have the homozygous c.1528G>C (E510Q) mutation, with strict dietary treatment and develop further their treatment and follow-up strategies. A total of 47 patients with LCHADD caused by a homozygous c.1528G>C mutation were diagnosed in Finland from 1976 through 2014. In our study, the outcome and course of the disease of the LCHADD patients born between 1997 and 2010 (Study II), were compared with an earlier Finnish study of LCHADD patients born 1976 to 1996. In 1976-1996 (N=28) the ten-year survival rate was 14.3%, while in 1997-2010 (N=16) at the end of the study 62.5% were alive. Patients born between 1997 and 2010 presented at the age of 0 to 5 months with hypoketotic hypoglycemia, failure to thrive, hypotonia, hepatomegaly, metabolic acidosis, and cardiomyopathy. The therapy was started 0 to 30 days after diagnosis. Gastrostoma prove beneficial during infancy, ensuring continuous night-time feeding. Most long term survivors were in good overall condition. All except one, who had metabolic crises leading to resuscitation before diagnosis, had a normal intelligence quotient (IQ). We studied the development of polyneuropathy (PNP) by clinical neurophysiologi-cal methods in the LCHADD patients diagnosed between 1965 and 2014 (Study III). Electroneurography (ENG) was performed 1 to 12 times for 12 patients. The first abnormality was reduction in the sensory amplitudes of the sural nerves. During follow-up, progression extended to the upper limbs. Despite good compliance with the diet, of the 10 younger patients, 6 developed polyneuropathy but in a milder form than reported earlier. Their polyneuropathy had been detected at the ages of 6-12 years. To improve the ophthalmological follow-up, we rated the fundus images from seven children in stage 2 retinopathy to create a grading system to monitor retinopathy development (Study I). According to this rating the original staging was divided into three substages of pigmentary deposits (P1-P3) and retinal pigment epithelial (RPE) atrophy (A1-A3). Three ophthalmologists expressed moderate agreement in the assessment of pigmentary deposits (combined weighted K statistic, 0.38), whereas the assessment of RPE atrophy showed poor agreement (combined K statistic 0.018). The visual assessment of fundus photographs based on reference images showed agreement identical to that reported for grading of retinopathy of prematurity, so fundus photography is the suggestion for ophthalmological follow-up of LCHADD retinopathy. We followed up 11 LCHADD patients treated with the current dietary regimen in Helsinki University Central Hospital between 2000-2014 during routine visits (Study IV). Their intake of essential fatty acids (EFAs) was within normal limits. The amount of long-chain triglyceride (LCT) was 5 to 9 percent of total energy intake (E%), consistent with dietary recommendations of LCHADD. We detected, surprisingly, that the patients received one third of their linoleic acid (LA) and α-linolenic (ALA) from their diet and two-thirds from the supplements. Acylcarnitine levels remain elevated, despite good compliance with the diet, which indicated that the diet was not optimal. This demonstrates that in order to keep LCTs as low as possible, we should monitor EFA intake carefully by dietary regimen and by measuring fatty acid profiles. To conclude, the outcome of LCHADD has improved, but the patients still need careful monitoring for dietary compliance in order to prevent retinopathy and neuropathy and especially during infections to prevent hypoglycemia and metabolic decompensation. Future challenges are to adjust the therapy to be lifelong and to find new treatment strategies.
  • Sääksjärvi, Katri (Helsingin yliopisto, 2016)
    Parkinson's disease (PD) is a movement disorder with progressive neurodegeneration. As populations continue to age, the burden of this disease will increase. Although both environmental and genetic factors seem to play a major role in the etiology of PD, the causes of the neuronal death underlying the disease remains largely unknown. As curative treatment for PD remains a challenge, knowledge to aid in the development of prevention tools is crucial. Thus, identifying risk factors, especially modifiable ones, is essential. The aim of the present study was to examine the prediction of dietary, lifestyle and metabolic factors on PD risk in a cohort study design. This study included men and women free from PD at baseline of the Finnish Mobile Clinic Health Examination Survey (FMC, 1966-1972, n=4,524), the Follow-up study of the FMC (FMCF, 1973-1975, n=6,715), and the Mini-Finland Health Survey (1978-1980, n=4,828). Over a follow-up of 22 41 years, the PD cases (International Classification of Diseases 10, code G20, from the World Health Organization) in the FMC (n=85), the FMCF (n=101), and the Mini-Finland Health Survey (n=89) were identified through linkage with the nationwide Drug Reimbursement Register of Social Insurance Institution. Information on lifestyle factors, socioeconomic background, and physiological and biochemical determinants were collected by questionnaires, interview, and health examinations at the baselines of these three surveys. Dietary intake was assessed with a 1-year dietary history interview in the FMC. The exposure factors studied here were diet (individual food groups and items, as well as a diet quality index), leisure-time physical activity, smoking, alcohol consumption, coffee consumption, body mass index, blood pressure, as well as concentration of serum HDL cholesterol, serum triglyceride, fasting plasma glucose, and serum total cholesterol. Cox's proportional hazards model was used to estimate the strength of association between the exposure factors and PD risk. Potential confounding factors were adjusted by inclusion in multivariate models. Most of the individual food groups and items, as well as the diet quality, did not predict the risk of PD. There were few exceptions however, e.g. high consumption of milk was associated with increased PD risk in women. Smoking, heavy consumption of coffee, as well as heavy leisure-time physical activities were associated with decreased PD risk. Alcohol consumption had no clear association with PD risk. Those who had metabolic syndrome had a reduced risk of PD, mainly due to high serum triglyceride and fasting plasma glucose concentration. Serum HDL cholesterol, serum total cholesterol concentration, and blood pressure, however, had no association with PD risk. After taking into account the potential preclinical disease phase, we observed that obesity might increase the risk of PD. In conclusion, diet and other lifestyle factors, as well as metabolic health, may predict the risk of PD. The prospective design of this study is a strength when providing etiologic clues for unraveling the mystery of PD. The overall evidence from the literature is, however, currently too sparse for making recommendations for public health purposes to prevent PD. In the future, perhaps, the results of the present study, as well as previous studies, may be used in clinical practice when planning tools for the early identification of PD patients. Keywords: Parkinson s disease, cohort study, diet, lifestyle factors, metabolic health
  • Tähtinen, Siri (Helsingin yliopisto, 2016)
    According to latest estimates, cancer is becoming an increasing health risk on a global scale. Consequently, novel cancer treatment modalities are urgently needed, especially for the treatment of metastatic solid tumors that are refractory to standard therapies. One promising approach in the treatment of such advanced cancers is immunotherapy which aims to elicit de novo immune responses and/or to boost pre-existing anti-tumor immunity. Different forms of cancer immunotherapy include oncolytic viruses, which selectively replicate in and destroy cancer cells, and adoptive T-cell therapy, in which the patient is given vastly amplified numbers of tumor-targeting T-cells. Both of these have shown capacity to elicit anti-tumor immunity but efficacy in clinical settings has been suboptimal due to different resistance mechanisms employed by solid tumors. Anti-viral resistance represents a major hurdle in oncolytic virotherapy, as repeated administration of the same virus can lead to induction anti-viral rather than anti-tumor immunity. Moreover, cancer cells in some tumors may intrinsically be resistant to virus infection. In study I, we examined whether this could be circumvented by heterologous prime-boost setting, i.e. by switching between oncolytic adenovirus (Ad) and vaccinia virus (VV) during therapy. The results showed that presence of one virus does not preclude the infection of another and treatment with heterologous Ad-VV therapy can delay the onset of anti-viral resistance. Moreover, we found that restricted replication of the priming (adeno)virus can affect the efficacy of heterologous virotherapy. In study IV, we studied the role of anti-viral signaling in adenovirus replication in cancer cells and whether this could be augmented with Janus Kinase 1/2 inhibitor Ruxolitinib. Interestingly, we found that although exposure to type I interferon does not inhibit progressive Ad replication in vitro, significant improvement in anti-tumor efficacy of the virus was observed in vivo when combined to concomitant Ruxolitinib treatment. These results underline the possible approaches that could be taken to reduce naturally acquired or therapy-induced resistance, which interferes with viral spread and may hinder the therapeutic efficacy. Adoptive T-cell therapy (ACT) can be a potent form of immunotherapy. Despite the large number of anti-tumor T-cells infused during ACT, immunosuppression and immune evasion of advanced tumors can render tumor-infiltrating lymphocytes (TILs) inactive. In study II, we examined whether oncolytic adenovirus could increase anti-tumor efficacy of adoptively transferred T-cell receptor (TCR) transgenic T-cells. Indeed, intratumoral injections of adenovirus were able to counteract immunosuppression by activating antigen-presenting cells (APCs) and anti-tumor T-cells. Moreover, an endogenous T-cell response against other, non-related tumor antigens was detected and this polyclonal response contributed to systemic anti-tumor immunity. In study III, we analyzed whether cellular composition of tumor microenvironment could be modified by local administration of immunostimulatory recombinant cytokines. When combined to adoptive T-cell transfer, intratumoral injections of interleukin 2 (IL-2), interferon α (IFN-α) and interferon γ (IFN-γ) resulted in significant anti-tumor efficacy, increased tumor-levels of stimulatory immune cells and reduced exhaustion of CD8+ TILs. In contrast, administration of granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced tumor growth and recruited immunosuppressive cell types such as monocytic myeloid-derived suppressor cells (MDSCs) and M2 macrophages to the tumor bed. These results indicate that immunomodulation by carefully selected cytokines and/or oncolytic adenovirus can sensitize the tumor in favor of adoptively transferred anti-tumor T-cells. In conclusion, different combinatorial approaches can be employed to overcome intrinsic, naturally acquired or therapy-induced resistance to anti-tumor T-cells or oncolytic adenovirus. These advances enable significant improvement in the treatment of solid cancers and can potentially lead to development of curative cancer immunotherapies.
  • Haapamäki, Carola (Helsingin yliopisto, 2016)
    Extrahepatic biliary strictures are mainly managed using stents when treated endoscopically. At present, the main stent types in clinical practice are non expandable plastic stents (NEPS) and self-expandable metallic stents (SEMS), with an up to tenfold cost for the latter. In current praxis, SEMS are widely used for palliative management of malignant biliary strictures as they have longer patency.The role of SEMS in preoperative stenting and the management of benign biliary strictures (BBS) are unclear. The main purpose of this study was to describe the therapy outcome of metallic stenting in anastomotic strictures of liver transplanted (LT) patients, to compare stenting with NEPS and SEMS preoperatively preceding pancreaticoduodenectomy and in BBS caused by chronic pancreatitis (CP) and, finally, to describe stenting of BBS using covered SEMS (cSEMS), a new technique that has not previously been possible, particularly in patients with surgically altered anatomy. The therapy outcome of 17 LT patients with anastomotic biliary stricture or leakage treated with cSEMS was retrospectively analyzed in the piloting study (I). In Study II, the stent success and the surgical outcome of 191 patients preoperatively stented, with either NEPS or SEMS who had undergone pancreatic oduodenectomy or total pancreatectomy were analyzed in a retrospective manner. As a supplementary group, 166 preoperatively unstented and 9 percutaneously, transhepatically drained patients were evaluated concerning surgical outcome. A prospective, randomized, controlled trial was conducted in Study III to compare multiple NEPS with cSEMS therapy in biliary strictures caused by CP, with 30 patients in each group. Study IV presented three patient cases along with detailed description of equipment, devices, technique and outcome, when using cSEMS for BBS in patients with altered anatomy. The median stenting time for the LT patients (I) was 6.8 months (0.9–10.1). The overall stent migration rate was 24%; 100% for Wallstent™ (n=3), 4% for Allium™ (n=13) and 0% for a custom-made Micro-Tech (n=2) stent. There were two recurrences, but eventual stricture resolution was achieved in all patients after restenting. The median follow-up was 21.7 months (6.6–32.0) after stent removal. For the preoperatively stented patients (II), the stent failure rate was 7.4% (95% confidence interval [CI] 4.0%–12.3%) for NEPS and 3.4% (95% CI 0.1%–17.7%) for SEMS, (p=0.697). Among the NEPS stented patients, 45% with a pre-stent bilirubin level exceeding 50 μmol/l reached a preoperative level of 20 μmol/l or less, compared with 26% in the SEMS group (p=0.110). A level lower than 50 μmol/l was achieved by 80% of patients in the NEPS group and by 61% in the SEMS group (p=0.058). The bile juice bacterial scores did not differ between the differently stented patients but a statistically significant difference was found when the proportion of sterile bile juices in unstented patients with biliary obstruction (100%; n= 7/7) was compared with that of the stented patients (1%; n=1/155; p<0.001). Postoperative infection complications did not show any significant difference when comparing these stented and unstented groups with biliary obstruction. However, the number of unscented patients with biliary obstruction was very small. Overall postoperative infections, postoperative pancreatic fistulas or reoperations showed no significant difference between study groups. For the patients with CP and BBS (III), the median follow-up was 40 months (range 1–66 months). The stricture-free success rate at two years was 90% (95% CI 72%–97%) in the NEPS group and 92% (95%CI 70%–98%) in the cSEMS group (p=0.405). One late recurrence in the NEPS group, 50 months after stent removal, decreased the success rate to 72% (95% CI 27%–92%). The migration rate was 10%in the NEPS group and 7% in the cSEMS group (p=1.000). Three patients with altered anatomy and BBS successfully received an endoscopically deployed cSEMS, two of them utilizing the rendezvous technique, as the percutaneous transhepatic cholangiogram (PTC) route was available when the procedure was started (IV). In conclusion, endoscopic therapy with cSEMS is safe and efficient both regarding anastomotic complications after LT and CP-induced BBS. Progressive stenting with NEPS is a good alternative in BBS caused by CP, however, with impaired patient comfort. In ordinary preoperative stenting, SEMS do not seem to offer any advantage over NEPS. In patients with altered anatomy, endoscopic deployment of cSEMS in BBS has become possible, as equipment and devices have evolved.
  • Andrade Barazarte, Hugo (Helsingin yliopisto, 2016)
    Objective Multiple intracranial aneurysms are frequent, with an incidence of 15-40% among intracranial aneurysms carriers. Of these carriers, 20-40% have bilateral intracranial aneurysms. The rupture risk is higher for patients with multiple intracranial aneurysms. For those patients, several treatment options are available (microsurgery comprising a unilateral-contralateral approach, bilateral craniotomies in one-stage or two stages surgery, and endovascular methods) varying from institution s resources and surgeon s experience. The present study focuses and analyses the angiographic characteristics, specific parameters, and surgical results of the unilateral-contralateral approach for ICA-opht segment and MCA aneurysms. In addition, it describes and analyses the proximal vascular control by transient cardiac arrest induced by adenosine during the contralateral clipping of ICA-opht segment aneurysms. Patients and Methods We retrospectively reviewed 68 patients with ICA-opht segment and bMCA aneurysms treated through a contralateral approach at the department of neurosurgery of the University of Helsinki, between January 1998 and December 2013. A detailed analyses of the aneurysms characteristics and constrains of the contralateral surgical corridor was performed. A further subgroup analysis of 8 patients harboring ICA-opht segment aneurysms approached through a contralateral craniotomy and requiring intravenous adenosine administration to induce transient cardiac arrest during microsurgical clipping was performed as well. Results ICA-opht segment aneurysms: All the 30 ICA-opth aneurysms were small (less than 7 mm), unruptured, saccular, and had no wall irregularities, calcifications or secondary pouches. Microsurgical clipping of these aneurysms was possible when the prechiasmatic distance had a median of 5.7 mm (range 3.4-8.7 mm) and the interoptic distance a median of 10.5 mm (range, 7.6-15.9). The most frequent aneurysm dome projection was superomedial (77%). Of the patients with ICA-opht segment aneurysms approached through a contralateral craniotomy, 93% had good postoperative outcome at 3-month follow-up. bMCA aneurysms: The contralateral approach for bMCA aneurysms was possible in 38 patients. All the 38 contralaterally approached MCA aneurysms were unruptured and had saccular shape (expect one with bilobular shape). The majority (97%) of contralateral aneurysms were small to medium in size. The median length of the contralateral A1 was 13.2 mm (range: 6-19.8 mm), and the median length of the contralateral M1 was 14.2 mm (range: 4.6-21 mm). Of the patients with unruptured bMCA aneurysms treated through a contralateral approach, 24 (86%) patients had good outcome and 4 (14%) had poor outcome at 3-month follow-up, 1 patient was lost to follow-up. There were 9 patients harboring bMCA aneurysm presented with SAH due to a ruptured ipsilateral aneurysm. Of these patients, 7 (78%) had good outcomes, and 2 (22%) had poor outcomes at 3 months. Olfactory disturbances were present in 21% of cases treated through a contralateral approach. Transient cardiac arrest induced by adenosine during contralateral clipping of ICA-opht aneurysms: 8 patients received intravenous bolus of adenosine to induce transient cardiac arrest during clipping. Of the total patients, 5 received single bolus of adenosine, and 3 patients received multiple doses. The median single dose of adenosine was 22.5 mg (range, 5-50 mg). The asystole time range between 20-40 seconds after adenosine administration. All the 8 patients showed good surgical outcomes at 3-month and 1-year follow-up, and showed no procedure-related complications. Conclusion: The contralateral approach remains as a feasible option for microsurgical treatment of ICA-opht segment aneurysms, and bMCA aneurysms. Its feasibility depends on general parameters related to the aneurysm itself (shape, morphology, size, status and projection), and specific parameters that varies according to the vascular segment to be treated (prechiasmatic and interoptic distances, length of A1 and M1). Transient cardiac arrest induced adenosine represents a useful tool to obtain proximal vascular control while performing a contralateral approach for ICA-opth segment aneurysms in selected patients.
  • Göhre, Felix (Helsingin yliopisto, 2016)
    Objective: Aneurysms of the posterior cerebral artery are rare vascular lesions. The overall incidence is less than 1%, representing around 7% of posterior circulation aneurysms. Due to this low incidence, most of the institutional series on PCA aneurysms are small and contain less than 25 patients. Only one other series comparable in scope to ours has been previously published. The presented study analyzes and describes the characteristic features of PCA aneurysms as well as investigates the relevant treatment strategies and their outcomes. A particular focus is in the description and analysis of PCA aneurysms treated from a subtemporal approach and the pres - entation of an associated aneurysm treatment from a lateral supraorbital approach. Patients and Methods: We reviewed 121 patients diagnosed with 135 PCA aneurysms, all of whom were treated between 1980 and 2012 at two Finnish neurosurgical units (Department of Neurosurgery at the University of Eastern Finland, Kuopio and Department of Neurosurgery at the University of Helsinki). Additionally, twelve historical (pre-1980) cases were presented. Detailed analyses of cerebral angiographies were conducted for 93 PCA aneurysms in 81 patients. A further subgroup analysis of 34 patients diagnosed with 37 PCA aneurysms treated via subtemporal approach was also performed. Results: Of the 121 patients with 135 PCA aneurysms, 52 (39%) aneurysms were ruptured and 83 (61%) unruptured . The following distribution along the PCA segments was observed: P1 segment (n=53), P1/2 junction (n=39), P2 segment (n=28), and P3 segment (n=15); no P4 segment aneurysms were found. Saccular aneurysms were more common than fusiform PCA aneurysms (76% vs. 24%). The detailed angiographic analysis showed that the median aneurysm size was 7 mm for ruptured PCA aneurysms and 4 mm for unruptured aneurysms. Saccular aneurysms (n=69, 74%) had a characteristic dome projection for each location: P1 segment, upward (67%); P1/P2 junction, anterior/upward (80%); P2 segment, lateral (67%); and P3 segment, posterior (50%). The following treatment results at 1-year follow-up were achieved for patients with: unruptured PCA aneurysms (n=19; 12 good outcomes, 63%; 6 moderate, 31%; 1 poor, 5%), ruptured PCA aneurysms (n=27; 10 good, 37%; 9 moderate, 33%; 8 poor, 30%), and patients with complex neurovascular pathologies and PCA aneurysms (n=96; 42 good, 43%; 40 moderate, 42%; 14 poor, 15%). Analyzing the subtemporal approach we found that most complications were not related to the subtemporal approach itself but to the specific nature of the PCA aneurysms treated and the chosen strategy. The most common (12 out of 34; 35%) serious complication in this series was an ipsilateral PCA infarction after parent vessel occlusion. Conclusion: PCA aneurysms are infrequent vascular lesions that are often associated with other vascular pathologies. Most ruptured PCA aneurysms are smaller than 10 mm and distally located. The saccular PCA aneurysms have a typical dome orientation at each PCA segment. Microsurgery and endovascular treatment are effective options for the occlusion of PCA aneurysms. As a result, individual treatment strategies are required. Despite commonly adequate vessel collateralization of the distal PCA territory, preservation or reconstruction of the parent vessel is crucial for favorable treatment outcomes. The subtemporal approach is favorable for the treatment of PCA aneurysms in proximity to the tentorium. Frontolateral approaches allow the treatment of proximal PCA aneurysms and ipsilateral anterior circulation aneurysms inside the Circle of Willis.
  • Laulajainen-Hongisto, Anu (Helsingin yliopisto, 2016)
    Acute otitis media (AOM) is an infection that is particularly common in children. The bacterial etiology of AOM, in both children and adults, affects its clinical picture. While in some cases the infection can simply be carefully monitored without treatment, antimicrobials are often prescribed. Caution is required, however, when prescribing antimicrobials as their excessive use has led to antimicrobial resistance; this resistance has been seen among some of the causative pathogens for these infections. Before the development of antimicrobial treatment, complications due to middle ear infections were common, potentially causing severe symptoms or even death. Some middle ear infections spread into surrounding structures, leading to intratemporal or extratemporal (extracranial or intracranial) complications. This thesis focuses on complicated otitis media and the causative factors of its complications. In the first two studies, we evaluated the medical records of all (n=100) children (0-16 years old) hospitalized at the Department of Otorhinolaryngology in the Helsinki University Hospital from 2003 to 2012 for acute mastoiditis (AM) or AOM, as well as the infection s bacteriology in relation to the patient s clinical findings and treatment. Using this information, we analyzed the differences in the etiologies and clinical pictures of those children hospitalized due to AOM compared to AM. In our third study, we examined the medical records of all (n=166) patients hospitalized at our institution from 1970 to 2012 due to intracranial abscesses (IA), including those of otogenic background (oIA). In the fourth study, we evaluated the bacteriology in relation to the patients clinical findings and treatment in all (n=160) adult patients treated at our institution from 2003 to 2012 for AOM or acute mastoid infection. In adults, acute mastoid infection was subclassified into AM, latent mastoiditis (LM), and AM following chronic middle ear infection (AMc). The clinical picture of AM in children differed according to the causative pathogen. Streptococcus pneumoniae (Pnc), especially its resistant strains, caused severe symptoms and often led to mastoidectomies. Pseudomonas aeruginosa (Ps) typically affected older children with prior tympanostomy tubes and generally caused milder symptoms. Furthermore, the bacteriological etiology of hospitalized AOM and AM patients was different compared to outpatient AOM. Two of the typical AOM pathogens, Haemophilus influenzae (Hi) and Moraxella catarrhalis (Mc), were uncommon among the hospitalized patients. Pnc, especially its resistant strains, was less common in children hospitalized for AOM compared to AM, and less common in adults than children. Streptococcus pyogenes (StrA) and Ps were both linked to otorrhea and were found only in older children. Over our 42 year study period, oIAs became less common and typically developed following chronic middle ear infections, often in connection with cholesteatoma. In adults, the bacteriological etiology and clinical picture of AMc differed from AOM as well as the other acute mastoid infection types (AM, LM). AOM and AM led to less surgical procedures than the more prolonged forms of acute mastoid infection (LM, AMc). In children, the hospitalized cases of AOM and AM differed from outpatient AOM. In adults, severe AOM, AM, and LM seem to compose a continuum that may lead to chronic otitis media and its acute complications, including oIAs. Otogenic IAs are quite rare, however, and became less common over our study period.