The type I interferons (IFN) are a family of cytokines with pleiothropic activities that include inhibition of viral replication, cell proliferation and activation of the immune system. These properties give the IFNs important physiological and pathological roles in infection and cancer and have led to their therapeutic use for many clinical conditions. In humans, the type I IFNs consist of 12 different IFNa subtypes as well as single IFNb, w and k subtypes. They all compete for binding to a common receptor, consisting of two subunits, IFNAR1 and IFNAR2. In almost all cell types proliferation is inhibited by IFNs as a consequence of the antiviral properties. However, previous studies on human peripheral B-lymphocytes have shown increased survival as well as proliferation upon IFN treatment. We established a purification system for extraction of B-lymphocytes from buffy-coat, utilizing density centrifugation in combination with anti-CD19 magnetic beads. In an attempt to identify the molecular mechanisms of increased survival, the expression and/or activation pattern of different signaling proteins were analysed by Western blot. It was previously reported that phosphatidylinositol 3’-kinase (PI3K) physically interacts with the IFNAR complex, via adaptor proteins. Activated PI3K indirectly activates Akt/PKB, a kinase involved in a pathway leading to both survival and proliferation signals. We were able to show a novel signaling pathway - IFN treatment activated Akt/PKB as well as a downstream effector, one member of the Forkhead family (FKHR) was inactivated by phosphorylation and as a consequence p27/Kip1 expression was downregulated. Activation of this pathway resulted in increased survival as measured by TUNEL assay, an effect efficiently counteracted by the the synthetic PI3K inhibitor, LY294002. In additional experiments we investigated the molecular mechanisms of proliferation. Activation of B-cells was ensured by using limiting concentrations of anti-IgM antibodies, mimicing natural activation. Using thymidine incorporation, we discovered that IFN treatment increased the sensitivity to anti-IgM stimulation. As a consequence, more cells proliferated as measured by CFSE staining. However, on its own, IFN was unable to induce proliferation. IFN turned out to be as efficient as IL-2, a classical B-lymphocyte growth factor. In order to distinguish proliferation from increased survival, Rb phoshorylation was analysed by Western blot. Phosphorylation induced by anti-IgM was further enhanced by IFN. As we determined earlier, p27/Kip1 expression was downregulated, releasing the cell cycle block. However, p21/Cip1 expression was upregulated but almost exclusively localised to the cytoplasm, therefore unable to perform the classical growth inhibitory functions. We conclude that type I interferons contribute to increased survival as well as proliferation of human primary B-lymphocytes. The IFN receptor subunits was studied in a human myeloma cell line (U266), using a variant of which that are totally resistant towards the anti-proliferative properties of IFN. The reason for resistance in clinical situations is seldom elucidated, but is often believed to be due to development of antibodies against interferon. The resistant cells were unable to bind radio-labelled IFN, and through Southern Blot we could determine that the IFNAR1 gene was not functional. Also the IFNAR2 gene was affected, since Northern blot and sequencing detected an aberrant transcript not present in the wild type cells. Karyotyping showed that the cells had 3-4 copies of chromosome 21, but Southern blot did not detect any cytoplasmic region of IFNAR2. The IFN receptors are close to each other on the genome, and a deletion affecting one receptor gene is likely to affect the other as well. We conclude that the IFN resistance in U266Res cells is due to lack of functional receptor subunits.

Multiple myeloma (MM) is a hematological tumor of plasma blast/plasma cell origin heterogeneous with respect to the morphological differentiation stage of the tumor cells, genetic alterations and course of disease. A challenge in MM research is to overcome resistance to therapy, which inevitably arises. In this thesis, we have used different strategies to sensitize MM cells to apoptosis and explored possible mechanisms of apoptotic control by the insulin-like growth factor-1 receptor (IGF-1R) survival pathway. mTOR is a key molecule in the regulation of translation activated by survival signaling pathways in MM. We demonstrate that the mTOR-inhibitor rapamycin alone induced apoptosis in primary MM cells. In addition, rapamycin sensitized MM cells to apoptosis induced by dexamethasone, a glucocorticoid frequently used in MM therapy. MM survival factors IGF-1 and IL-6 could neither restore phosphorylation of the mTOR target p70S6K, nor cell growth inhibited by rapamycin and dexamethasone. To study the regulation of inhibitors of apoptosis (IAP), we induced apoptosis and cell cycle arrest with dexamethasone and simultaneously abrogated IGF-1R signaling using the antagonistic antibody αIR3 or the selective IGF-1R inhibitor picropodophyllin (PPP). Dexamethasone transiently up-regulated c-IAP2. The subsequent down-regulation of c-IAP2 and XIAP was associated with the onset of apoptosis. c-IAP2 and XIAP levels further decreased when enhancing dexamethasone-induced apoptosis using αIR3 or PPP indicating a role for IAPs in regulating resistance to apoptosis in MM. Finally, we explored glycogen synthase kinase (GSK)3 as a possible pro-apoptotic molecule and its role in regulating sensitization to apoptosis. We show that inhibition of GSK3 counteracts growth inhibition induced by dexamethasone alone and in combinatorial treatments with inhibitors against PI 3-kinase, mitogen-activated protein kinase (MEK), mTOR and IGF-1R. CT99021 also reversed cell cycle arrest induced by LY294002 or rapamycin. Importantly, the GSK3 inhibitor CT99021 sustained viability in untreated and dexamethasone-treated primary MM cells.

Multiple myeloma (MM) is an incurable plasma cell malignancy. NK cells have demonstrated anti-MM activity in allogeneic transplants and donor lymphocyte infusions, and may provide a more effective therapy for MM. This work demonstrates cytotoxicity of NK-92 and KHYG-1 against MM cells in chromium release and flow cytometry cytotoxicity assays. At a 10:1 effector to target ratio, the cytotoxicity of NK cell lines against MM cells is 50-90%. Blocking NKp30 significantly reduces the cytotoxicity of NK-92 and KHYG-1, while blocking NKG2D and DNAM-1 only reduces the cytotoxicity of NK-92. Notably, NK-92 and KHYG-1 have shown preferential cytotoxicity against the clonogenic population, killing 89-99% in a methylcellulose cytotoxicity assay. Preliminary results in a xenograft bioluminescent mouse model show that NK-92, but not KHYG-1, reduces the tumor burden detected by bioluminescence imaging and bone marrow engraftment by flow cytometry. Therefore, NK cell lines may offer a more effective therapy for MM.

Multiple myeloma (MM) is an incurable plasma cell malignancy. NK cells have demonstrated anti-MM activity in allogeneic transplants and donor lymphocyte infusions, and may provide a more effective therapy for MM. This work demonstrates cytotoxicity of NK-92 and KHYG-1 against MM cells in chromium release and flow cytometry cytotoxicity assays. At a 10:1 effector to target ratio, the cytotoxicity of NK cell lines against MM cells is 50-90%. Blocking NKp30 significantly reduces the cytotoxicity of NK-92 and KHYG-1, while blocking NKG2D and DNAM-1 only reduces the cytotoxicity of NK-92. Notably, NK-92 and KHYG-1 have shown preferential cytotoxicity against the clonogenic population, killing 89-99% in a methylcellulose cytotoxicity assay. Preliminary results in a xenograft bioluminescent mouse model show that NK-92, but not KHYG-1, reduces the tumor burden detected by bioluminescence imaging and bone marrow engraftment by flow cytometry. Therefore, NK cell lines may offer a more effective therapy for MM.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010. / Härtill 5 uppsatser.

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Myleom är en cancersjukdom som uppkommer i benmärgen. I Sverige insjuknar ungefär 500-600 personer i myelom per år. Syftet med litteraturstudien var att med hjälp av de sex dimensionerna av smärta belysa upplevelsen av att leva med sjukdomen myelom. Studien genomfördes utifrån en teoretisk referensram. För att få en helhetsbild av individernas upplevelser av sjukdomen redovisas resultatet utifrån sex dimensioner av smärta. Resultatet visar att individer med myelom får ett inskränkt socialt liv, förändrade vanor och både fysisk och psykisk ohälsa. Individerna påverkas av smärta, trötthet och upprepade infektioner som orsakas av ett nedsatt immunförsvar. Då kunskap om sjukdomen i samhället är begränsad får individer med myelom minskad förståelse när det gäller sjukdomsbilden. För att sjuksköterskan skall få förståelse för individerna och deras livskvalitet krävs en adekvat smärtbedömning, som bör beröra hur individen påverkas av sin sjukdom i det vardagliga livet. Mer forskning kring sjukdomen, behandlingar och mediciner behövs och det vore önskvärt om målet vore att sjukdomen skall kunna botas. / Multiple myeloma is a cancer illness that arises in the bone marrow. In Sweden 500-600 people per year get diagnosed with multiple myeloma. The purpose of the literature study was to highlight the experiences of individuals with multiple myeloma through the six dimension of pain. The study is based on a theoretical frame of reference. To get at overall picture of the individuals experiences of living with multiple myeloma, the result was described through the six dimensions of pain. The result shows that multiple myeloma patient´s social life becomes reduced, their habit changes and booth their physical and psychological health reduces. Pain, fatigue and infections caused by lower immune system affects the individuals. The knowledge of the illness in society is trivial and this makes the understanding for people with multiple myeloma and their illness slight. A proper pain assessment is necessary for the nurse to complete, to get a full understanding about the individuals and their quality of life. More research about the illness, treatments and medications is needed and the goal should be to cure the disease.

A possible treatment for patients with lymphoma and myeloma is stem cell transplantation (SCT). SCT is preceded with cytostatic treatment. There are several side effects related to this treatment, for example fatigue, nausea, constipation/diarrhoea, pain, mucositis and loss of appetite. Aim: Investigate which side effects related to the treatment where most troubling after SCT and if nurse assessment and patient assessment differ. Methods: A quantitative empirical study with repeated measuring. The two groups of nurses and patients answered a form independently. Results: Loss of appetite and fatigue are the most troubling side effects according to both nurse and patient. Older patients tended to be more affected by fatigue. The nurses estimated the side effects such as loss of appetite, fatigue, diarrhoea and nausea lower than the patients did. Conclusion: No definitive conclusion could be made because of the small patient/nurse sample. However, there is a tendency showing difficulty for nurses to estimate correctly the side effects suffered by the patients. The nurses tend to estimate the side effects lower than the patients do.

Analysis of M-components is an important part of the diagnosis of monoclonal gammopathies and for the evaluation of disease response during treatment. In this project, two widely used electrophoresis methods and their corresponding immunotyping method were compared to evaluate the sensitivity of each method for the detection of small M-components. The project included 30 plasma samples from patients with identified M-components; 10 samples containing each IgG, IgA and IgM, respectively. All samples were diluted with normal EDTA plasma to achieve M-components of 5,00g/L. The samples were then serially diluted to achieve M-component concentrations of; 5,00, 2,50, 1,25, 0,63, 0,31 and 0,16g/L. All 180 samples were analysed with agarose gel electrophoresis and capillary electrophoresis. The dilutions above and below the detection level of each method were then analysed with immunofixation and immunosubtraction. The results showed good agreement between agarose gel electrophoresis and capillary electrophoresis in the highest concentrations of IgG and IgM. With agarose gel electrophoresis, IgA was detected in the same location as transferrin and the lowest concentration detected were therefore 1,25g/L. Besides the samples containing IgG, immunofixation was the most sensitive method.

Šioje disertacijoje aprašyti tyrimai, atlikti siekiant įvertinti plazminių ląstelių imunotipavimo, taikant tėkmės citometriją, prognostinį potencialą. Patikrinome hipotezę, jog cirkuliuojančių plazminių ląstelių kinetika gydymo metu gali, anksčiau, nei standartiniai metodai įvertinti atsaką į gydymą. Tyrime naudojome iki tol neaprašytą plazminių ląstelių imunofenotipavimo metodiką. Mėginiai dažyti dviem skirtingais žymenų deriniais: CD56/CD138/CD45/CD19/CD38/CD20 ir cLambda/cKappa/CD138/CD19/CD38/CD56. Nustatėme, kad sveikų donorų kaulų čiulpuose aptinkama ~30% plazminių ląstelių, turinčių atipine CD56 ir/ar CD19 žymenų raiška. Optimizavome tėkmės citometrijos metodiką normalių ir piktybinų plazminų ląstelių aptikimui. Nustatėme, kad piktybinių cirkuliuojančių plazminių ląstelių proporcijos nesumažėjimas po pirmojo chemoterapijos kurso, su 91,7% jautrumu bei 93,3% specifiškumu prognozuoja ankstyvą progresiją. Pacientų, kuriems cirkuliuojančių plazminių ląstelių proporcija nesumažėjo, laikas iki progresijos ir bendras išgyvenamumas buvo statistiškai patikimai trumpesnis, nei pacientų, kuriems piktybinių cirkuliuojančių plazminių ląstelių proporcija sumažėjo ar šios ląstelės buvo neaptinkamos. Ištyrėme normalių plazminių ląstelių populiacijos klinikinę vertę pacientams, sergantiems mielomine liga. Apibendrinant, plazminių ląstelių imunofenotipavimas taikant tėkmės citometrijos metodą suteikia prognostiškai reikšmingos informacijos. Svarbiausias radinys, jog cirkuliuojančių... [toliau žr. visą tekstą] / The investigations presented in this dissertation were initiated with the intention to evaluate the prognostic value of plasma cells immunophenotypic analysis in multiple myeloma patients. We tested the hypothesis that kinetics of peripheral blood circulating plasma cells in response to first chemotherapy cycle could identify patients refractory to given treatment. We employed novel original methodology for plasma cells immunophenotyping: cells were stained in two tubes with antibody combinations CD56/CD138/CD45/CD19/CD38/CD20 and cLambda/cKappa/CD138/CD19/CD38/CD56. We found that ~30% of all plasma cells in bone marrow of healthy donors are immunophenotypically aberrant by CD56 and/or CD19 marker expression. We optimized immunophenotypic differentiation between malignant and normal plasma cells. Non reduction of malignant circulating plasma cells in response to first chemotherapy cycle predicted early progression with sensitivity and specificity of 91.7% and 93.3%, respectively. Time to progression and overall survival were significantly shorter in these patients as compared to patients with undetectable or reduced malignant circulating plasma cells. We also evaluated the clinical value of normal plasma cell subpopulation detection in peripheral blood and bone marrow of multiple myeloma patients. In summary, we demonstrated that immunophenotyping of plasma cells using multiparameter flow cytometry provides important prognostic information. The major finding was that the... [to full text]