The aim of this project was to explore the role of the tyrosine kinases JAK1 and JAK3 in cytokine signalling, focusing on interleukin-2 signalling in CD8⁺ effector T lymphocytes. Initial experiments compared the effects of the pan JAK1/JAK3 inhibitor tofacitinib, the selective JAK1 inhibitor GSK186, and the selective JAK3 inhibitor GSK192 on IL-2 control of effector CD8+ cytotoxic T cells (CTL). On the basis of these preliminary data, a detailed analysis of the effect of tofacitinib on effector CD8⁺ T lymphocytes was performed. Phosphorylation events regulated by tofacitinib were identified using mass spectrometry analysis of SILAC (stable isotope labelling with amino acids in cell culture) labelled CTL. Tofacitinib regulated a selective number of phosphorylation sites, with less than 1.2% of the CTL phosphoproteome significantly regulated by tofacitinib treatment following 4hrs tofacitinib treatment. Proteins with downregulated phosphorylation sites were enriched in functions related to the Jak-STAT signalling, regulation of gene expression, and MAPK signalling cascades. Proteins with upregulated phosphorylations were also enriched in functions related to regulation of gene transcription. The proteome of tofacitinib treated CTL was defined by label free mass spectrometry. Approximately 4.5% of the CTL proteome was significantly regulated following 24 hours tofacitinib treatment, suggesting tofacitinib regulates the expression of a selective subset of proteins. Tofacitinib treatment resulted in the downregulation of proteins involved in ribosome biosynthesis, steroid biosynthesis, regulation of transcription and the cell cycle; and the upregulation of proteins with hydrolase activity, and with roles in the lysosome and extracellular exosomes. The phosphoproteomic and proteomic data demonstrates that JAK kinase dependent IL-2 signalling regulates essential processes in CTL by controlling a selective number of phosphorylation events and proteins. Validation of proteins identified as regulated following tofacitinib treatment identified new targets of IL-2 signalling in CTL, including the transcription factor NFIL3. NFIL3 was shown to be upregulated in CD8⁺ T lymphocytes following stimulation with IL-2 and regulated perforin and CD62L expression, suggesting a role in the regulation of CTL effector function.

O tráfego de leucócitos é um processo complexo, dependente da ação de inúmeras substâncias químicas, além da perfeita interação celular. Desta forma, este estudo teve como objetivo avaliar a ação dos GCe e da ANXA1 sobre o eixo SDF-1α/CXCR4 e IL-17/IL-23/G-CSF e sobre a expressão de moléculas de adesão CD18, CD49d e CD62L. Foram utilizados camundongos machos Balb/C selvagens (WT) ou ANXA1-/-. As avaliações foram realizadas em condições basais, na presença de altas concentrações de GCe e na vigência de processo inflamatório, induzidos pela administração de ACTH (5 µg/animal, i.p.) ou pela injeção de LPS (100 µg/kg, i.p.), respectivamente, ou na ausência da ação dos GCe, pela ação do RU 38486 (RU, 10 mg/kg, i.p.). A participação da ANXA1 e do receptor FPR2 foi avaliada pelo pré-tratamento com Ac2-26 (1 mg/Kg, i.p.) ou com BOC2 (10 µg/animal, i.p.) durante 4 dias, 1 vez ao dia. A quantificação total e diferencial das células foi realizada em câmara de Neubauer e em esfregaços corados por May-Grunwald ou citometria de fluxo. As quantificações de CXCR2, CXCR4, FPR2, CD18, CD49d, CD62L e maturação granulocítica (CD11b/Ly6G) em células da medula e da circulação foram realizadas por citometria de fluxo. A expressão de ANXA1 nos tecidos do estomago e do baço foi realizada por western blotting e nas células da medula óssea e sangue circulante foi realizada por imunofluorescência. As quantificações de IL-17, IL-23, G-CSF, SDF-1α e corticosterona foram realizadas por ELISA. A quimiotaxia de neutrófilos da medula óssea e sangue periférico foi ensaiada na placa de quimiotaxia com filtro de poro de 8 µm. A fagocitose de neutrófilos apoptóticos por macrófagos da medula óssea foi avaliada por ensaio in vitro. Para verificar os efeitos do ACTH na migração de neutrófilos no processo inflamatório, foi empregado o modelo de bolsa de ar (100 µg/mL; LPS); e o comportamento dos leucócitos circulantes de animais tratados com ACTH foi avaliado pela técnica de microscopia intravital. Os resultados obtidos, que estão apresentados em quatro temáticas, mostraram que: 1) neutrófilos da medula óssea e sangue periférico expressam ANXA1 no citoplasma e membrana, bem como o receptor FPR2, constitutivamente, e a expressão de ambos é regulada pelos GCe. A ANXA1, via receptor FPR2 expresso em células da medula óssea, controlam a maturação neutrofílica e o tráfego destas células da medula óssea para o sangue. A ANXA1, via interação ao FPR2, controla o clearance de neutrófilos do sangue para a medula óssea, modulando o eixo SDF-1α/CXCR4; 2) A administração do ACTH causa neutrofilia e os neutrófilos circulantes são ANXA1+, CD18+, CD49d+, CD62L+, mostrando que injeção do ACTH in vivo altera o fenótipo destas células na circulação. Estas modificações alteram o comportamento dos neutrófilos na circulação, bem como a migração para a bolsa de ar na vigência de inflamação e para os tecidos de clearance. Estes efeitos podem ser dependentes, pelo menos em parte, da inibição de migração orientada, já que quimiotaxia frente ao fMLP ou ao SDF-1α estavam reduzidas. Ainda, o clearance de neutrófilos é reduzido em animais tratados com o ACTH pela menor atividade fagocítica e secretora dos macrófagos medulares; 3) Animais tratados com RU 38486 e ANXA1-/- mobilizam granulócitos da medula óssea para o sangue circulante e, deste compartimento para o foco de inflamação com maior intensidade que o observado em animais controles. O eixo IL-17/IL-23/G-CSF parece estar envolvido na granulopoiese e na mobilização de neutrófilos para o sangue durante a inflamação, mas não é alvo de ação da ANXA1 e o GCe nesta etapa do processo inflamatório. Adicionalmente, foi observado que na vigência de peritonite, as moléculas de adesão, CD49d e CD62L estão envolvidas no processo de migração de neutrófilos da medula óssea para o sangue. Os resultados aqui obtidos permitem concluir que os GCe e a ANXA1 são relevantes para granulopoiese e tráfego dos neutrófilos da medula óssea em condições fisiológicas e na vigência de processo inflamatório. Ainda, em conjunto com os dados da literatura, os nossos resultados podem sugerir a participação da ANXA1 dos GCe na plasticidade fenotípica dos neutrófilos de acordo com os estímulos a que são submetidos, e podem auxiliar na compreensão dos novos conceitos sobre a produção, tempo de vida, localização e funções de neutrófilos. / The traffic leukocytes is a complex process dependent on the action of severals chemical mediators, in addition to perfect cell interaction. Therefore, this study aimed to evaluate the effect of GCe and ANXA1 on SDF-1α/CXCR4 and IL-17/IL-23/G-CSF and on the expression of adhesion molecules CD18, CD49d and CD62L. Balb/C wild type and ANXA1-/- male mice were employed. The analysis were performed at physiological conditions, in the presence of high concentrations of GCe and during of inflammatory process induced by ACTH administration (5 µg/animal, i.p.) or LPS injection (100 µg/kg, i.p.), respectively or in the absence of GCe action, by the action of RU 38486 (RU, 10 mg/kg , i.p.). The involvement of the receptor FPR2 and ANXA1 was assessed by pre-treatment with Ac2-26 (1 mg/kg, i.p.) or BOC2 (10 µg/animal, i.p.) for 4 days, once a day. The quantification of total and differential cell was performed in a Neubauer chamber and stained smears by May-Grunwald and flow cytometry. Quantification of expression of CXCR2, CXCR4, FPR2, CD18, CD49d, CD62L and granulocytic maturation (CD11b/Ly6G) in the bone marrow and circulation were performed by flow cytometry. The expression of ANXA1 on tissues was performed by western blotting and on cells from bone marrow and blood by immunocytochemistry. Quantification of IL-17, IL-23, G-CSF, SDF-1α and corticosterone were performed by ELISA. The chemotaxis of neutrophils from the bone marrow and blood was tested in the chemotaxis chamber with filter pore of 8 microns. The phagocytosis of apoptotic neutrophils by bone marrow macrophages was assessed by in vitro assay. To investigate the effects of ACTH in the migration of neutrophils in the inflammatory process, the model employed was air pouch (100 µg/ ml, LPS), and the behavior of circulating leukocytes from animals treated with ACTH were evaluated by intravital microscopy. The results obtained, which are presented in three sections, showed that: 1) neutrophils from the bone marrow and blood expressed ANXA1 in the cytoplasm and membrane, as well as FPR2, constitutively and the expression of both is regulated by GCe. The ANXA1 via FPR2 receptor expressed in bone marrow cells, controls the neutrophilic maturation and traffic of these cells from the bone marrow into the blood. The ANXA1 via interaction to FPR2 controls the clearance of neutrophils from the blood to the bone marrow by modulating the SDF-1α/CXCR4 axis; 2) the administration of ACTH induces neutrophilia and the circulating neutrophils are ANXA1+, CD18+, CD49d+ and CD62L+, showing that the injection of ACTH in vivo alters the phenotype of these cells in the blood. These modifications alter the behavior of neutrophils in the blood, as well as the migration to the air pouch in the presence of inflammation and to the tissue clearance, and these effects may be dependent, at least in part, on inhibition of migration oriented events, as chemotaxis in response to fMLP or SDF-1α were reduced. Further, the clearance of neutrophils is reduced in animals treated with ACTH due to the lower phagocytic and secretory activity of medullary macrophages; 3) Animals treated with RU 38486 and ANXA1-/- mobilize granulocytes from bone marrow into the blood, and from this compartment to the focus of inflammation with higher intensity than that observed in the control group. The axis IL-17/IL-23/G-CSF seems to be involved in granulopoiesis and mobilization of neutrophils into the blood during inflammation, but it is not the target of action of ANXA1 and GCe at this step of inflammatory process. Additionally, it was observed that in the presence of peritonitis, the adhesion molecules, CD49d and CD62L are involved in the migration of neutrophils from the bone marrow into the blood. The results obtained allow concluding that the GCe and ANXA1 are relevant to the granulopoiesis and the traffic of neutrophils from bone marrow under physiological conditions and in the presence of inflammation. Furthermore, together with literature data, the data presented here may suggest the involvement of ANXA1 the GCe in phenotypic plasticity of neutrophils according to the stimuli that are submitted, and may support to understand the new concepts of production, half-life, location and function of neutrophils.

Due to the growing burden of malignancy and chronic infections, manipulating CD8+ T cell responses for clinical use has become an important goal for immunologists. CD8+ T cells have the unique capacity to recognize and kill tumor cells and intracellular pathogens. Thus far, failed or only minimally effective T cell vaccines against chronic pathogen infections and tumors have highlighted basic knowledge gaps for eliciting memory CD8+ T cell protection. Defining the immunological mechanisms that determine protective capacity and longevity in T cells will be critical to both therapeutic and prohylactic vaccine efficacy. My studies focus on strategies to manipulate effector and memory CD8+ T cell responses, including their mechanisms of action. Specifically, I show that dendritic cell (DC) immunization coupled with relatively early (days 1-3) or late (days 4-6) administration of enhanced IL-2 signals drive either effector or memory programs. DC + IL-2c administered 4-6 days post-DC transfer is shown to enhance Ag-specific effector CD8+ T cell responses; this approach is further explored in the context of a cancer immunotherapy, demonstrating effective control of tumor burden in multiple murine models of cancer. Temporal alterations of IL-2 signaling from day 4-6 to day 1-3 post-DC immunization is shown to increase memory potential and memory CD8+ T cell numbers long-term. Additional studies reveal CTLA-4-mediated down-regulation of B7-ligands on DCs after IL-2c treatment, demonstrating that weaker or more transient signaling through the CD28-B7 axis may favor memory CD8+ T cell programs. My work contributes valuable concepts in memory CD8 T cell generation to develop T cell vaccines that are both safe and predictable.

Emerging evidence indicates that 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) and computed tomography (CT) are useful imaging modalities for evaluating tumor progression in transgenic mouse models of solid human cancers, but the potential of integrated FDG-PET/CT for assessing tumor development in genetically engineered mouse models of liquid human cancers - including neoplasms of immunoglobulin (Ig)-producing plasma cells - has not been established. Here we use a double-transgenic strain of laboratory mice, designated C.IL6Myc, that recapitulates key features of human plasma cell myeloma (a.k.a. multiple myeloma [MM]) to demonstrate that FDG-PET/CT affords a useful research tool for assessing plasma cell tumor (PCT) development in a serial, objective and, importantly, stage- and lesion-specific manner. Supported by serum biomarker analyses (Ig level, paraprotein) and histopathological findings in C.IL6Myc mice undergoing PCT development, the newly generated FDG-PET/CT data set demonstrates the potential of this imaging modality for preclinical basic and translational MM research. PET imaging of genetically engineered mice in which MM-like tumors arise predictably in an intact immunocompetent microenvironment may facilitate the design and testing of new approaches to the treatment and prevention of MM in humans.

Opioids are powerful analgesics. Despite their high efficacy for the management of moderate to severe pain, their clinical utility is limited due to the occurrence of adverse effects. The main problem associated with opioid use is the differential rate of tolerance development to the various pharmacological effects of opioids, with tolerance to respiratory depression occurring at a slower rate than analgesic and euphoric effects. The development of analgesic tolerance, where the efficacy of the drug progressively diminishes with repeated administration, requires higher doses of the drug to achieve a maximum effect. Reports have implicated inflammation as a major driver of analgesic tolerance development. With surmounting evidence that the prototypical opioid, morphine induces pro-inflammatory cytokine release in the brain, spinal cord, and gastrointestinal tract, a question arises of whether pro-inflammatory cytokine release in the gut as a result of chronic morphine treatment is paralleled with the development of morphine antinociceptive tolerance. This dissertation investigated the rate at which antinociceptive tolerance to various doses of morphine developed to a different degree in the presence of colonic inflammation. Using a mouse model, colonic inflammation was induced with 2,4,6-Trinitrobenzenesulfonic acid (TNBS) and then the mice were pelleted with 25 mg, 50 mg (2x25), or 75 mg morphine pellet. Antinociceptive tolerance to morphine was determined in a warm-water tail-immersion assay upon an administration of a morphine challenge dose (10 mg/kg). Inflammatory cytokine expressions and protein levels were measured from whole colon using qPCR and ELISA, respectively. Morphine antinociceptive tolerance was significantly enhanced in the presence of colonic inflammation in a dose and time dependent manner. With a daily injection of 0.5 mg/kg peripheral opioid receptor antagonist 6β-N-heterocyclic substituted naltrexamine derivative (NAP), mice pelleted with 25 mg, 50 mg (2x25), or 75 mg morphine pellets were tested on day 5, 4, or 3, respectively. Tolerance to morphine as well as the enhanced tolerance observed in the presence of colonic inflammation was prevented with daily NAP treatment. However, NAP did not block morphine-induced or TNBS-induced inflammation. Collectively, our findings indicate that inflammation is a major modulator of morphine antinociceptive tolerance and peripheral opioid receptors may be responsible for mediating antinociceptive tolerance.

Mast cells are known to play an important role in allergic events and in other inflammatory reactions through varied intracellular signaling transduction proteins. RasGRP4 is a mast cell-restricted guanine nucleotide exchange factor (GEF) and diacylglycerol (DAG)/phorbol ester receptor. Interleukin (IL) -13, a critical cytokine for allergic inflammation, exerts its effects through a complex receptor system including IL-4Rα, IL-13Rα1 and IL-13Rα2. IL-13Rα2 has been reported to be a decoy receptor for IL-13. My experiments indicate that the mast cell specific RasGRP4 protein regulates the level of IL 13Rα2 and controls IL-13/ IL 13Rα1-mediated intracellar signaling events in mast cells. Phosphorylation of STAT6 plays an important role in airway hyperresponsiveness and asthma. The development of therapeutics that can regulate RasGRP4 could be used to modulate the IL-13-induced phosphorylation of STAT-6 that may be used as therapy in patients with asthma. SLE is a complex, heterogeneous systemic autoimmune disease characterized by the presence of high levels of autoantibodies. Dysregulation of RasGRP1, a Ras active gene, in mice resulted in a SLE-like disorder. Yasuda and coworkers demonstrated that a defective isoform of RasGRP1 (Δ11) was present in a subset of patients with SLE. My experiments indicate that RasGRP1 upregulates the expression of IL2RG in T cells. In contrast the Δ11 RasGRP1 isoform expressed in a subset of SLE patients leads to defective expression of IL2RG. The IL2RG chain is a common chain which forms part of a number of different receptors eg. IL-2, 4, 7, 9, 15, 21. IL-2 as well as IL-21, which shares sequence homology with IL-2, has been reported to be involved in the generation of regulatory T cells (Tregs). In SLE patients, CD4 + CD25+ Tregs, which play an essential role in controlling immunologic tolerance to self-antigens and preventing autoimmunity, are significantly decreased when compared with healthy controls. The accumulative evidence suggests that the defective isoform of RasGRP1 (Δ11) downregulates expression of IL2RG in SLE patients?? T cells and this could effect the generation of CD4 + CD25+ Tregs. This may be another immunological mechanism in loss of tolerance observed in patient with SLE.

It has been recently demonstrated that the cytokine interleukin (IL)-6 is unique among the so called

It is currently believed that the etiology of inflammatory bowel disease involves an aberrant immune response towards the gastrointestinal microbial flora. In addition, an increase in intestinal paracellular permeability may also be a contributing factor of disease, as it precedes disease in several animal models. However, it remains unclear whether increased intestinal permeability is an epiphenomenon of disease or if it can lead to it. The goal of this thesis is to elucidate this cause-effect relationship. The IL-10-/- mouse is a model of IBD that spontaneously develops colitis after 12 weeks of age. We measured intestinal permeability in this mouse from 4-17 weeks of age and observed that there was a significant increase in small intestinal permeability early in life and before the onset of colitis. When small intestinal permeability was selectively decreased with AT-1001 (a ZOT antagonist peptide) colitis was significantly ameliorated. In contrast, when it was increased with AT-1002 (a ZOT agonist peptide) colitis worsened, indicating that modifications in the paracellular traffic of the small intestine had a significant effect on the severity of colonic disease. In order to study the possible mechanisms by which small intestinal permeability modulated disease in the colon, we measured the effect of increasing small intestinal permeability on the colonic microbial flora of IL-10-/- mice. After AT-1002 treatment from 4-12 weeks of age, there was an evident shift in colonic adherent flora. This effect was not a consequence of inflammation as there was a similar effect in wild type mice. We also studied the effect of increasing small intestinal permeability in the development of oral tolerance to dietary antigens. When wild-type mice were fed OVA under conditions of increased small intestinal permeability there was a significant increase in the proliferation of B cells in the spleen and an increase in OVA-specific humoral response, compared to animals fed OVA alone. Moreover, the production of IL-10 in response to oral OVA was prevented when OVA was given with AT-1002, both in the small intestine and the colon. The studies presented in the doctoral thesis suggest that small intestinal permeability has a critical role in the development of colitis in IL-10-/-mice, and that increasing paracellular traffic in the small intestine may lead to changes in colonic bacterial flora and the abrogation of tolerance to oral antigens, two features of inflammatory bowel disease in humans. / Experimental Medicine

Interleukin-21 (IL-21), a recently discovered member of the IL-2 cytokine family, has been shown to have diverse regulatory effects on B cells including the induction of antibody secretion, differentiation, or apoptosis depending on the cell milieu and activation status. However, the effects of IL-21 on B cell neoplasms such as diffuse large B cell lymphoma (DLBCL) are largely unknown. Our research uncovered the widespread expression of the IL-21 receptor (IL-21R) in B cell lymphomas including DLBCL and that IL-21 stimulation resulted in potent phosphorylation of STAT1 and 3 and weak activation of STAT5. However, our findings also showed that treatment of DLBCL cell lines with IL-21 induced cell cycle arrest and apoptosis. The cell death was caspase-dependent and evident in a majority of DLBCL cell lines. To further examine the potential therapeutic applicability of IL-21, we assessed the effects of IL-21 on primary DLBCL tumors and in vivo DLBCL xenografts in mice. In primary tumors, IL-21 induced apoptosis in five of five DLBCLs compared to two of three follicular lymphomas and two of seven chronic lymphocytic leukemias. No apoptosis or cell death was induced in normal peripheral B lymphocytes. In mice bearing DLBCL xenograft tumors, in situ IL-21 injections induced tumor regression and dramatically extended the overall survival of mice (P<0.001). To elucidate the mechanism of IL-21-induced cell death we analyzed the expression of apoptosis-regulating proteins and observed a strong downregulation of anti-apoptotic Bcl-2 and Bcl-XL and an upregulation of pro-apoptotic Bax post IL-21 stimulation. Subsequent experiments showed that ectopic expression of Bcl-2 or Bcl-XL was able to partially reduce cell death induced by IL-21 while Bax knockdown with siRNA blocked apoptosis completely. To gain insight into the signaling pathways shifting the expression of these proteins toward cell death we performed microarray analysis on sensitive and resistant DLBCL cell lines. The most striking difference in gene expression was observed in C-MYC which was only induced in cell lines exhibiting apoptosis upon IL-21 treatment. Previous reports have shown that c-Myc, which has been studied extensively for its oncogenic properties, can induce apoptosis via downregulation of its transcriptional targets Bcl-2 and Bcl-XL. We then showed that IL-21-induced cell death is dependent on c-Myc by utilizing specific siRNA and shRNA to block the upregulation of this transcription factor and prevent apoptosis. Since c-Myc is a bona-fide target of STAT3 we also showed that siRNA-mediated knockdown of STAT3 abrogated apoptosis by preventing c-Myc upregulation and its subsequent effects on apoptosis-regulating proteins. Our results delineate a novel IL-21 pro-apoptotic signaling pathway and one of the first examples in which the STAT3 - c-Myc pathway, which usually promotes B cell survival and oncogenesis, can be exploited for treatment of cancer. Furthermore, our findings demonstrate that IL-21 is a highly potent anti-DLBCL agent in vitro and in animal models and should be examined in clinical studies of DLBCL.

Vid beskattning finns det två sidor med olika intressen. Dels den enskilde individen som bevakar sitt intresse av att minimera sin skattebelastning och dels staten med ett intresse för skyddet av skattebasen. Vid en flytt från Sverige kan obegränsad skattskyldighet aktualiseras på grund av att den skattskyldige har väsentlig anknytning till Sverige. I svensk skatterätt har legalitetsprincipen, ingen skatt utan lag, en stark ställning. Detta ställer krav på lagstiftningen att den skall uppfylla ett visst mått av förutsägbarhet. Den skattskyldige skall kunna förutse konsekvenserna av sitt handlande. 3:7 IL innehåller tio anknytningsmoment som vid en sammanvägd bedömning skall ligga till grund för graden av skattskyldighet. Ur lagtexten går det dock inte att utläsa vad dessa anknytningsmoment i praktiken innebär. Ledning för ett beslut bör då sökas i förarbete, praxis och doktrin. Praxis på området om väsentlig anknytning är inte enhetlig och gällande en del anknytningsmoment följer inte praxis de kriterier förarbetena stadgat som norm för bedömning. Detta gör att rättssäkerheten såväl som effektiviteten gällande lagrummet 3:7 IL som det är utformat idag kan ifrågasättas. Vidare saknar många domar från Regeringsrätten motiveringar till varför utgången i målet blev som den blev, vilket ytterligare bidrar till en osäkerhet och otydlighet på området. Någon lagstadgad rangordning av de tio anknytningsmomenten finns inte utan det är genom praxis viktningen av anknytningsmomenten har skett. Ett av de viktigaste anknytningsmoment är bostad inrättad för åretruntbruk. Praxis på området har tenderat att förskjutas från att objektivt värdera standard och läge på fastigheten till att subjektivt bedöma hur fastigheten nyttjas. Detta innebär att fritidsbostäder, oavsett läge och standard, inte medför väsentlig anknytning, medans fall där den skattskyldige behåller en permanentbostad alltid betraktas ha väsentlig anknytning. Familj utgör ett starkt skäl till att obegränsad skattskyldighet består, särskilt om familjen kvarstannar. Regelbundna besök till Sverige ses som en indikation på att den skattskyldige inte har för avsikt att bryta banden med Sverige. Vidare utgör ekonomisk engagerad här genom att inneha tillgångar som, direkt eller indirekt, ger honom väsentligt inflytande och om han bedriver näringsverksamhet här starka grunder för fortsatt obegränsad skattskyldighet i Sverige trots en flytt härifrån. Gränsdragningen för storleken på tillgångar i relation till skattskyldighet är svårbedömd. Generellt kan sägas att ett innehav som inte överstiger 10 % av tillfångarna i ett fåmansföretag bör inte medföra väsentlig anknytning medans ett innehav över 15 % gör det. Gällande anknytningsmomentet om han bedriver näringsverksamhet här antas vanligen att den skattskyldige har så täta kontakter med Sverige att det medför väsentlig anknytning. För att öka både rättssäkerheten och effektiviteten kan förespråkas att domstolen blir tydligare i sin utformning av domar och beslut. Likaså att domarna motiveras väl och att det klart och tydligt framgår varför beslutet blev som det blev. I ett steg i att förbättra både rättssäkerheten och effektiviteten kan förslagsvis ett införande av en ”specialdomstol”, vars ansvarsområde bland annat berör frågor runt väsentlig anknytning, motiveras. Ur ett effektivitetsperspektiv skulle detta troligen förkorta handläggningstider men även höja kompetensen hos domstolen och på så sätt öka förutsägbarheten, minska godtycklighet på området och då minska antalet situationer som kommer till prövning i domstolen.