No description available.

Mouse models (that develop or can be stimulated to develop lymphomas) are used to examine cancer-related processes. Mouse models can be effective tools used to identify new, early, and pre-malignant markers of lymphomas. Signal Transducer and Activator of Transcription (STAT) 6 is a transcription factor activated through the Jak-Stat pathway. Transgenic mice expressing a constantly activated Stat6 (Stat6VT) were previously generated and characterized to have altered lymphocyte homeostasis. Some of these Stat6VT mice developed a lymphoproliferative disorder (LPD). LPD, including lymphomas, develops when lymphocytes are overproduced or act abnormally. These Stat6VT mice may serve as a model for examining lymphoma development. In order to characterize the altered lymphocytes and determine if LPD observed in the Stat6VT mice is characteristic of lymphoma, RT-PCR analysis and Western analysis were done to examine if the presence of Stat6VT alters the expression of the cell cycle genes Gfi-1 and Gfi-1b and if these genes differ in LPD Stat6VT verses control mice. / Department of Biology

No description available.

BACKGROUND: In their lifetime, 42% of men and 38% of women will be diagnosed with cancer. One of the most prominent cancers in women is breast cancer, which represents 14.8% of newly diagnosed cancers. Research has demonstrated the importance of the lysyl oxidase (LOX) gene, more specifically the lysyl oxidase propeptide (LOX-PP), in the attenuation of breast cancer and in mouse xenograft models, making it an important and ongoing area of investigation. In recent, unpublished experiments, mice with a variant of LOX-PP were treated with 7,12-Dimethylbenz[a]anthracene (DMBA) to induce breast cancer. However, some of the treated mice developed poor body conditions without any signs of having developed breast cancer. Published studies have demonstrated that, following treatment with DMBA, some mice generate leukemia or other lymphoproliferative diseases. Research has shown that identification of these diseases is possible via histological analyses or immunohistochemistry. Objective: To determine the cause of poor body conditions in mice treated with DMBA that did not develop mammary tumors by histological analyses and immunohistochemistry. Additionally, to determine the difference between a variant of LOX-PP (LOX-PPv) knock-in (Ki) and wild type (WT) mice in the generation of poor body conditions or leukemia/lymphoproliferative disease. METHODS: A total of 48 mice, 24 LOX-PPv Ki and 24 WT, were treated with DMBA and observed for mammary tumor formation. Body conditions of the mice were observed and noted, and liver samples from these mice were obtained and analyzed via histological and immunohistochemistry interventions. In Hematoxylin & Eosin (H&E) assessments of mice livers, mice were classified as normal or as having possible lymphocyte infiltration, mild lymphocyte infiltration, or massive lymphocyte infiltration. Staining with the Ki-67 antibody in immunohistochemistry allowed for classification of mouse livers as normal or as having slightly positive or massively positive expression of the Ki-67 antigen. The degree to which each liver sample was positive for both readings was compared and further analyzed. RESULTS: A total of 9 mice (30.4% of Ki mice and 8.7% of WT mice) displayed poor body conditions following DMBA treatment. Of the 9 (7 Ki and 2 WT) mice having poor body condition, 2 Ki mice suffered from severe dermatitis. For the 7 remaining mice, no cause for such poor body conditions was obvious. 6 of these mice (85.7%) were diagnosed with leukemia. In all other DMBA-treated mice, 52.6% of Ki and 23.8% of WT displayed some degree of abnormal lymphocyte infiltration in the liver, while a total of 39.1% of the K¬I mice and 69.6% of the WT mice had normal liver histologies. 60.9% of Ki and 45.5% of WT mice displayed some degree of positivity for expression of Ki-67. CONCLUSIONS: Mice not diagnosed with breast cancer but that were suffering from poor body conditions likely had developed leukemia or some other lymphoproliferative disease. In the mice not definitively diagnosed with leukemia by a pathologist, H&E analyses and immunohistochemistry of the livers showed lymphocyte infiltration and cellular proliferation possibly indicative of leukemia., but further tests are necessary to confirm this. Furthermore, LOX-PPv Ki mice appeared to have a greater likelihood of generating poor body conditions or some observable sign of abnormal lymphocyte infiltration in the liver versus their WT counterparts when treated with DMBA. / 2018-06-16T00:00:00Z

<p>Immune complexes (ICs) are produced during normal immune responses and facilitate clearance of foreign antigens. ICs not efficiently cleared from the circulation can cause tissue damage. This might happen if ICs are formed with autoantibodies and autoantigens. Well described effects of ICs are neutralization of antigen, classical complement activation or FcR-mediated phagocytosis, whereas cytokine inducing effects of ICs in human clinical settings are less well described. I have investigated cytokine-inducing properties <i>in vitro</i> of ICs from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and cryoglobulinemia in association with lymphoproliferative diseases. </p><p>Cryoglobulin (CG)-induced cytokine production varied with changes in temperature and ionic strength in parallel to CG precipitation. IgG CG-induced cytokine production was also mediated via FcγIIa on monocytes. Blockade of the complement system, resembling the <i>in vivo</i> situation of complement consumption in CG-associated diseases, increased IgG CG induced IL-10 and decreased TNF-α production. This represents hitherto not described mechanisms for CG-associated inflammation.</p><p>ICs from SLE patients induced IL-10 and IL-6 production from PBMC cultures via FcγRIIa. Occurrence of anti-SSA autoantibodies and signs of <i>in vivo</i> complement activation contributed to increased levels of circulating ICs in SLE patients, corresponding to increased amounts of IC-induced IL-10 <i>in vitro</i>. This represents a possible vicious cycle that might perpetuate antibody dependent pathology in SLE, and put anti-SSA in a new pathological context.</p><p>RF-associated ICs from RA joints and ICs formed with antibodies against collagen type II from RA serum induced pro-inflammatory cytokine production from monocytes via FcγRIIa, showing how specific autoantibodies might induce or perpetuate joint inflammation in RA. </p><p>I have described how ICs can induce significant amounts of pathophysiologically important monocyte-derived cytokines in three major IC-dependent diseases. Blockade of FcγRIIa and suppression of monocytes/macrophages might be a means of reducing pathogenic IC-induced cytokine production in these diseases. </p>

The aim of this thesis was to determine the relevant dose of arbepoetin-alfa (DA) in patients with lymphoproliferative diseases (LPD) and chemotherapy induced anemia (CIA), to study the clinical impact of intravenous (IV) iron supplementation combined with epoetin beta treatment, to identify factors that might predict hemoglobin (Hb) response to treatment with epoetins and to investigate safety of DA. A dose-finding phase II study was able to assess a reasonable DA dose of 2.25 μg/kg once weekly for the treatment of CIA in patients with LPD. Dose-response trends were observed for the different dose cohorts although not statistically significant for any of the endpoints. However a significantly higher proportion of patients achieved Hb response (increase ≥2 g/dL) in the combined DA groups than in placebo (P<0.001). A larger pivotal phase II trial was performed in a similar setting o confirm that the dose 2.25μg/kg once weekly was appropriate and safe. The proportion of patients achieving Hb response was significantly higher in the DA group (60%) than in the placebo group (18%) (P<0,001) and resulted in higher mean changes in Hb than placebo from baseline, 2.66 g/dl versus 0.69 /dl. Also a significantly lower proportion of patients in the DA group (31%) received RBC tranfusions than in the placebo group (48%). The short-term safety of DA with the tested dose was confirmed. The efficacy of DA was consistent for all end points independent of malignancy type or baseline endogenous erythropoietin serum levels. The correction of moderate anemia in truly iron repleted patients with clinically stable LPD not receiving hemotherapy or RBC transfusions with epoetin beta treatment, with or without IV iron treatment was studied in an open label randomized trial. Also the impact on iron kinetics was assessed. The mean change in Hb concentration from baseline to end of treatment (EOT ) was 2.91 versus 1.50 g/dL respectively (P<0.0001). There was a significant (P<0.0001) difference in mean Hb at EOT between the iron and no-iron groups (13.0 g/dL versus 11.8 g/dL). Hb response was achieved by significantly more patients in the iron group (P=0.0012)than in the no-iron group (93% versus 53%) and the median time to achieve a Hb response was 6 weeks in the iron group compared with 12 weeks in the no-iron group. The mean weekly epoetin dose per patient was statistically significant lower in the iron group at week 13 (P =0.029) and at least 25% lower at EOT. To investigate the long-term safety of DA in cancer patients with CIA four previously published double blind, randomized placebo-controlled phase II -III studies were analysed (n = 1.129). Median durations of progression-free survival and overall survival was comparable between DA and placebo for lung cancer (median follow up 15.8 months), for LPD (median follow up 32.6 months) and in the pooled population (follow up 4 months).

Immune complexes (ICs) are produced during normal immune responses and facilitate clearance of foreign antigens. ICs not efficiently cleared from the circulation can cause tissue damage. This might happen if ICs are formed with autoantibodies and autoantigens. Well described effects of ICs are neutralization of antigen, classical complement activation or FcR-mediated phagocytosis, whereas cytokine inducing effects of ICs in human clinical settings are less well described. I have investigated cytokine-inducing properties in vitro of ICs from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and cryoglobulinemia in association with lymphoproliferative diseases. Cryoglobulin (CG)-induced cytokine production varied with changes in temperature and ionic strength in parallel to CG precipitation. IgG CG-induced cytokine production was also mediated via FcγIIa on monocytes. Blockade of the complement system, resembling the in vivo situation of complement consumption in CG-associated diseases, increased IgG CG induced IL-10 and decreased TNF-α production. This represents hitherto not described mechanisms for CG-associated inflammation. ICs from SLE patients induced IL-10 and IL-6 production from PBMC cultures via FcγRIIa. Occurrence of anti-SSA autoantibodies and signs of in vivo complement activation contributed to increased levels of circulating ICs in SLE patients, corresponding to increased amounts of IC-induced IL-10 in vitro. This represents a possible vicious cycle that might perpetuate antibody dependent pathology in SLE, and put anti-SSA in a new pathological context. RF-associated ICs from RA joints and ICs formed with antibodies against collagen type II from RA serum induced pro-inflammatory cytokine production from monocytes via FcγRIIa, showing how specific autoantibodies might induce or perpetuate joint inflammation in RA. I have described how ICs can induce significant amounts of pathophysiologically important monocyte-derived cytokines in three major IC-dependent diseases. Blockade of FcγRIIa and suppression of monocytes/macrophages might be a means of reducing pathogenic IC-induced cytokine production in these diseases.

Stat6 is a protein that activates the transcription of IL-4-stimulated genes. Amino acids critical for Stat6 function were examined in a mutational analysis of the Src homology (SH2) domain of the Stat6 protein. One mutation, substitution of two Alanines for Valine and Threonine in the N-terminal portion of the SH2 domain, produced a constitutively active form of the molecule that did not require IL-4 for activation. This mutant was named Stat6VT. Mice expressing Stat6VT in lymphocytes were generated, and it was found that approximately 10% of the population of Stat6VT mice, a lympho-proliferative disorder (LPD) occurred. In this study, we are examining genes that have a possible role in the development of this proliferative condition. Specifically, we examined the expression levels of Tiam1, Tacstdl, and Gfi-1 and Gfi-1B (genes known to regulate cellular proliferation and survival) in wildtype, normal Stat6VT and Stat6VT/LPD splenocytes by RT-PCR. Tiam1 results were inconclusive, and Tacstdl was not expressed at levels different from those seen in controls. Interestingly, Gfi-1 B, the homolog of Gfi-1, was expressed at increased levels in a specific subpopulation of cells from Stat6VT/LPD mice. Taken together, these data suggest that in cells expressing a constitutively active Stat6, increased expression of Gfi-1B may play a role in the mechanism of lymphoma development. / Department of Biology

No description available.

Ovine herpesvirus 2 (OvHV-2) is a gamma herpesvirus and is the causative agent of lymphoproliferative disease – sheep-associated malignant catarrhal fever in susceptible ruminants, including cattle. Sheep become persistently infected but do not show apparent clinical infection. MCF is characterized by marked T cell hyperplasia and proliferation of unrestricted cytotoxic large granular lymphocytes (LGLs) which leads to necrosis of infiltrated tissues and generally causes death of the host. Little is known about the underlying molecular basis of MCF pathogenesis or what controls the differences in clinical outcome of infection in two closely-related host species. MicroRNAs (miRNAs) constitute a large family of small, ~22nt, noncoding RNA molecules that regulate gene expression by targeting messenger RNAs post-transcriptianally in eukaryotes and viruses. Herpesvirus encoded miRNAs have been shown to play a role in regulating viral and cellular processes including cell cycle and may have a role in pathogenesis. OvHV-2 has also been found to encode for at least 46 OvHV-2 miRNAs in an immortalized bovine LGL cell line. 23 of these miRNAs have also been validated by northern blot analysis and RT qPCR. It was hypothesised that these OvHV-2 miRNAs may regulate viral and cellular genes expression and may play a role in MCF pathogenesis. The aim of this project was to determine if OvHV-2 miRNAs have functional targets within viral and host cell genes. Bio-informatic analysis has predicted several targets for these OvHV2 miRNAs in the 5’ and 3’ UTRs of several virus genes. Luciferase inhibition assay confirmed that out of 13 selected predicted targets, three (two targets ORF73 and one within ORF50) were positive and functional. A fourth predicted target was also found functional (ORF20), but its functionality could not be confirmed by knocking out the target site. A newly developed technique Crosslinking, Ligation And Sequencing of Hybrids (CLASH) was also used to identify miRNAs bound targets within cattle and sheep genome. High throughput sequencing and analysis of the hybrid data revealed many target genes. Four of those targeted genes, were validated by luciferase inhibition assays and three were found to be targeted by OvHV-2 miRNAs. This study gives the first evidence of viral miRNAs bound to their targets in cattle and sheep cells, by a highly sensitive technique-CLASH and provides a tool for studying differences in pathogenesis of two closely-related host species.