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Novel diagnostic and prognostic tools in infiltrative cardiomyopathy
No full textBackground: Heart failure is a severe medical condition, increasingly common in the aging population, causing both vast morbidity and mortality. Amongst different etiologies of heart failure, infiltrative cardiomyopathies are relatively rare but associate with worse prognosis. The two most common infiltrative cardiomyopathies, cardiac amyloidosis (CA) and cardiac sarcoidosis (CS) are underdiagnosed. Treatments addressing their specific pathophysiologic mechanisms may be considered in both conditions. Increased awareness of these diseases, better tools to establish an early diagnosis and improved understanding of prognostication is of value to optimize treatment and potentially improve outcomes in both CA and CS. Previous studies indicate that lumbar spinal stenosis (LSS) associate with transthyretin (ATTR) amyloidosis and that LSS might be a possible early sign of disease, preceding CA. In sarcoidosis, variants of the HLA-DRB1 gene associate with different phenotypes, but the association with cardiac involvement is less studied.The aims of this thesis are to 1) Investigate the prevalence, type, and grade of amyloid deposits in ligamentum flavum in LSS, explore signs of systemic amyloidosis and determine the prevalence of manifest CA, at time of surgery and at six years follow up. 2) Characterize Swedish CS patients, assess if variants of the HLA-DRB1 gene associate with cardiac involvement and explore baseline characteristics in association with severe outcomes.Methods and Results: Study I. 250 consecutive patients that underwent surgery for LSS at Stockholm Spine Center were included. Connective tissue removed at surgery was analyzed for the presence, grade, and type of amyloid deposits. 37% were found to have ATTR amyloid deposits in ligament tissue. Subjects with higher grades of ATTR deposits were evaluated with cardiac investigations. Several findings indicate systemic disease but none of the patients had significant cardiac involvement, at time of surgery. Study II. Retrospective data on 87 patients diagnosed with CS at Karolinska University Hospital and Sahlgrenska University Hospital were collected from the electronic medical records, including HLA-DRB1 allele genotyping. The majority (59%) presented with cardiac symptoms as first sign of sarcoidosis (de novo CS). CS as first manifestation associated with worse cardiac function than those previously diagnosed with sarcoidosis in other organs. We did not observe significant differences in HLA-DRB1 allele frequencies in patients with CS compared to sarcoidosis in general, but trends towards significant differences in subgroups were observed. Study III. Data on the combined endpoint of death, heart transplantation (HTx) or sustained ventricular arrhythmias (VT/VF) were extracted from the electronic medical records. De novo CS and initial presentation of VT/VF associated with worse outcomes. 42 different possible predictors were assessed using a machine learning algorithm to decide their relative importance in the prediction of the combined endpoint. De novo CS and right ventricular (RV) dysfunction displayed the highest relative importance in predicting outcomes in this model. Study IV. Prospective follow-up study with multimodality cardiac imaging of 21 patients, with high grades of amyloid deposits in ligament tissue, six years after surgery for LSS. 16% (3/19) were diagnosed with age-related transthyretin (ATTRwt) CA. 48% (10/21) had been diagnosed with other tenosynovial disorders that associate with ATTR amyloidosis. Changes in cardiac function and signs of interstitial pathology in the overall cohort were assessed with echocardiography and cardiac magnetic resonance imaging.Conclusion: ATTR amyloid deposits are common in ligament tissue in LSS. Several findings support the hypothesis that LSS is a possible systemic manifestation of ATTRwt and longitudinal cardiac follow up after surgery is a promising diagnostic pathway to find early cases of ATTRwt-CA. Around half of the CS patients presented with de novo CS and this presentation associated with more severe cardiac dysfunction and increased risk of death, cardiac transplantation, and VT/VF. Increased awareness of CS as a primary manifestation may improve detection and enable earlier treatment. In a machine learning model, cardiac symptoms as first presentation together with RV dysfunction displayed the highest relative importance in predicting worse outcomes. In CS, HLA-DRB1 allele frequencies did not differ from the sarcoidosis population in general.List of scientific papersI. Eldhagen P, Berg S, Lund LH, Sörensson P, Suhr OB, Westermark P. Transthyretin amyloid deposits in lumbar spinal stenosis and assessment of signs of systemic amyloidosis. J Intern Med. 2021 Jun ;289(6):895-905. https://doi.org/10.1111/joim.13222 II. Eldhagen P, Bobbio E, Darlington P, Grunewald J, Eklund A, Polte CL, Bergh N, Bollano E, Sörensson P, Kullberg S. Phenotypic and HLA-DRB1 allele characterization of Swedish cardiac sarcoidosis patients. Int J Cardiol. 2022 Jul 15;359:108-112. https://doi.org/10.1016/j.ijcard.2022.04.006 III. Bobbio E, Eldhagen P, Polte CL, Hjalmarsson C, Karason K, Rawshani A, Darlington P, Kullberg S, Sörensson P, Bergh N, Bollano E. Clinical Outcomes and Predictors of Long-Term Survival in Patients With and Without Previously Known Extracardiac Sarcoidosis Using Machine Learning: A Swedish Multicenter Study. J Am Heart Assoc. 2023 Aug ;12(15):e029481. https://doi.org/10.1161/JAHA.123.029481 IV. Eldhagen P, Tzortzakakis A, Lund LH, Söderström L, Berg S, Westermark P, Sörensson P. Prevalence of cardiac amyloidosis and other signs of systemic amyloidosis six years after lumbar spinal stenosis surgery, in patients with transthyretin amyloid deposits in ligamentum flavum. [Manuscript]</p
Advancements in detection of performance enhancing drugs in dried blood spots : focusing on erythropoietin
No full textThe use of erythropoietin (EPO) for performance enhancing purposes is detected mainly in urine and serum samples in anti-doping laboratories. However, dried blood spots (DBS) have emerged as a potential additional sample for EPO and other various doping substances. The aims of these studies were to develop a method for EPO analysis from DBS (study I) and use this method to sensitively detect endogenous EPO, micro-doses of exogenous EPO, and the EPO c.577del protein VAR-EPO (studies I-III). I also observed the stability of EPO and insulin-like growth factor 1 (IGF-I) in DBS (study I and V), along with investigations into the relationship between EPO and testosterone (study IV).Sample collection: Capillary blood (for DBS), venous blood (for DBS and serum) and urine were collected from healthy volunteers. The DBS devices used were Capitainer®B 50, Mitra® VAMS, Tasso-M20, and Whatman 903 filter paper cards for EPO and IGF-I detection. Urine and serum were also collected from 30 self-reported anabolic androgenic steroid (AAS) users to examine the presence of EPO in such samples.Methods: The primary method I used for detecting EPO and VAR-EPO in the collected samples was immunopurification with the EPO Purification Gel Kit, SAR- PAGE, and Western blot. EPO concentrations were measured in serum using a commercial immunoassay kit. IGF-I was analyzed by LC-MS/MS and with the automated immunoassay system IDS-iSYS. Serum AAS was quantified using an immunoassay.Results: Endogenous EPO was sensitively detected on both polymer and paper blood supports (studies I, II, III), providing stable results (study I), while urinary EPO fluctuated more (study I) and occasionally showed degradation or undetectable bands (studies I and III). The instability of IGF-I in DBS at room temperature (study V) and the slight variations of EPO detection indicate that currently DBS is not suited for longitudinally monitoring these markers. In addition, micro-doses of various recombinant EPOs (rEPO) and VAR-EPO were well-detected in 4 DBS devices from healthy volunteers (studies II, III). When rEPO micro-doses were administered with testosterone to healthy volunteers, as expected, there was no increase in EPO signal intensity in DBS, but in AAS users who were positive for testosterone, serum EPO concentrations were slightly elevated, along with some hematological parameters (study IV).Conclusion: The detection and knowledge of EPO in DBS has made progress, but DBS detection methods, sample collection, and storage strategies still require further discussion and investigation.List of scientific papersI. Heiland CE, Ericsson M, Pohanka A, Ekström L, Marchand A. Optimizing detection of erythropoietin receptor agonists from dried blood spots for anti-doping application. Drug Testing and Analysis. 2022;14(8): 1377-1386. https://doi.org/10.1002/dta.3260 II. Heiland CE, Martin L, Zhou X, Zhang L, Ericsson M, Marchand A. Dried blood spots for erythropoietin analysis: Detection of micro- doses, EPO c.577del variant and comparison with in-competition matching urine samples. Drug Testing and Analysis. 2023;1-5. https://doi.org/10.1002/dta.3596 III. Heiland CE, Lehtihet M, Börjesson A, Ekström L. Evaluation of a single Eporatio® micro-dose in urine and dried blood spots. Drug Testing and Analysis. 2024;1-4. https://doi.org/10.1002/dta.3651 IV. Heiland CE, Schickel Y, Lehtihet M, Börjesson A, Ekström L. Supra- physiological doses of anabolic androgenic steroids impact erythropoietin and blood parameters. Drug Testing and Analysis. 2023;15(6):599-604. https://doi.org/10.1002/dta.3452 V. Heiland CE, Mongongu C, Semence F, Pohanka A, Ericsson M, Marchand A, Ekström L. IGF-I intra-individual variation in serum and DBS using immunoassay and LC-HRMS methods. [Manuscript]</p
Appeasing the wheezing : determinants and outcomes of respiratory disease in childhood
No full textThe aim of this thesis was to study the determinants and outcomes asthma and cystic fibrosis in childhood by using study populations of persons born in Sweden and data from a clinical cohort and national health and demographic registers and a quality register.In Paper I and Paper II we explored determinants of respiratory disease.In Paper I we studied if there was association between maternal asthma, allergic asthma, and lung function during pregnancy and childhood asthma and growth using a clinical cohort of mothers and their children born from 2012 to 2017, retrieved from the MAESTRO (Maternal Asthma Events, Stress and Offspring) and MAESTRO-Child studies. Results showed that higher lung function values in the mother was associated with a lowered risk of childhood asthma. Further, there was no statistically significant association between maternal asthma/maternal allergic asthma/lung function during pregnancy and childhood growth.In Paper II we studied the relationship between parental socioeconomic status (SES, education and income) measured at birth and at five years and childhood asthma using data from national registers and a cohort born from 2001 to 2013. We found that parental education and income at birth was associated with childhood asthma with onset under one year of age and that only parental education at birth was associated with an increased risk of childhood asthma with onset over one year of age. When this was studied in a cousin-comparison we found that the association between parental education persisted, implying that confounding by shared factors in the family do not fully account for the association between SES and asthma Finally low parental SES measured at five years was associated with an increased risk of asthma at five years.In Paper III and Paper IV, we explored the outcomes of respiratory disease.In Paper III we investigated the association between asthma and all-cause mortality in children and young adults born from 1986 to 2012 using data from national registers. Asthma was associated with an increase in all-cause mortality and the highest mortality rate was for children aged five to 15 years with asthma. The estimates remained increased when this association between asthma and all- cause mortality was studied in a sub-group analysis of children born from 2000. Further, the estimates differed depending on if the person also had a life-limiting condition, but not on parental SES at birth.In Paper IV we studied the association between parental SES (measured at birth and at five years) and severe disease, lung function and growth in persons with cystic fibrosis born 1973 to 2019 using a cohort from the Swedish Cystic Fibrosis Register and data from the Swedish Cystic Fibrosis Register and national registers We found some evidence that parental SES measured at birth and at five years was associated with severe disease and increased lung function decline, however there was no statistically significant relationship between low parental SES and growth decline.In conclusion factors associated with respiratory disease in childhood include maternal asthma, maternal lung function and parental SES. Asthma is associated with all-cause mortality, where comorbidity but not parental SES play an import role.List of scientific papersI. Caffrey Osvald E, Lundholm C, Brew BK, Karim H, Rejnö G, Rhedin S, Almqvist C. Maternal asthma and lung function during pregnancy and asthma and growth in the offspring, the MAESTRO-Child study. [Manuscript]II. Caffrey Osvald E, Gong T, Lundholm C, Larsson H, Brew BK, Almqvist C. Parental socioeconomic status and asthma in children: Using a population-based cohort and family design. Clinical & Experimental Allergy. 2022 Jan;52(1):94-103. https://doi.org/10.1111/cea.14037 III. Osvald EC, Bower H, Lundholm C, Larsson H, Brew BK, Almqvist C. Asthma and all-cause mortality in children and young adults: a population-based study. Thorax. 2020 Dec 1; 75(12):1040-6. https://doi.org/10.1136/thoraxjnl-2020-214655 IV. Caffrey Osvald E, Lundholm C, Rhedin S, Smew AI, Brew BK, de Monestrol I, Almqvist C. Parental socioeconomic status, lung function and growth in children and young adults with cystic fibrosis – a Swedish cohort study. [Manuscript]</p
Diverticular disease of the colon - risk factors and validation of diagnosis
No full textDiverticular Disease (DD) is a common gastrointestinal disease, particularly among the population in western countries. The aetiology of DD is complex and have not yet been established conclusively. Dietary habits and lifestyle choices such as, physical inactivity, obesity, and smoking, affects the development of the disease. The prevalence of DD is increasing with age, at the age of 65 years, 66% have DD. Inflammation (diverticulitis) is the most common complication with up to 5%, which may result in bowel perforation, obstruction, and/or bleeding. The aim of this thesis was to identify risk factors for DD, validate the quality of registered ICD-codes in the Swedish National Patient Register and compare rates of anastomotic leakage (AL) and other possible risk factors for AL between patients with planned surgery for DD versus colonic cancer.In Paper I, the intake of oral/inhaled corticosteroids, indometacin or aspirin and if they influence the risk of diverticular disease were evaluated. A population-based prospective cohort of middle-aged women in the Swedish Mammography Cohort were assessed. Use of corticosteroids, indometacin or aspirin in 1997 was determined from questionnaires. Cases of diverticular disease were identified from the Swedish national registers until the end of 2010. The total cohort consisted of 36 586 middle-aged women, out of these, 674 (1.8 %) were admitted to hospital at least on one occasion with DD. A significant correlation was observed between corticosteroids, especially those inhaled, and hospitalization due to diverticular illness.In Paper II, the association between dietary fibre intake and hospitalisation due to DD was investigated in a cohort study. The Swedish Mammography Cohort and the Cohort of Swedish Men were linked to the Swedish Inpatient Register and the Causes of Death Register. Data of dietary fibre consumption were gathered through questionnaires. The risk for hospitalisation was reduced by 30% in the women with the highest intake (median 12.6 g/day) of fruit and vegetable fibres compared to those with the lowest intake (4.1 g/day). The results were replicated in the male population, where the men with the highest intake (median 10.3 g/day) had a 32% lower risk of hospitalization than the men with the lowest intake (median 2.9 g/day).In Paper III, the validity of the coding for DD in the Swedish National Patient Register was studied. The study included 323 and 327 patients from the years 2002 and 2010, respectively. The overall Positive Predictive Value for both years and all diagnoses was high, 95% (95% CI: 93-96). The Positive Predictive Value for the year 2010 was slightly higher 98% (95% CI: 95-99) than for the year 2002, 91% (95% CI: (87-94). The higher Positive Predictive Value for the year 2010 may be due to increased use of computed tomography in the diagnosis work.In Paper IV, the rates of anastomotic leakage and potential risk factors for anastomotic leakage between patients undergoing surgery for diverticulosis versus colonic cancer was studied within an Enhanced Recovery After Surgery protocol. The study population consisted of patients operated on left sided colonic / sigmoid resection with diverticulosis and colonic cancer in Sweden between the years 2010-2020. There was no difference in anastomotic leakage between both diagnosis groups, colon cancer (4%) versus diverticulosis (3.8%) (p =0.992). When compared to laparoscopic surgery, there was more leakage among patients with diverticulitis who had open surgery. Anastomotic leakage was more common among cancer patients who had undergone laparoscopic surgery (adjusted OR 4.02, 95% CI (1.35- 11.91), p=0.01).List of scientific papersI. Cohort study of corticosteroid use and risk of hospital admission for diverticular disease. Hjern F, Mahmood MW, Abraham-Nordling M, Wolk A, Håkansson N. Br J Surg. 2015 Jan;102(1):119-24. https://doi.org/10.1002/bjs.9686 II. High intake of dietary fibre from fruit and vegetables reduces the risk of hospitalisation for diverticular disease. Mahmood MW, Abraham-Nordling M, Håkansson N, Wolk A, Hjern F. Eur J Nutr. 2019 Sep;58(6):2393–2400. https://doi.org/10.1007/s00394-018-1792-0 III. Identification of diverticular disease in Swedish healthcare registers: a validation study. Mahmood MW, Schmidt PT, Olén O, Hellsing C, Hjern F, Abraham-Nordling M. Scand J Gastroenterol. 2023 Nov 7:1-7. https://doi.org/10.1080/00365521.2023.2278422 IV. Anastomotic leakage in patients with diverticular disease undergoing surgery within an ERAS-protocol. Mahmood MW, Abraham-Nordling M, Löf-Granström A, Hjern F, Gustafsson UO. [Manuscript]</p
Neuromuscular electrical stimulation in physical inactivity
No full textPhysical inactivity and immobilization have emerged as major health issues. Neuromuscular electrical stimulation (NMES) is a potential treatment to prevent the negative effects of physical inactivity, such as muscle atrophy and poor venous circulation. However, current NMES application is limited by poor compliance. Thus, the overarching aim of this thesis was to improve the efficacy of NMES targeting compliance by developing wearable NMES-pants. Development and testing of the NMES-pants on healthy, voluntary participants explored the effects on comfort, global muscle mRNA-expression, venous hemodynamics and blood coagulation.To optimize electrode dimensions and positioning within the NMES-pants, the intensity (mA) needed for a visible muscle contraction (ML I) and comfort during NMES when applied with different electrode sizes and placements on the quadriceps (Q), hamstrings (H), and gluteus (G) muscles were tested. The NMES-pants were then created based on the combination of electrode size and placement that provided the best comfort and that required the lowest current intensity for ML I.Subsequently, the textile electrodes in the NMES-pants were compared to commercial self-adhesive electrodes, by investigating the knee extensor force production created with NMES measured in an isokinetic dynamometer. We demonstrated that a contraction at 20% of the participant’s maximal voluntary contraction (MVC) could be reached at an acceptable level of discomfort with both methods. A large inter-individual variation in regards of comfort, intensity required, and force production was demonstrated, highlighting the importance of individually adjusted NMES for optimal compliance.To further investigate the NMES-pants, the effects of Q-NMES on muscle activation were assessed by examining vastus lateralis muscle gene expression using RNA-sequencing before and three hours after a 30-minute Q-NMES-pants session and/or regular exercise. The NMES-intensity was set to 20% of each participant’s MVC and the EX-protocol was performed at 80% of 1-repetition-maximum. NMES induced 4448 differentially expressed genes (DEGs) with an 80%-overlap of the 2571 DEGs observed with EX. Genes well-known to be upregulated by EX were also upregulated by NMES to a lesser extent. Gene set enrichment analysis demonstrated many common pathways affected by both NMES and EX, but also some pathways exclusive to NMES, such as connective tissue proliferation.Next, the effects of NMES-pants on venous hemodynamics and coagulation were explored. The peak venous velocity (PVV) in the femoral vein was assessed at ML I, and with an additional increase of six NMES-levels (ML II). NMES-pants significantly increased PVV from baseline by 93% at ML I and 173% at ML II. Additionally, levels of different coagulation factors and inflammatory markers were assessed with blood samples before and immediately after a 2-hour QHG-NMES-pants session. The NMES resulted in a small but significant increase in overall hemostatic potential, fibrinogen, and factor VIII, as well as a decrease in overall fibrinolytic potential. Furthermore, proteins which regulate inflammation and extracellular matrix degradation were differentially expressed by NMES.The present findings of this thesis demonstrate that NMES-pants can be used, with single or combined thigh-gluteal muscle-stimulation, with a high comfort at low-intensity and with an acceptable discomfort at submaximal intensity. Treatment with NMES-pants produced intensity-dependent increases in venous hemodynamics and induced more DEGs, but with a significant overlap, as compared to exercise. These findings suggest that NMES can induce exercise-like molecular effects, with potential health and performance benefits in individuals unable to perform resistance exercise and that textile-based NMES-wearables have the potential to improve compliance with treatment.List of scientific papersI. Flodin J, Juthberg R, Ackermann PW. Effects of electrode size and placement on comfort and efficiency during low-intensity neuromuscular electrical stimulation of quadriceps, hamstrings and gluteal muscles. BMC Sports Sci Med Rehabil. 2022 Jan 16;14(1):11. https://doi.org/10.1186/s13102-022-00403-7 II. Flodin J, Mikkelsen C, Ackermann PW. Knee extensor force production and discomfort during neuromuscular electrical stimulation of quadriceps with and without gluteal muscle co-stimulation. Eur J Appl Physiol. 2022 Jun;122(6):15211530. https://doi.org/10.1007/s00421-022-04949-9 III. Flodin J, Wallenius P, Guo L, Persson NK, Ackermann P. Wearable Neuromuscular Electrical Stimulation on Quadriceps Muscle Can Increase Venous Flow. Ann Biomed Eng. 2023 Aug 19. https://doi.org/10.1007/s10439-023-03349-0 IV. Flodin J, Reitzner SM, Emanuelsson EB, Sundberg CJ, Ackermann P. The effect of neuromuscular electrical stimulation on the human skeletal muscle transcriptome. Acta Physiol. 2024 Mar 8 240:e14129. https://doi.org/10.1111/apha.14129 V. Flodin J, Reitzner SM, Mahmoud Hourani Soutari N, Ahmed AS, Guo L, Persson NK, Antovic JP, Ackermann PW. The effect of Neuromuscular Electrical Stimulation on Overall Hemostatic Potential and Coagulation Factors. [Manuscript]</p
Child health in the Democratic Republic of the Congo : exploring subnational disparities and inequities in illnesses and mortality
No full textBackground: Today, sub-Saharan Africa accounts for a disproportionally high burden of deaths in children, with the Democratic Republic of the Congo (DRC) being one of the countries with the highest mortality burden. Ample evidence exists on what protective, preventive, and curative services need to be in place to end preventable deaths in children. However, context-specific sub-national data that can effectively guide public health interventions is scarce for the DRC. This thesis first explored causes of death and coverage of preventive interventions to give a broad understanding of inequities around child mortality. Based on these findings, a case study approach was taken to look in depth at system factors that contribute to diarrhoeal deaths.Methods: In Paper I, household survey and health facility data were used to compare child mortality rates and coverage of key indicators known to lower child mortality at the provincial level. Conflict event data was further used to classify provinces according to conflict intensity to explore conflict exposure as a predictor for child health. Paper II used demographic and verbal autopsy data to empirically estimate the causes of and circumstances around deaths. Papers III and IV used focus group discussions, health facility audits, and knowledge questionnaires to understand the health system’s capacity for cholera surveillance and diarrhoeal disease case management in cholera hotspots.Results: Paper I identified large disparities in child mortality and indicator coverages. Conflict was found to be a poor predictor of child health. Provinces classified as conflict-affected had both the highest and lowest under-five mortality rates and indicator coverages. Paper II (manuscript) identified infectious diseases as the main cause of death in children and adolescents 1month to 19 years of age. Most deaths occurred at home; however, the majority sought care in the days preceding death. Papers III and IV found low capacity for diarrhoea surveillance and case management, and that health facility providers, drug shop vendors, and traditional health practitioners are perceived as important providers of care and should be considered in health policy plans.Conclusions: Sustained, and more equitable, health system strengthening efforts are needed to decrease sub-national disparities and end preventable deaths in children in the DRC.List of scientific papersI. Schedwin M, Furaha AB, Kapend R, Akilimali P, Malembaka EB, Hildenwall H, Alfvén T, Tylleskär T, Mapatano MA, King C. Under-five mortality in the Democratic Republic of the Congo: secondary analyses of survey and conflict data by province. Bull World Health Organ. 2022 Jul 1;100(7):422-435. https://doi.org/10.2471/BLT.22.287915 II. Fumwakwau JK & Schedwin M, Hildenwall H, Alfvén T, Mapatano MA, King C, Mavinga DP. Mortality in children and adolescents in Western Democratic Republic of Congo – Analysis of verbal autopsy- and demographic data. [manuscript]III. Schedwin M, Furaha AB, Elimian K, King C, Malembaka EB, Yambayamba MK, Tylleskär T, Alfvén T, Carter SE, Okitayemba PW, Mapatano MA, Hildenwall H. Facility capacity and provider knowledge for cholera surveillance and diarrhoea case management in cholera hotspots in the Democratic Republic of Congo - a mixed-methods study. Glob Health Action. 2024 Dec 31;17(1):2317774. https://doi.org/10.1080/16549716.2024.2317774 IV. Schedwin M, Furaha AB, Hildenwall H, Elimian K, Malembaka EB, Yambayamba MK, Forsberg BC, Damme WV, Alfvén T, Carter SE, Okitayemba PW, Mapatano MA, King C. Exploring different health care providers´ perceptions on the management of diarrhoea in cholera hotspots in the Democratic Republic of Congo: A qualitative content analysis. PLOS Glob Public Health. 2024 Mar 19;4(3):e0002896. https://doi.org/10.1371/journal.pgph.0002896 </p
The dimensions of human and murine CD8 T lymphocyte diversity
No full textThe adaptive immune system generates and maintains a pool of CD8+ T cells with an almost limitless specificity with the purpose of maintaining homeostasis. Upon antigen encounter, CD8+ T cells undergo functional diversification and give rise to daughter cells. The daughter cells adopt one of many distinct states along a functional gradient from stem-like towards highly cytotoxic. After antigen clearance, a small fraction of antigen-experienced CD8+ T cells survives and constitutes a long-lived memory population that conveys long-term protection. The likelihood of a T cell acquiring or possessing the ability to maintain a long-term presence is related to the functional characteristics of that T cell during the active response phase. This thesis aims to unravel the biological dimensions underlying CD8+ T cell diversity and fate determination.Recent developments in high dimensional proteome and transcriptome analysis revealed that antigen- experienced T cells form a continuum of functional states. This continuum is in contrast with the established subsetting into discrete subpopulations. We identified the chemokine receptor CX3CR1 as a graded, cross-species, pan T cell differentiation marker. CX3CR1 can be used to, in a convenient and simple manner, capture a principal component of T cell diversity. Furthermore, the CX3CR1 expression levels reflected similar functional states across species, enabling cross-species comparison of T cell properties. The enhanced insight into differentiation revealed that CX3CR1high CD8+ T cells uniquely patrol the luminal arteriolar surface. These CD8+ T cells scan the arteriolar surface for antigen, which supported their long-term survival. Finally, we unraveled T cell activation and proliferation as principal aspects of the T cell diversity in addition to differentiation. From this data, we constructed transcriptomic- and protein-based scores to provide a precise yet practical tool to identify and isolate differentiation and phase-specific CD8+ T cells.In this thesis, we moved beyond binary classification of CD8+ T cell states and identified transcriptomic and functional gradients of CD8+ T cell diversity. These axes of diversity precisely described the position of individual CD8+ T cells within the T cell heterogeneity found in the effector and memory phase. By leveraging these axes, we identified markers and marker panels that easily, practically and precisely characterized CD8+ T cells.List of scientific papersI. Graded expression of the chemokine receptor CX3CR1 marks differentiation states of human and murine T cells and enables cross-species interpretation. Anthonie J. Zwijnenburg, Jyoti Pokharel, Renate Vernaitė, Wenning Zheng, Elena Hoffer, Iman Shryki, Natalia Ramirez Comet, Marcus Ehrström, Sara Gredmark-Russ, Liv Eidsmo and Carmen Gerlach. Immunity. 2023 Augus 8;56:1955-1974.e10. https://doi.org/10.1016/j.immuni.2023.06.025 II. The cellular microenvironment regulates CX3CR1 expression on CD8+ T cells and the maintenance of CX3CR1+ CD8+ T cells. Jyoti Pokharel*, Iman Shryki*, Anthonie J. Zwijnenburg, Ioana Sandu, Laura Krumm, Christina Bekiari, Rebecka Heinbäck, Victor Avramov, Josefin Lysell, Liv Eidsmo, Helena Erlandsson Harris and Carmen Gerlach. European Journal of Immunology. 2023 October;10:e2350658. *Equal contribution. https://doi.org/10.1002/eji.202350658 III. Microvascular immune surveillance by CX3CR1hi effector and memory CD8+ T cells. Olga Barreiro*, Scott M. Loughhead*, Paris Pallis, Anthonie J. Zwijnenburg, Jasper van den Ende, Rodrigo González, Mahmoud Eljably, Victor Collado, Carly Ziegler, Bishan Bhattarai, Nir Yosef, Alex Shalek, Carmen Gerlach and Ulrich H. von Andrian. *Equal contribution. [Manuscript]IV. CD8+ T cell differentiation and activation represent distinct axes of diversification. Anthonie J. Zwijnenburg*, Adam Gayoso*, Natalia Ramirez Comet, Maria-Nefeli Christakopoulou, Nir Yosef°, Aaron Streets° and Carmen Gerlach°. *Equal contribution, °Equal contribution. [Manuscript]</p
Mechanisms driving notch disorders and therapeutic implications
No full textBiliary tree is an intricate tubular structure lined by cholangiocytes or biliary epithelial cells. The differentiation, development and morphogenesis of bile ducts is controlled by Notch signaling and other pathways. Alagille syndrome (ALGS) is among many diseases associated with aberrant Notch signaling. ALGS is caused due to mutations in JAG1 (97%) and NOTCH2 (2-3%), core components of Notch signaling pathway. ALGS is a multisystemic genetic disease. Patients have short stature with hepatic, vascular, ocular, renal, and vertebral anomalies. Hepatic disease is due to peripheral bile ducts paucity, due to which bile gets accumulated in the liver leading to cholestasis and liver damage. In some patients, the biliary tree is recovered spontaneously by adulthood. The mouse model of ALGS, Jag1Ndr/Ndr mimic human disease in many systems. Similarly, some Jag1Ndr/Ndr mice regenerate bile ducts by adulthood. Our lab has shown previously that regeneration in hilum and periphery of biliary tree occur through different architectural mechanisms, but it remains poorly understood whether the regenerated bile ducts exhibit molecular differences as compared to native bile ducts. Furthermore, our lab has shown that in both humans and mice IGF-1 is downregulated at RNA and protein level, thereby identifying it as a potential therapeutic target.In Paper I, we have addressed the molecular differences between hilar and peripheral intrahepatic bile ducts using organoids culture and RNA sequencing. Proliferation in Jag1Ndr/Ndr ICOs is far less as compared to Jag1+/+ ICOs as evaluated by organoid size analysis and EdU incorporation assay. Among Jag1Ndr/Ndr ICOs, hICOs were least proliferative which was also confirmed by transcriptional analysis as cell cycle genes were significantly downregulated. Treatment with IGF-1 specifically rescues the survival and proliferation of Jag1Ndr/Ndr pICOs, while hICOs remains unresponsive. Jag1Ndr/Ndr hICOs exhibited less Notch signaling and hepatocyte like nature with enrichment of pathways in IGF uptake and transport proteins, which may explain non-responsive behavior towards IGF-1 treatment. Analysis of portal tract near hilum in vivo revealed ectopic hepatocyte like nuclei which were Hnf4α and IGF-1 positive.Paper II describes a technique developed by our lab, double resin casting micro computed tomography (μCT) (DUCT) for visualizing and quantifying the 3D architecture of liver vasculature and biliary tree. This method can be used to assess the efficacy of potential therapeutic target for ALGS. The protocol entails two parts. In the first part, two tubular systems, PV and bile ducts are injected with two different radiopaque resins followed by fixation. In second part, the hardened liver is subjected to μCT. The data is then segmented by the custom-written pipeline and analyzed. DUCT injection at P15 and adult Jag1+/+ liver shows that both vascular and biliary systems are well-aligned from hilum towards periphery until adulthood. The biliary tree and vasculature must expand and lengthen further to fill the whole liver lobe to the periphery. DUCT can also be used to study tubular systems in other organs, for example, lung airways.In Paper III, we determine whether an integrated assessment of multiple liver architecture parameters would correlate better with serum markers of cholestasis than bile duct paucity (BD/PV) alone. In this regard, we suggest an improved immunofluorescence staining method to visualize and quantify portal veins independent of any associated structure. We also developed pipeline for automated quantification of liver architecture. At P30, Jag1Ndr/Ndr displays a wide spectrum of disease as seen in human patients. The Jag1Ndr/Ndr mice can be classified into mild, intermediate, or severely affected categories based on their body size and presence or absence of bile infarcts. Bile ducts/mm2 near hilum and bile acid levels in mild/intermediate Jag1Ndr/Ndr mice positively correlate indicating ongoing regeneration associated with peak bile acid levels, but rescued bilirubin levels. However, the periphery of intermediate and hilar/periphery in severe Jag1Ndr/Ndr mice did not correlate with any of the parameters indicating low biliary regeneration. Manual quantification was compared with the results from automated analysis, which supported these findings, yet it is an ongoing project and needs some modifications to improve the analysis by automated quantification pipeline.In summary, my thesis shows that hilar and regenerated peripheral bile ducts in adult Jag1Ndr/Ndr mice are distinct. Our findings, both in vitro and in vivo, indicate that hilar bile ducts formed in the absence of Notch signaling exhibit reduced commitment to biliary fate. Additionally, this thesis demonstrates the potential of IGF-1 to rescue bile ducts in ALGS. Our research lays the groundwork for further studies to assess the effectiveness of IGF-1 in vivo. Furthermore, we have introduced an innovative technique for studying tubular systems in three dimensions within the liver, which can also be applicable to other organs. Additionally, we emphasize the significance of the P30 stage in mice as it serves as a representative stage of disease in humans, exhibiting a broad spectrum of liver disease. The excessive number of bile ducts near the hilum in Jag1Ndr/Ndr mice indicates an ongoing ductular reaction.List of scientific papersI. Afshan Iqbal , Noemi Van Hul, Lenka Belicova, Agustin A. Corbat, Simona Hankeova, Emma R. Andersson. Spatially segregated defects and IGF-1-responsiveness of hilar and peripheral biliary organoids from a model of Alagille syndrome. Liver International. 2024, Volume 44(2): pp 541-558. https://doi.org/10.1111/liv.15789 II. Simona Hankeova, Jakub Salplachta, Noemi Van Hul, Michaela Kavkova, Afshan Iqbal, Tomas Zikmund, Josef Kaiser, Emma R. Andersson. DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis. Journal of Visualized Experiments. 2021 (175). Volume 175:e62941. https://doi.org/10.3791/62941 III. Afshan Iqbal, Noemi Van Hul, Agustin A. Corbat, Lukas Poppe, Linde Sevenants, Petter Ranefall, Emma R. Andersson. Hepatic vascular and biliary density determines liver disease severity in a model of Alagille syndrome. [Manuscript]</p
Exploring signaling pathways in endothelial mechanotransduction
No full textMechanotransduction, the process by which cells sense and convert mechanical forces from their surroundings, plays a pivotal role in cellular function. For vascular endothelial cells, coping with the continuous and significant shear stress caused by blood flow is essential for the stability of the circulatory system. Dysfunction in this mechanism can lead to various vascular pathologies, including atherosclerosis and aneurysms. Therefore, understanding how endothelial cells form mechanosensory complexes and thereby respond to external forces is crucial.This thesis aims to study the involvement of the Angiomotin protein family in endothelial mechanotransduction. The Amot protein family, including Angiomotin (Amot), Angiomotin-Like 1 (AmotL1), and Angiomotin-like 2 (AmotL2), shares common structures as well as protein interaction motifs. However, they exhibit significantly different roles in vascular functions. In Paper I, we elucidate how Amot binds to Talin within the integrin adhesome and regulates force transmission between fibronectin and the cytoskeleton in migrating endothelial cells. Additionally, we demonstrate that deletion of Amot impairs both physiological and pathological angiogenesis. In Paper II, AmotL2 isshown to bind VE cadherin through p120 catenin and connect to the nuclear membrane via actin filaments in aortic endothelial cells, thereby transmitting junctional mechanical signals. Depletion of AmotL2 resulted in a pro-inflammatory response and, in severe cases, leads to the spontaneous formation abdominal aortic aneurysms (AAAs) in male adult mice. In Paper III, it is demonstrated that AmotL1 not only binds to N-cadherin but is also associated with focal adhesion proteins. This suggests that AmotL1 functions may extend beyond endothelial cell junctions to include interactions with the extracellular matrix. Additionally, we provide a comprehensive summary of the protein binding profiles of all Amot proteins, as obtained from BioID-MS analysis, thus offering a global perspective on this protein family.In conclusion, Amot family proteins, despite their involvement in separate cellular processes, share common connections with a set of junction-related proteins. Furthermore, they exhibit unique, specific binding partners, offering mechanistic insights into the distinct activities of individual Amot proteins.List of scientific papersI. The Amot/integrin protein complex transmits mechanical forces required for vascular expansion. Yuanyuan Zhang, Yumeng Zhang, Sumako Kameishi, Giuseppina Barutello, Yujuan Zheng, Nicholas P. Tobin, John Nicosia, Katharina Hennig, David Kung Chun Chiu, Martial Balland, Thomas H. Barker, Federica Cavallo, Lars Holmgren#. Cell reports. 2021, 36(8), 109616. #Corresponding authors. https://doi.org/10.1016/j.celrep.2021.109616 II. The VE-cadherin/AmotL2 mechanosensory pathway suppresses aortic inflammation and the formation of abdominal aortic aneurysms. Yuanyuan Zhang, Yumeng Zhang, Evelyn Hutterer, Sara Hultin, Otto Bergman, Solrun Kolbeinsdottir, Hong Jin, Maria J. Forteza, Daniel F. J. Ketelhuth, Joy Roy, Ulf Hedin, Martin Enge, Ljubica Matic, Per Eriksson & Lars Holmgren#. Nature Cardiovascular Research. 2023, 2, 629–644. #Corresponding authors. https://doi.org/10.1038/s44161-023-00298-8III. Mapping of the Angiomotin protein family Adhesome by BioID Analysis. Yumeng Zhang, Lars Holmgren# and Yuanyuan Zhang. #Corresponding authors. [Manuscript]</p
Benefit-harm assessment in childbirth care, with breech presentation as an illustration
No full textIntroduction: Childbirth care is aiming at safe births and positive birth experience. Obstetric decision-making is often complicated. Breech presentation is an example of such complexity. Judgements about individual cases and quality of care in general can be affected by choice of outcome measures. The aim of this project was to study use of obstetric outcome measures, and to explore if changes in the choice can lead to re-evaluation of results of some interventions for breech presentation and help to improve obstetric research, audit, and counselling.Methods: I. Guidelines for external cephalic version (ECV) from all Swedish labour wards were analysed, and effectiveness of the procedure was compared between hospitals with liberal and restrictive approach. II. All births 2017-2020 recorded in the Swedish Pregnancy Register were stratified by Robson classification. Five outcomes were assessed: caesarean section, operative vaginal delivery, postpartum haemorrhage, obstetric anal sphincter injury, and Apgar score Results: I. Large differences between hospitals were found in several aspects of ECV. In more liberal hospitals, success rate was significantly higher (54.0% vs 50.5%, p=0.015), and the proportion of remaining breech births significantly lower (2.81% vs 3.01%, p=0.009) compared to restrictive hospitals. II. We identified Robson groups with the highest rate of each outcome. The largest contribution to four of the five outcomes was made by nulliparous women with spontaneous or induced labour and, notably, multiparous women with spontaneous labour. III. Our search resulted in 806 Cochrane Systematic Reviews, of which we included 141. We identified 348 unique outcome measures, of which only Conclusions: I. A liberal approach to ECV is more effective than restrictive in reducing breech births and is preferable to ungrounded restrictivity. II. Parallel interpretation of a balanced set of important outcomes of births stratified by Robson groups has potential to improve childbirth care. III. There is too large variation in the choice of outcome measures for labour management and discrepancy between those preferred by reviewers and reported by trialists. Harmonization is needed to facilitate research synthesis. IV. An agreement was reached on a Swedish Perinatal Core Outcome Set to be recommended in future studies on management of term birth, regardless of the specific population or condition studied. V. Vaginal compared with caesarean breech birth of the first child was associated with higher risks for this child, but lower risks for its younger sibling, giving equal outcome burden in total for the two children together. Benefit-harm assessment in childbirth care includes many interconnected aspects. A careful choice of outcome measures facilitates overview and ability to shift focus from the immediate results to ultimate goals. Several management options of breech presentation are reasonable, enabling individualised decisions.List of scientific papersI. Savchenko J, Lindqvist PG, Wendel SB. External cephalic version for breech presentation: The guideline landscape and a quest for an optimal approach. Eur J Obstet Gynecol Reprod Biol. 2020 Dec;255:197-202. https://doi.org/10.1016/j.ejogrb.2020.10.019 II. Savchenko J, Ladfors L, Hjertberg L, Hildebrand E, Brismar Wendel S. A step towards better audit: The Robson Ten Group classification system for outcomes other than cesarean section. Acta Obstet Gynecol Scand. 2022 Jul;101(7):827-835. https://doi.org/10.1111/aogs.14350 III. Savchenko J, Lindqvist PG, Brismar Wendel S. Comparing apples and oranges? Variation in choice and reporting of short-term perinatal outcomes of term labor: A systematic review of Cochrane reviews. Eur J Obstet Gynecol Reprod Biol. 2022 Sep;276:1-8. https://doi.org/10.1016/j.ejogrb.2022.06.017 IV. Savchenko J, Asp M, Blomberg M, Elvander C, Hagman A, Pegelow Halvorsen C, Lindqvist P, Nelander M, Skiöld B, Brismar Wendel S. Key outcomes in childbirth: Development of a perinatal core outcome set for management of labor and delivery at or near term. Acta Obstet Gynecol Scand. 2023 Jun;102(6):728-734. https://doi.org/10.1111/aogs.14560 V. Savchenko J, Pegelow Halvorsen C, Lindqvist P, Brismar Wendel S. Happy family after breech – outcomes of the two first consecutive births whereof the first one in breech presentation: a Swedish nationwide register-based cohort study. [Submitted]</p