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Perfil oxidativo e bioquímico em pacientes que apresentam anemia falciforme ou traço falciforme / Oxidative and biochemistry profile in sickle cell trait subjects and sickle cell anaemia patientsManfredini, Vanusa January 2008 (has links)
A Anemia Falciforme (AF) é uma doença autossômica recessiva e, dentre as hemoglobinopatias, é a mais comum das alterações hematológicas hereditárias conhecidas no homem. Sua distribuição é ampla, abrangendo todos os continentes. AF foi a primeira doença caracterizada em nível molecular. É causada por uma mutação no gene beta da globina, produzindo uma alteração estrutural na molécula. No gene da globina beta S, há a substituição de uma base nitrogenada do códon GAG para GTG, resultando na substituição do ácido glutâmico (Glu) pela valina (Val) na posição número seis da globina beta. Essa troca dos aminoácidos que resulta na HbS, altera estruturalmente a molécula e, sob determinadas condições, ocorre a polimerização, trazendo graves conseqüências ao indivíduo sintomático. As espécies reativas de oxigênio (ERO) podem causar profundas lesões em eritrócitos, diminuindo seu período de vida útil, em especial nos pacientes com AF. Acredita-se que, os eritrócitos falcizados estejam sob constante estresse oxidativo e, assim, liberem produtos de degradação da HbS, contribuindo para a progressão da doença. Dessa forma, o dano oxidativo agrava a fisiopatologia dos doentes falciformes. Utilizando técnica espectrofotométrica, foram determinadas as atividades das enzimas antioxidantes catalase (CAT), glutationa peroxidase (GPx) e superóxido dismutase (SOD) e quantificada a glutationa total (GSH) nos eritrócitos dos pacientes. Também determinou-se o dano oxidativo nas proteínas plasmáticas e no hemolisado celular pelo método do carbonil a 360 nm. Os níveis da peroxidação lipídica (MDA) e da vitamina C foram determinados por cromatografia líquida de alta performance (HPLC). Quantificou-se, por fim, os níveis de proteína C reativa ultra-sensível (CRPus) por técnica turbidimétrica. Os participantes da pesquisa (30 HbAA, 28 HbAS e 20 HbSS) foram selecionados junto ao Centro de Apoio do Portador de Anemia Falciforme (CAPAF/RS) e/ou cadastrados no Laboratório de Hematologia da Faculdade de Farmácia (UFRGS). Os doentes falciformes foram identificados por HPLC e confirmados por estudo molecular, utilizando a reação da polimerase em cadeia (PCR). Todos os indivíduos assinaram o termo de consentimento livre e esclarecido e foram submetidos a um questionário nutricional. Os dados obtidos foram expressos como médias ± desvio padrão e analisados utilizando-se o Teste ANOVA de uma via com posterior teste ad hoc. Os resultados do trabalho mostram que os indivíduos traço falciforme (HbAS) apresentam atividade significativamente elevada da CAT em relação aos indivíduos controle. Por outro lado, os doentes falciformes possuem maior atividade da GPx e SOD. O nível de GSH foi proporcionalmente maior nos HbSS seguido dos HbAS e HbAA. Observamos também, dano oxidativo em proteínas plasmáticas, mas não no hemolisado celular. Os HbSS possuem dano oxidativo em proteínas plasmáticas significativamente maior que nos demais grupos. Um aumento significativo da produção de MDA no soro dos HbSS foi observado, como um indicativo do aumento da auto-oxidação dos lipídios sob condições de estresse oxidativo. Os níveis séricos da vitamina C foram significativamente maiores nos HbSS que nos indivíduos controle. A CRPus apresentou-se significativamente elevada nos HbSS em relação aos HbAA. Esses resultados reforçam a idéia de que os pacientes com AF estão sujeitos a um estresse oxidativo crônico, o que contribui para a progressão das complicações dessa anemia hemolítica. Já, os indivíduos traço possuem elevada atividade das defesas antioxidantes capazes de reduzir o dano oxidativo em biomoléculas como proteínas e lipídios. / Sickle cell anaemia (SCA) is an autossomal recessive disease and, among the hemoglobinopaties, is the most common of the known hereditary hematologic alterations in man. Its distribution is ample, enclosing all the continents. Sickle cell anaemia (SCA) was the first disease to be characterized on the molecular level. It is basically a red blood cell (RBC) disorder in which the gene encoding the human β-globin subunit presents a mutation with the resulting replacement of β6 glutamic acid (Glu) by valine (Val). This exchange of the amino acids that results in the HbS modifies the molecule structurally, and under determined conditions, the polymeration occurs, bringing serious consequences to the symptomatic individual. The reactive oxygen species (ROS) can cause deep injuries in erythrocytes, diminishing its period of useful life, specially in patients with sickle cell anaemia. Sickled erythrocytes are under constant oxidative stress and thus liberate products of degradation of the HbS, contributing for the progression of the disease. Because of the oxidative damage aggravates the pathophysiology of sickle cell patients. Using spectrophotometrically technique, catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), were determined the activities of antioxidant enzymes and total glutathione (GSH) quantified in the erythrocytes of the patients. We also determined oxidative damage of plasma proteins and in hemolysate using carbonyl assay at 360 nm. The levels of lipid peroxidation (MDA) and vitamin C were determined by high-performance liquid chromatography (HPLC). Finally, we quantified the levels of high sensitivity Creactive protein (hsCRP) using turbidimetry technique. The participants of the research (30 HbAA, 28 HbAS and 20 HbSS) were selected from Centro de Apoio ao Portador de Anemia Falciforme (CAPAF/RS) and/or registered in cadastre in Laboratório de Hematologia da Faculdade de Farmácia (UFRGS). Sickle cell patients had been identified by HPLC and confirmed by molecular study, using the polymerase chain reaction amplification (PCR). All individuals signed the term of free and clarified assent and were submitted to a nutricional questionnaire. Data were expressed as average ±SD and analyzed using ANOVA test of one way via with the post ad hoc test. Results show that sickle cell trait subjects (HbAS) had significantly high activity of CAT when compared to healthy controls. On the other hand, sickle cell patients had greater activity of GPx and SOD. The GSH levels was proportionally higher in the followed HbSS of the HbAS and HbAA. We also observe oxidative damage in plasma proteins, but not in the cellular hemolysate. HbSS have significantly higher oxidative damage in plasma proteins than other groups. A significant increasing of the production of MDA in the serum of the HbSS was observed as an indicative of the increasing of the auto-oxidation of the lipids under oxidative estress. Serum vitamin C levels in HbSS were significantly higher than healthy control. The hsCRP was presented significantly higher in HbSS than HbAA. These results reinforce the idea that patients with SCA are subjected to chronic oxidative stress, that contributes for the progression of this hemolytic anaemia. On the other hand, sickle cell trait subjects have higher antioxidant defenses that are able to reduce oxidative damage in biological macromolecules such as proteins and lipids.
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Crianças e adolescentes portadores de anomia falciforme: os significados das relações estabelecidas com os profissionais no âmbito dos serviços de saúdeSousa, Eulange de 29 August 2014 (has links)
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Previous issue date: 2014-08-29 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Sickle cell anemia is an inherited disease considered a public health problem
in Brazil. In Brazil, it is estimated that there are more than 2 million people affected
by some form of this anemia, with 3.500 new cases occurring every year. This
dissertation aims to understand the meanings present in the relations established
between children and adolescents with sickle cell anemia and professionals within
the health services. The methodological choice of this study was the Social Strategic
Qualitative Research and it took place at a Teaching Hospital. Data were collected
through semi-structured interviews with 3 children, 5 adolescents and 9 health
professionals. Data were analyzed through the content analysis technique. From the
interviews with children and adolescents, the following categories emerged: the
disease, the professionals, and the treatment in other services. From the interviews
with health professionals, the following categories emerged: child, adolescent,
mother-family, professionals from other services, the disease, and what to do. The
research conducted indicated that it is necessary to engage children and adolescents
in decisions related to their health-disease process. It also indicated that, in order to
achieve it, the health professionals training have to include elements that enable
professionals to consider children and adolescents as protagonists in their healthdisease
process. In addition, it demonstrated the occurrence of institutional violence
in the health treatment that patients with sickle cell anemia receive, as they have
their autonomy, their subjectivity and their speech prevented and annulled. / A Anemia falciforme é uma doença hereditária considerada um problema de
saúde pública no Brasil. Estima-se que existem, no Brasil, mais de 2 milhões de
pessoas afetadas por alguma forma desta anemia, com uma ocorrência de 3.500
novos caso por ano. Este trabalho tem por objetivo compreender os significados
presentes nas relações estabelecidas entre as crianças e adolescentes portadores
de anemia falciforme e os profissionais no âmbito dos serviços de saúde. Esta
pesquisa foi realizada tendo como opção metodológica a Pesquisa Social
Estratégica de abordagem qualitativa, e como local de estudo optou-se por um
hospital de ensino. Os dados foram obtidos por meio de entrevistas semiestruturadas
com três crianças, cinco adolescentes e nove profissionais de saúde.
Os dados foram analisados pela técnica de análise de conteúdo. A partir das
entrevistas com as crianças e adolescentes, emergiram as seguintes categorias:
doença, os profissionais e o atendimento em outros serviços. Das entrevistas com
os profissionais, emergiram as categorias: criança, adolescente, mãe–família,
profissionais de outros serviços, a doença e o que fazer. O estudo realizado indicou
que é necessário envolver as crianças e adolescentes nas decisões relativas a seu
processo saúde-doença e que a formação de profissionais de saúde deve incluir
elementos que os instrumentalizem no sentido de considerar crianças e
adolescentes como protagonistas em seu processo saúde-doença. Indicou também
a ocorrência de violência institucional no atendimento que os portadores de anemia
falciforme recebem, pois têm sua autonomia, sua subjetividade e sua fala impedidas
e anuladas.
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INHIBITION OF ERYTHROCYTE BAND 3 TYROSINE PHOSPHORYLATION: CHARACTERIZATION OF A NOVEL THERAPY FOR SICKLE CELL DISEASE AND MALARIAPanae Noomuna (10716546) 29 April 2021 (has links)
While the molecular
defect that cause sickle cell disease has well been established, the cause of
vaso-occlusive crisis remains elusive and largely debated upon. Majority of
studies have linked the painful episodes to polymerization of sickle hemoglobin
following its deoxygenation. The variability of the disease symptoms among
patients, compounds efforts for a holistic therapy. Hydroxyurea, a stimulator
of Hb F induction and a widely used treatment, has ameliorated the complication
of SCD but it is only effective in 50% of the patients. Expression of Hb F
lowers the content of Hb S in blood and hence reduces oxidative stress caused
by Hb S denaturation. Sickle cell disease severity depends on several factors.
Most importantly, the ability of red cell to sickle dominates all other
determinants. While deoxygenation of sickle hemoglobin may be inevitable, the
duration with which the red cell remains in the deoxygenated state can be
manipulated. Deoxygenation is a transient process that when compared to the
time taken to develop the long filaments of deoxyhemoglobin to causes severe
sickling, the red cell would have been cycled back to the lungs and
re-oxygenated to restore the healthy conditions of the cell. In fact, if sickle
cells would flow as fast as healthy erythrocytes, the detrimental impacts of
sickling such as vaso-occlusive crisis, would not be a concern for this
disease. Unfortunately, the unstable sickle hemoglobin undergoes denaturation
through auto-oxidation, which imposes oxidative stress to the cells. The
oxidative stress inhibits erythrocytes tyrosine phosphatases, a course which
subsequently impair their constitutive action against the tyrosine kinases. In
the end, a net tyrosine phosphorylation state in the red cell membrane
proteins, most notably the transmembrane protein band 3, succeeds. Band 3
tyrosine phosphorylation abrogates the protein’s interaction with ankyrin and
spectrin-actin cytoskeleton, hence the cytoskeleton loses its major anchorage
to the membrane thus engendering membrane destabilization. A destabilized
erythrocyte sheds membrane fragments in form of microvesicles/microparticles
and discharges free hemoglobin into the extra cellular matrix. In consequence,
the microparticles power initiation of coagulation cascade through activation of
thrombin, while free Hb inflicts inflammation, scavenges nitric oxide which is
necessary for vasodilation and induces further oxidative stress within the
microvasculature, and activates expression of adhesion receptors on the
endothelium. Taken together, these events culminate in entrapment of red cells
(not naming leucocytes and platelets) in the microvasculature, blockade of
blood vessels and further damage of erythrocytes through prolonged deoxygenated
state thus terminating in tissue injury, strokes, and organ damage, amid
vaso-occlusive episodes which always require hospitalization and extensive
medical care for survival. Band 3 tyrosine phosphorylation and membrane
weakening is not unique just to SCD, but also a druggable target for malaria.
Malaria, a disease that is touted as the evolutionary cause of sickle cell
disease, surprisingly thrives through the same mechanism. Briefly, malaria
parasite consumes hemoglobin for its DNA synthesis, and in the process generate
reactive oxygen species from denatured hemoglobin that feeds into the oxidative
stress which triggers band 3 tyrosine phosphorylation. In this case however, a
destabilized membrane offers perfect conditions for merozoites’ (malaria
daughter parasites) egress/exit out of the cell to begin infecting other red
cells. Ultimately, the ensuing anemia and organ dysfunction leads to patient’s
death. Treatment of diseased cells with imatinib and other Syk inhibitors
effectively reversed membrane weakening. A stabilized membrane not only
survives longer in circulation to alleviate SCD symptoms but also traps and
starves malaria parasite leading to termination of the parasitic infection.
With band 3 tyrosine phosphorylation at center stage, this dissertation
explores the above events in an effort to unveil a novel therapy for sickle
cell and malaria diseases. First, the therapeutic strategy regarding SCD is
discussed in detail beginning with non-transfused patients and ending in
additional mechanistic study on inactivation of the principal erythrocyte’s protein
tyrosine phosphatase 1 B, PTP1B. The dissertation then provides an initial
proof of concept on efficacy of imatinib in treatment of malaria as a
monotherapy and its efficacy when used in a triple combination therapy with the
standard of care treatment. Finally, I outline an alternative possible
mechanism of action of quinine against malaria.
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Contribution à l'étude du mécanisme d'action anti-drépanocytaire du cromoglycate disodiqueBizumukama, Leonidas 22 December 2011 (has links)
La drépanocytose est une maladie génétique touchant l'hémoglobine, de transmission autosomique récessive, caractérisée par trois grandes manifestations cliniques :anémie hémolytique chronique, phénomènes vaso-occlusifs et susceptibilité accrue aux infections. Dans diverses régions du monde et particulièrement en Afrique subsaharienne, cette maladie est très fréquente et constitue un problème crucial de santé publique. Sa physiopathologie complexe est basée sur la polymérisation de l’hémoglobine anormale (Hb S) qui provoque une falciformation et une déshydratation du globule rouge. Les hématies falciformées sont impliquées dans les phénomènes de vaso-occlusion. Le traitement et la prise en charge de la maladie reste toujours problématique. A l’heure actuelle, le seul traitement curatif est la transplantation de moelle osseuse mais les donneurs compatibles sont assez rares et les coûts élevés. Des traitements symptomatiques et préventifs (principalement la transfusion et l’hydroxyurée) existent toutefois.<p>Des études in vitro et in vivo ont démontré les possibilités thérapeutiques de certaines molécules dont les cibles sont les transports membranaires impliqués dans la déshydratation cellulaire.<p>Depuis les années 1990, le cromoglycate de sodium, un médicament anti-allergique et anti-asthmatique, a montré un intérêt potentiel dans le traitement de la drépanocytose. Néanmoins, son mode d’action n’est actuellement pas connu. Notre travail a pour but de contribuer à l’étude du mécanisme d’action anti-drépanocytaire de la molécule.<p>Dans un premier temps, des globules rouges drépanocytaires préincubés en absence ou présence de cromoglycate ont été désoxygénés par un flux d’azote. Ensuite, les concentrations intracellulaires en Na+ et en K+ ont été mesurées. Les résultats de ces investigations ont montré un effet inhibiteur du cromoglycate sur l’efflux de K+ et l’influx du Na+ provoqués par la désoxygénation.<p>Sur base de ces observations, des expériences testant l’action du cromoglycate sur le canal K+ dépendant du Ca2+ (canal de Gardos) ont été effectuées. Dans des globules rouges normaux et drépanocytaires, ce canal a été activé par augmentation de la concentration intra-cellulaire en Ca2+. L'effet du cromoglycate a été comparé à celui d'un inhibiteur connu, le clotrimazole. Les résultats ont montré que 1e cromoglycate n'exerce pas d'effet inhibiteur sur le canal de Gardos, au contraire du clotrimazole. Il est également sans effet significatif sur la Ca2+-ATPase.<p>Nous avons ensuite investigué l’implication du Ca2+ dans les perturbations du flux des ions K+ et Na+. Des globules rouges drépanocytaires ont été incubés en absence et présence d’EGTA 5 mmol/l ou de BAPTA 10 µmol/l, respectivement chélateurs du Ca2+ extra et intracellulaire. Après désoxygénation, les concentrations intracellulaires en Na+ et K+ ont été mesurées. Les résultats de ces expériences montrent que seul le chélateur du Ca2+ extracellulaire bloque les perturbations ioniques causées par la désoxygénation. Ces résultats viennent donc confirmer les observations d’autres auteurs quant à l’implication du Ca2+ extracellulaire dans la fuite de K+ des globules drépanocytaires soumis à la désoxygénation. <p>Enfin, l’effet du cromoglycate sur l’influx de Ca2+ extracellulaire et sur la falciformation induits par le métabisulfite a été mesuré et comparé à celui du clotrimazole. Des globules rouges drépanocytaires, prélablement chargés en Fura Red, un indicateur fluorescent du Ca2+, ont été exposés au métabisulfite, un puissant réducteur provoquant une falciformation rapide. L’influx de Ca2+ a été mesuré par la diminution de la fluorescence du Fura Red. Parallèlement, la falciformation a été suivie en mesurant la lumière diffractée à 90° par les cellules. Les résultats de ces investigations montrent que le cromoglycate (1 µmol/l) et le clotrimazole (10 µmol/l) ont des effets inhibiteurs comparables sur la falciformation mais que le cromoglycate freine significativement plus l'influx de Ca2+ au cours de ce processus.<p>En conclusion, sur base de ces différents tests in vitro, le cromoglycate inhibe la falciformation induite par la désoxygénation. Cette inhibition résulte du blocage des perturbations ioniques induites par la désoxygénation en empêchant l’influx du Ca2+ extracellulaire et secondairement la fuite du K+ intracellulaire, ce qui inhibe la déshydratation cellulaire.<p>La diminution des crises vaso-occlusives observée chez les patients drépanocytaires traités par le cromoglycate s’expliquerait donc par ces effets. En présence de cromoglycate, les globules rouges sont moins déshydratés et falciforment moins rapidement. Ils sont dès lors moins impliqués dans les phénomènes de vaso-occlusion, ce qui améliore l’état des patients.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished
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Obstacles and Circumvention Strategies for Hematopoietic Stem Cell Transduction by Recombinant Adeno-associated Virus VectorsMaina, Caroline Njeri 18 March 2009 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / High-efficiency transduction of hematopoietic stem cells (HSCs) by recombinant adeno-associated virus serotype 2 (AAV2) vectors is limited by (i) inadequate expression of cellular receptor/co-receptors for AAV2; (ii) impaired intracellular trafficking and uncoating in the nucleus; (iii) failure of the genome to undergo second-strand DNA synthesis; and (iv) use of sub-optimal promoters. Systematic studies were undertaken to develop alternative strategies to achieve high-efficiency transduction of primary murine HSCs and lineage-restricted transgene expression in a bone marrow transplant model in vivo. These included the use of: (i) additional AAV serotype (AAV1, AAV7, AAV8, AAV10) vectors; (ii) self-complementary AAV (scAAV) vectors; and (iii) erythroid cell-specific promoters. scAAV1 and scAAV7 vectors containing an enhanced green-fluorescent protein (EGFP) reporter gene under the control of hematopoietic cell-specific enhancers/promoters allowed sustained transgene expression in an erythroid lineage-restricted manner in both primary and secondary transplant recipient mice.
Self complementary AAV vectors containing an anti-sickling human beta-globin gene under the control of either the beta-globin gene promoter/enhancer, or the human parvovirus B19 promoter at map-unit 6 (B19p6) were tested for their efficacy in a human erythroid cell line (K562), and in primary murine hematopoietic progenitor cells (c-kit+, lin-). These studies revealed that (i) scAAV2-beta-globin vectors containing only the HS2 enhancer are more efficient than ssAAV2-beta-globin vectors containing the HS2+HS3+HS4 enhancers; (ii) scAAV-beta-globin vectors containing only the B19p6 promoter are more efficient than their counterparts containing the HS2 enhancer/beta-globin promoter; and (iii) scAAV2-B19p6-beta-globin vectors in K562 cells, and scAAV1-B19p6-beta-globin vectors in murine c-kit+, lin- cells, yield efficient expression of the beta-globin protein. These studies suggest that the combined use of scAAV serotype vectors and the B19p6 promoter may lead to expression of therapeutic levels of beta-globin gene in human erythroid cells, which has implications in the potential gene therapy of beta-thalassemia and sickle cell disease.
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The Relationships Among Health Literacy, Stigma, Self-efficacy, Self-care, and Health Outcomes in Patients with Sickle Cell DiseaseO'Brien, Julia Ann 21 June 2021 (has links)
No description available.
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Evaluating Bilateral Phenomena: The Case of Pain in Sickle Cell DiseaseDahman, Bassam 01 January 2007 (has links)
Symmetry in biological systems is the occurrence of an event on both sides of the system. The term bilateralism was introduced to represent this phenomenon, and it was defined as the conditional co-occurrence of two events given that at least at one of them has occurred. This phenomenon is highly associated with the prevalence of each of the events. Two parameters were developed to evaluate the presence of this phenomenon, testing whether events co-occur with higher probability than would be expected by chance. Nonparametric confidence intervals were constructed using the bootstrap percentile method. These non parametric confidence intervals were used in testing the null hypothesis of no bilateralism.A simulation study was performed to examine the properties of the two bilateralism parameters' estimates. The size and power of the tests of bilateralism were examined under a variety of sample sizes and prevalences of the two events. The simulation study showed that both parameter estimates have similar properties, and the tests have similar size and power. The power of the test was affected by the prevalence of either event, the differences in the prevalences, the sample size and by number of events that occur simultaneously. The methodology of testing for bilateralism was applied on data from the Pain in Sickle Cell Epidemiology Study (PiSCES). This study collected up to 6 months worth of daily diaries about pain and medical utilization from patients with sickle cell disease. Each diary recorded the body site and side where pain was experienced over the past 24 hours. The sample consists of 119 subjects who completed at least 50 daily pain diaries (reference). Information about the subjects age, gender and sickle cell genotype were also available. Nine body sites (5 upper peripheral, and 4 lower peripheral site) were analyzed to test for bilateralism. Bilateralism was tested for each subject and each site separately. The associations of prevalence of bilateralism on each site, and percentages of sites that hurt bilaterally with age, gender and genotype where studied.The results show a high prevalence of bilateral pain among sickle cell patients at all sites. Age gender and genotype were associated with higher prevalence in bilateral pain in some, but not all sites. The percentage of sites that have bilateral pain is also associated with the number of sites that have pain.
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IN-VITRO PK/PD PROFILING AND MODELING OF THE ANTI-SICKLING AGENTS, 5-HYDROXYMETHYL FURFURAL (5-HMF) AND NOVEL SYNTHETIC ALLOSTERIC EFFECTORS OF HEMOGLOBIN (AEH) IN HUMAN WHOLE BLOODParikh, Apurvasena 01 January 2013 (has links)
Introduction. 5-HMF and novel INN-compounds are left-shifting AEH, shown to have anti-sickling action by forming transiently covalent Schiff-base adducts with hemoglobin (Hb), thereby increasing the Hb O2-affinity. They are hypothesized to be substrates for aldehyde dehydrogenase (ALDH) in the liver and red blood cells (RBC). Methods. Biopharmaceutical assessments were made for AEH, using calculated physicochemical properties. Their in-vitro hepatic metabolism (mediated by ALDH) was characterized using hepatic cytosol, and in-vitro-in-vivo extrapolations (IVIVE) were made. Inter-species differences in hepatic cytosolic ALDH activity were investigated using acetaldehyde as a model substrate in different mammalian species. Time- and concentration-dependent in-vitro disposition of 5-HMF in human whole blood was fully characterized and quantitatively modeled. In-vitro time- and concentration-dependent pharmacodynamic (PD) profiling of AEH (0.5 – 5 mM) was carried out in normal whole blood. 5-HMF binding to (normal) HbA and (sickle) HbS was studied in systematic time- and concentration-dependency studies using isolated Hb solutions. Quantitative PK/PD models were developed to fit the experimental data by nonlinear regression (Scientist®). Results. 5-HMF and the two INN-compounds were classified as BCS-I and BCS-II, respectively. All AEH were substrates for hepatic ALDH, with predicted low/intermediate hepatic extraction. Intrinsic ALDH activity varied significantly between mammalian species. In whole blood, 5-HMF plasma concentrations declined rapidly (t1/2 of 0.8 – 4 hrs), with nonlinear kinetics, due to saturable Hb-binding. AEH showed a time-dependent, biphasic PD effect in whole blood, suggesting transiently covalent Hb binding, with slow recovery to the baseline, corresponding to dissociation from Hb and subsequent metabolism by RBC-ALDH. Binding studies with HbA and HbS demonstrated slight differences in binding affinity, but sustained adduct formation - with slow dissociation t1/2. A novel semi-mechanistic target-site drug disposition (TSDD)/PD model was developed, integrating the information, for simultaneous modeling of 5-HMF concentrations in plasma, and its effect in whole blood. Conclusions. This translational research investigated in detail the in-vitro PK/PD of AEH, and systematically compared findings with older generation compounds. A (generic) novel TSDD/PD model was developed for disposition of AEH, identifying k-1 (dissociation constant of AEH from Hb) and kmet (RBC-ALDH metabolism rate constant) as key properties for the time course of PD effect.
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Molecular mechanisms of bio-catalysis of heme extraction from hemoglobinSakipov, Serzhan, Rafikova, Olga, Kurnikova, Maria G., Rafikov, Ruslan 04 1900 (has links)
Red blood cell hemolysis in sickle cell disease (SCD) releases free hemoglobin. Extracellular hemoglobin and its degradation products, free heme and iron, are highly toxic due to oxidative stress induction and decrease in nitric oxide availability. We propose an approach that helps to eliminate extracellular hemoglobin toxicity in SCD by employing a bacterial protein system that evolved to extract heme from extracellular hemoglobin. NEAr heme Transporter (NEAT) domains from iron-regulated surface determinant proteins from Staphylococcus aureus specifically bind free heme as well as facilitate its extraction from hemoglobin. We demonstrate that a purified NEAT domain fused with human haptoglobin beta-chain is able to remove heme from hemoglobin and reduce heme content and peroxidase activity of hemoglobin. We further use molecular dynamics (MD) simulations to resolve molecular pathway of heme transfer from hemoglobin to NEAT, and to elucidate molecular mechanism of such heme transferring process. Our study is the first of its kind, in which simulations are employed to characterize the process of heme leaving hemoglobin and subsequent rebinding with a NEAT domain. Our MD results highlight important amino acid residues that facilitate heme transfer and will guide further studies for the selection of best NEAT candidate to attenuate free hemoglobin toxicity.
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Non-Pharmacological Approaches for Pain Management in Sickle Cell Disease: Development of a Mindfulness-Based InterventionWilliams, Hants January 2016 (has links)
<p>Background: Sickle Cell Disease (SCD) is a genetic hematological disorder that affects more than 7 million people globally (NHLBI, 2009). It is estimated that 50% of adults with SCD experience pain on most days, with 1/3 experiencing chronic pain daily (Smith et al., 2008). Persons with SCD also experience higher levels of pain catastrophizing (feelings of helplessness, pain rumination and magnification) than other chronic pain conditions, which is associated with increases in pain intensity, pain behavior, analgesic consumption, frequency and duration of hospital visits, and with reduced daily activities (Sullivan, Bishop, & Pivik, 1995; Keefe et al., 2000; Gil et al., 1992 & 1993). Therefore effective interventions are needed that can successfully be used manage pain and pain-related outcomes (e.g., pain catastrophizing) in persons with SCD. A review of the literature demonstrated limited information regarding the feasibility and efficacy of non-pharmacological approaches for pain in persons with SCD, finding an average effect size of .33 on pain reduction across measurable non-pharmacological studies. Second, a prospective study on persons with SCD that received care for a vaso-occlusive crisis (VOC; N = 95) found: (1) high levels of patient reported depression (29%) and anxiety (34%), and (2) that unemployment was significantly associated with increased frequency of acute care encounters and hospital admissions per person. Research suggests that one promising category of non-pharmacological interventions for managing both physical and affective components of pain are Mindfulness-based Interventions (MBIs; Thompson et al., 2010; Cox et al., 2013). The primary goal of this dissertation was thus to develop and test the feasibility, acceptability, and efficacy of a telephonic MBI for pain catastrophizing in persons with SCD and chronic pain. </p><p>Methods: First, a telephonic MBI was developed through an informal process that involved iterative feedback from patients, clinical experts in SCD and pain management, social workers, psychologists, and mindfulness clinicians. Through this process, relevant topics and skills were selected to adapt in each MBI session. Second, a pilot randomized controlled trial was conducted to test the feasibility, acceptability, and efficacy of the telephonic MBI for pain catastrophizing in persons with SCD and chronic pain. Acceptability and feasibility were determined by assessment of recruitment, attrition, dropout, and refusal rates (including refusal reasons), along with semi-structured interviews with nine randomly selected patients at the end of study. Participants completed assessments at baseline, Week 1, 3, and 6 to assess efficacy of the intervention on decreasing pain catastrophizing and other pain-related outcomes. </p><p>Results: A telephonic MBI is feasible and acceptable for persons with SCD and chronic pain. Seventy-eight patients with SCD and chronic pain were approached, and 76% (N = 60) were enrolled and randomized. The MBI attendance rate, approximately 57% of participants completing at least four mindfulness sessions, was deemed acceptable, and participants that received the telephonic MBI described it as acceptable, easy to access, and consume in post-intervention interviews. The amount of missing data was undesirable (MBI condition, 40%; control condition, 25%), but fell within the range of expected missing outcome data for a RCT with multiple follow-up assessments. Efficacy of the MBI on pain catastrophizing could not be determined due to small sample size and degree of missing data, but trajectory analyses conducted for the MBI condition only trended in the right direction and pain catastrophizing approached statistically significance. </p><p>Conclusion: Overall results showed that at telephonic group-based MBI is acceptable and feasible for persons with SCD and chronic pain. Though the study was not able to determine treatment efficacy nor powered to detect a statistically significant difference between conditions, participants (1) described the intervention as acceptable, and (2) the observed effect sizes for the MBI condition demonstrated large effects of the MBI on pain catastrophizing, mental health, and physical health. Replication of this MBI study with a larger sample size, active control group, and additional assessments at the end of each week (e.g., Week 1 through Week 6) is needed to determine treatment efficacy. Many lessons were learned that will guide the development of future studies including which MBI strategies were most helpful, methods to encourage continued participation, and how to improve data capture.</p> / Dissertation
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