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Thesis (Ph.D.)--Boston University / The purpose of this investigation was to study the metabolism of normal human leukocytes. Leukocytes were incubated in vitro with c14-labeled acetate, and the fate of the radioactive carbon was determined by fractionation and analysis of the major, cell components. Leukocytes were obtained from whole blood by fibrinogen sedimentation and differential centrifugation. Optimal conditions for isolation and incubation of viable cells were developed and assessed using phase microscopy for direct observation of their morphological integrity and by their oxidative metabolism. Control experiments to determine the possible effect of erythrocyte utilization of acetate were run using twice the number of cells ordinarily found in leukocyte suspensions. No utilization of acetate by red blood cells was observed. Respiration studies were performed using standard Warburg manometry. Otherwise, incubations were carried out in modified Erlenmeyer flasks equipped with center wells for C02 collection and stoppered with serum bottle caps. Flasks were equilibrated and various additions were made to the suspensions using hypodermic needles. Total cells per flask varied from 1 to 5 X 108 with concentrations ranging from 60 to 80 X 106 cells per ml. of suspension. Response of respiration and combustion of acetate were directly proportional to cell number and no detrimental effects due to cell crowding were detectable within this range. [TRUNCATED]

The genetic relationship between the serologically detectable antigens of the HLA-A and B loci and the mixed lymphocyte culture (MLC) reaction is of utmost importance in the understanding of histocompatibility. From the literature it appeared that neither the HLA-A, B genotype nor the MLC reaction is the complete answer to donorrecipient selection in organ transplantation. This study was therefore initiated in June 1971 in an attempt to clarify the problem. It is inherent in a project of this nature that much time is spent in collecting samples from donors in diverse areas. Most experiments had to be repeated two or three times, therefore some of the problems we set out to resolve were clarified by other workers before we were able to complete our investigations.

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Stroke is the third most common leading cause of death worldwide and is a major cause of serious long-term disability among adults. Platelet activation and it¡¦s interaction with leukocytes plays an important role in the pathophysiology of ischemic stroke. Anti-platelet drugs are widely used for secondary prevention after cerebral ischemia of non-cardioembolic origin and different anti-platelet drugs exert different pharmacologic effects on platelets. Several stimulations cause elevation of cytosolic Ca2+ level ([Ca2+]i), activating the secondary messenger in the platelet, and then leading to platelet activation. The majority of damage following acute stroke does not occur immediately, but rather develops gradually over the course of the following hours. Leukocytes are believed to liberate inflammatory cytokines and other neurotoxins in the ischemic brain and to promote microvascular occlusion through platelet-leukocyte-endothelium interactions in the ischemic penumbra. In this thesis, we tested the serial changes of platelet [Ca2+]i movement in patients after acute ischemic stroke. We evaluated platelet activation markers, leukocyte adhesion molecules and platelet-leukocyte interaction in patients with acute ischemic stroke. We also analyzed the relationship between these biomarkers and the clinical outcome. Furthermore, we compared the antiplatelet effect of aspirin and clopidogrel in patients after acute stroke Dysregulation of Ca2+ Movement in Platelets from Patients with Acute Ischemic Stroke Thirty-one patients with acute ischemic stroke and 27 at-risk controls were enrolled in this study. The platelet [Ca2+]i was measured using a fluorescent dye fura-2 after stimulation with arachidonic acid (AA), adenosine diphosphate (ADP), platelet-activation factor (PAF), and thrombin. The basal [Ca2+]i was higher in the stroke group than in the at-risk controls irrespective of the presence or absence of extracellular Ca2+. In the Ca2+-containing medium, both PAF and ADP, but not AA and thrombin, significantly increased the platelet [Ca2+]i in the stroke patients than in the at-risk controls. However, in the Ca2+-free medium, only PAF significantly increased the platelet [Ca2+]i in the stroke patients than in the at-risk controls. The basal [Ca2+]i and PAF-induced platelet [Ca2+]i rises were still higher in the stroke patients at the subacute stage than in the at-risk controls. Levels and Value of Platelet Activation Markers in Different Subtypes of Acute Non-Cardio-Embolic Ischemic Stroke Platelet activation markers (CD62P, CD63, and CD40L) and platelet-leukocyte interaction were measured by flow cytometry at different time points in 54 (32 small-vessel and 22 large-vessel diseases) acute ischemic stroke patients, 28 convalescent stroke patients (3 to 9 months after acute stroke), and 28 control subjects. Patients with ischemic stroke had significantly increased circulating CD62P, CD63, platelet-monocyte interaction, and platelet-lymphocyte interaction in the acute stage compared with the convalescent stage and control groups. Levels of CD62P and CD63 were significantly higher in the large-vessel disease group than in the small-vessel disease group, and differences in CD62P were significant even at one month. The CD40L level in the poor outcome group was significantly higher than that in the good outcome group. The Value of Leukocyte Adhesion Molecules in Patients after Ischemic Stroke We examined serially the change of PSGL-1, Mac-1, and LFA-1 expression on leukocytes by using flow cytometry at various time points in 65 acute ischemic stroke patients and 60 controls. PSGL-1 expression on neutrophils and monocytes was significantly higher from Day 1 to Day 90 after stroke as compared with control subjects. The expression of monocyte Mac-1, LFA-1, and neutrophil Mac-1 were more significantly increased on Day 1 and 7 after stroke as compared with the control subjects. Furthermore, the neutrophil PSGL-1 expression on admission was independently associated with early neurologic deterioration. Serial Changes in Platelet Activation Markers with Aspirin and Clopidogrel after Acute Ischemic Stroke We designed a prospectively randomized case-control study and 70 patients with non-cardioembolic stroke who were treated with either aspirin (100 mg/d) or clopidogrel (75 mg/d) after acute ischemic stroke were evaluated. Ischemic stroke patients had significantly increased circulating CD62P, CD63, and CD40L in the acute stage as compared to the control group. Levels of CD62P, CD63 and CD40L were more significantly reduced in the clopidogrel group than in the aspirin group in the first week after stroke. Furthermore, differences in CD62P and CD63 levels were significant even at one-month post-stroke. Conclusion Stroke patients have enhanced platelet activity and platelet-leukocyte interaction in acute and convalescent phase of ischemic stroke compared with controls. Large-vessel cerebral infarction elicits higher platelets activation than small-vessel infarction in the acute phase of stroke. The dysregulation of Ca2+ movement, through the PAF- and ADP- receptor mediated pathway, in platelets may persist up to the subacute stage of ischemic stroke. It is possible that clopidogrel, an ADP-receptor inhibitor, elicit stronger antiplatelet effect than aspirin in the acute and convalescent phases after ischemic stroke. Sustained leukocyte-endothelium interaction, as reflected by expression of Mac-1 and LFA-1 on circulating leukocytes, may cause substantial inflammatory reaction and lead to secondary injury of potentially salvageable neurons after cerebral infarction. Ischemic stroke patients presenting with a higher CD40L level on admission were associated with a 3-month poor outcome.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 74-80).

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