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To supplement and modify the diagnosis and clinical research of B-cell Chronic Lymphocytic Leukemia (B-CLL), a new method based on cell imaging and image processing was developed and applied to the B-CLL patient samples. The fluorophore-labelled leukemia cells were clearly visualized, reflecting the positive/negative expression of the corresponding surface markers and their distribution. Computer algorithms were devised and used to analyze a large number of images. The fluorescence intensity of the labelled antibodies on a given cell directly reflects the expression of the corresponding surface markers. The morphology and size of leukemia cells were not identical even in the same patient’s sample and the size variation does not correlate with the number of surface markers. The amount of each surface marker was approximately fixed for each patient, but there were some relationships, for instance, the number of CD19 and CD38 markers were correlated to each other. The heterogeneous expression of surface markers confirmed an assumption that surface markers have their preferred membrane positions. One of the most important results is that the cell imaging and our image processing method has provided an alternative and reliable way to diagnose B-CLL and new insights in the prognosis of subtype of B-CLL.

Phosphoinositide 3-kinase is a family of lipid kinases that function by phosphorylating the D3 position of phosphoinositide (PI) lipids generating PI(3)P, PI(3,4)P2 and PI(3,4,5)P3. These D3 phosphoinositides regulate various cellular processes through the recruitment of effector proteins containing lipid specific pleckstrin homology (PH) domains. PI phosphatases such as PTEN and SHIP function to restrain PI3K signaling by limiting the amount of D3 PI available for binding. Deletion of either PTEN or SHIP significantly alters B cell function and humoral immune responses. TAPP1 and TAPP2 are dual PH domain containing adaptors which selectively bind the phosphoinositide PI(3,4)P2 via their C-terminal PH domains. PI(3,4)P2 is a lipid messenger generated by PI3K and through the inositol phosphatase activity of SHIP. The function of PI(3,4)P2 remains incompletely understood. To identify the functional role of TAPP-PI(3,4)P2 interactions, we utilized a knock-in (KI) mouse bearing mutations within the PI-binding pocket of both TAPPs. Our study assessed the effect of PI3K dependent KI mutation on B lymphocyte development, activation and antibody production. Flow cytometry analyses of lymphoid tissues found that TAPP KI mice develop relatively normal frequencies of mature B cell populations with the exception of peritoneal B1 cells, which are increased by approximately 50%. Strikingly, TAPP KI mice developed substantially elevated serum antibody levels. TAPP KI mice were able to generate high affinity antigen-binding antibodies upon immunization with NP-OVA in alum adjuvant; however, total immunoglobulin production was markedly increased under this immunization condition. We further assessed the germinal centre (GC) response, which are known to require PI3K signaling and a hallmark of T cell dependent (TD) antibody responses. TAPP KI mice generated larger germinal centers (GC) upon immunization, which was associated with increased GC B cell survival. We further assessed whether uncoupling of TAPPs from PI(3,4)P2 alters B cell signaling and functional responses in vitro. B cells purified from TAPP KI mice were found to have altered functional responses in vitro, with significantly increased survival and cell division following antigen receptor cross-linking. Consistent with increased cell survival, TAPP KI B cells show increased Akt phosphorylation on Ser473 and Thr308 after antigen receptor cross-linking. However, reconstitution of B cell deficient mice with either WT or TAPP KI B cells was found to generate similar GC responses, suggesting that activation of other cells may contribute to the enhanced in vivo responses. Consistently, when we examined the CD4+ T follicular helper cells, a subset providing critical cues to GC responses, we found increased expression of ICOS activation marker. Our results indicate the interactions of TAPP adapters with PI(3,4)P2 serve to restrain lymphocyte activation and limit antibody production, providing the first in vivo evidence that this interaction is important for immune function.

Cell migration is critical to a wide range of physiological and pathological events and is central to disease progression of B lymphocyte (B cell)-derived leukemia and lymphoma as well as many other types of cancer. It is extensively controlled by phosphoinositide 3-kinase (PI3K), which generates PI(3,4,5)P3 (PIP3) and PI(3,4)P2, lipid messengers that recruit pleckstrin homology (PH)-domain-containing signaling proteins. While PIP3 is known to regulate cell migration, it remains a major unanswered question in the field whether PI(3,4)P2 is also implicated in this cellular function. A series of investigations here on PI(3,4)P2-specific lipid phosphatases and binding proteins in the context of chemotaxing malignant B cells provide the first insights into a previously unappreciated role of PI(3,4)P2 signaling in cell migration. First, I used physiological regulators of PI(3,4)P2, the inositol polyphosphate 4-phosphatase (INPP4) enzymes, as tool to manipulate PI(3,4)P2 levels to determine the function of this lipid second messenger. PI(3,4)P2 depletion by INPP4A or INPP4B relative to phosphatase-dead mutants indicated an essential role of PI(3,4)P2 in mediating both the speed and directionality of chemotaxis. Gene silencing of the authenticated PI(3,4)P2-specific binding protein TAPP2 leads to reduced migration speed and directionality, similar to PI(3,4)P2 depletion. The impaired migration is underlain by alterations in chemokine-induced rearrangement of the actin cytoskeleton, loss of migratory polarity and dysregulation of the leading edge activator Rac. A putative PI(3,4)P2-binding protein, lamellipodin (Lpd), is found to strongly colocalize with PI(3,4)P2 depending on the Lpd PH domain. Lpd knock-down rescue experiments indicated that PI(3,4)P2 controls directionality through Lpd, while Lpd also promotes motility independently of PH domain binding to PI(3,4)P2. The PI(3,4)P2-binding protein kinase Akt/PKB (also binds to PIP3) is found to play a positive role in the B cell context. Here, PI(3,4)P2 depletion does not inhibit phosphorylation of Akt but seemingly reduces its activity. It is likely that PI(3,4)P2 mediates malignant B cell migration in part through promoting Akt activity. Taken together, the thesis work establishes the PI(3,4)P2 pathway as a novel branch of the PI3K signaling network controlling cell migration and suggests that PI(3,4)P2 may integrate diverse downstream migratory pathways to impact on cell migration.

Identification of the complement C4d fragment in peritubular capillaries as a specific marker for antibody mediated rejection in renal transplantation revealed the critical role of antibodies in graft survival. In this thesis, I document the design and findings of studies performed to investigate the clinical impact of anti-HLA antibodies present before and/or after transplantation. Over time, the detection techniques for anti-HLA antibodies has evolved from the less sensitive complement-dependent lymphocytotoxicity (CDC) crossmatching (XM) to more sensitive solid phase assays such as Luminex®. Studies have been conducted to compare the predictive value of different antibody detection techniques. The first result chapter presents antibody specificity in positive CDC B-cell crossmatch (BXM), analysed with highly specific Luminex® assays. The study also investigates the predictive value of BXM in the general transplant population. I found that donor-specific anti-HLA antibodies (DSA) are only present in one third of positive BXM and are associated with poor outcomes. The novel finding is that >80% of the DSA detected by BXM are complement-fixing IgG₁ and IgG₃ subclasses. Transplant glomerulopathy (TG) is type of chronic renal graft rejection. The pathogenesis of TG is unclear. In the second result chapter, I report risk factors and involvement of anti-HLA antibodies in the development of TG. This study shows that glomerular rejection, delayed graft function, HLA presensitization and DSA have a univariate effect on TG development. Multivariate analysis revealed that DSA are an independent predictor of TG, after adjustment for other risk factors. I have further investigated the role of BXM in a unique, well-matched, highly sensitized patient group transplanted under the national renal exchange programme. I compared Luminex® antibody analysis with BXM in predicting transplant outcomes. In highly sensitized patients, DSA are found in two thirds of positive BXM. In univariate analyses, BXM is associated with humoral rejection whereas DSA defined by Luminex® are associated with total and all rejection types. The major finding is that, by multivariate analysis, DSA defined by Luminex® are an independent predictor of total and humoral rejection, but BXM are not. These interesting findings are reported in the third result chapter. Studies reported in this thesis define the clinical significance of anti-HLA antibodies in renal transplant outcomes. Method comparison studies provide useful information on antibody specificity and their impact on graft survival. Collectively, a better understanding of alloantibodies associated with graft rejection and limitation of antibody detection methods may facilitate donor selection and choice of immunosuppressants, and consequently improve transplant outcomes. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1379925 / Thesis (Ph.D.) - University of Adelaide, School of Medicine, 2010

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 6 uppsatser.