Development of the ProPal-COPD tool to identify patients with COPD for proactive palliative care.

BACKGROUND: Our objective was to develop a tool to identify patients with COPD for proactive palliative care. Since palliative care needs increase during the disease course of COPD, the prediction of mortality within 1 year, measured during hospitalizations for acute exacerbation COPD (AECOPD), was used as a proxy for the need of proactive palliative care. PATIENTS AND METHODS: Patients were recruited from three general hospitals in the Netherlands in 2014. Data of 11 potential predictors, a priori selected based on literature, were collected during hospitalization for AECOPD. After 1 year, the medical files were explored for the date of death. An optimal prediction model was assessed by Lasso logistic regression, with 20-fold cross-validation for optimal shrinkage. Missing data were handled using complete case analysis. RESULTS: Of 174 patients, 155 patients were included; of those 30 (19.4%) died within 1 year. The optimal prediction model was internally validated and had good discriminating power (AUC =0.82, 95% CI 0.81-0.82). This model relied on the following seven predictors: the surprise question, Medical Research Council dyspnea questionnaire (MRC dyspnea), Clinical COPD Questionnaire (CCQ), FEV1% of predicted value, body mass index, previous hospitalizations for AECOPD and specific comorbidities. To ensure minimal miss out of patients in need of proactive palliative care, we proposed a cutoff in the model that prioritized sensitivity over specificity (0.90 over 0.73, respectively). Our model (ProPal-COPD tool) was a stronger predictor of mortality within 1 year than the CODEX (comorbidity, age, obstruction, dyspnea, and previous severe exacerbations) index. CONCLUSION: The ProPal-COPD tool is a promising multivariable prediction tool to identify patients with COPD for proactive palliative care.

Human erythrocytes as drug carriers: loading efficiency and side effects of hypotonic dialysis, chlorpromazine treatment and fusion with liposomes.

Human red blood cells (RBCs) are emerging as a highly biocompatible microparticulate drug delivery system. So far, drugs have commonly been loaded into freshly isolated RBCs using rather disruptive methods based on hypotonic shock, and assessment of damage was restricted to hemolysis. Here, we investigated loading of RBCs from blood bank units with enzymes of various molecular weights using hypotonic dialysis (HD), pretreatment with chlorpromazine (CPZ) and fusion with liposomes. The latter two techniques have received little attention in RBC loading so far. Along with loading efficiency, all methods were tested for the induction of side effects. Very importantly, next to hemolysis, we also addressed morphological changes and phosphatidyl serine (PS) exposure, which has been recognized as a critical parameter associated with premature RBC removal and induction of transfusion-related pathologies. The efficiency of loading using hypotonic dialysis decreased with the molecular weight of the enzyme. For liposomes and chlorpromazine, loading efficiencies were higher and independent of enzyme molecular weights. While hypotonic dialysis always induced a high degree of hemolysis, irreversible modifications in the morphology of the cells and PS exposure, the side effects that were induced by loading using CPZ and liposomes were limited. In particular, PS exposure, although high immediately after treatment, returned to physiological levels after recovery. Retention and deformability studies using a spleen-mimicking device showed that RBCs treated with CPZ and liposomes behave like physiological RBCs, while HD led to very fragile and poorly deformable RBCs.

Functional consequences of sphingomyelinase-induced changes in erythrocyte membrane structure.

Inflammation enhances the secretion of sphingomyelinases (SMases). SMases catalyze the hydrolysis of sphingomyelin into phosphocholine and ceramide. In erythrocytes, ceramide formation leads to exposure of the removal signal phosphatidylserine (PS), creating a potential link between SMase activity and anemia of inflammation. Therefore, we studied the effects of SMase on various pathophysiologically relevant parameters of erythrocyte homeostasis. Time-lapse confocal microscopy revealed a SMase-induced transition from the discoid to a spherical shape, followed by PS exposure, and finally loss of cytoplasmic content. Also, SMase treatment resulted in ceramide-associated alterations in membrane-cytoskeleton interactions and membrane organization, including microdomain formation. Furthermore, we observed increases in membrane fragility, vesiculation and invagination, and large protein clusters. These changes were associated with enhanced erythrocyte retention in a spleen-mimicking model. Erythrocyte storage under blood bank conditions and during physiological aging increased the sensitivity to SMase. A low SMase activity already induced morphological and structural changes, demonstrating the potential of SMase to disturb erythrocyte homeostasis. Our analyses provide a comprehensive picture in which ceramide-induced changes in membrane microdomain organization disrupt the membrane-cytoskeleton interaction and membrane integrity, leading to vesiculation, reduced deformability, and finally loss of erythrocyte content. Understanding these processes is highly relevant for understanding anemia during chronic inflammation, especially in critically ill patients receiving blood transfusions.

Comparative proteomics of erythrocyte aging in vivo and in vitro.

During aging in vivo and in vitro, erythrocytes display removal signals. Phagocytosis is triggered by binding of autologous IgG to a senescent cell antigen originating on band 3. Erythrocytes generate vesicles as an integral part of the aging process in vivo and in vitro, i.e. during storage. These vesicles display senescent cell antigens as well as phosphatidylserine, that is recognized by scavenger receptors. Recent comparative proteomic analyses of erythrocytes and their vesicles support the hypothesis that aging is accompanied by increased binding of modified hemoglobins to band 3, disruption of the band 3-mediated anchorage of the cytoskeleton to the lipid bilayer, vesicle formation, and antigenic changes in band 3 conformation. Proteomic data also suggest an, until then unknown, involvement of chaperones, stress proteins, and proteasomes. Thus, the presently available comparative proteomic analyses not only confirm previous immunochemical and functional data, but also (1) provide new clues to the mechanisms that maintain erythrocyte homeostasis; (2) open new roads to elucidate the processes that regulate physiological erythrocyte aging and removal, and thereby; (3) provide the foundation for rational interventions to prevent untimely erythrocyte removal, and unwanted interactions between the erythrocyte and the immune system, especially after transfusion.

Production of yeastolates for uniform stable isotope labelling in eukaryotic cell culture.

Preparation of stable isotope-labelled yeastolates opens up ways to establish more cost-effective stable isotope labelling of biomolecules in insect and mammalian cell lines and hence to employ higher eukaryotic cell lines for stable isotope labelling of complex recombinant proteins. Therefore, we evaluated several common yeast strains of the Saccharomycetoideae family as a source of high-quality, non-toxic yeastolates with the major aim to find a primary amino acid source for insect and mammalian cell culture that would allow cost-effective uniform stable isotope labelling (13C, 15N). Strains of the facultative methylotrophic yeasts Pichia pastoris and Hansenula polymorpha (Pichia angusta) as well as a strain of the baker's yeast Saccharomyces cerevisiae were compared as a source of yeastolate with respect to processing, recovery and ability to sustain growth of insect and mammalian cell lines. The best growth-supporting yeastolates were prepared via autolysis from yeast obtained from fed-batch cultures that were terminated at the end of the logarithmic growth phase. Yeastolates obtained from H. polymorpha performed well as a component of insect cell cultures, while yeastolates from S. cerevisiae and H. polymorpha both yielded good results in mammalian cell cultures. Growth of yeasts in Heine's medium without lactic acid allows relatively low concentrations of 13C and 15N sources, and this medium can be reused several times with supplementation of the 13C source only.

Erythrocyte ageing in vivo and in vitro: structural aspects and implications for transfusion.

Erythrocyte transfusion is essential in conditions of large blood loss, of inadequate bone marrow production and of increased erythrocyte breakdown. The structural and biochemical changes that erythrocytes go through during storage, probably associated with the disappearance of up to 30% of the erythrocytes within 24 h after transfusion, are likely to contribute to the transfusion side effects: iron overload, erythrocyte adhesion to the endothelial surface with proinflammatory consequences, autoantibody formation, endothelial damage by released erythrocyte constituents, a hampered microcirculation and oxygen delivery. In vivo, senescent erythrocytes are marked for removal by binding of autologous immunoglobulin G to ageing antigens, which arise by changes in the conformation of the membrane domain of band 3. Also, vesicle formation has been described as an integral part of the erythrocyte ageing process. Comparable changes occur during erythrocyte storage. This review describes the current state of knowledge of the mechanism of erythrocyte ageing in vivo, ageing-related changes occurring during erythrocyte storage in blood bank conditions and their possible relation with the transfusion side effects. In view of the key position of band 3 in the maintenance of erythrocyte structure and function, elucidation of the pathways that control posttranslational modification of band 3 during storage may lead to new approaches towards maintaining ATP concentration and cellular integrity. This review concludes with the challenge to further explore the underlying processes of erythrocyte ageing in order to provide physiologically relevant tools for assessing and predicting erythrocyte homeostasis in vitro and in vivo and thereby to contribute to the development of rational transfusion protocols for various patient categories.

Storage-related changes in erythrocyte band 3: not a case for the Diego blood group antigens.

Removal of erythrocytes from the circulation is mediated by the immune system. Changes in structure and function of band 3, a major membrane protein of the erythrocyte, trigger the binding of antibodies to a band 3-derived neoantigen, senescent cell antigen, on erythrocytes aged in vivo. This mechanism probably is also involved in determining the survival of erythrocytes after transfusion. Band 3 is the carrier of the Diego blood group system, and subtle changes in the three-dimensional conformation of the same extracellular loops of band 3 determine Diego blood group activity as well as senescent cell antigen activity. Therefore we used the Diego blood group system to probe these changes with a combination of serological and immunochemical methods. Our data indicate that changes in band 3 structure during storage under blood bank conditions, as shown by immunoblot analysis, are not detectable as changes in expression of Diego antigens in intact cells. This makes it unlikely that immunological removal of erythrocytes after transfusion is mediated by reactions involving the Diego blood group system.

The red cell revisited--matters of life and death.

An erythrocyte-fractionating method combining volume and subsequent density separation is described. Iron isotope (59Fe)-validation proved this combination of methods to be complementary. By deploying HbA1c as cell age marker, obtained fractions demonstrated that circulating erythrocytes lose 20% of hemoglobin and membrane by shedding vesicles. Vesiculation from older cells proved to be facilitated by the spleen. Animal studies revealed that such vesicles are rapidly removed from the circulation by scavenger receptors on Kupffer cells with phosphatidylserine acting as the principal ligand. These studies reveal the existence of an alternative pathway of erythrocyte breakdown. This means that the premortal substrate of 20% of any erythrocyte is at our disposal. As this kind of vesiculation takes place during the entire erythrocyte lifespan, loss and sometimes reutilisation of marker substances limits the usefulness of isotope studies to the first half of the erythrocyte lifespan, thereby putting the dogmatic lifespan of 120 days into question. Furthermore, these studies add to the understanding of hemoglobin A1c (HbA1c) metabolism and the origin of the wide variation of erythrocyte parameters in peripheral blood. Removal of old erythrocytes from the circulation and from donor blood may open new ways into the treatment of both bilirubin and secondary iron overload.

Survival of the fittest?--survival of stored red blood cells after transfusion.

During the last 90 years many developments have taken place in the world of blood transfusion. Several anticoagulants and storage solutions have been developed. Also the blood processing has undergone many changes. At the moment, in The Netherlands, red blood cell (RBC) concentrates (prepared from a whole blood donation and leukocyte-depleted by filtration) are stored for a maximum of 35 days at 4 degrees C in saline adenine glucose mannitol (SAGM). Most relevant studies show that approximately 20% of the RBCs is lost in the first 24 hr after transfusion. Even more remarkable is that the average life span is 94 days after a storage period of 42-49 days. Such observations create the need for a parameter to measure the biological age of RBCs as a possible predictor of the fate of RBCs after transfusion. The binding of IgG to RBCs can lead to recognition and subsequent phagocytosis by macrophages. This occurs during the final stages of the RBC life span in vivo. We determined the quantity of cell-bound IgG during storage, and found considerable variation between RBCs, but no significant storage-related change in the quantity of cell-bound IgG. The significance of this finding for predicting the survival of transfused RBCs in vivo remains to be established. Hereto we developed a flow cytometric determination with a sensitivity of 0.1% for the measurement of survival in vivo based on antigenic differences. This technique has various advantages compared with the 'classical' 51Cr survival method.

Erythrocyte aging in sickle cell disease.

Physiological removal of old erythrocytes from the circulation by macrophages is initiated by binding of autologous IgG to senescent cell antigen (SCA). SCA is generated from the anion exchanger band 3. This process is accompanied by a number of alterations in the function and structure of band 3. We measured these aging-related parameters in erythrocytes from individuals with sickle cell anemia. Most sickle erythrocytes have characteristics that are also found in senescent normal erythrocytes, such as an increased density and considerable concentrations of cell-bound IgG. Together with the concomitant changes in structure and function of band 3, these data suggest that most sickle erythrocytes have undergone a process of accelerated aging. Preliminary results indicate that this process is reversed upon vitamin E supplementation. These data show that the erythrocyte aging paradigm may provide a useful conceptual framework for the study of the pathophysiology and the evalution of therapeutic intervention in sickle cell disease, and support the view that oxidation can generate neoantigens that are recognized by autoantibodies.

Molecular characterization of anion exchangers in the cochlea.

Anion exchange proteins (AE) in the inner ear have been the focus of attention for some time. They have been suggested to play a role as anion exchangers for the regulation of endolymphatic pH or as anion exchangers and anchor proteins for the maintenance of the shape and turgor of outer hair cells, and they also have been discussed as a candidate protein for motile hair cell responses that follow high-frequency stimulation. The existence of anion exchangers in hair cells and the specific isoforms which are expressed in hair cells and the organ of Corti is controversial. Using a polyclonal antibody to AE1 (AB 1992, Chemicon), we immunoprecipitated a 100 kDa AE polypeptide in isolated outer hair cells which, due to its glycosylation, is comprised of AE2 than AE1 isoforms. We confirmed AE2 expression in outer hair cells with the help of subtype-specific monoclonal and polyclonal antibodies to AE, AE subtype-specific primers and AE subtype-specific cDNA and found glycosylated truncated as well as full-length AE2 isoforms. No AE1 or AE3 subtypes were noted in outer hair cells. In contrast, AE2 and AE3 but not AE1 subtypes were seen in supporting cells of the organ of Corti. Their expression preceded the development of cochlear function, coincident with the establishment of the endocochlear potential and the differentiation of supporting cells. While most developmental processes in the inner ear usually begin in the basal cochlear turn, the AE2 expression in outer hair cells (but not that of AE2 and AE3 in supporting cells) progressed from the apical to the basal cochlear turn, reminiscent of the maturation of frequency-dependency. Irrespective of their presumed individual role as either anion exchanger, anchor protein or motility protein, the differential expression and developmental profile of these proteins suggest a most important role of anion exchange proteins in the development of normal hearing. These findings may also provide novel insights into AE function in general.

Dementia, gliosis and expression of the small heat shock proteins hsp27 and alpha B-crystallin in Parkinson's disease.

Cognitive impairment and dementia are common in the later stages of Parkinson's disease (PD). Neuropathological examination of demented PD (PDD) patients often reveals changes that are typical of Alzheimer's disease (AD). In AD, there is a massive reactive gliosis and increased expression of the small heat shock proteins (hsp) hsp27 and alpha B-crystallin. Since these proteins are characteristic for reactive astrocytes in AD, we investigated their expression in the brains of PDD patients. The results were compared with those obtained in the brains of non-demented PD patients. We found (1) no detectable expression of hsp in PD without dementia, and low expression in PD with mild dementia; (2) reactive gliosis and increased expression of hsp in the cortex of PDD brains; (3) a strong association between hsp immunoreactivity and the severity of the AD-specific changes, especially with the number of tangles in the hippocampus; (4) a distinct immunoreaction of alpha B-crystallin in microglia in the substantia nigra and in the hippocampus in PDD. These results indicate that astrocytes react to the disease conditions in AD and in PDD in a similar way, namely by the increased expression of small heat shock proteins, and present additional evidence for the thesis that the pathology of the dementia in PD is related to that in AD.

The molecular chaperone alphaB-crystallin enhances amyloid beta neurotoxicity.

Amyloid beta (Abeta) is a 40- to 42-residue peptide that is implicated in the pathogenesis of Alzheimer's Disease (AD). As a result of conformational changes, Abeta assembles into neurotoxic fibrils deposited as 'plaques' in the diseased brain. In AD brains, the small heat shock proteins (sHsps) alphaB-crystallin and Hsp27 occur at increased levels and colocalize with these plaques. In vitro, sHsps act as molecular chaperones that recognize unfolding peptides and prevent their aggregation. The presence of sHsps in AD brains may thus reflect an attempt to prevent amyloid fibril formation and toxicity. Here we report that alphaB-crystallin does indeed prevent in vitro fibril formation of Abeta(1-40). However, rather than protecting cultured neurons against Abeta(1-40) toxicity, alphaB-crystallin actually increases the toxic effect. This indicates that the interaction of alphaB-crystallin with conformationally altering Abeta(1-40) may keep the latter in a nonfibrillar, yet highly toxic form.

The biochemistry of Alzheimer's disease.

In the course of the biochemical efforts devoted to elucidation of the cause(s) and mechanism(s) of neurodegeneration in Alzheimer's disease (AD), much attention has been given to the processes by which amyloid is generated from amyloid precursor protein, notwithstanding the finding that mutations in 2 other proteins, presenilin 1 and 2, are associated with early-onset, familial AD in the majority of patients. In addition, the reason why the apolipoprotein E epsilon4 allele is over-represented in patients with the sporadic form of AD is unknown. Furthermore, the degree of dementia is clearly associated more with the degree of neurofibrillary pathology than with the amyloid plaque burden. In general, amyloid formation may very well be at the end of a pathophysiological cascade, set in motion by many different triggers. This cascade could involve excessive apoptosis, followed by necrosis and inflammation. In this process, microglia as well as astrocytes are involved. Disturbance of I or more critical signal transduction processes, especially at the level of the plasma membrane, may be an important trigger. The pathogenesis of AD is complicated, but further identification of the processes of neurodegeneration will also lead to identification of the factors that make specific neurons vulnerable and, hopefully, point the way to a means to prevent neuronal degeneration at an early stage.

Neuronal anion exchange proteins in Alzheimer's disease pathology.

Anion exchange (AE) proteins are present in human neurons in the brain. Immunohistochemical data indicate that their apparent expression level increases with age, and especially with degeneration in Alzheimer's disease-affected brain areas. The increase in immunoreactivity is probably caused by changes in AE structure that lead to an increased accessibility of hitherto hidden epitopes. These epitopes correspond to regions in the membrane domain that are involved in generation of senescent cell-specific antigen from AE1 in aging erythrocytes. Elucidation of the molecular nature of these changes and the underlying mechanisms will lead to insight in the processes that govern aging- and degeneration-associated perturbation of membrane integrity. The functional consequences of changes in AE structure may range from acidosis and disturbance of cytoskeleton integrity to untimely or impaired recognition of neurons by microglia.

Heat shock, but not the reactive state per se, induces increased expression of the small stress proteins hsp25 and alpha B-crystallin in glial cells in vitro.

The stress proteins hsp25 and alpha B-crystallin are found in increased concentrations in reactive astrocytes of brains undergoing neurodegeneration. In order to characterize this reaction, we investigated the expression of hsp25 and alpha B-crystallin during growth and after stress (heat shock) in glial cells in vitro. In primary rat brain cultures, hsp25 was present in actively dividing astrocytes that were positive for glial fibrillary acidic protein. alpha B-crystallin was found predominantly in oligodendrocytes. Heat shock resulted in increased concentrations of hsp25 and alpha B-crystallin in astrocytes, without any detectable changes in intracellular localization, as detectable with confocal laser microscopy. These results indicate that a neurodegeneration-related increase of the small stress proteins in astrocytes in independent of gliosis per se, and may be a disease-related event.

Erythrocyte aging in the demented elderly: a fluctuating process?

Measurement of erythrocyte aging parameters in patients with dementia indicates that an Alzheimer-related disturbance of the erythrocyte aging process may not be detectable until in the more advanced stages of the disease. Also, a strong fluctuation in the values of erythrocyte aging parameters, over a period of 15 months, was observed in patients with dementia, but not in age-matched control donors. This fluctuation was independent of the type and stage of dementia, and its cause remains to be elucidated. Such variability hampers the use of erythrocyte aging characteristics for the diagnosis of dementia. On the other hand, the aging-related erythrocyte IgG content may be a sensitive biomarker for disturbed systemic homeostasis in the elderly.

Erythrocyte aging characteristics in elderly individuals with beginning dementia.

An increase in erythrocyte-bound IgG and enhanced breakdown of the erythrocyte anion exchanger band 3, characteristics of normal erythrocyte aging are observed in old, healthy individuals when compared with young donors. These findings indicate that the rate of cellular aging increases with organismal aging. Results from previous studies on the same parameters have suggested that the erythrocyte aging process is disturbed in patients in advanced stages of Alzheimer type dementia, and in individuals with Down's syndrome who show no signs of dementia. In this study we find no changes in erythrocyte aging parameters in old individuals in beginning stages of dementia of various etiologies. We conclude that, in general, characteristics of disturbed erythrocyte aging cannot serve as presymptomatic markers of Alzheimer-type dementia.

Involvement of neuronal anion exchange proteins in cell death in Alzheimer's disease.

Anion exchange (AE) proteins are present in human neurons in the brain. Immunohistochemical data indicate that their apparent expression level increases with age, and especially with degeneration in Alzheimer's disease-affected brain areas. The increase in immunoreactivity is probably caused by changes in AE structure that lead to an increased accessibility of hitherto hidden epitopes. These epitopes correspond to regions in the membrane domain that are involved in generation of senescent cell-specific antigen from AE1 in aging erythrocytes. Elucidation of the molecular nature of these changes and the underlying mechanisms, will lead to insight in the processes that govern aging- and degeneration-associated perturbation of membrane integrity. AE-mediated chloride/bicarbonate exchange is a major component in the regulation of intracellular pH. The functional consequences of changes in AE structure may range from acidosis, disturbance of cytoskeleton integrity, and untimely or impaired recognition of cells by components of the immune system, such as microglia. A molecular and physiological description of these changes will establish AE proteins as valuable tools in elucidating the processes of normal aging, and the disturbances in aging-related diseases such as Alzheimer's disease.