Somatic mutations in lymphocytes in patients with immune-mediated bone marrow failure

Somaattisten mutaatioiden rooli syövän kehityksessä on osoitettu kiistattomasti, mutta niiden yleisyys ja vaikutukset terveissä soluissa tunnetaan vielä puutteellisesti. Tutkimushypoteesimme mukaan somaattiset mutaatiot voivat edistää T-solukloonien kasvua ja selviytymistä kroonisessa inflammaatiossa, kuten autoimmuunisairauksissa. Tutkimme somaattisia mutaatioita immuunivälitteisissä luuydinkato-oireyhtymissä (aplastisessa anemiassa ja hypoplastisessa myelodysplastisessa oireyhtymässä), joissa sytotoksiset T-solut tuhoavat luuytimen kantasoluja. Aplastista anemiaa sairastavilla potilailla on aiemmin osoitettu esiintyvän STAT3-mutaatioita. Kartoitimme ei-pahalaatuisten T-solujen somaattisia mutaatioita 2533 geenin paneelilla. Sekvensoimme CD4+ ja CD8+ T-solut 29 potilaalta, jotka sairastivat immunologista luuydinkato-oireyhtymää, ja vertasimme tuloksia 20 terveeseen sekä aiemmin julkaistuun aineistoon aplastista anemiaa sairastavista potilaista. Somaattiset muutokset T-soluissa olivat yleisiä niin terveillä kuin potilaillakin, mutta ne rikastuivat aplastista anemiaa sairastavien potilaiden CD8+ T-soluihin. Näiden T-solujen mutaatiot kasautuivat voimakkaimmin JAK-STAT ja MAP kinaasi -signalointireiteille. Havaitsemiemme somaattisten mutaatioiden määrä oli yhteydessä T-solujen klonaalisuuteen, jota tutkimme sekvensoimalla T-solureseptoreita. Ymmärtääksemme somaattisten mutaatioiden roolia T-soluissa, analysoimme yksisolu-RNA- sekvensointimenetelmällä yhteensä 6 pitkittäisnäytettä kahdelta potilaalta, joiden CD8+ T-soluklooneissa oli STAT3, KRAS, NFATC2 ja PTPN22 geenien mutaatioita. Mutatoituneet kloonit osoittautuivat fenotyypiltään sytotoksisiksi, ja muuttuivat immunosuppressiivisen hoidon myötä. Tutkimustulostemme mukaan somaattiset mutaatiot lymfosyyteissä ovat yleisiä, liittyvät klonaalisuuteen ja voivat muuttaa T-solujen fenotyyppiä. Lisätutkimusta tarvitaan selvittämään, ovatko somaattiset mutaatiot T-soluissa syy vai seuraus klonaalisesta kasvusta, ja mikä on näiden solujen rooli autoimmuniteetin kehittymisessä.The role of somatic mutations in cancer development is undisputable, but our knowledge of the prevalence and function of somatic mutations in healthy cells is yet unresolved question. In immune-mediated bone-marrow failure (such as aplastic anemia and hypoplastic myelodysplastic syndromes) hematopoietic stem cells are destroyed by cytotoxic T cells, which have been shown to be oligoclonal and in some cases harbor STAT3 mutations. We addressed clonality of non-malignant T cells by investigating somatic mutations with a custom- made gene panel of 2533 genes. We sequenced CD4+ and CD8+ cells of 29 patients with immune-mediated bone marrow failure and compared the results to 20 healthy controls as well as previously published data on patients with aplastic anemia. Somatic variants in lymphocytes were common both in patients and healthy controls, but especially enriched in aplastic anemia patients and CD8+ T cells. CD8+ T cells of aplastic anemia patients accumulated most mutations on JAK-STAT and MAPK pathways. The number of somatic mutations was associated with CD8+ T cell clonality, assessed by T cell receptor beta sequencing. To further understand the role of mutations in T cells, we performed single-cell RNA sequencing from 6 longitudinal samples of 2 aplastic anemia patients with STAT3, KRAS, NFATC2 and PTPN22 mutations in CD8+ T cell clones. Mutated clones showed cytotoxic phenotype, which was altered by successful immunosuppressive treatment. Our results suggest that somatic mutations in T cells are common, associate with clonality and may alter T cell phenotype. The role of somatic mutations in the initiation and persistence of autoreactive clones needs to be further investigated

Motion asymmetries in horses at trot – biological variation or lameness?

Tidigare studier har funnit att hästar som ansetts ohalta av sina ägare och fungerat väl i träning och tävling har haft ett rörelsemönster som vid en objektiv mätning med ”Lameness Locator” har överstigit gränsvärdena för vad systemet klassar som en rörelseasymmetri. Dessa asymmetrier har varit i samma storleksgrad som hos hästar som utreds för lindrig hälta på klinik. Detta examensarbete har studerat hästar som ansetts som ohalta av sina ägare men som vid en objektiv rörelsemätning med ”Lameness Locator” befunnits asymmetriska. Syftet med studien var att ta reda på om dessa asymmetrier var smärtutlösta och hur asymmetrierna påverkades av hårt respektive mjukt underlag. Det fanns redan insamlat material från sex hästar som rörde sig asymmetriskt och under hösten har ytterligare sex hästar studerats. Hästarnas rörelsemönster har registrerats innan och efter behandling med NSAID samt innan och efter placebo. I och med detta har varje häst varit sin egen kontroll. Av de tolv hästar som ingick i studien hade fem hästar bakbensasymmetrier, 3 hästar frambensasymmetrier och 4 hästar hade fram – och bakbensasymmetrier vid den första mätningen. Det kunde inte visas någon statistiskt signifikant skillnad innan och efter placebo eller innan och efter behandling på någon typ av asymmetri. Däremot var hästarna signifikant mer frambensasymmetriska på hårt underlag jämfört med mjukt underlag men denna skillnad kunde dock inte visas för bakbensasymmetrierna. Antalet hästar är för litet för att kunna uttala sig om den svenska ridhästpopulationen. Studien indikerar dock att alla lindriga hältor inte behöver vara smärtutlösta på grund av en inflammation. Istället kan det finnas andra smärtmekanismer och/eller kan det tänkas att hästar kan ha en stor biologisk normalvariation vad gäller rörelsemönstret och att denna även kan variera beroende på underlag.Previous studies have found that horses, considered sound by their owners and well functioning in training and competition have, by an objective measurement equipment “Lameness Locator", exceeded the limits to be classified as lame by the system. These asymmetries have been of the same magnitude as for horses examined for mild lameness in the clinic. This thesis has studied horses that were considered as sound by their owners but asymmetric by an objective motion analysis with "Lameness Locator". The aim of the study was to investigate if these asymmetries were pain induced and whether asymmetries were changed depending on hard or soft surfaces. Previous collected data from six horses with motion asymmetries and data from six horses collected during the autumn were analyzed. The motion pattern of the horses was measured before and after treatment and placebo respectively. Of the twelve horses that were included in the study five horses showed hind limb asymmetries, three horses showed forelimb asymmetries and four horses showed both hind limb and forelimb asymmetries. There were no significant differences in asymmetries before and after NSAID/placebo treatment. The horses trotted significant more asymmetric on hard compared to soft ground surfaces for the forelimbs. This difference could not be shown for the hind limb asymmetries. The number of horses is too small to draw conclusions about the Swedish horse population. The study indicates that subtle lameness doesn´t always has to be triggered by pain caused by an inflammation. Instead, there may be other pain mechanisms and / or it is possible that horses can have a great biological normal variation in the pattern of movement and that this also can vary depending on the surface

Factors during the dry period that can predispose for mastitis during the next lactation period

Sinperioden är bra då den dels ökar mjölkavkastningen och dels är en period då infektioner kan läka ut. Det är dock en känslig period för kon då hon också är infektionskänslig och många nyinfektioner kan ske. Det är viktigt att ge kon en bra start på sinperioden genom att minska foder och mjölkning så att mjölkproduktionen avtar innan hon sinläggs helt, detta för att spenkanalen ska kunna sluta sig och juvret börja tillbakabildas. Kon ska under perioden hållas i en ren miljö, det är extra viktigt under de två första veckorna och i slutet när kalvning närmar sig då kon är mer infektionskänslig. Under sinperioden är det viktigt att ge en bra foderstat men man måste se upp så att kon inte blir fet då detta predisponerar för mastit. Sinperiodens längd verkar inte ha betydelse på juverhälsan. Celltalen under sinperioden korrelerar med mastitförekomst i föregående laktation. Höga celltal under sinperioden ökar risken för mastit i nästkommande laktation. Studier tyder på att intramammära infektioner, IMI, som uppstår i början av sinläggningen inte kvarstår till nästa laktation men ändå predisponerar för mastit i nästkommande laktation. IMI som uppstår sent i sinläggningen ger ofta mastit med samma bakterie men här vet forskarna inte om det rör sig om en persisterande infektion eller en nyinfektion, dock skiljer sig litteraturen lite när det gäller detta och det verkar som att förmågan att överleva under sinperioden skiljer sig mellan olika bakteriearter

Aikuisten aplastinen anemia

Vertaisarvioitu. English abstract.Aplastinen anemia on harvinainen hematologinen autoimmuunisairaus, jossa verisolujen tuotanto vähenee, mikä aiheuttaa anemian, vuototaipumuksen ja infektioalttiuden. Aplastinen anemia on itsenäinen sairaus sytopenioita aiheuttavien, osin päällekkäisten, luuytimen vajaatoimintatilojen kirjossa. Sen syntymekanismi on monimutkainen, mutta immunosuppressiivisen hoidon yleensä hyvä teho on osoitus taudin immunologisesta luonteesta. Keskeisiä ovat autoreaktiiviset sytotoksiset T-solut. Aplastinen anemia diagnosoidaan ja hoidetaan pääasiassa yliopistosairaaloissa. Ikä on tärkein taudin hoitoa määrittävä tekijä, mutta lähes kaikille potilaille on tarjolla tehoavaa hoitoa. Ilman hoitoa potilaat menehtyvät verenvuotoihin ja infektioihin.Peer reviewe

Somatic compensation of inherited bone marrow failure

Inherited bone marrow failure syndromes (IBMFS) are a heterogeneous group of genetic disorders characterized by insufficient blood cell production and increased risk of transformation to myeloid malignancies. While genetically diverse, IBMFS are collectively defined by a cell-intrinsic hematopoietic stem cell (HSC) fitness defect that impairs HSC self-renewal and hematopoietic differentiation. In IBMFS, HSCs frequently acquire mutations that improve cell fitness, a phenomenon known as somatic compensation. Somatic compensation can occur via distinct genetic processes such as loss of the germline mutation or somatic alterations in pathways affected by the disease-causing gene. While the clinical implications of somatic compensation in IBMFS remain to be fully discovered, understanding these mutational processes can help understand disease pathophysiology and may inform future diagnostic and therapeutic approaches. In this review, we highlight current understanding about somatic compensation in IBMFS. (c) 2022 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )Peer reviewe

Construct Validity of the Swedish Version of the Revised Piper Fatigue Scale in an Oncology Sample—A Rasch Analysis

AbstractObjectivesFatigue is a common and distressing symptom in cancer patients due to both the disease and its treatments. The concept of fatigue is multidimensional and includes both physical and mental components. The 22-item Revised Piper Fatigue Scale (RPFS) is a multidimensional instrument developed to assess cancer-related fatigue. This study reports on the construct validity of the Swedish version of the RPFS from the perspective of Rasch measurement.MethodsThe Swedish version of the RPFS was answered by 196 cancer patients fatigued after 4 to 5 weeks of curative radiation therapy. Data from the scale were fitted to the Rasch measurement model. This involved testing a series of assumptions, including the stochastic ordering of items, local response dependency, and unidimensionality. A series of fit statistics were computed, differential item functioning (DIF) was tested, and local response dependency was accommodated through testlets.ResultsThe Behavioral, Affective and Sensory domains all satisfied the Rasch model expectations. No DIF was observed, and all domains were found to be unidimensional. The Mood/Cognitive scale failed to fit the model, and substantial multidimensionality was found. Splitting the scale between Mood and Cognitive items resolved fit to the Rasch model, and new domains were unidimensional without DIF.ConclusionsThe current Rasch analyses add to the evidence of measurement properties of the scale and show that the RPFS has good psychometric properties and works well to measure fatigue. The original four-factor structure, however, was not supported

High prevalence of low-allele-fraction somatic mutations in STAT3 in peripheral blood CD8+ cells in multiple sclerosis patients and controls

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Clonal hematopoiesis in patients with rheumatoid arthritis

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Somatic mutations and T-cell clonality in patients with immunodeficiency

Common variable immunodeficiency (CVID) and other late-onset immunodeficiencies often co-manifest with autoimmunity and lymphoproliferation. The pathogenesis of most cases is elusive, as only a minor subset harbors known monogenic germline causes. The involvement of both B and T cells is, however, implicated. To study whether somatic mutations in CD4(+) and CD8(+) T cells associate with immunodeficiency, we recruited 17 patients and 21 healthy controls. Eight patients had late-onset CVID and nine patients other immunodeficiency and/or severe autoimmunity. In total, autoimmunity occurred in 94% and lymphoproliferation in 65%. We performed deep sequencing of 2,533 immune-associated genes from CD4(+) and CD8(+) cells. Deep T-cell receptor b-sequencing was used to characterize CD4(+) and CD8(+) T-cell receptor repertoires. The prevalence of somatic mutations was 65% in all immunodeficiency patients, 75% in CVID, and 48% in controls. Clonal hematopoiesis-associated variants in both CD4(+)and CD8(+) cells occurred in 24% of immunodeficiency patients. Results demonstrated mutations in known tumor suppressors, oncogenes, and genes that are critical for immuneand proliferative functions, such as STAT5B (2 patients), C5AR1 (2 patients), KRAS (one patient), and NOD2 (one patient). Additionally, as a marker of T-cell receptor repertoire perturbation, CVID patients harbored increased frequencies of clones with identical complementarity determining region 3 sequences despite unique nucleotide sequences when compared to controls. In conclusion, somatic mutations in genes implicated for autoimmunity and lymphoproliferation are common in CD4(+) and CD8(+) cells of patients with immunodeficiency. They may contribute to immune dysregulation in a subset of immunodeficiency patients.Peer reviewe

Somatic mutations in lymphocytes in patients with immune-mediated aplastic anemia

The prevalence and functional impact of somatic mutations in nonleukemic T cells is not well characterized, although clonal T-cell expansions are common. In immune-mediated aplastic anemia (AA), cytotoxic T-cell expansions are shown to participate in disease pathogenesis. We investigated the mutation profiles of T cells in AA by a custom panel of 2533 genes. We sequenced CD4+ and CD8+ T cells of 24 AA patients and compared the results to 20 healthy controls and whole-exome sequencing of 37 patients with AA. Somatic variants were common both in patients and healthy controls but enriched to AA patients' CD8+ T cells, which accumulated most mutations on JAK-STAT and MAPK pathways. Mutation burden was associated with CD8+ T-cell clonality, assessed by T-cell receptor beta sequencing. To understand the effect of mutations, we performed single-cell sequencing of AA patients carrying STAT3 or other mutations in CD8+ T cells. STAT3 mutated clone was cytotoxic, clearly distinguishable from other CD8+ T cells, and attenuated by successful immunosuppressive treatment. Our results suggest that somatic mutations in T cells are common, associate with clonality, and can alter T-cell phenotype, warranting further investigation of their role in the pathogenesis of AA.Peer reviewe