Observations on haemolytic disease

1. The thesis has considered the investigation of haemolytic disease and has discussed observations on seventy-six patients regarding the pathogenesis, natural history and management of excessive red cell destruction in the three conditions that are most commonly encountered in Great Britain, namely autoimmune haemolytic anaemia, symptomatic non-immune haemolytic anaemia and congenital spherocytosis. 2. The clinical features and the diagnostic value of routine examination of the peripheral blood, bone marrow and urine for signs of anaemia, reticulocytosis and acholuric jaundice, the classical indicators of haemolysis, are described in each group. Where applicable, study has been made of the role of serological investigation and of tests of red cell fragility. Since measurements of red cell survival using chromium - 51 as an erythrocyte label have formed a major interest, they have been considered in detail. The technique has been examined for reproducibility, studied in normal subject and various chronic and haematological diseases and compared simultaneously with the Ashby method. It was decided to use the TIC/. (the time in days when 50 per cent of the injected 51Cr has been cleared from the circulation) as the basis for comparison between data. The procedure for estimating the role of the spleen in a haemolytic process by measuring its uptake of radioactivity, due to sequestration of 51Cr labelled red cells, has been widely applied. The spleen sequestration index (the percentage increment in radioactivity over the surface of the spleen between the start of the test and T2Cr) and the spleen /liver ratio at TzCr have been used, but neither adequately describes the implication of the spleen; the former ignores any liver uptake of radioactivity and the latter does not take account of high blood flow through the spleen. Accordingly a new measurement, the Spleen Number, a multiple of the spleen sequestration index and the spleen/ liver ratio at TiCr has been proposed. 3. In the group of patients with autoimmune haemolytic anaemia, apart from two subjects with the cold haemagglutin syndrome, the nature of the red cell antibody has not clearly influenced the course of disease or its response to treatment. Blood transfusion has been used freely without incident; one patient required more than 40 litres in six weeks for survival. All patients received steroid therapy at some stage of their management; the dilemma that arises when response is poor or when the steroid preparation cannot be withdrawn to an acceptable maintenance level has been well illustrated; approximately one third of the patients had major complications from steroid therapy. Splenectomy was recommended in nine of the twenty-three patients; only one of these had a good result from operation while three others had a partial remission. From the data that are available a high Spleen Number (exceeding 300) appears to offer prospect of remission from splenectomy; it is also of interest that high Spleen Numbers were seen in those who had the best remissions from steroid therapy. The course of autoimmune haemolytic anaemia followed three paths; self limiting disease, chronic relapsing disease and fulminating, potentially fatal, disease. Three unique patients were considered in detail. The first died in infancy of a peculiar haemophagocytic reticulosis that had earlier killed two of his three siblings. The second was observed for several years; she was a good example of the chronic relapsing variety of illness and required sterol therapy only during exacerbations of anaemia; the haemolyti process was constantly active and during this time she had two normal pregnancies, in the second of which she was given large doses of prednisone in the first trimester without ill effects; she had two crises of anaemia, one while receiving folic acid supplements and one which was believed to be due to folic acid deficiency. The third patient developed a violent haemolytic process while being treated with prednisone for idiopathic thrombocytopenia, previously cured by splenectomy. Some influence on the condition was achieved by irradiation of the thymus; haemolysis was eventually arrested and the thrombocytopenia corrected by the combined use of the new antineoplastic agents, Actinomycin-C and BW 57 -322 (Imuran). 4. Studies on symptomatic non - immune haemolytic anaemia have been confined to patients with leukaemia, reticulosis and myeloid metaplasia. The usual signs of overt haemo- lysis, reticulocytosis and acholuric jaundice, were found in only one patient, despite markedly reduced red cell survival in many in the group. Neither splenomegaly nor its degree correlated with severity of red cell destruction or with the splenic uptake of 51Cr labelled red cells; all grades of splenic sequestration were found, the Spleen Numbers ranging from 26 to 1056. Irradiation of the spleen caused a valuable reduction in haemolysis in some patients, but this effect was not related to the Spleen Number; the role of this form of treatment has been discussed. Two patients in this group were subjected to autopsy shortly after red cell survival measurement with chromium -51 and it was possible to estimate the content of radioactivity in most body tissues. There was good agreement between the spleen /liver ratio at autopsy and that obtained earlier in vivo at T2Cr. The data from one of the patients suggest that the marrow may be a much more important site of erythroclasis than is generally realised. 5. Congenital spherocytosis was encountered over a wide range of age from the first months of life to sixty -two years. There were all grades of severity and many differen modes of presentation; there was a small incidence of menta defect, developmental anomaly, miscarriages and chronic dermatosis of the legs and this has been described. Only five patients (23 %) had gall stones. One simple point emerged that might be useful in the early steps towards diagnosis; the E.S.R. tends to be low in this condition and very high in autoimmune haemolytic anaemia, the only other haemolytic disorder which is commonly accompanied by sphero- cytosis. Splenectomy has been undertaken in all but four patient who have mild well compensated disease. Removal of the spleen appears to have arrested haemolysis in all but one patient who died in the post -operative period; red cell survival has returned to normal in the eight patients in whom this has been measured. Observations have been made which support the view that the role of the spleen in congenital spherocytosis is to "condition" the red cells and critically increase their spheroidicity and fragility rather than to destroy them; these show improvement in the osmotic fragility of red cells after splenectomy, some delay in the correction of red cell survival after operation and lower Spleen Numbers than have been encountered in some of the patients with acquired disease. Hypocholesterolaemia in untreated patients with congenital spherocytosis has been an incidental finding. The values for plasma cholesterol after the haemolytic process has been arrested by splenectomy are normal. 6. Megaloblastic erythropoiesis has been observed in seven patients, four with autoimmune haemolytic anaemia (one in early pregnancy and one with a previous history of gastroenterostomy) and three with congenital spherocytosis. While the available data suggest that conditioned deficiency of folic acid is most usually implicated and that the giving of folic acid supplements will correct leucopenia, reticulocytopenia and thrombocytopenia, if these be present, three of the patients (including the patient with previous gastro- enterostomy) had low levels of vitamin B12 in the serum

Skylab experiments. Volume 4: Life sciences

The life sciences experiments conducted during Skylab missions are discussed. The general categories of the experiments are as follows: (1) mineral and hormonal balance, (2) hematology and immunology, (3) cardiovascular status, (4) energy expenditure, (5) neurophysiology, and (7) biology. Each experiment within the general category is further identified with respect to the scientific objectives, equipment used, performance, and data to be obtained

Of macrophages and red blood cells; a complex love story

Macrophages tightly control the production and clearance of red blood cells (RBC). During steady state hematopoiesis, approximately 10(10) RBC are produced per hour within erythroblastic islands in humans. In these erythroblastic islands, resident bone marrow macrophages provide erythroblasts with interactions that are essential for erythroid development. New evidence suggests that not only under homeostasis but also under stress conditions, macrophages play an important role in promoting erythropoiesis. Once RBC have matured, these cells remain in circulation for about 120 days. At the end of their life span, RBC are cleared by macrophages residing in the spleen and the liver. Current theories about the removal of senescent RBC and the essential role of macrophages will be discussed as well as the role of macrophages in facilitating the removal of damaged cellular content from the RBC. In this review we will provide an overview on the role of macrophages in the regulation of RBC production, maintenance and clearance. In addition, we will discuss the interactions between these two cell types during transfer of immune complexes and pathogens from RBC to macrophages

Blood protein and platelet interactions on surface engineered biomaterials

Modification of surfaces to improve the thrombo-resistance of a synthetic biomaterial is a vital aspect in the design of haemocompatible surfaces. Recent work suggests a non-haemocompatible surface may ubiquitously adsorb specific plasma proteins that form a proteinacious layer which mediates the adhesion and activation of platelets and rejection of the material and subsequently, the implanted medical device. Currently, apart from surface chemistry and wetability of the surface, preferential adsorption of specific proteins, their exact interaction and the effect of physical and spatial cues from the nano-environment prevents us from acknowledging a general interplay between biomaterials, proteins and platelets. Thus this study aims to investigate the effect of plasma protein adsorption and subsequent platelet interactions on the smooth and nano-patterned commercially used surface coatings such as hydrogenated amorphous carbon (a-C:H), tetrahedral amorphous carbon (ta-C) and titania (TiO2) surfaces. Results have shown a-C:H and ta-C surfaces exhibited increased affinity to fibrinogen than TiO2, while facilitating similar levels of platelet attachment. A-C:H resulted in decreased cellular spreading when compared with ta-C and TiO2, while same level of platelet activation was detected indicating that platelets could exist in their activated state without spreading. When platelet interactions on nano-patterned surfaces (RMS 5-8nm) were compared against flat surfaces, nano-rough surfaces presented with increased levels of platelet attachment as well as its spreading, while similar levels of platelet activation was detected amongst the smooth and rough substrates. Increased levels of platelet adhesion and spreading were positively correlated with increased fibrinogen adsorption, reinforcing the crucial role of fibrinogen in platelet binding but also its possible role in platelet spreading

The influence of space flight on erythrokinetics in man. Space Life Sciences Missions 1 and 2. Experiment E261

The purpose of this contract was to design and conduct experiments that would increase our understanding of the influence of space flight on erythrokinetics and the rapid change that occurs in the red blood cell mass during spaceflight. The experiment designated E261, was flown on Space Life Science missions SLS-1 and SLS-2 (STS 40 and STS 58). Unique features of this experiment included radionuclide tracer studies during flight and frequent in-flight blood samples specifically for the first three or four days of the mission. Plasma volume measurements were made early and late in the missions. Radioactive iron kinetics studies were initiated after one or three days in microgravity since the magnitude of the red blood cell mass decrease dictated that bone marrow production must be decreased very early in the flight. The schedule was designed to study the time course of the changes that occur during spaceflight and to possibly define a mechanism for the rapid reduction in red blood cell mass

Modulation Of Monoamine Transporters By Palmitoylation In Human Health And Disease

The projects discussed in this dissertation outline the physiologic consequences of S-palmitoylation, a lipid-based post-translational modification, on the serotonin and norepinephrine transporters (SERT and NET, respectively). SERT and NET are trans-membrane proteins that belong to the SLC6 family of secondary active transporters. During neuro- and neuro-hormonal transmission, SERT and NET utilize the differential ionic concentrations of the extracellular and intracellular spaces to remove their corresponding substrates (serotonin [5HT] and norepinephrine [NE]) from the extracellular space. Each monoamine modulates distinct and overlapping physiologic functions, with dysregulation driving the pathogenesis of neuro-cognitive, neuro-humoral, sympathetic, cardiac, and vascular diseases. Because of this, these transporters are a target in the development of therapeutics to battle human disease associated with monoamine signaling. Prescription therapeutics that target SERT and NET include methylphenidate (Ritalin), bupropion (Wellbutrin), selective-serotonin (Lexapro, Prozac, Lustral), and serotonin-norepinephrine reuptake inhibitors (Pristiq, Cymbalta). SERT and NET are also targets for drugs of abuse like cocaine (COC), amphetamine (AMPH), methamphetamine (METH), and methylenedioxymethamphetamine (MDMA). As a medical student, I approached my graduate research training from the point-of-view that dysregulated physiology precedes pathophysiology, which can be observed through clinical phenotypes and rectified by intelligent chemo-therapeutic design and selection. More clearly, this is the pathogenesis-to-presentation model of medicine. The experimental design of this dissertation seeks to (1) outline the normal physiologic consequences of S-palmitoylation for SERT and NET, (2) describe how these processes become dysregulated and facilitate disease pathogenesis, and (3) use this knowledge to outline how therapeutics can potentially rectify these pathophysiology’s and re-justify monoamine homeostasis. The first project of this dissertation explores the regulation of SERT by S-palmitoylation and how this process is altered by therapeutic manipulation with escitalopram (Lexapro). Our studies revealed that when SERT-expressing cells are acutely challenged with the irreversible palmitoyl acyl-transferase (DHHC) inhibitor, 2-bromopalmitate (2BP), SERT palmitoylation was reduced in a time-wise fashion without changing surface or total SERT expression. Acute inhibition of SERT palmitoylation decreased SERT Vmax without altering surface expression or relative affinity of the transporter for 5HT (Km). This suggests that palmitoylation regulates SERT acutely by adjusting SERT kinetics without altering levels of SERT at the cellular surface. In longer time intervals with higher 2BP concentrations, inhibition of SERT palmitoylation promoted a loss in SERT surface density and total protein, suggesting that palmitoylation is involved in trafficking of SERT through cell surface recruitment or endocytosis, dependent on SERT’s state-of-palmitoylation. Additionally, palmitoylation may prevent a loss of total SERT protein by opposing lysosomal degradation or supporting biogenesis. When treated with escitalopram, SERT palmitoylation was reduced alongside 2BP inhibition. These results correlate with losses in surface SERT and 5HT uptake under the same conditions, suggesting that escitalopram may bind and configure SERT to a conformation that discourages palmitoylation, leading to internalization and downregulation of SERT activity. The second project explored the pathogenesis of autism and how escitalopram may be efficacious in its treatment. In previous research, functionally-rare SERT coding variants associated with autism and obsessive-compulsive disorders (ASD and OCD, respectively) exhibit increased surface expression and transport capacity. ASD is a disorder of developmental delay in cognitive processes characterized by difficulties in communication, interaction in social settings, and obsessive-compulsive patterns in thought and behavior. Here, we reveal that the ASD SERT coding variant, F465L, confers an increase in palmitoylation and confirm from previous studies an increase in F465L cell surface levels and Vmax when compared to WT hSERT. Promising studies from clinical and functional magnetic resonance imaging (fMRI) data describe therapeutic approaches for adults with severe forms of ASD/OCD that consist of SSRIs like escitalopram. Acyl-biotinyl exchange (ABE) and cell surface analysis of WT and F465L hSERTs treated with 2BP or escitalopram reveal reductions in both palmitoylation and SERT surface levels to basal WT conditions. These results suggest dysregulated palmitoylation is a step in the pathogenesis of autism and obsessive-compulsory based cognitive illnesses, and escitalopram may be effective in rectifying this process. The third project investigated the family of enzymes responsible for protein palmitoylation called palmitoyl-acyl transferases or DHHCs. We have previously published that 5 members of this family (DHHC2, 3, 8, 15, and 17) catalyze palmitoylation of the dopamine transporter (DAT). This information, alongside our data from project one, led us to hypothesize that these enzymes may play a role in regulating SERT trafficking, activity, and serotonergic tone. When SERT was co-expressed with 9 different DHHCs, we observed an increase in SERT palmitoylation from DHHC1, 8, 15, and 17. Increased SERT palmitoylation status was consistent with increased levels of SERT surface expression, transport capacity, and total cellular SERT protein. From this group, SERT saturation analysis was performed in the absence or presence of DHHC 1 and 8 with transport capacity normalized to total cellular protein (pmol/min/mg) and SERT surface density. We identified that DHHC 1 and 8 modulate SERT activity differently, with DHHC1 directing a trafficking-dependent increase in transport capacity, while DHHC 8 directly enhanced SERT Vmax independent of cell surface levels. These results outline the diversity of DHHC outcomes for SERT trafficking, activity, and expression. It is possible that DHHCs may operate independently to palmitoylate SERT on different intracellular cysteines, and at different points in SERTs life-cycle, to accomplish the cell’s current physiologic objectives. The fourth project examined palmitoylation of NET and how perturbance of this process controls NET processing, trafficking, and may be involved in the development of a vascular disorder termed orthostatic intolerance (OI). NET is a catecholamine transporter that facilitates the reuptake of epinephrine, norepinephrine, and/or dopamine from pre-synaptically stimulated neurons, controlling cognitive functions and sympathetic tone. In this study, we demonstrate that native rat and heterologous human NETs are palmitoylated. Treatment of heterologous cells expressing NET with 2BP resulted in acute time-dependent decreases in Net palmitoylation, surface density, transport capacity, and total NET protein levels. As inhibition of NET palmitoylation was increased by 2BP concentration and time of exposure, we observed losses of total NET protein without loss of beta-actin, suggesting no changes in cellular cyto-toxicity. Physiologically, OI is a syndrome characterized by hyperadrenergic symptoms involving postural tachycardia, syncope, and excessive plasma NE. We have demonstrated that the OI coding variant, A457P has reduced palmitoylation and total expression compared to WT. These processes were partially recovered upon co-expression of DHHC1, an ER bound DHHC, suggesting that co-translational palmitoylation may facilitate NET biogenesis and that its dysregulation may be a mechanism in the pathogenesis of orthostatic intolerance

Doctor of Philosophy

dissertationPlatelet-surface interactions are a key mediator in a host's response to vascular biomaterials. Directly after implantation, a wide variety of serum proteins are adsorbed onto the surface of vascular devices, many of which activate platelets. Serving as the first step in the coagulation cascade, the local activation of platelets sets off a chain of events that can ultimately determine the fate of implanted vascular devices. Many material engineering approaches have been taken in an effort to reduce this activation; however, the most successful method to date remains the systemic delivery of antiplatelet and anticoagulant agents. The prevalence of antiplatelet pharmaceutics, coupled with a variation in efficacy across a diverse population, has led to an industry focused on the personalized assessment of platelet function. In this dissertation, methods were developed to address a deficiency in the current approach to platelet function testing. No current assays take into account the transient interactions of platelets with agonist surfaces, interactions that have the capability to prime a platelet population for enhanced adhesion and activation downstream of a stimulus. This phenomenon was leveraged here to create a flow cell in which the upstream priming of a platelet population by a surface-bound agonist could be assessed by downstream capture assay. It was demonstrated that this device is capable of determining the response of a platelet population to a variable priming stimulus, both alone and in the presence of common antiplatelet agents. To further investigate the priming response of platelets, it was of interest to develop a method by which to pattern multiple proteins on the same surface, thus enabling the measurement of the platelet population response to multiple surface bound agonists. Taking advantage of advances in microcontact printing and modern light microscopy, a new method by which to deposit multiple aligned patterns of agonists on a single substrate was developed. This patterning method was then used to investigate the ability of multiple agonists to prime platelets in flowing blood and observe the synergistic response that platelets exhibit when primed by multiple pathways. Taken collectively, these experiments contribute to the field of platelet activation by providing a controlled environment in which to study the priming response of platelets following transient exposure to surface bound agonists. This assay provides a platform in which the platelet response to a variety of surface coatings, biomaterials, and antiplatelet agents can be explored, and establishes a foundation for the further investigation of priming pathways in platelets

Vascular smooth muscle cell heterogeneity and plasticity in models of cardiovascular disease

Vascular smooth muscle cell (VSMC) accumulation is a hallmark of atherosclerosis and vascular injury. However, fundamental aspects of proliferation and the phenotypic changes within individual VSMCs, which underlie vascular disease remain unresolved. In particular, it is not known if all VSMCs proliferate and display plasticity, or whether individual cells can switch to multiple phenotypes. To assess whether proliferation and plasticity in disease is a general characteristic of VSMCs or a feature of a subset of cells, multi-colour lineage labelling is used to demonstrate that VSMCs in injury-induced neointimal lesions and in atherosclerotic plaques are oligo-clonal, derived from few expanding cells, within mice. Lineage tracing also revealed that the progeny of individual VSMCs contribute to both alpha Smooth muscle actin (aSma)-positive fibrous cap and Mac-3-expressing macrophage-like plaque core cells. Co-staining for phenotypic markers further identified a double-positive aSma+ Mac3+ cell population, which is specific to VSMC-derived plaque cells. In contrast, VSMC-derived cells generating the neointima after vascular injury generally retained expression of VSMC markers and upregulation of Mac3 was less pronounced. Monochromatic regions in atherosclerotic plaques and injury-induced neointima did not contain VSMC-derived cells expressing a different fluorescent reporter protein, suggesting that proliferation-independent VSMC migration does not make a major contribution to VSMC accumulation in vascular disease. Similarly, VSMC proliferation was examined in an Angiotensin II perfusion model of aortic aneurysm in mice, oligo-clonal proliferation was observed in remodelling regions of the vasculature, however phenotypic changes were observed in a large proportion of VSMCs, suggesting that the majority of VSMCs have some potential to modulate their phenotype. To understand the mechanisms behind the inherent VSMC heterogeneity and observed functionality, the single cell transcriptomic techniques Smart-seq2 and the Chromium 10X system were optimized for use on VSMCs. The work within this thesis suggests that extensive proliferation of a low proportion of highly plastic VSMCs results in the observed VSMC accumulation after injury, and the atherosclerotic and aortic aneurysm models of cardiovascular disease

Synthesis of bio-functional nanomaterials in reactive plasma discharges

Plasma processing technologies have been extensively used as surface modification platforms in many biomedical applications. Particularly, plasma polymerization (PP) is a versatile deposition technology which has the potential to deliver biocompatible interfaces for a myriad of medical devices. To successfully translate new materials for specific clinical applications, the plasma process needs to be scalable and incorporate appropriate control feedback strategies. However, the plasma medium in PP is exceptionally complex and identifying the main physical quantities that allow a suitable formulation and description of the interface growth mechanisms is challenging. The first part of the thesis reports the design and optimization of a single step ion assisted PP process to create plasma-activated coatings (PAC) that meet the extreme mechanical demands for cardiovascular implants and in particular stents. An ideal working window in the parameter space is identified, and found suitable for the synthesis of PAC interfaces that are mechanically robust, hemocompatibility and allow one step covalent protein immobilization without the need for chemical processes. This window is identified by combining plasma optical emission spectroscopy (OES) with a comprehensive macroscopic process description that isolates key coating growth mechanisms. During process scalability, OES diagnostics revealed the formation of plasma polymer nanoparticles (nanoP3), usually known as plasma dust, in parallel with the deposition of PAC coatings. The second part of the thesis reports the demonstration of carbonaceous plasma nanoparticles for nanomedicine applications. By controlling nanoparticle formation and collection, nanoP3 were engineered with unique immobilization capabilities facilitating multifunctional nanocarriers. The unique surface chemistry of nanoP3, allowing a robust immobilization of the cargo without the need for intermediate functionalization strategies, has great potential to overcome major limitations of currently proposed platforms. As many of the favorable characteristics of nanoP3 are inherent to the fabrication process, this work proposes PP as a nanoparticle synthesis route with valuable potential for broad clinical and commercial applications