Search

Regulatory T cells (Tregs) are key players in controlling immune responses, limiting autoimmune disease and allergies, and attenuating immune responses to tumours and pathogens. Understanding and harnessing the suppressive effects of Tregs in autologous and allogeneic haematopoietic progenitor cell (HPC) transplantation presents a significant challenge due to lack of consensus over optimal markers to uniquely identify Tregs and variation in centre-specific factors including disease mix, conditioning regimens, graft origin and manipulation and prophylaxis and treatment of graft versus host disease (GVHD). This study aimed to determine if CD3+CD4+CD125highCD127lowFoxP3+ Treg quantification, assessed flow cytometrically, in grafts or in the post-transplant peripheral blood of patients who received transplants for malignant disease, could provide a useful predictor for disease relapse in autologous (n=85) and allogeneic patients (n=75) and falling chimerism and/or incidence of GVHD in the latter group. The impact of Treg numbers were quantified in HPC harvests, in transplant grafts and in recipients’ peripheral blood during immune reconstitution. Additionally, a simplified Treg assessment protocol using the marker tumour necrosis factor receptor-2 (TNFR2) with CD3, CD4 and CD25 was assessed. In autologous donors, significantly higher Tregs relative to CD34 HPCs were noted in harvests mobilised with the more novel regimen, granulocyte-colony stimulating factor (G-CSF) plus Plerixafor than with G-CSF alone or used in combination with cyclophosphamide. In allogeneic harvests Treg numbers following G-CSF mobilisation were significantly lower than in non-mobilised harvests. Lower absolute Treg numbers in donor lymphocyte infusion (DLI) doses were significantly associated with successful outcome in terms of restoration of donor chimerism and resolution of relapse. Cryopreservation of mobilised cells at the time of initial transplant for later use for DLI has thus been incorporated into practice at this Trust as this is expedient in terms of clinical result, convenience and cost. Interestingly although mobilisation regimens influenced Treg levels in harvests, no correlation was apparent between Treg doses transplanted or peripheral blood levels during immune reconstitution post autologous or allogeneic transplantation or with falling chimerism and/or incidence and severity of GVHD in allogeneic patients during the first year post transplant. Extending this follow-up time would be an interesting area of further study as the majority of patients who relapse do so beyond one year.

AimThe broad aim of this thesis was to explore the risk of malignancy in people with rheumatoid arthritis (RA), treated with anti-tumour necrosis factor (TNF) drugs.MethodsThis thesis used data from patients with RA registered with the British Society of Rheumatology Biologics Register-RA. The risk of cancer in biologic-naive patients treated with traditional disease modifying drugs (nbDMARD) was compared to that in the general population by calculating standardised incidence ratios (SIR). The influence of anti-TNF on cancer risk was then explored by comparing the risk in the anti-TNF cohort to that in the nbDMARD cohort using Cox proportional hazard models.ResultsThe risk of cancer was increased in the nbDMARD cohort by 28% compared to the general population (SIR 1.28, 95% confidence interval (CI) 1.10, 1.48). Risks of lung cancer (SIR 2.39, 95% CI 1.75, 3.19), Hodgkin lymphoma (SIR 12.82, 95% CI 4.16, 29.92) and Non-Hodgkin Lymphoma (SIR 3.12, 95% CI 1.79, 5.07) were increased compared to the general population and both prostate cancer and cancers of the female genital organs reduced; SIRs 0.35 (95% CI 0.11, 0.82) and 0.35 (95% CI 0.10, 0.90) respectively. There was no difference in the risk of cancer in patients treated with anti-TNF compared to nbDMARD, after adjusting for differences in baseline characteristics; Hazard ratio for lymphoma: 1.00 (95% CI 0.49, 2.05); cancers of the solid organs: 0.83 (95% CI 0.64, 1.07); and keratinocyte skin cancer: basal cell carcinoma 1.06 (95% CI 0.64, 1.75), squamous cell carcinoma 1.62 (95% CI 0.44, 5.90). ConclusionsSubjects with RA, treated with nbDMARD were at increased risk of cancer compared to the general population. In particular, lung cancer, lymphoma and KSC were increased. Treatment with the TNF inhibitors ETA, INF or ADA was not associated with a difference in relative risk of lymphoma, solid cancer or skin cancers when compared to nbDMARD.

Many haematological malignancies are associated with widespread alterations of the transcriptional and epigenetic programmes. Changes in DNA methylation provide the clearest example of epigenetic changes, but the mechanism(s) underlying such changes is unknown. To investigate this I studied DNA methylation across an ~80kb segment of the genome which is not known to be mutated in haematological malignancies. Methylation was perturbed in 35-100% of samples of DNA from individuals with a wide range of haematological malignancies but not in non-malignant haematological disorders. DNA methylation was comprehensively assessed by Southern blot analysis, classical bisulphite sequencing and using a newly developed capture bisulphite sequencing protocol. The results were also compared with analysis by MeDIP, an immunoprecipitation-based technique. These analyses provide methylation status at various levels including individual CpG resolution. This showed both gain and loss of methylation at CpG dinucleotides. Of interest, hypomethylation was most frequently seen in intergenic regions corresponding to transcription factor binding sites and areas of increased chromosome accessibility. These observations suggested that hypomethylation of the genome in haematological malignancies could arise from aberrantly expressed DNA binding proteins which, recruited to sequences in regions of open chromatin, would protect the underlying CpG dinucleotides from the methylation machinery. This, in turn, could lead to passive demethylation accumulating with increasing cell divisions. This hypothesis was tested with electrophoretic mobility shift assays using oligonucleotides representing the DNA underlying one such region. This showed that, compared to nuclear extracts from the lymphocytes of normal individuals, those from patients with CLL were enriched for a protein which binds to oligonucleotides containing the underlying sequence. Using a mass spectrometry approach, I identified a variety of proteins that may bind such regions and account for their passive demethylation in haematological malignancies.

Aims: To elucidate the aetiopathological mechanisms underlying the IIMs, through a combination of genotyping, serotyping and clinical phenotyping in a large cohort of Caucasian idiopathic inflammatory myopathy (IIM) patients. Methods: A cross-sectional study of prevalent IIM cases, ascertained through the Adult Onset Myositis Immunogenetic Collaboration, was performed. Cases were confirmed as possessing myositis according to Bohan and Peter (Bohan and Peter 1975a; Bohan and Peter 1975b). IIM clinical subtypes studied included polymyositis (PM), dermatomyositis (DM) and myositis associated with other connective tissue disease (myositis/CTD-overlap). Genotyping of major histocompatibility complex genes, including HLA-B, -DR, -DQ, tumour necrosis factor alpha (TNF-α), was performed using commercial kits. Serotyping of a comprehensive range of myositis specific/associated antibodies (MSA/MAAs) was undertaken. Results: Clinical subsets are described within the serological groupings, suggesting that the classification of the IIMs appears to be better served by the serotype than by the clinical subgrouping of disease. The IIMs possess HLA class I and II haplotype associations and genetic differences observed between PM and DM are accounted for by serological differences. The TNF-308A association is not independent of HLA class I, due to the strong LD within the MHC, but does form part of a haplotype with these factors. An absence of routinely tested for MSA/MAAs makes cancer associated myositis (CAM) more likely, especially in the DM subgroup. An antibody against a 155 and 140kDa doublet is associated with the development of CAM. Outcome measures in the IIMs show construct validity. HLA-DRB1*07 appears to predict a milder clinical phenotype with less disability. No convincing gene-environmental interaction was found capable of altering disease susceptibility or clinical phenotype. Conclusions: Myositis disease subtypes therefore appear to be defined by specific haplotypes acting as risk factors for the development of various MSAs and MAAs.

All epithelia are characterized by keratins, which make up a type of intermediate filament (IF). In epithelial tumors, which account for the majority of clinical cancers, the loss of cytoskeletal integration is considered one of the first alterations in epithelial metaplasia. This may have something to do with the expression of keratins or rearrangement of keratin filaments. In this study, I employed shotgun proteomic analysis and bioinformatic tools to identify proteins that interact with keratin filaments and thus may contribute to the disintegration of cytoskeleton. Using four airway epithelial cell lines in culture, I confirmed they highly expressed Keratin 14 (K14) and its obligatory partners, Keratin 5 (K5) or Keratin 6A (K6A). This suggests that the predominant IF is made up of K14 paired with K5/K6A. Although samples were enriched in keratin-associated proteins by immunoprecipitation (IP) with an antibody directed against K14 and K17, additional keratins not specifically targeted were also captured. Proteomic analysis revealed a list of non-keratin proteins enriched by IP. Some were associated with actin and microtubules, 23 and 6 proteins, respectively. Most of these were not linearly related to keratin content by abundance, but the motor protein, dynein I heavy chain, showed a Pearson correlation coefficient (CC) of -0.84 with keratin. Similarly, of 54 proteins associated with focal adhesions, intercellular junctions, or membranes, only septin-9 had a CC suggesting its abundance tracked with that of keratins. Finally, I analyzed IP-specific proteins that were cytosolic or had unknown subcellular distribution. A CC of -0.91 was found for one of these proteins, namely 26S proteasome regulatory subunit 8 (Psmc5). Further investigation and validation of the dataset was done by GO Enrichment Analysis. Using a subset of proteins highly concentrated by IP, compared to controls, I found the GO functions predicted were intracellular transport, translation initiation, epithelial cell migration, and filopodium assembly. This result suggests that the method may have value for unraveling the functions of the proteins and the pathways in which they operate. Integration of proteomic analysis and bioinformatic approaches may have promise for unraveling the molecular mechanisms underlying disintegration of cytoskeletal integrity in highly malignant cells.

Background: Mucinous cysts of the pancreas (MCN) are infrequent, usually unilocular tumors which occur in postmenopausal women and are located in the pancreatic body/tail. The risk of malignancy is low. The objective is to define preoperative risk factors of malignancy in pancreatic MCN and to assess the feasibility of the laparoscopic approach. Methods: Retrospective multicenter observational study of prospectively recorded data regarding distal pancreatectomies was carried out at seven HPB Units between 01/01/08 and 31/12/18 (the ERPANDIS Project). Results: Four hundred and forty-four distal pancreatectomies were recorded including 47 MCN (10.6%). Thirty-five were non-invasive tumors (74.5%). In all, 93% of patients were female, and 60% were ASA (American Society of Anaesthesiology) II. The mean preoperative size was 46 mm. Patients with invasive tumors were older (54 vs. 63 years). Invasive tumors were larger (6 vs. 4 cm), although the difference was not significant (P=0.287). Sixty percent was operated via laparoscopic approach, which was used in 74.6% of non-invasive tumors and in 16.7% of the invasive ones. The spleen was not preserved in 93.6% of the patients. R0 resection was obtained in all patients. Two patients with invasive tumors died. Conclusions: In our surgical series of MCN, patients with malignancy were older and presented larger tumors, although the difference was not statistically significant. Laparoscopy is a safe and feasible approach for MCN. Prospective studies are now needed to define risk factors that can guide the decision whether to administer conservative treatment or to operate.

Delayed identification and treatment of septic shock is associated with increased mortality. A retrospective chart review was performed on patients admitted with hematologic malignancies in 2010 (188 control patients and 7 patients that developed septic shock). A mixed effects multivariate logistic regression model was used to determine if typical clinical biomarkers (vitals signs and routine laboratory studies) could be used to predict the development of septic shock in patients with hematologic malignancies prior to transfer to the intensive care unit. While routine vital signs could be used to differentiate control patients from patients that developed septic shock even at 4 hours prior to intensive care transfer (AUC of 0.967), routine laboratory studies performed significantly worse (AUC of 0.761).

Hypercoagulable states can be detected by measuring activation peptides, enzyme-inhibitor complexes, and fibrin/fibrinogen degradation products, which are markers of hemostatic activation. A series of these prethrombotic markers has been evaluated in the elderly, pregnancy, diabetes and acute myocardial infarction patients (n=30 in each group) as well as in hematologic malignancies (n=42). The parameters assayed were: prothrombin fragment 1+2 (F1+2), thrombin-antithrombin III complexes (TAT), fibrinopeptide A (FPA), plasmin-alpha2 antiplasmin complexes (PAP) and D-Dimer. Results were compared with those obtained in a group of 30 healthy subjects. We found a significant increase of F1+2, TAT and FPA in elderly (p

Different coagulation and fibrinolysis parameters were investigated in 149 patients with metastatic and non-metastatic tumours and results were compared with those obtained in a healthy population. Results showed a significant increase of thrombin-antithrombin complexes, fibrinopeptide A (FPA) and fibrin monomers in the group of patients (p less than 0.001). There was also a significant prolongation of euglobulin lysis time (p less than 0.005) and an increase of plasminogen activator inhibitor activity (p less than 0.0001), fibrinogen degradation products (p less than 0.001), and D-dimer (p less than 0.05) in the group of patients as compared to controls; FPA levels were also increased in patients with metastases (p less than 0.005). This study demonstrates clotting activation, at the level of fibrinogen to fibrin conversion, and impairment of fibrinolysis in patients with malignancy.

This investigation was concerned with characterizing effects of this tumor line on lipid metabolism in DBA/lJ mice and serum protein levels and cellular changes in DBA/lJ and Swiss white mice. Total lipids, lipid phosphorus, neutral lipids, and changes in fatty acids were determined in liver, spleen, skin, and tumor of DBA/lJ mice bearing the lymphosarcoma at various days after injection of tumor cells.