Morphometric studies on the pathology of chronic airways obstruction

The work submitted in this thesis consists of seven individual and inter-related studies, all of which are concerned with aspects of the pathology of chronic airways obstruction. This is a disease complex made up of pulmonary emphysema, chronic bronchitis and bronchiolitis. In all the studies quantitation of the extent and severity of the disease processes was carried out. In the first study the incidence of pulmonary emphysema was established in a series of 50 autopsies on men, performed in a general hospital in Glasgow. Emphysema was present in more than trace amounts in 32 lungs (64%). Centrilobular emphysema was the commonest variety found, being the only or predominant type present in 17 lungs (34%), panlobular emphysema was the only or predominant type in 5 lungs (10%) and in 15 lungs (30%) significant amounts of both centrilobular and panlobular emphysema were present. These results were compared to the published incidences of emphysema in other British industrial centres. Secondly the incidence of bronchial mucous gland hypertrophy, consistent with chronic bronchitis, was ascertained in 359 consecutive autopsies performed in the same Glasgow hospital. Hypertrophy of the bronchial mucous glands was measured by two techniques which showed good correlation. Using the Reid Index 31 per cent of bronchi showed mucous gland hypertrophy in the chronic bronchitis range as compared to 33 per cent by a point counting technique. Changes of this degree were commoner in men than women, but were not related to age or smoking habits. The mean Reid Index was significantly greater in smokers (0.46 +/- 0.11) than non-smokers (0.41+/- 0.09) and there was a significant relationship between the presence of a chronic productive cough during life and the size of the bronchial mucous glands at autopsy. The third paper involved a comparison of the changes in the lungs and heart of patients dying from chronic airways obstruction and those with non-fatal chest disease. A series of 50 autopsies was performed in Sheffield, to determine the size of the right ventricle, the amount, type and distribution of emphysema, the size of the bronchial mucous glands and the proportion of the lung occupied by the lumen of small airways of less than 2 mm. in diameter. Eighteen patients died as a result of chronic airways obstruction, 17 had symptoms of chest disease but died from some unrelated cause and 15 had no symptoms related to the respiratory system. A positive correlation was found between the total amount of emphysema, the amount of panlobular emphysema, the reduction in small airways lumen and both the clinical severity of disease and the weight of the right ventricle. No relationship was found between clinical severity and the amount of centrilobular emphysema or the bronchial mucous gland size. The fourth project was concerned with the changes in the pulmonary arterioles in the same 50 cases. The number of thick-walled peripheral lung vessels (defined as vessels less than 100 u in diameter, which had two distinct elastic laminae) was calculated in all the cases. The mean number of these vessels was found to be significantly greater both in the fatal disease group, as compared to both other groups, and in cases with right ventricular hypertrophy as compared to those with normal hearts. Significant correlations were found between the number of thick-walled vessels and the right ventricular weight, the total amount of emphysema, the amounts of centrilobular and panlobular emphysema and the proportion of the lung occupied by the lumen of small airways. The fifth study involved the examination of small airway changes in chronic airways obstruction by a radiographic method. (Abstract shortened by ProQuest.)

Chronic bronchitis in the dog

Abstract Not Provided

New formats for affinity selection of human cells

Despite recent advances in stem cell biology, immunotherapy and transplantation, substantial barriers still exist in the large-scale specific separation of a discrete population of human therapeutic cells from a cell suspension. The ideal purification technique should combine high cell purity, yield and function, with fast processing and affordability. Currently, fluorescence-activated cell sorting with flow cytometry (FACS) and magnetic activated cell sorting (MACS®) are the most used methods for cell separation and purification and have been employed extensively in molecular biology, diagnostic and cell sorting applications, because they are considered to be gentle, fast and scalable. However, these methods have several key disadvantages; they are invariably expensive, yield low log cell reduction (LCR) rates, and suffer from drawbacks when applied to niche cell populations, such as those requiring multiple tandem separation steps and/or involving combined positive and negative cell selection steps. To address this challenge, a new cell affinity selection system was developed. The selectivity is based on the reversible monomeric avidin biotin interaction and it is primary designed for positive selection. The initial studies were performed on flat, nonporous, glass coverslips and the technology was then successfully transferred on high grade smooth non-porous glass beads (with a diameter of 79.12 to 118.59 μm). The multi-step layer-by-layer deposition procedure culminating in dextran-coated supports bearing monomeric avidin was rigorously characterized and subsequently employed in packed bed chromatography experiments with human erythrocytes isolated from cord blood and B lymphocytes from cell lines. The developed affinity selection platform was highly selective, efficient and, most importantly, resulted in high yields, cell purity and viability comparable with MACS® technology. Additionally scale up is possible and could be easily transferred to another chromatographic matrix with the appropriate structure

EGFR Regulation of Epidermal Barrier Function

Keratinocyte terminal differentiation is the process that ultimately forms the epidermal barrier that is essential for mammals to survive in the ex utero environment. This process is tightly controlled by the expression of many well-characterized genes. Although a few of these genes are known to be regulated by the epidermal growth factor receptor (EGFR), an important regulator of multiple epidermal functions, neither the genome-wide scale of EGFR-mediated regulation nor the mechanisms by which EGFR signaling controls keratinocyte differentiation are well understood. Using microarray analysis we identified 2,676 genes that are regulated by EGF, a ligand of the EGFR. We further discovered, and separately confirmed by functional assays, that EGFR activation abrogates all essential metabolic processes of keratinocyte differentiation by (1) decreasing the expression of lipid matrix biosynthetic enzymes, (2) regulating numerous genes forming the cornified envelope, and (3) suppressing the expression of tight junction proteins. In organotypic cultures of skin, the collective effect of EGF impaired epidermal barrier integrity, evidenced by increased transepidermal water loss. As defective epidermal differentiation and disruption of the epidermal barrier are primary features of many human skin diseases, we used bioinformatics analysis to identify genes that are known to be associated with human skin diseases. In comparison to non-EGF-regulated genes, the EGF-regulated gene list was significantly enriched for disease genes. Further validation of the expression profiles of many of the 114 identified skin disease genes included the transcription factors GATA binding protein 3 (GATA3) and Kruppel-like factor 4 (KLF4), both required for establishing the barrier function of the skin in developing mice. These results provide a new systems level understanding of the actions of EGFR signaling to inhibit keratinocyte differentiation. As the overall effect of this inhibition is to impair epidermal barrier integrity, this study clarifies how dysregulation of the EGFR and its ligands may contribute to diseases of the skin

The dermal delayed type hypersensitivity reaction in sheep naturally infected with maedi-visna virus

Maedi Visna virus (MVV) is the prototype lentivirus, capable of infecting cells of the monocyte/macrophage series in sheep. Infection is associated with pathological changes characterised by dysfunction of the cell mediated immune system. The work described in this thesis was undertaken to study in vivo cell mediated immune function in sheep infected with MVV using the tuberculin driven delayed type hypersensitivity (DTH) reaction as the experimental system.The gross and immunohistological characteristics of the DTH were firstly evaluated in control sheep (CD4+, CD8+, y8, and B lymphocytes, macrophages and MHC class II expression). Grossly, the reaction consisted of an indurative plaque, maximal in size at 48-72 hours post challenge. Histologically, there was an early infiltrate of polymorphonuclear neutrophils (PMNs), with a subsequent influx of CD4+ and CD8+ T cells. Macrophages and MHC class II bearing cells left the lesion at the later stages. In comparison, MVV infected sheep exhibited a reduction in the size of the gross DTH which was significantly associated with a decreased density of PMNs and CD4+ cells in the early reaction, but not with the degree of classical pathological change evaluated at subsequent post mortem or the presence of viral RNA in the skin.Depletion of circulating PMNs using cytotoxic drugs in control sheep resulted in the depression in the size of the DTH and a reduced influx of CD4+ cells, confirming the importance of PMNs in the development of the DTH lesion.The migratory ability of PMNs and CD4+ cells to the sites of dermally injected proinflammatory mediators (IL-8, TNF-a, and zymosan activated plasma) was subsequently shown to similar in control and MVV infected sheep.Evaluation of the levels of circulating anti-PPD antibodies in the MVV infected and control groups provided evidence for a negative association between antibody levels and DTH size suggesting a switch to a Th2 type response in the MVV infected sheepFinally, the presence of cytokine mRNA (IL-10, TNF-a, IFN-y and IL2-R) in the early lesions of both groups was assessed using reverse transcription polymerase chain reaction (RT-PCR) technology. This indicated that the mRNA expression pattern of these cytokines was not significantly different in control and MVV infected sheep.In conclusion, the work has shown a significant depression of the DTH response in sheep infected with MVV. This depression is associated with decreased density of PMNs and CD4+ cells in the early reaction, but is not associated with abnormalities in the trafficking of these cells to dermally injected proinflammatory mediators, the presence of antibody/antigen complexes, the presence of viral RNA, or difference in the cytokine mRNA production profile. The importance of the PMN in the DTH has been indicated, and a defect in the in vivo immune response of the PMN in MVV infected sheep has been described

Investigation of the Mechanism of the Beneficial Effect of Blood Transfusion on Rat Renal Allograft Survival

In the rat renal allograft model, pre-operative administration of donor strain blood is sufficient to produce long-term renal allograft survival even in the absence of adjunctive immunosuppression. The precise mechanisms underlying this enhancing effect are unclear but possible mechanisms include elimination or clonal deletion of alloreactive lymphocytes, removal of passenger leucocytes, blocking of effector cells by antibody or antigen and the induction of alloantigen-specific suppressor cells. Rejecting rat renal allografts are characterised by a heterogeneous mononuclear cell and it was therefore important to determine whether pre-operative blood transfusion affected the infiltration of allografts. A detailed analysis was made of the pattern and phenotype of mononuclear cells infiltrating rejecting DA strain allografts in unmodified PVG recipients and non-rejecting DA allografts from PVG recipients which had been actively enhanced by injection of 1 ml of DA blood intravenously 7 days before transplantation. Excised grafts were stained with a range of monoclonal antibodies to rat cell surface antigens on day 1, 3, 5 and 7 post transplantation. Paradoxically pre-operative blood transfusion caused accelerated cellular infiltration of the graft and the rapid disappearance of graft interstitial dendritic cells. The phenotype of cells found within both enhanced and rejecting grafts was similar except for a reduced number of CD8+cells and IL-2 receptor positive cells in the enhanced compared with rejecting grafts at day 5 after transplantation. However,there was no concomitant decrease in levels of in vitro cytotoxicity, since graft infiltrating cells and splenocytes from transfused animals had comparable levels of both specific and non-specific cytotoxicity to those found in rejecting animals. Class I and class II MHC antigen expression within enhanced and rejecting grafts was studied since the distribution and density of MHC expression may influence the vulnerability of the graft to host effector responses. Unexpectedly it was found that enhanced grafts underwent an accelerated and extensive induction of both donor class I and class II MHC antigens. This suggested that pre-operative blood transfusion initially causes sensitisation of the recipient resulting in an accelerated immunological response to the allograft, but that this is rapidly and effectively suppressed by immunoregulatory mechanisms. The possibility that humoral factors may play a role in the beneficial effect of blood transfusion on graft survival was then investigated. Because of reports suggesting an association between serum Fc blocking activity and renal allograft survival, Fc blocking activity was measured in the serum of unmodified and transfused PVG recipients of a DA renal allograft. Serum harvested on day 5 from actively enhanced PVG recipients of a DA renal allograft was shown to specifically inhibit erythrocyte-anti body (EA) rosette formation with donor strain, but not third-party strain, splenocytes, while the levels of EA rosette inhibition in day 5 serum from rejecting rats was markedly lower. This FcR-blocking activity was present in enhanced serum fractions, prepared by discontinuous density gradient centrifugation, which corresponded to the 7S peak. Purified IgG prepared from enhanced serum was also found to inhibit EA rosette formation with donor splenocytes, and absorption of the IgG preparations with donor-strain erythrocytes failed to abrogate EA rosette inhibition. Further experiments, in which absorbed IgG from enhanced animals was tested for FcR-blocking activity against splenocytes of defined major histocompatibility complex (MHC) subregion specificities, established that FcR blocking activity was mediated by IgG alloantibodies directed against donor MHC class II antigens. This finding suggested that there might be important differences in the nature and timing of the alloantibody response to a renal allograft in rejecting and enhanced recipients. (Abstract shortened by ProQuest.)

Studies to allow the consistent fabrication of many units of engineered tissue.

Tissue engineering is the regeneration of tissues through the use of cells with the aid of scaffolds. Such in vitro fabrication of tissues is beginning to become an option for the management of patients who have irreversible failure of an organ. There are many technical challenges to overcome before 'off-the-shelf' tissues that represent the translation of scientific discoveries into routine treatments (or millions of patients becomes a reality. To meet this demand, the products must be manufactured reproducibly and in large numbers employing current good tissue practice (cGTP). This thesis takes a whole bioprocess approach and includes: Bioreador design A new and novel methodology is described which potentially allows any tubular organ to be fabricated using an easily scalable liquid based technique suited to automated mass- production. It is novel in that it employs a single multitasking chamber which functions as both a "dynamic former" to create a precise tubular cell/polymer construct and then acts as the core of the bioreactor in which to grow the tissue. Optical coherence tomography (OCT) OCT is an advanced non-invasive high-resolution clinical imaging technique. This thesis describes both its initial application to the engineering of tissue as a means of imaging a construct within its individual bioreactor and also the use of Doppler enhanced OCT to qualitatively and quantitatively image the flow of culture medium through the construct. Polymer scaffold Typically, tissue engineers construct a scaffold and then seed it with cells. This has inherent difficulties including the achievement of homogeneous seeding densities. An alternative is described whereby cells and liquid polymer are mixed prior to scaffold formation. Stem cells Because of the novel approach to construct fabrication, rat smooth muscle cells were used for "range-finding" experiments. Later experiments deployed human adult mesenchymal stem cells (MSCs) which were shown to network within the tubular constructs

An investigation of the relationship between the inflammatory response and outcome in patients with breast cancer

Therefore, the value of a systemic inflammation-based score (Glasgow Prognostic Score, GPS) was evaluated in patients with metastatic breast cancer (n=96). The GPS was constructed as follows; patients with both an elevated C-reactive protein (>10 mg/1) and hypoalbuminaemia (<35g/l) were allocated a score of 2. Patients in whom only one or none of these biochemical abnormalities was present were allocated a score of 1 or 0, respectively. The minimum follow-up was 7 months and the median follow-up of the survivors was 16 months. During this period 51 patients died of their cancer. On multivariate analysis, only the GPS (HR 2.26, 95% CI 1.45-3.52, P<0.001) remained significantly associated with cancer-specific survival. The median survival in these patients was 24 months, 13 months and 1 month for a GPS of 0, 1 and 2, respectively. Therefore, the presence of a systemic inflammatory response, the GPS that based on simple routinely available and well-standardised measurements, appeal's to be a useful independent indicator of poor outcome in patients with metastatic breast cancer. In patients with early-staged invasive disease, the prognostic value of the relationship between the systemic inflammatory response; as evidenced by elevated C-reactive protein and lowered albumin concentrations that were measured prior to surgery, standard clinico-pathologic factors and cancer outcome was examined in 300 patients with operable primary cancer. The results of the studies suggest that the systemic inflammatory response, as measured by GPS, may be a useful tool in the assessment of survival in patients with systemic metastatic breast cancer. Moreover, the host inflammatory responses are closely related to poor tumour differentiation and hormone-negativity, and malignant disease progression in patients with early-staged invasive breast cancer. Furthermore, that the tumour-based cell mediated immune response and pathological factors are subordinate to the systemic factors, such as albumin, in determining survival in patients with primary loco-regional operable disease. Future prognostic studies should be large enough and over adequate follow-up time and should include all potential prognostic factors in the multivariate survival analysis. (Abstract shortened by ProQuest.)