Faculty of Medicine

 

Recent Submissions

  • Lahelma, Mari (Helsingin yliopisto, 2022)
    Background: Non-alcoholic fatty liver disease (NAFLD) and the metabolic syndrome (MetS) have become major health concerns due to the global obesity epidemic. Both conditions increase the risk of atherosclerotic cardiovascular disease (ASCVD), and their mechanisms partially lie in dysregulated lipid metabolism. Obesity is the leading risk factor for NAFLD and the MetS, but not all obese individuals develop these metabolic abnormalities. The risk may be modulated by lifestyle factors, which also remain the cornerstone of the treatment of NAFLD, the MetS, and ASCVD. However, their role has not been fully elucidated. Aims: The present series of studies was undertaken to better understand the role of lifestyle factors and lipid metabolism in NAFLD, the MetS, and ASCVD. Specifically, we wished to determine i) whether the characterization of lifestyle factors can help identify obese patients who have advanced NAFLD with fibrosis (NAFLD-fibrosis), ii) how physical training using a weighted hula-hoop influences abdominal obesity and the associated metabolic parameters compared to walking, iii) how the lipid composition of the liver is related to the lipid composition and aggregation susceptibility of low-density lipoprotein (LDL) particles, and iv) how overfeeding different macronutrients influences the proatherogenic properties of LDL particles. Participants and methods: We cross-sectionally examined a group of 100 (Study I), and 40 (Study III) obese individuals. Fifty-five abdominally obese participants underwent a cross-over designed trial addressing different forms of physical activity (PA) (Study II), and 36 participants underwent a three-week overfeeding intervention comparing three different diets (Study IV). The participants were randomized to hula-hooping for six weeks using a 1.5 kg weighted hula-hoop followed by energy expenditure matched walking for another six weeks or vice versa (Study II), and to daily consuming an extra 1000 kilocalories from either saturated fat, unsaturated fat, or simple sugars (Study IV). Physical and biochemical parameters were measured during clinical visits (Studies I–IV), and liver histology was assessed from liver biopsies (Studies I, III). Lifestyle factors (PA, diet, sleep, fatigue, smoking) were assessed using questionnaires and face-to-face interviews, and PA was also assessed objectively using accelerometer recordings (Study I). The lipid compositions of the liver (Study III) and isolated LDL particles (Studies III–IV) were measured using ultra-high performance liquid chromatography–mass spectrometry. The aggregation susceptibility of LDL particles was measured ex vivo by inducing aggregation with human recombinant acid sphingomyelinase (Studies III–IV). The proteoglycan-binding of plasma lipoproteins and the concentrations of oxidized LDL were also measured (Study IV). Abdominal obesity was measured using dual-energy X-ray absorptiometry (Study II). Results: Low levels of self-reported moderate-to-vigorous PA, high red meat intake, low relative carbohydrate intake, and smoking independently were associated with the presence of NAFLD-fibrosis in obese participants. The characterization of these lifestyle factors, in addition to known physical, biochemical, and genetic risk factors, significantly improved the identification of patients with NAFLD-fibrosis (Study I). Weighted hula-hooping decreased abdominal fat % and lowered LDL-cholesterol in abdominally obese individuals in comparison to walking (Study II). Walking, on the other hand, decreased blood pressure and increased high-density lipoprotein (HDL)-cholesterol. The fatty acid total carbon number and double bond compositions of the triglycerides, sphingomyelins, and phosphatidylcholines in the liver were strongly associated with those in LDL particles. Hepatic dihydroceramides and ceramides correlated positively with the concentrations of the corresponding sphingomyelin species in LDL, as well as with increased LDL aggregation susceptibility (Study III). Overfeeding saturated fat increased the content of sphingolipids and saturated triglycerides in LDL as well as LDL aggregation susceptibility. Overfeeding unsaturated fat reduced the proteoglycan-binding of plasma lipoproteins, whereas no changes were observed during carbohydrate overfeeding (Study IV). Conclusions: Lifestyle factors, that is, both diet and PA, are associated with and modulate the features of NAFLD, the MetS, and ASCVD in obese individuals. Assessing these lifestyle habits helps estimate the risk of advanced NAFLD in obese patients. Weighted hula-hooping reduces abdominal obesity, and excess intake of saturated fat modifies the composition of LDL particles, increasing their aggregation susceptibility. The lipid composition of the liver and LDL particles are closely related, and patients with aggregation-susceptible LDL particles show changes in hepatic sphingolipid metabolism. In line with current treatment recommendations, the present series of studies supports the metabolic and cardiovascular benefits of PA, also in their untraditional forms, and the replacement of dietary saturated fat with unsaturated fat in cases of obesity.
  • Lemma, Aurora (Helsingin yliopisto, 2022)
    Background: Acute mesenteric ischemia (AMI) is a disease with high mortality. The exact incidence is still unclear as data mostly come from studies based on patient records. These do not include those that are not correctly diagnosed with AMI while alive. Mortality of AMI rises concomitantly with door to operation time, but the factors contributing to these delays remain largely unknown. Portal vein thrombosis (PVT) is usually treated with anticoagulation and only in a relatively small portion of patients the thrombus extends to the superior mesenteric vein (SMV) or splenic vein (SV) which may lead to the development of bowel ischemia. However, even without serious complications an untreated PVT can lead to portal hypertensive complications that can cause significant morbidity. The risk factors for insufficient recanalization and indicators for more invasive treatment methods for PVT are still unclear. Material and methods: This thesis included four original retrospective studies. The first three studies concerning AMI focused on arterial occlusive AMI, therefore, AMI caused by strangulation and non-occlusive, traumatic, and iatrogenic AMI were excluded. Study I included patients who were diagnosed with or died of AMI during 2006-2015 in the Hospital District of Helsinki and Uusimaa (HUS). The study also included AMI patients diagnosed postmortem. This data was collected from death certificates. Study II included patients treated for arterial AMI in Meilahti hospital during 2006-2015. Study III included patients who were diagnosed with arterial AMI between 2014 and April 2020 in the HUS area and were operated on in Meilahti. The patients were divided into pre-and postpathway groups according to the time of diagnosis in relation to the implementation of a treatment pathway and care bundle (from 2014 to 2017 and from May 2018 to April 2020). Study IV included patients diagnosed with non-cirrhotic and non-malignant PVT in the HUS area between 2006 and 2015. Results: In study I, 29% of patients were diagnosed postmortem (out of a total of 470). The overall incidence did not change during the study period, although some subgroups, such as the working age population, showed an increase. The overall incidence rate was 3.1/100 000 PY during the study period and 26.7 in those aged 70 years or more. Study II included 81 patients, of whom 65% died within 90 days of diagnosis. If the patient arrived first at a surgical emergency room (SER), first door to operation time was shorter (10h vs. 15h, p = .025), hospital stay was shorter (7 vs. 11 days, p = .045) and 90-day mortality lower (50% vs. 75%, p = .025). Study III included 145 patients who were divided into pre- and postgroups. The groups were comparable in disease acuity and baseline characteristics. In the postgroup, more patients were diagnosed with contrast enhanced CT (74% vs. 94%, p = .001), the in-hospital delay to operating room was shorter (7h vs. 3h, p = .023), more revascularizations were done (68% vs. 84%, p = .030), especially endovascular interventions (33% vs. 64%, p <.001) and 30-day mortality was lower (51% vs. 25% p = .001). In multivariate analysis belonging to the postgroup was a protective factor for 30-day mortality. Study IV included 145 patients with PVT, the majority of whom (92%) were treated primarily with anticoagulation. The 5-year cumulative incidence of complete recanalization was 42% and 31% for portal hypertensive complications (PHC). A non-acute thrombus was an independent risk factor for insufficient recanalization (HR 3.1, 95% CI 1.6-5.8) whereas acute pancreatitis was a protective factor (HR 0.3, 95% CI 0.2-0.7). Risk factors for PHC were non-acute thrombus (HR 2.9, 95% CI 1.6-5.3), ascites at baseline (HR 3.3, 95% CI 1.7-6.7), thrombus extending to the SMV or SV (HR 2.6, 95% CI 1.2-5.7), myeloproliferative disease (HR 3.0, 95% CI 1.4-6.5) and anemia (HR 2.1, 95% CI 1.1-3.9), whereas acute pancreatitis was a protective factor (HR 0.1, 95% CI 0.03-0.5). Conclusions: The overall incidence of AMI did not change during the study period, but there was an increase in the working age population. A third of patients were only diagnosed postmortem. The specialty of the first emergency room an AMI patient encounters crucially affects the delays in treatment as well as early survival. Delays were decreased by implementing a treatment pathway and care bundle, which also led to decreased mortality, as well as an increase in appropriate CT imaging and an increased rate of revascularizations. Thrombus etiology and age affected recanalization in PVT patients, whereas thrombus age, etiology and extent, ascites at baseline and anemia affected development of portal hypertensive complications. The age, extent and etiology of a PVT might be of use in determining which patients benefit from more invasive treatment options.
  • Ahonen Mehmeti, Maria (Helsingin yliopisto, 2022)
    Adipose tissue (AT) dysfunction is a hallmark of unhealthy obesity and links it to metabolic comorbidities. Dysfunctional AT exhibits inhibited adipogenesis, impaired lipid metabolism, and increased inflammation. However, we are still lacking a sufficient understanding of the mechanisms of adipocyte dysfunction and how they contribute to conditions such as insulin resistance, type 2 diabetes, non-alcoholic fatty liver disease, and cancers. MiR- 107, miR-221-3p, and thyroid hormone responsive protein (THRSP) have been shown to play a role in murine models of metabolic disease in AT or other tissues, but the data in human adipocytes was limited or function unknown. Increasing knowledge of adipocyte dysfunction is critical for the development of new approaches to treat obesity and prevent its metabolic comorbidities. This thesis investigates the differentiation and function of human adipocytes and how they are altered in metabolic disease. The aim was to modulate the expressions of miR-107, miR-221-3p, or THRSP and study how adipocyte differentiation and metabolic functions were affected. In study I, we revealed miR-107 to inhibit Simpson-Golabi Behmel syndrome (SGBS) adipogenesis and lipid storage. We showed that the differentiation defect is mediated via downregulation of cyclin-dependent kinase 6 (CDK6) and Notch3. In mature adipocytes, miR-107 impaired glucose uptake and triglyceride (TG) synthesis. Our results suggest that by distinct mechanisms, it promotes insulin resistance may contribute to ectopic lipid storage by impairing function of pre- and differentiated adipocytes. Study II focused on the role of miR-221-3p in the terminal differentiation of adipocytes and in cancer-associated adipocytes (CAAs). We showed that it inhibited terminal differentiation and identified 14-3-3γ as a potential mediator of the differentiation defect. We measured the lipidome in miR-221- 3p overexpressing adipocytes and analyzed the mechanisms behind the alterations. MiR-221-3p inhibited de novo lipogenesis (DNL) of TG, but increased ceramide and sphingomyelin concentrations while reducing diglycerides (DG). MiR-221-3p is connected to many cancers; here we showed that miR-221-3p expression was elevated in tumor proximal adipose tissue from patients with invasive breast cancer. Moreover, conditioned medium from miR-221-3p transfected adipocytes increased invasion and proliferation of cancer cells in vitro. Taken together, we characterized the role of miR-221- 3p in adipocyte dysfunction and showed that the alterations in CAAs are relevant for cancer progression. In study III, we found THRSP to be induced by insulin in humans by using the euglycemic insulin clamp technique in 36 subjects. We measured a 5- and 8-fold increase in THRSP mRNA after 180 and 360 min of in vivo euglycemic hyperinsulinemia. We identified functions of THRSP by conducting a transcriptomic analysis of THRSP-silenced adipocytes. We measured mitochondrial respiration, oxidation of radiolabeled oleate, and uptake of glucose, observing that THRSP silencing impaired mitochondrial function. Lipidomic analysis of THRSP-silenced adipocytes exhibited decreased hexosylceramide concentrations, accompanied by altered expression of sphingolipid metabolism genes. In summary, THRSP induction is impaired in insulin resistance. Its downregulation in human adipocytes could promote mitochondrial dysfunction and impaired sphingolipid metabolism may contribute to metabolic dysfunction. In conclusion, these findings reveal and demonstrate different mechanisms of adipocyte dysfunction. Adipocyte differentiation and function are impaired by overexpression of miR-107 and miR-221-3p, and downregulation of THRSP expression.
  • Lähteenmäki, Hanna (Helsingin yliopisto, 2022)
    Chronic periodontitis and peri-implantitis are complex inflammatory conditions in the periodontium. Periodontal tissues are mainly made up of type I collagen, which is a major component of the extracellular matrix of both soft and hard periodontal tissues, and collagen breakdown is one of the main events in periodontal destructive lesions. Oral bacteria and their metabolites can penetrate peri-implant tissues more easily than permanent teeth tissues, and the inflammatory state progresses more rapidly to destruction of bone tissue in peri-implantitis. This is mainly due to differences between natural dental tissues and peri-implant tissues. The treatment of periodontitis and peri-implantitis is challenging, and therefore, prevention and early diagnoses are crucial. Traditional methods are used to assess the health status of periodontal and implant tissues. However, these measures assess past disease rather than the present pathology of these tissues. Current clinical assays do not work properly in the early diagnosis of peri-implant diseases. This thesis demonstrated the active form of matrix metalloproteinase (aMMP-8) is possible to be determined from the gingival crevicular fluid (GCF)/peri-implant sulcus fluid (PISF) in the initial and real-time diagnosis. Elevated levels of aMMP-8 occur at the onset of periodontal tissue inflammation and can found in GCF/PISF or saliva, contributing to the progression of the disease. Study I aimed to investigate the strongest possible biomarker that can be linked to periodontitis and peri-implantitis. This study confirmed that the elevated level of aMMP-8 is associated with connective tissue diseases, with Study II subsequently focusing on the validation of the aMMP-8 enzyme test in PISF. The hypothesis was that the aMMP-8 enzyme assay provides reliable results for detection of early inflammation in patients and for determination of the rate of progression of peri-implant disease. Studies III and IV investigate whether the aMMP-8 point-of-care (PoC) /chairside test can be implemented beyond the dentist´s office. Studies V and VI investigate the applicability of the aMMP-8 enzyme test in evaluation of the health effects of different home therapies such as fermented lingonberry and dual-light therapy. This thesis showed that aMMP-8 PoC tests used to diagnose periodontal disease can reliably detect and predict both early and ongoing disease and monitor the efficacy of various substances or devices used at home.
  • Gunnar, Riikka (Helsingin yliopisto, 2022)
    Background: Intestinal Failure (IF) is a multifactorial chronic disease requiring long-term parenteral nutrition (PN) to maintain daily energy and fluid balance, and to provide nutrients required for expected growth in children. Recent decades have improved the prognosis of children with IF. Modern multidisciplinary care and changes in PN regimes, new surgical technics, and improving medical care enable living with home PN for years, saving the intestinal transplantation for most severely affected patients. At the same time, the quality of life and the future health and developmental consequences of the pediatric IF emerge as important factors. Aims: Early life events and the necessary treatment may have effects on long term outcome of the pediatric IF patients. In this thesis cognitive and motor development, the risk factors for essential fatty acid deficiency and the risk factors for PN associated steatosis during infancy were investigated. Methods: Essential fatty acid deficiency (EFAD) among 49 IF patients between 0 to 18 years old was investigated. For this retrospective study fatty acid fractions were analyzed and examined against preceding nutrition, the type of parenteral lipid emulsion and patient characteristics. A prospective cross-sectional study evaluated cognitive and motor development of 30 children with IF using validated tests: Wechsler Preschool and Primary Scale of Intelligence, 3rd edition, Wechsler Intelligence Scale for Children, 4th edition, and Movement Assessment Battery for Children, 2nd edition. To study early steatosis in infants with short bowel syndrome (SBS) a retrospective study comprising 31 patients was conducted. Patients had liver biopsy taken at the median age of 5 (IQR 3-8) months. Biopsy results were compared to PN composition and patient characteristics. Results: Altogether 8 patients out of 49 were deemed at risk of developing EFAD with Triene: tetraene ratio (TTR) ≥0.1, including three patients with biochemical EFAD (TTR≥ 0.2). The patients with supplemental PN (P = 0.0016), current PN with low lipid content (P = 0.0003), younger age (P=0.0291) and shorter small bowel (P =0.0013) were at increased EFAD risk. Median Intelligence quotient (IQ) among 26 IF patients in the study at the median age of 7.5 years was 78 (IQR 65–91). Ten patients had an IQ under 70 (-2 SD). Significant motor impairment was detected in 10, and milder difficulties in 8 out of 28 patients. Poor cognitive outcome was associated with shorter remaining small bowel (P=0.005), number of laparotomies (P=0.007) and general anesthesia (P=0.010), and length of inpatient status (P=0.047), while weaker motor outcome was associated with prematurity (P= 0.029), lower birth weight (P=0.024), and lower IQ scores (P=0.020). Steatosis was found in 8 out of 31 diagnostic biopsies taken. Early steatosis associated with higher parenteral glucose (P=0.04), amino acids (P=0.03), and energy (P=0.01) content at the age of three months compared to study patients without early steatosis. Increased serum bile acid (P=0.02), alanine aminotransferase (P=0.0002) and aspartate aminotransferase (P=0.001) levels associated with early steatosis. Conclusions: Children with IF are at risk of EFAD while undergoing intestinal rehabilitation from parenteral to enteral nutrition, especially if lipid infusions are terminated while PN support is still required. Children with neonatal IF commonly have unfavorable cognitive and motor outcome at early school age. Neurological follow up visit for neurodevelopmental evaluation is recommended for all children with clinically significant IF. Early liver steatosis in infants with SBS associated with high parenteral glucose, amino acid, and energy content. Adhering to current pediatric guidelines when administering parenteral nutrition to infants with IF, and monitoring transaminase and bile acid levels for avoidance and early suspicion of liver steatosis is recommended.
  • Lommi, Sohvi (Helsingin yliopisto, 2022)
    Globally excess weight and dental caries are highly prevalent chronic conditions both in children and adults, suggested as sharing risk factors, amongst them dietary sugar. Notably, sugar intake in children and adolescents appears high in many developed countries. The oral microbiota – that is, the microbial communities in the oral cavity – contribute to oral health as well as contributing to systemic health, and changes to the saliva microbiota might depend upon sugar consumption. This doctoral thesis aimed to 1) examine the relationship between the consumption frequency of foods and drinks generally high in added sugar (‘sweet treats’) and the risk of excess weight, 2) examine changes in sweet treat consumption patterns during early adolescence and whether these changes associate with changes in weight status and central obesity or the risk of developing excess weight or central obesity, 3) examine the relationship of sweet treat consumption frequency and other dietary factors to the risk of caries experience and 4) assess the diversity, composition and functional capacities of the saliva microbiota in children with low and high frequency levels of sweet treat consumption. The studies in this doctoral thesis consist of four original research publications and utilise material from the Finnish Health in Teens (Fin-HIT) study. The Fin-HIT is an ongoing prospective cohort of over 11 000 children aged mostly 9–12 years at enrolment. Baseline data collection was conducted between 2011 and 2014, and children were followed for nearly three years. Study I (n = 8461), study III (n = 6660) and study IV (n = 453) utilised cross-sectional data, whereas study II (n = 4237) was longitudinal. The research data includes self-reported questionnaire data on the consumption of indicative food items and other lifestyle factors, measured and self-reported anthropometrics as well as saliva samples. Sweet treats consisted of chocolate and sweets, sweet pastries, biscuits and cookies, ice cream, sugary soft drinks and sugary juice drinks. A sweet treat index (STI) was calculated to indicate the sum of the weekly consumption frequencies of sweet treats. Based on the STI and change in STI, children fell into low, medium and high as well as decreased, stable and increased STI groups, respectively. Weight status was defined as thin, normal weight, overweight or obese (that is, excess weight) using age- and sex-specific cut-offs, and as without central obesity and with central obesity based on the waist–height ratio (WHtR). The decayed, missing and filled teeth (DMFT) index for permanent teeth was used to indicate a history of cavitated caries lesions (caries experience). The saliva microbiota was characterised using 16S rRNA gene sequencing. We performed statistical analyses using the chi-square test, t-test, analysis of variance (ANOVA), logistic regression, Cox regression, permutational multivariate analysis of variance (PERMANOVA) and general linear model. Less than one in six children had excess weight. Children with a low consumption frequency of sweet treats were at an increased risk of having excess weight, whereas children with a high consumption were at an increased risk of thinness. On average, STI decreased during early adolescence, although the consumption frequency of chocolate and sweets increased in girls and boys, and that of sugar-sweetened soft drinks increased in boys. Similarly, decreasing trends in the mean STI were observed in groups of children who developed excess weight or central obesity. A decrease in the STI was associated with the risk of developing excess weight. Two-thirds of children had no caries experience. A high sweet treat consumption associated with an increased risk of a caries experience, whilst a low consumption associated with a decreased risk. Diversity in the saliva microbiota did not differ between a low and high sweet treat consumption, although differences in the composition and functional capacities were observed; several bacterial genera were differentially abundant, and pathways relating to nitrate reduction and gondoate biosynthesis were enriched in the saliva of children with the highest consumption frequency of sweet treats. The highest consumption frequency of sweet treats did not associate with excess weight cross-sectionally nor longitudinally, suggesting that sugary product consumption alone may not be relevant as a target of obesity prevention programmes amongst school-aged children. Although the overall sweet treat consumption frequency decreased during early adolescence, the increase in the consumption frequency of chocolate and sweets and sugary soft drinks requires attention. Specifically, we observed differences between girls’ and boys’ consumption patterns. A high consumption frequency of sweet treats associated positively with the risk of caries experience, highlighting the need to focus on sugar consumption to prevent caries even in an affluent Nordic country. Moreover, findings regarding the different saliva microbiota profiles in children with the lowest and highest sweet treat consumption frequencies can be utilised in future studies to further our knowledge about the influence of sugary diets on the saliva microbiota and, consequently, on oral and systemic health.
  • Moura Baronio, Diego (Helsingin yliopisto, 2022)
    Brain amines are neurotransmitters that modulate important functions in the central nervous system, including behavior and brain development. Several brain disorders are characterized by impairments in aminergic systems, including autism spectrum disorder (ASD). ASD is characterized by impaired social behavior, difficulty in communication and stereotypies. The etiology of ASD is poorly understood, but both environmental and genetic risk factors are known to play a role on it. Valproic acid (VPA), a drug commonly used to treat bipolar disorder, and mutations in the monoamine oxidase a gene are both environmental and genetic risk factors. Zebrafish shares relevant neurochemical aspects with humans and display a wide range of behaviors, which contribute to its appreciation as a model organism in neuroscience. Thus, this study characterized the phenotypes of zebrafish pharmacologically and genetically challenged with ASD risk factors. Additionally, functional aspects of aminergic neurotransmission in the zebrafish brain were studied through the characterization of animals that lacked vesicular monoamine transporter 2 (vmat2, also known as slc18a2) and monoamine oxidase (mao), relevant genes for aminergic vesicular transport and metabolism, respectively. In the first publication of this thesis, larval zebrafish embryonically exposed to VPA showed a reduction in the number of histaminergic neurons and in the levels of histamine when compared to control animals. The histaminergic system of VPA-exposed larvae was also affected by a downregulation of histidine decarboxylase and histamine receptors. Some of these abnormalities persisted until adulthood along with impaired social behavior. This study brings more attention to a possible involvement of the histaminergic system in the outcomes related to ASD. In the second article, the levels of dopamine, noradrenaline, serotonin and histamine of vmat2 mutants were decreased, whereas levels of dopamine and serotonin metabolites were increased, indicating elevated amine turnover. There were also fewer aminergic immunoreactive cells. Further, in mutants notch1a and pax2a were downregulated in brain proliferative zones. This mutant line may be used in the investigation of how amines transport affects brain development and function, and for use in high-throughput and drug screening. In the third article, mao−/− larvae showed a hyperserotonergic phenotype characterized by extracellular serotonin immunoreactivity that was associated with damage in aminergic systems. They also showed weaker responses to visual and acoustic stimuli, abnormal expression of developmental markers and died within 20 days post-fertilization. mao+/- fish were viable and demonstrated impaired social interactions compared with adult mao+/+ siblings. These mutants could be used in investigations aiming to assess the roles of MAOA/B and amines during brain development and to study the behavioral outcomes associated with MAOA/B deficiency. Collectively, the results of the present thesis support zebrafish as a tool to investigate mechanisms underlying ASD. Additionally, it presented two new models to study important aspects of aminergic neurotransmission in zebrafish and its role in brain function and behavior.
  • Gustafsson-Silén, Charlotta (Helsingin yliopisto, 2022)
    Background. Treatment protocols for orofacial clefts (OFC) have evolved over the years. Modern outcome goals of primary repair have shifted from solely achieving an intact palate repair towards successful long-term outcomes, with focus on speech development, occlusion, hearing, appearance, and maxillary growth. An anatomically and functionally intact palate are vital for development of proper speech; this is among the most important aspects of cleft repair. Unsuccessful primary repair may result in velopharyngeal insufficiency (VPI) due to a structural deficit in the palate or an oronasal fistula (ONF). These complications may be severe enough to have a detrimental impact on the growing child’s psychological health and social relationships and to require a secondary surgical (SS) intervention in the form of speech-correcting surgery (SCS), fistula repair, or both. These complications not only create a surgical dilemma (particularly ONFs) but also cause additional pain and suffering for the child and increased medical costs. Although a myriad of surgical treatment protocols and techniques for OFCs has been developed due to the controversies concerning speech and maxillofacial growth, the optimal protocol regarding both timing and repair method remains unclear. The Cleft and Craniofacial Center, Department of Plastic surgery, Helsinki University Hospital has a history of various surgical protocols and techniques that have been utilized over the past three decades. Aims. The main aim with this thesis was to retrieve an overview of the long-term incidence for SS, with focus on SCS and fistula repair in different types of cleft palates (CP), including patients with Robin sequence (RS), treated at the Cleft and Craniofacial Center, Department of Plastic surgery, Helsinki University Hospital. The different surgical protocols and techniques utilized since the 1990s at the cleft center were also assessed and compared. Methods. This thesis consists of four separate studies, thus creating a study series involving the most common types of OFCs classified by the Veau system (isolated cleft palate [ICP], unilateral cleft lip and palate [UCLP], and bilateral cleft lip and palate [BCLP]), including RS. All studies were retrospective single-center follow-up studies. Only non-syndromic (ns-CP) subjects born between 1990 and 2011 were included, and all were treated and followed by the multidisciplinary cleft team as a part of the standard treatment protocol at the time of the cleft center. Only children with frequent follow-up to at least 8 years of age or longer were included in the ICP and RS studies (Studies I-II), whereas results were analyzed at alveolar bone grafting (ABG) and post-ABG in the UCLP and BCLP studies (Studies III-IV). The primary palatoplasty protocols utilized throughout the years involved four different single-stage techniques and two different two-stage protocols, early soft palate repair combined with short delayed hard palate repair, and early hard palate repair combined with short delayed soft palate repair. Results. The thesis included altogether 872 patients with ns-CP: ICP 423 (SCP 123 and HSCP 300), RS 78, UCLP 290, and BCLP 81. The long-term incidence for SS was high, and ranged between the Veau hierarchy from 23.6% to 46.9% (mean 34.5%) for SCS and 0.8-53.1% (mean 20.9%) for fistula repair. Extensive clefts, partly or totally detached from the vomer as the HSCP (inclusive RS) and BCLP were more likely to require SCS. Fistula repair followed the severity of the Veau hierarchy. The outcome differences between the surgical protocols in CLP (UCLP and BCLP) were small, indicating that none of the treatment protocols was clearly superior to another. However, for both SCS and fistula repair, attention was drawn to the somewhat more favorable outcomes of the single-stage protocol. Moreover, no single-stage technique was clearly superior to another. A substantial number of fistulas were closed at ABG, particularly the single-stage protocol with a high rate of anterior fistulas, especially in connection to a perialveolar fistula. RS is associated with a high incidence of SCS and fistula formation. Gender, age at primary palatoplasty, surgical technique, surgeon, cleft severity, and airway management at infancy do not appear to be associated with need for SS. Conclusion. As SS (SCS and fistula repair) are common in patients with OFCs in the long-term, these surgeries are particularly anticipated in extensive clefts and in patients with associated RS. No surgical protocol or technique is superior to another regarding the long-term need for SCS or fistula repair. Proper fistula assessment requires long-term follow-up, as a substantial part of the ONFs is repaired at ABG.
  • Kask, Gilber (Helsingin yliopisto, 2022)
    Background Soft-tissue sarcomas (STS) represent approximately 1% of all solid malignancies in adults. More than 50% of STSs are localized in the extremities. The aim of extremity STS treatment is survival and limb salvage with the best possible quality of life (QoL) and functional outcome. There has been increasing interest to report the health-related quality of life (HRQL) and functional outcome after treatment. Measures should be valid, reliable, accurate, and clinically meaningful for the population in question. A few studies have focused on patient-related factors or assessment using multiple metrics in analyzing long-term functional outcome and HRQL in surgically treated lower-extremity STS patients. Materials and methods The dissertation is based on four papers that are focused on different aspects of functional outcome and HRQL in lower-extremity STS. The first paper was a pilot study in which the Toronto Extremity Salvage Score (TESS) and the Musculoskeletal Tumor Society (MSTS) score were translated to Finnish language according to accepted guidelines. The second paper was a systematic literature review that was based on the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. Results on measurement types, measures, and time of measurement of functional outcome were compiled from the included studies. Validity of the used measures was reported. The third paper was a cross-sectional validation study that was based on STS limb-salvage patients. Assessments of functional outcome and HRQL were performed using the TESS, the QLQ-C30 Function and QoL modules, the 15D, and the MSTS score. The TESS was repeated twice at a 2- to 4-week interval. The fourth paper was a cross-sectional study in which functional outcome and HRQL analysis were performed using STS limb-salvage patients. The TESS, QLQ-C30, and 15D measures were used. Functional outcome and HRQL data were collected prospectively and other data retrospectively. Results Finnish versions of the TESS and the MSTS questionnaires were translated and culturally adapted. No problems or difficulties were reported by the patients in completing the Finnish versions of TESS or MSTS questionnaires. The systematic literature review search found 3461 publications, of which 37 met the inclusion criteria. The measurement types used were clinician-reported outcomes (n=27), patient-reported outcomes (PRO) (n=20), and observer-reported outcomes (n=2). The most frequently used measures were the TESS (n=16) and the MSTS (1993) (n=12) scores. Three of the ten previously reported measures provided validated functional outcome scores. The TESS questionnaire’s ceiling effect was noted, as 21% of the patients scored maximum points. The intraclass correlation coefficient between first and second administration of the TESS was very high (0.96). The results of exploratory factor analysis together with high Cronbach’s α (0.98) supported a unidimensional structure. The TESS correlated moderately with the MSTS score ( = 0.59, p < 0.001) and strongly with the mobility dimension in the 15D HRQL instrument ( = 0.76, p < 0.001) and physical function in QLQ-C30 ( = 0.83, p < 0.001). A total of 141 patients undergoing limb-salvage surgery were included in the fourth study. Based on our sample, lower functional outcome was statistically significantly associated with older age, higher body mass index (BMI), and need for reconstructive surgery and radiotherapy. Lower HRQL was statistically significantly associated with older age, higher BMI, and reconstructive surgery. Conclusion Translation and cultural adaptation of questionnaires should be performed according to internationally accepted guidelines. The current thesis is the first to validate the TESS distinctly for lower-extremity STS patients. In addition, the thesis demonstrates that the TESS measure is valid and reliable in Finnish. Based on the systematic review, several different methods exist for assessing functional outcome in patients with lower-extremity sarcoma. The most frequently used measure is a validated TESS. The postoperative functional outcome in patients with lower-extremity STSs seems to increase back to the preoperative baseline level during long-term follow-up evaluation. Post-treatment functional outcome of patients with lower-extremity STS is affected by BMI, age, and need for reconstructive surgery and radiotherapy. The most significant predictors affecting HRQL are BMI, age, and need for reconstructive surgery. Although tumor- and treatment-related factors had an impact, patient-related factors such as age and BMI were the most consistent and significant determinants of both functional outcome and HRQL.
  • Sorto, Pia (Helsingin yliopisto, 2022)
    Background: Stroke is the third leading cause of death, and a significant cause of disability. Approximately 20% of ischemic strokes are due to carotid atherosclerosis, characterized by the formation of lipid-rich inflammatory cell collections inside the intima of the carotid bifurcation – carotid plaques. The main cell type is a macrophage foam cell, the archetypal cell of the atherosclerotic plaques. When a plaque proceeds to a vulnerable phenotype, it manifests itself with clinical symptoms. The most common mechanism is the rupture of the fibrous cap overlying the lipid core of the plaque, followed by local thrombosis and embolization, ultimately leading to cerebral infarction. Plaques are common, but most will remain clinically silent. The main treatment of carotid atherosclerosis is primary and secondary prevention with pharmacotherapy and lifestyle interventions. In advanced symptomatic atherosclerosis, carotid endarterectomy can be used for stroke prevention. However, selecting the treatment option is not clear-cut. For example, many currently surgically treated patients are potentially curable by modern pharmacotherapy alone, while other asymptomatic patients – those who had a catastrophic plaque-originated ischemic stroke – would have benefited from more accurate assessment of the vulnerability of the carotid plaque and surgery as the primary prevention. The pathobiology behind the progression of the carotid plaque into a vulnerable phenotype is still unclear, and there are no clinical methods to evaluate it. This is a core question not only in carotid atherosclerosis, but also in other cardiovascular diseases such as coronary artery disease. This study aims at characterizing selected symptom-associated genes inside carotid plaques to generate hypotheses about the cellular events associated with plaque vulnerability. Methods: 92 clinically well-characterized stroke, TIA or asymptomatic patients underwent carotid endarterectomy due to high-grade (>70%) carotid stenosis in 1997–2000 at the Helsinki University Central Hospital, and their plaques were collected. A DNA microarray analysis in a sub-group of radiologically confirmed asymptomatic and stroke-causing plaques showed 60 genes with significant expression differences between the symptomatology groups. Some of the genes were previously almost unknown for clinically symptomatic carotid atherosclerosis. The protein products of six of these genes – ADFP, CD36, ABCA1, CD163, HO-1 and GLUL – were studied with a detailed map-style immunohistochemical methodology, which combined semi-quantitatively assessed protein expression level in each plaque (1–2 mm2 area), amount of the most common molecular and cellular stressors inside the plaques, and the histopathological features of the plaques. This analysis was supported by protein quantification in some of the studies. The immunohistochemical findings were compared with macroscopic plaque data and clinical data, as well as with the previously published data from the same patients on other genes found in the original DNA microarray work. An advanced statistical methodology was designed for each study by a professional statistician. Results: We found meaningful spatial associations between macrophage protein expression and different local pathological plaque constituents, in line with previous evidence. The lipid droplet protein ADFP associated with erythrocytes and cholesterol clefts, indicating their role in foam cell formation. The relationship between CD36 and ABCA1 seemed to favor lipid loading in ruptured carotid plaque macrophages, supporting the theory of dysregulated lipid loading as a factor in plaque progression into a vulnerable phenotype. GLUL, CD36, ABCA1, CD163 and HO-1 also associated with intraplaque hemorrhages. The local GLUL association with the studied local pathological factors was more pronounced in symptom-causing plaques. Higher local GLUL associated with a thinner fibrous cap, a phenomenon linked with plaque vulnerability. Based on the results, we formulated the first theory about the significance of GLUL in atherosclerosis. Conclusions: The pattern of how plaque pathology-promoting factors and molecules such as iron and erythrocytes associated with most of the studied genes suggested that antiatherosclerotic macrophages were found in many pathological plaque regions. The previous literature from GLUL in other disease states and cell cultures supported the role of GLUL as an evolutionarily highly conserved, life sustaining molecule. On the contrary, it was highly associated with multiple types of different pathology-promoting features, indicating that it might be linked with plaque pathogenesis rather than antiatherogenic macrophage activity. This led to a shift in the view about atherosclerosis. The question “What is wrong with the biology when atherosclerosis develops?” transformed into the question “Why do biologically meaningful responses produce atherosclerosis?” This question was analyzed in light of evolution. Certain pathological alterations and cellular problems might be due to evolutionarily ‘outdated’ mechanisms in the human. The ancient macrophage may not be programmed for phagocytosing decades of excess lipoproteins, a threat faced by man in the modern era of overnutrition, eventually leading to heightened vulnerability of the plaques.
  • Ranjit, Anu (Helsingin yliopisto, 2022)
    It is understood that cigarette smoking is associated with depressive symptoms, and depression can also be associated with quitting smoking. Longitudinal twin studies provide genetically informative data and a better understanding of such complex relationships; however, the causal nature of this relationship is still poorly understood. Longitudinal data helps to confirm the direction of the association between smoking status and depressive symptoms. It is crucial to account for confounding by measured covariates and by underlying shared genetic and environmental factors affecting smoking behaviour and depression. The genetically informative data allows us to control for possible mechanisms underlying the associations and strengthens causal inference. This thesis aimed to study the complex relationships between depressive symptoms and various aspects of cigarette smoking behaviour. Further, by using data from twins who grew up together this thesis sought to control for shared genetic and environmental factors underlying the association. All sub-studies utilised population-based Finnish Twin Cohort data collected at the University of Helsinki. The FinnTwin12 cohort data were used for study I (N=4,152) and study II (N=2,954), where adolescents and young adults were studied at the ages of 14, 17, and 22 years. Study III utilised the Older Twin Cohort data, where participants at the baseline (in 1990) were linked with antidepressant prescription data from the Finnish Social Insurance Institution (in 1995–2004) (N=10,652). Study IV also utilised Older Twin Cohort data where daily smokers in 1990 were followed-up for their smoking status in 2011 (N=1,438). The General Behaviour Inventory (GBI) and the Beck Depression Inventory (BDI) were used to measuring self-reported depressive symptoms. Participants self-reported their smoking behaviour in the questionnaire surveys. Cigarette smoking at age 14 was associated with subsequent depressive symptoms at age 17. Regular smokers and those consuming a higher number of cigarettes had higher depression scores at the age of 17. Further, there existed a reciprocal association between cigarette smoking and depressive symptoms when studied among twins from adolescence (age 17) to young adulthood (age 22). Daily smoking at the age of 17 predicted higher depression scores at the age of 22, and depressive symptoms in adolescence were associated with an increased risk of cigarette smoking in young adulthood. However, the associations were not independent of shared familial factors. Among older twins, daily smokers at baseline had an elevated likelihood of having antidepressant prescriptions in follow-up, even after controlling for essential confounders and familial factors. Furthermore, daily smokers reporting depressive symptoms at baseline had a lower likelihood of smoking cessation during a 20-year follow-up period. A higher number of cigarettes smoked at baseline, and familial factors partly explained the observed association between depressive symptoms and smoking cessation. These studies concluded that there is a reciprocal relationship between cigarette smoking and depressive symptoms among the young population. Later in life, daily smoking predicts a higher likelihood of anti-depressant prescriptions, and depressive symptoms predict a lower likelihood of smoking cessation. Shared familial factors (genetic and environmental) and measured confounders (for example, alcohol use) partly explain the observed associations between smoking behaviour and depression. Thus, findings from genetically informative twin data bring us a step closer to the causal inference and provide additional evidence for scientific use. In a public health setting, the results highlight the need to consider the complex nature of the relationship between smoking and depressive symptoms in prevention, treatment, and rehabilitation efforts. This complexity seems to involve reciprocal association, which is likely to be partly causal but partly explained by familial and/or other factors. Further, intervention efforts should consider that smoking may be an indicator of liability to depressive symptoms, and likewise, depression an indicator of liability to smoking.
  • Nummi, Annu (Helsingin yliopisto, 2022)
    Cardiovascular diseases are the leading causes of death worldwide. Ischemic heart failure due to coronary artery disease is one of the most common reasons for mortality. Drug therapy, transcatheter procedures, and surgery cannot restore the functional cell loss of the dead myocardium. Instead, the traditional treatment consensus focuses on delaying the progression of the disease and diminishing the symptoms. Eventually, ventricular dysfunction develops when the heart suffers from inadequate blood flow. Only a ventricular assist device or heart transplantation can treat end-stage heart failure. The vision of restoring structural and functional myocardium after ischemic damage and scarring has fascinated the cardiac regeneration research community for decades and driven the search for a clinically feasible, reliable, and effective treatment for repairing and replacing damaged heart tissue. Epicardial transplantation of atrial appendage micrografts (AAMs) is a newly adopted surgical technique with cardio regenerative potential and can, as a straightforward method, be readily adopted worldwide. As myocardial support therapy, performing AAMs transplantation with open heart surgery is feasible—such as coronary artery bypass grafting (CABG) surgery. This PhD thesis aims to assess the safety of the epicardial delivered AAMs with CABG surgery and evaluate the procedure’s feasibility in a clinical setting. Patients and methods Study I assessed the safety and feasibility of epicardial transplanted AAMs in pigs after coronary artery ligation. First, the right atrial appendage was harvested and mechanically processed into AAMs. The left descending coronary artery was ligated to experimentally model myocardial infarction and heart failure. AAMs with fibrin gel were placed on an extracellular matrix patch and transplanted epicardially onto the infarct area. Four pigs received an AAMs patch, and four received similarly prepared patches without AAMs. Open heart echocardiography was performed preoperatively and at three weeks follow-up to evaluate the cardiac function and dimensions of the infarcted ventricular wall. The primary outcome measures were the safety and feasibility of therapy administration during open heart surgery. Studies II, III, and IV included 12 ischemic heart failure patients scheduled for elective CABG surgery. Late gadolinium enhancement cardiac magnetic resonance imaging (CMRI) was performed preoperatively and six months postoperatively. As well as the standard clinical treatment protocol, six patients received an AAM patch, and six were operated on according to the standard clinical treatment protocol without AAMs patch transplantation. Clinical data of 30 elective CABG patients, without any additional follow-up or imagining, was collected from hospital records; this group formed another control group. A sample of the right atrial appendage was mechanically processed to generate the AAMs. The micrografts were applied with a fibrin gel onto a tissue-engineered extracellular matrix sheet and further epicardially onto the infarct scar site identified in the preoperative CMRI. Laboratory tests and quality-of-life questionnaires (QoL) were performed preoperatively and at a six-month follow-up. The primary outcome measures were I) patient safety concerning hemodynamic and cardiac function over the follow-up time and II) feasibility of therapy administered in a clinical setting. Secondary outcome measures were left ventricular wall thickness, change in myocardial scar tissue volume, changes in left ventricular ejection fraction, plasma concentrations of N-terminal pro-B-type natriuretic peptide levels, NYHA class, number of days in the hospital, and changes in QoL. Results Study I showed no arrhythmias, complications, or increased mortality among the pigs receiving epicardial AAMs or control patches. According to processing time and sterility, AAMs patch preparation was feasible to be conducted simultaneously with heart surgery. AAMs suppressed the overall inflammation (foreign body reaction) caused by the matrix as measured by the amount of infiltrated inflammatory cells and the size of the inflammation area. Immunohistochemistry showed increased CD3+ cell (T-lymphocyte) density in the AAMs patch group. In Studies II, III, and IV, epicardial transplantation of AAMs for patients was safe and feasible to be performed with open heart surgery. Patient recovery was the same in all groups. CMRI demonstrated that patients receiving AAMs treatment had a greater volume of viable cardiac tissue at follow-up. Conclusions According to the results, epicardial transplantation of AAMs is safe, clinically feasible, and shows good clinical applicability during open heart surgery. As the results from the safety and feasibility trial suggest, the therapy’s regenerative efficacy should be evaluated in a larger randomized clinical trial. Moreover, epicardial transplantation of AAMs may be a delivery platform for future cell-, drug-, or gene-based cardiac therapies.
  • Rautalin, Mervi (Helsingin yliopisto, 2022)
    Breast cancer patients’ health-related quality of life (HRQoL) is an important quality indicator in breast cancer care. HRQoL is recommended to be studied with both generic and disease-specific HRQoL measuring tools. Different techniques in breast cancer surgery affect the patients’ well-being in different ways. Patient-dependent factors and tumour biology both have their impact on treatment choices. Knowledge of breast cancer patients’ HRQoL in relation to different surgical methods is still scarce. The method and timing of breast reconstruction is under debate between immediate breast reconstruction (IBR) and delayed breast reconstruction (DR). Pressure on societies caused by the rising incidence of breast cancer is associated with a need for health-economic evaluations to guide resource allocation on cancer treatment. This thesis evaluates breast cancer patients’ HRQoL in different states of the disease and compares different measuring tools to detect differences between them and produce data on their validity. Different surgical methods to treat breast cancer are studied in relation to HRQoL. All available breast reconstruction methods and the timing of reconstruction are evaluated. The cost of care from the care providers’ perspective is also described during a two-year prospective follow-up. This thesis produces new information concerning HRQoL measuring tools and their usability in breast cancer; how breast cancer treatments performed in Finland affect the patients’ HRQoL and how the costs of different surgical methods are generated. This thesis is based on two study populations. 840 breast cancer patients treated in the Helsinki and Uusimaa Hospital district from 2009 to 2011 were recruited to a cross-sectional observation study as part of a study of prostate, colorectal, and breast cancer patients. Patients filled in an informed consent form and three different HRQoL measuring tools: EQ5D-3L (including VAS), 15D and EORTC QLQ C-30 BR23. Their answers were analysed with linear, stepwise regression analyses. Individual study population of 1065 patients with primary breast cancer were recruited to a prospective study from year 2008 to 2015 in Helsinki and Uusimaa Hospital District. Recruited patients filled in an informed consent form and two HRQoL questionnaires: the generic 15D and breast-cancer specific EORTC QLQ C-30 BR23. The questionnaires were handed out at the first hospital visit and repeated via mail at 3, 6, 12 and 24 months later. Clinical data were collected from hospital records, combined with HRQoL results and then analysed with statistical methods. Data on cost of care were obtained from the ECOMED database, analysed and presented according to the surgical method. EQ5D was associated with a high ceiling effect with 41% of the patients reporting perfect health; other measuring tools performed with less ceiling. Breast cancer patients’ HRQoL deteriorated along the disease progression with patients reporting fatigue, pain and sleeping disorder symptoms. The prospective follow-up included 351 mastectomies, 415 breast resections, 248 oncoplastic resections and 51 immediate breast reconstructions (IBR). 402 patients went through axillary clearance. 840 patients (79% of all) received radiation therapy and 523 (49%) chemotherapy, 766 (72%) patients had endocrine treatment, and 119 (11%) patients had targeted therapy (anti HER2- medication). 41 patients had later corrective surgery and 34 patients had DR. HRQoL was affected by disease status and by the disease burden. Higher Grade, N-class and BMI (body-mass index) correlated with poorer HRQoL at 3 months. The effect of N- and M-class and receiving chemotherapy still correlated with poorer HRQoL at 24 months. Active smoking correlated with complications. Mastectomy patients had the poorest HRQoL throughout the study period and they reported the most pain and arm symptoms. Patients operated on with oncoplastic techniques had the best body image at 24 months. Reconstruction patients had the best physical and sexual functioning scores at 24 months. Reconstruction patients’ recovery after treatments was the slowest. No difference was found between different autologous reconstruction methods. The lowest costs from surgery were observed in BCS patients (mean 6015 euros). Mastectomy was associated with mean costs of 8114 euros, IBR with 18 217 euros, and DR with 19 041 euros. EQ5D-3L is associated with high ceiling effects. Consequently, care must be taken when choosing HRQoL measuring tools. Breast cancer patients frequently reported insomnia, pain and fatigue, indicating the main focus on symptom handling. Mastectomy patients are at risk of poor HRQoL and higher symptom burden. Oncoplastic techniques produce good HRQoL and body image. Breast reconstruction produces good HRQoL, physical - and sexual-functioning scores, but the improvement in HRQoL materialises later than in BCS patients. Breast reconstruction method and timing should be tailored individually, and no patient should be pushed toward reconstruction before being ready for it. The cost difference between IBR and DR is relatively small, so the cost of reconstructive surgery should not be a factor in the decision making.
  • Pakkanen, Pihla (Helsingin yliopisto, 2022)
    Laryngeal cancer is, worldwide, the 22nd most common cancer. In Finland, those diagnosed with laryngeal cancer account annually for approximately 130 patients. The most common histological subtype is squamous cell carcinoma (SCC), covering over 95% of cases. Approximately 70% of laryngeal squamous cell carcinomas (LSCC) are glottic; the other subtypes are supraglottic and subglottic. This thesis focuses on T1 glottic LSCC, in which recurrences are rarer than at advanced stages, developing in approximately 10% of cases. The early diagnosis of recurrence is essential; delay can cause major functional deficit or mortality. Treatment of T1 glottic LSCC with transoral laser microsurgery (TLM) or radiotherapy (RT) shows a favorable outcome. This thesis study examined the treatment, follow-up, and prognosis of T1 glottic LSCC patients at five Finnish university hospitals between 2003 and 2015. The patient total was 303. The primary treatment modality (surgery vs. RT) did not show any association with improved laryngeal preservation or 5-year disease-specific survival (DSS). The majority of the 38 recurrences were detectable during a routine follow-up visit, and of those 38, 21 (55%) of the recurrences were asymptomatic, and 15 of the recurrences were detectable more than 2 years following diagnosis of the primary LSCC. Routine post-treatment follow-up of T1 glottic LSCC seems beneficial. Furthermore, to examine the prognosis of T1a glottic LSCC in a randomized study, we recruited 56 male patients with T1a glottic LSCC. Of these 56, 31 we randomized for TLM, and 25 for RT. The prognoses in both treatment groups of T1a glottic LSCC were similar. Five-year recurrence-free survival (RFS) in patients treated with TLM was 81%, DSS 97%, and overall survival (OS) 87%. In patients treated with RT, five-year RFS was 88%, DSS 100%, and OS 92%. The laryngeal preservation rate in both treatment groups (TLM 97% vs. RT 92%) were similar. Studies comparing treatment modalities and prognosis of T1a glottic LSCC have been mainly retrospective. This study showed similar results in a randomized study setting. Immunological changes in the tumor microenvironment affect enormously both tumorigenesis and tumor spread. Programmed death-ligand 1 (PD-L1) is a transmembrane protein, the overexpression of which in tumor cells can lead to T cell exhaustion and immune escape. High tumor-infiltrating lymphocyte (TIL) density is associated in many solid tumors with improved prognosis. We investigated PD-L1 expression, stromal and intratumoral TIL density, and toll-like receptors (TLRs) 4 and 5 expression in T1 glottic LSCC patients treated at five Finnish university hospitals; these totaled 174. Over half the patients had positive PD-L1 expression, yet PD-L1 showed no prognostic relevance. Low stromal TIL density was associated with local recurrence and new primary tumors of the larynx. TLR4 and TLR5 expression were not connected with survival. Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that remodel the extracellular matrix. Tissue inhibitors of MMPs (TIMPs) can deactivate MMPs. MMPs play several roles in tumorigenesis, and MMP-8 has both tumor-promoting and tumor-suppressing effects. This study included 55 recurrent respiratory papillomatosis (RRP) patients and 59 with T1-T4 LSCC. Five of the RRP patients developed LSCC during follow-up. LSCC patients and RRP patients with malignant transformation had higher S-MMP-8 levels than did RRP patients without malignant transformation. High S-MMP-8 levels predicted malignant RRP transformation. In LSCC patients, an elevated level of S-TIMP-1 was associated with poorer RFS and OS.
  • Ylivuori, Maija (Helsingin yliopisto, 2022)
    Diseases of the nose and sinus (rhinologic diseases) are quite common. It is estimated that almost 11% of Europeans already suffer from chronic rhinosinusitis (CRS) and 23% from allergic rhinitis (AR) alone. Studies on the health-related quality of life (HRQoL) of rhinologic patients have been completed on specific diagnostic groups (CRS and AR patients), but, to our knowledge, none have evaluated unselected rhinologic patients. In our study, we examined the quality of life (QoL) of 337 patients referred to the Helsinki University Hospital’s (HUS’s) Department of Otorhinolaryngology – Head and Neck Surgery due to rhinologic disease or symptoms in 2014, and then compared the HRQoL for both the population and other patients with chronic diseases to that of rhinologic patients. The generic HRQoL of rhinologic patients was statistically and clinically significantly worse than that for the age- and sex-standardised control population. Moreover, their QoL was also lower than that of many other patient groups (e.g., patients with head and neck cancer or Addison’s disease). In addition, seasonal variation has been reported for many diseases, which may also be related to air temperature and humidity. Seasonal variation affects the morbidity, symptoms, and severity of an illness. Amongst the elderly, the risk of institutionalisation and even mortality increases during the cold winter months. Seasonality has been found in patients with rheumatic diseases, hypertension, and sleep apnoea. Seasonal trends can also be observed in the QoL of the general population. Some research suggests that the season should be taken into account, especially when conducting QoL research. To our knowledge, only one study of the seasonal variation in the HRQoL of rhinologic patients has been previously conducted and applied exclusively to CRS patients. Therefore, we recruited 301 patients from the rhinologic clinic at HUS in 2014 for our study and determined their disease-specific (Sino-Nasal Outcome Test 22, SNOT-22) and general (15-dimension questionnaire, 15D) HRQoL questionnaire during the four seasons. We found no significant seasonal variation in patients’ QoL amongst our study population. Notably, however, we identifed very few patients suffered from seasonal allergies (2.3% of study patients). However, given that limitation, we can conclude that these HRQoL questionnaires can be used during clinical follow-up, for example, after surgery, with no significant impact from the seasons. In addition, exposure to specific occupational sensitisers (low-molecular weight [LMW] and high-molecular weight [HMW] sensitisers) or irritants serves as a risk for the development of chronic rhinitis and rhinosinusitis. However, little is known about the role of occupational exposure on patients’ rhinologic symptoms. Moreover, the HRQoL of rhinologic patients and its relationship to occupational exposures has primarily been studied amongst patients with rhinitis. Our study, then, aimed to assess the extent to which patients referred to specialist care due to rhinologicF diseases exhibited worsening rhinologic symptoms at work and whether these were related to exposure to specific workrelated sensitisers or irritants. Exposure was also compared to both diseasespecific and generic HRQoL. In addition, we also examined the need for sick leave reported by patients due to their rhinologic disease during the preceding year. For the 177 unselected adult rhinologic patients in employment recruited for our study, we found that exposure to specific occupational exposures did not affect patients’ symptoms at work or their HRQoL. However, the majority of rhinologic patients reported disease exacerbation at work. It appears that their increasing symptoms were, therefore, more likely related to unspecific respiratory triggers. In our study, we found that rhinologic diseases cause substantial work absenteeism. Furthermore, HRQoL has traditionally been evaluated using paper questionnaires. However, in order to implement HRQoL measurements in everyday clinical practice and, thus, increase the amount of HRQoL data available, efforts have recently focused on converting the forms to electronic formats. Our study aimed to determine whether the generic 15D questionnaire can be converted to an electronic format without impacting its reliability and measurement equivalence or diminishing the response rates (RRs). We also assessed patient experiences and preferences between different formats. To do so, we recruited 159 patients from the Department of Otorhinolaryngology at HUS between April and June 2019 for our study and randomised them into four different study groups. In each group, patients completed the paper version of the 15D and one of two electronic versions (a web-based or an application-based format). Based on our findings, the 15D can be converted to an electronic version without compromising its reliability. Patients also preferred electronic formats (74.1%) over paper versions (16.5%). However, the RRs for the electronic surveys remained lower than that for the paper-based questionnaire. To conclude, we found that the HRQoL of rhinologic patients seems to decrease and does not show marked seasonal variation. Patients reported symptom exacerbation at work, but this was not clearly related to specific occupational exposures. Absenteeism due to rhinologic diseases remains high. As such, rhinologic diseases cause a remarkable burden both to the individual and to society as a whole.
  • Moshe, Isaac (Helsingin yliopisto, 2022)
    Background. Depression is the leading cause of disability worldwide. Although evidence-based treatments exist, less than one-in-five people in high-income countries and less than one-in-twenty-seven in low-income countries receive treatment, giving rise to a treatment gap in mental healthcare. Digital interventions have been proposed as a solution to address the treatment gap. As an increasing number of public and private healthcare providers adopt digital interventions to meet the growing demand for treatment, the current thesis set out to examine the latest evidence-base for digital depression interventions and the extent to which new technologies may be used to identify at-risk individuals. Methods. Study 1 assessed the efficacy of digital interventions for the treatment of depressive symptoms based on the largest meta-analysis of digital depression interventions conducted to-date. Databases were searched for RCTs of a computer-, internet-, or smartphone-based interventions for depression versus an active or passive control condition. Participants were individuals with elevated symptoms of depression at baseline. Using a random-effects multilevel metaregression model, we examined effect size of treatment versus control (Hedges’ g) and explored moderators of treatment outcome. Study II was a secondary analysis of data from two RCTs (N=253) of a digital intervention for the prevention and treatment of major depression. Using logistic regression, we first examined participant characteristics as potential predictors of intervention dropout. We then assessed to what extent dropout could be predicted following completion of the first module using a combination of participant characteristics and intervention usage data. Dropout was defined as completing less than six modules. Study III was an observational study of N = 60 adults (ages 24–68) who owned an Apple iPhone and Oura Ring. A smartphone app (Delphi) continuously monitored participants’ location and smartphone usage behavior over a 4- week period. The Oura Ring provided measures of activity, sleep and heart rate variability (HRV). Participants were prompted to report their daily mood and self-reported measures of depression, anxiety and stress were collected at baseline, midpoint and the end of the study using the DASS-21. Multilevel regression models were used to predict the association between smartphone and wearable data and mental health scores. Study IV was a secondary analysis of data from Study III in which we compared the accuracy of five supervised machine learning algorithms in the classification of individuals with normal versus above normal symptoms of depression, as defined by the DASS-21. Results. A systematic search of the literature in Study I identified 83 trials (N = 15,530). The overall effect size of digital interventions versus all controls was g = .52. Significantly lower effect sizes were found in studies conducted in real-world settings (effectiveness trials; g = .30) versus laboratory settings (efficacy trials; g = .59). Significantly higher effect sizes were found in interventions that involved human therapeutic guidance (g = .63) compared with unguided, self- help interventions (g = .34). Additionally, we found significant differences in effect size depending on the type of control used (WLC: g = .70; attention: g = .36; TAU: g = .31). No significant difference in outcomes was found between human-guided digital interventions and face-to-face therapy, although the number of studies was low. In Study II we found that lower level of education (OR=3.33) and both lower and higher age (a quadratic effect; age: OR=0.62, age^2: OR=1.55) were significantly associated with higher risk of dropout. In the analysis that aimed to predict dropout following completion of the first module, lower and higher age (age: OR=0.61, age^2: OR=1.58), medium versus high social support (OR=3.40) and a higher number of days to module completion (OR=1.05) predicted higher risk of dropout, whilst a self-reported negative event in the previous week was associated with lower risk of dropout (OR=0.22). In Study III, we found a significant negative association between the variability of locations visited and symptoms of depression (b = −0.21, p = 0.037) and significant positive associations between total sleep time and depression (b = 0.24, p = 0.023) and time in bed and depression (b = 0.26, p = 0.020). Additionally, we found that wake after sleep onset significantly predicted symptoms of anxiety (b = 0.23, p = 0.035). Study IV revealed that a Support Vector Machine using only sensor-based predictors had an accuracy of 75.90% and an Area Under the Curve of 74.89%, whilst an XGBoost model that combined mood and sensor data as predictors classified participants as belonging to the group with normal or above normal levels of depressive symptoms with an accuracy of 81.43% and an Area Under the Curve of 82.31%. Conclusion. The current thesis provided evidence of the efficacy of digital interventions for the treatment of depression in a variety of populations. Importantly, we provided the first meta-analytic evidence that digital interventions are effective in routine healthcare settings, but only when accompanied by human guidance. Notwithstanding, adherence to digital interventions remains a major challenge with little more than 25% of patients completing the full intervention on average in real-world settings. Finally, we demonstrated that data from smartphone and wearable devices may provide valuable sources of data in predicting symptoms of depression, thereby helping to identify at-risk individuals.
  • Turunen, Katri (Helsingin yliopisto, 2022)
    BACKGROUND The extensive rate of international travels indicates hundreds of millions of travellers with an increased risk of acquiring infectious diseases. For visitors to low- and middle-income countries, travellers’ diarrhoea (TD) is the most common health problem encountered. It is also the most common reason for contacting health care providers or taking antibiotics. At pre-travel consultations, doctors may prescribe stand-by antibiotics to be used in case of (severe) TD. In some countries, this has been the standard approach. Today, with the increasing antimicrobial resistance as a global health threat, antibiotic prescribing policies should be based on scientific data assessing the need for antibiotic treatment. This is of particular concern for travellers, since antibiotic use increases the risk of acquiring multidrug resistant intestinal bacteria and transporting these to the travellers’ home countries. Here, we examined the specifics of travellers’ illnesses, the use of antibiotics to treat TD, and the consequences of prescribing stand-by antibiotics. METHODS Volunteers of this prospective study were recruited at a pre-travel consultation at the Aava Travel clinic in Helsinki. Inclusion criteria comprised a journey outside the Nordic countries with a duration between four days and six months. Participants were asked to fill in three questionnaires (before travel, within two days, and within three weeks after return) and a diary of symptoms while abroad. Stool samples were collected pre- and post-travel and analysed by qPCR for nine bacterial TD pathogens. Univariable and multivariable models were used for the statistical analysis of the data. RESULTS The total population recruited comprised 620 travellers, from whom the final participants for each study were selected. A strikingly high rate of 79% of our travellers reported health problems while travelling and 32% during follow-up. The most common health problems were TD, skin problems, and fever, reported by 69%, 17%, and 17%, respectively. TD was mostly mild (63%). Severe TD was associated with findings of enterotoxigenic Escherichia coli’s STh toxin and campylobacter. Of the travellers with a more severe disease 41% resorted to antibiotic treatment. However, when stand-by antibiotics were prescribed, travellers used them also for non-severe TD: 29% of stand-by antibiotic carriers compared to 5% of non-carriers. CONCLUSIONS Due to the ever increasing international travel, high rates in travel-related health problems are inevitable. Our study showed the great variety of health problems that travellers encounter and presented two tools that are suitable for combatting a major concern, the transmission of multi-drug resistant bacteria across the world by travellers colonised by the bacteria at their destination. In order to decrease the risk of colonisation, the use of antibiotics should be reduced. Two approaches were identified: the first is to avoid prescribing stand-by antibiotics and the second to target antibiotics as a treatment for only the most severe cases of TD.
  • Pohju, Anne (Helsingin yliopisto, 2022)
    Severe reduction of bowel function, such that long-term parenteral support (PS) is necessary to maintain health, defines chronic intestinal failure (CIF). This rare gastroenterological condition covers a wide range of underlying diseases; thus, its onset as well as course vary greatly. The multidisciplinary treatment of CIF includes providing PS, dietary therapy, and medical and surgical interventions. Despite specialised multidisciplinary team taking care of CIF patients, difficult complications may arise from CIF and long-term PS. Overall, the CIF treatment is burdensome and expensive, both to the patient and the health care system. Data regarding CIF in Finnish adults has so far been limited. This study, Treatment of Adult Patients with Intestinal Failure in Finland (TAPIFF), aimed to fill this gap in knowledge. The specific objectives of the TAPIFF study were I) to evaluate current management of long-term PS across Finland; II a) to investigate the intestinal failure (IF) prevalence among Finnish adults; II b) to describe clinical details of Finnish adults with IF; and III) to investigate catheter-related bloodstream infection (CRBSI) rate, longitudinal changes in biochemical liver and kidney tests, and their explanatory factors. As part of the TAPIFF study, we also gathered an institutional cohort of adult IF patients in Helsinki University Hospital to especially investigate liver status with non-invasive methods. In September 2017, all Finnish health care units with the potential of longterm PS provision to adult IF patients received an electronic survey investigating the local practices of PS management. The survey also inquired whether the unit had provided long-term PS to any adult(s) during the preceding year. All units that responded they had managed at least one such patient, as well as those units which did not respond to the survey, were recontacted. Patient records of the enrolled patients were obtained. The inclusion criteria were age at least 18 years, PS duration at least 120 consecutive days, IF as the indication for PS, and the availability of patient records. Clinical data was manually collected from the hospital patient records from the start of PS (baseline) up to the latest record entry in 2017 (data collection). The statistical software was IBM SPSS statistics versions 24, 25 and 27 (IBM Corp., Armonk, NY). In study I, 71 health care professionals from 52 different units responded to the survey, resulting in an overall response rate of 47% of the invited units. The responses revealed that three out of four units had some experience in managing long-term PS. This experience was, however, very limited, because most units managed currently only 0–2 patients. Hospital-at-home services had primary responsibility for the practical administration of PS, as well as for the supply of PS equipment and admixtures. In most units, assigned teams managing patients on PS, and written protocols on the practical PS management were lacking. Study II identified 52 adult patients who fulfilled the inclusion criteria. The calculated IF prevalence in adults in 2017 was 11.7 per million (95% confidence interval 8.9–15.3). Most patients in this national cross-sectional cohort were women (69%), and the median age was 62 years (interquartile range, IQR 45– 72). Short bowel syndrome was the most frequent (73%) pathophysiological mechanism. The most frequent underlying conditions, in order of prevalence, were surgical complications, Crohn’s disease, and mesenteric ischaemia. The median duration of PS was 27.5 (IQR 11.3–57.3) months. Patients received a median of 7 (IQR 3.5–7) parenteral infusions per week with a median weekly volume of 7.3 (IQR 4.4–14) litres and a median weekly energy supply of 6100 (IQR 3900–9800) kcal. The daily volume of parenteral nutrition was 2 litres or less in 66% patients, and 15% received fluids and electrolytes only. Ten patients (19%) ceased PS treatment during 2017 after a median PS duration of 20.0 (IQR 9.0–40.3) months. Of these ten, eight were successfully weaned from PS, one lost venous access sites, and one died. The retrospective study III on the national cohort of 52 patients indicated a CRBSI rate of 1.35 per 1000 catheter days. In long-term catheters, CRBSI led to catheter removal in 73% of cases. A statistically significant median change occurred both in estimated glomerular filtration rate (eGFR; -8.5 ml/min/1.73 m2, IQR -30–7, p=0.005), and in alkaline phosphatase (26 U/l, IQR -11–95, p=0.019) during a median PS treatment time of 27.5 (IQR 11.3–57.3) months. In a multiple regression model for eGFR at data collection, strong explanatory variables were age and baseline eGFR. In conclusion, the prevalence of IF in the Finnish adult population, as well as their clinical characteristics, are in line with reports from other Western countries. Over time, as the PS continues, abnormal biochemical liver tests and decreased kidney function become more frequent findings. The incidence of CRBSI in the Finnish adult IF population well represents a rate described in nonspecialised units. The experience of clinicians managing Finnish patients on long-term PS appears limited, and, on national level, the management of these patients seems fragmented.
  • Hisinger-Mölkänen, Hanna (Helsingin yliopisto, 2022)
    Asthma and chronic rhinitis are global health problems that cause major burden and disability. The prevalence of asthma has increased in recent decades, which has also been reported in Finland. To address this increasing burden, The National Asthma Programme was conducted in Finland in 1994-2004 and the Allergy Programme in 2008-2018. Both programs were successful as asthma costs and hospital admissions, as well as allergy diets in children and adolescents, decreased significantly. However, asthma and rhinitis symptoms are still a major burden in Finland both for society and patients. Information on preventable risk factors is needed to decrease the symptoms of asthma and rhinitis. Although occupational exposures are known to cause both asthma and rhinitis symptoms, the effect of combined exposure to vapours, gases, dusts, and fumes (VGDF) with environmental tobacco smoke (ETS) or smoking on asthma and chronic rhinitis has not been fully explored. In addition to exposures, age at asthma diagnosis is known to affect disease progression. Asthma diagnosed in adulthood is related to a poorer prognosis, whereas asthma diagnosed in childhood may even remit. However, studies on the effect of age at asthma diagnosis on asthma symptoms in study cohorts (including asthmatics with asthma diagnosed at all ages) are still scarce. We assessed the prevalence and risk factors of asthma and symptoms of asthma and chronic rhinitis. In addition, we investigated whether combined exposure to VGDF with ETS or smoking would increase the prevalence of asthma and the symptoms of asthma and rhinitis. We also evaluated if asthma diagnosed in adulthood is associated with a more symptomatic disease. A cross-sectional postal survey was conducted in a random population sample as a part of the FinEsS-study in Helsinki in 1996, 2006, and 2016. The questionnaire was sent to 8000 adults in 1996, 4000 in 2006, and 8000 in 2016. In 2016, the questionnaire was sent additionally to a similar random population sample of 8000 participants in Western Finland. The response rate declined by study year; the rate was 75.9% in 1996, 61.7% in 2006, and 50.3% in 2016. There was a 52.3% response rate in Western Finland in 2016. All responders (n=3998) were included in the analysis of asthma and chronic rhinitis prevalence. To assess the risk factors for asthma and rhinitis symptoms, we included responders with reported asthma diagnosis (n=836) or chronic rhinitis (n=3488), respectively, who also reported their exposure history of tobacco smoke and VGDF. For the analysis of age at asthma diagnosis, we included responders with physician-diagnosed asthma and reported age at asthma diagnosis and smoking habits (n=842). The increase previously seen in asthma prevalence in Helsinki plateaued from 2006 to 2016. The results were similar in responders who were ever diagnosed with asthma (6.6% in 1996, 10.0% in 2006 and 10.9% in 2016) and in those with current asthma (5.8% in 1996, 8.5% in 2006 and 8.8% in 2016). In addition to asthma prevalence, a similar decrease was also seen in respiratory symptoms. The prevalence of respiratory symptoms decreased both in the general population and in responders with physician-diagnosed asthma from 2006 to 2016. Fewer asthmatics reported multiple symptoms in 2016 compared to 2006 (71.8% in 2006 vs 62.2% in 2016). Combined exposure to VGDF with ETS or smoking increased the prevalence of current asthma in responders who were ever diagnosed with asthma by a physician. The highest prevalence figures were in responders with combined exposure to VGDF and ETS (89.5% vs 77.9% in responders with no exposure history; p=0.027). Combined exposure to VGDF with ETS or smoking was also associated with a higher symptom burden both in asthma and chronic rhinitis. Responders with asthma diagnosed in adulthood had a higher symptom burden than those diagnosed in childhood, as all analysed asthma symptoms were more often reported by those diagnosed in adulthood. Our findings on asthma and asthma symptom prevalence complement previous positive results of the Finnish Asthma and Allergy Programmes. However, further actions are needed as both asthma and chronic rhinitis continue to present challenges both in everyday clinical practice and on a national level despite these positive trends observed in Finland. Exposure to tobacco smoke and VGDF is preventable; therefore, our results highlight the importance of active measures in the prevention of current asthma and in treatment of asthma and chronic rhinitis. In addition, targeted identification and treatment of asthma patients diagnosed in adulthood is important to optimize resource utilization and to achieve better disease control.
  • Sieviläinen, Meri (Helsingin yliopisto, 2022)
    Head and neck squamous cell carcinoma (HNSCC) is a common and aggressive malignancy. It includes the tumors arising in the head and neck area with oral cancer representing one of the most common types. Intraorally, squamous cell carcinomas arising from the tongue (OTSCC) are the most frequent. The multimodality treatment regime includes surgery, radio and/or chemotherapy depending on the spread of the disease. Unfortunately, the five-year survival rates in the Nordic countries remain between 50-60% for oral cancer, with the incidence of OTSCC increasing without clear etiology. The newest addition to the treatment regime of recurrent and/or metastasized oral squamous cell carcinoma (OSCC) patients is immune checkpoint inhibitors (ICIs), namely anti-PD-1. These ICIs provide improved survival rates, and the expression of the immune checkpoints (ICs) are being investigated as potential prognostic factors. This dissertation aimed to investigate the role of ICs in the development of oral carcinogenesis. Our first project was to explore the expression of selected ICs, indoleamine 2,3-dioxygenase 1 (IDO1), and programmed death-ligand 1 (PD-L1) in oral dysplasia, a potentially malignant oral lesion. We retrospectively gathered 63 archival oral dysplasia samples from Oulu and Tampere University Hospitals from 2005 to 2016, with 9 samples extracted during wisdom tooth extractions to act as healthy controls. The samples were stained for anti-PD-L1 and anti-IDO1 and scored for the expression of both immune checkpoints, dysplastic grade, and immune cell infiltration. The expression of the ICs was modulated during the development of dysplastic changes associated with inflammatory cell infiltration. Eight patients were followed up to 36 months and biopsies were taken more than once. The expression of ICs fluctuated over time, suggesting that they are not useful as biomarkers for malignant transformation. Since the start of our first project, several articles have been published on the prognostic value of ICs in OSCC. Our second project aimed to systematically review the prognostic value of ICs in OSCC. By the end of 2017, 12 ICs had been studied for their prognostic value in OSCC. Seven of them (ALHD1, FKBP51, PD-L1, B7-H3, B7-H4, B7-H6, and IDO1) had been reported as adverse prognostic factors and five (CTLA-4, PD-1, PD-L2, TLT-2 and VISTA) did not have prognostic value. Only two molecules, PD-L1 and B7-H3, had sufficient data for conducting a meta-analysis. PD-L1 had controversial findings as a prognostic marker, with weak evidence suggesting high B7-H3 expression was an adverse prognostic factor for overall survival. Based on the systematic review results, we aimed to validate B7-H3 as an adverse prognostic factor in OTSCC. 323 OTSCC samples from Brazil, Finland, and Norway were retrospectively gathered and stained for B7-H3. High B7-H3 expression was not significantly associated with patient survival in the whole OTSCC cohort. The immune status/phenotype of the Brazilian and Finnish cases (n=191) was analyzed by the amount of tumor infiltrated lymphocytes (TILs), classifying the cases into ‘immune hot’ and ‘immune cold’. The immune status of the patient (n=191) was significantly associated with patient survival, with immune cold patients having an adverse outcome in univariate and multivariate survival. Our findings suggest that the immune status of the tumor should be considered when investigating the prognostic value of ICs and could be an individual prognostic factor aiding in identifying potential high-risk cases. Based on studies I and II, a research question arose regarding what would be the functional effect of the ICI on lymphocyte infiltration in the presence of cancer cells and what would happen to the cross-talk between lymphocytes and cancer cells. In a newly developed microfluidic chip model, we injected OTSCC cells (HSC-3) embedded in a 3D human tumor-derived matrix “myogel/fibrin” together with different lymphocyte populations (CD4+ T, NK and CD8+ T cells). The chips were incubated with different ICIs, anti-PD-1, nivolumab or the IDO1 inhibitor, epacadostat. Study IV revealed that each subpopulation of lymphocytes responded differently to ICI therapy with the release of specific cytokines. The IDO1 inhibitor significantly increased the migration of CD4+ T and NK cells towards the OTSCC cell line. This could potentially reverse the immune cell exclusion firing up cold tumors to hot. Anti-PD-1 significantly increased the release of IL-6, IL-8, and MIP-1α from CD4+ T, NK and CD8+ T cells, respectively. The elevation of IL-6 and IL-8 serum levels after ICI therapy in patients has been associated with nonresponding tumors. The blockage of these cytokines may be a promising direction for future studies.

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