Life Span Of Blood Cells Research Articles

3149 – COMPARISON OF PRE-ENRICHMENT STRATEGIES FOR SORTING OF MOUSE LIN-CKIT+SCA1+ CELLS

The short life span of blood cells requires hematopoiesis to be a continuous ongoing process. In adult mammals, blood production takes place in the bone marrow. This tissue is home to hematopoietic stem cells (HSCs) that possess the ability to both self-renew and differentiate into mature blood cells. HSCs are widely studied in cancer research, as they are the source of various hematological malignancies. Such cancers develop in response to a progressive accumulation of mutations that confer a selective growth advantage to HSCs. In the case of leukemia, the mutations will lead to a differentiation blockade, causing dysfunctional blood cells to accumulate. Mouse models are the system of choice to study blood cancers given the complexity of the hematopoietic system. Thus, in order to study HSCs, we need to be able to isolate them from mice. Several HSC subpopulations have been defined, however, the most widely studied are the Lin-cKit+Sca1+ (LKS) cells. The best way to isolate LKS cells is by FACS sorting. Yet, the frequency of LKS cells in the bone marrow being very low (< 0.1%), a pre-enrichment step is necessary to decrease the sorting time. With the aim of identifying the most suitable pre-enrichment strategy, we tested several magnetic kits and compared them based on enrichment efficiency and final LKS yield. Four approaches were tested: lineage depletion (Lin-), cKit-enrichment (cKit+), Sca1-enrichment (Sca1+) and a combination of lineage depletion followed by Sca1-enrichment (Lin- Sca1+). Our results show that the best strategy are lineage depletion and Sca1-enrichment. Combining these two methods highly increased the LKS enrichment, however, the final LKS yield was low due to increased cell loss from performing a second enrichment step.

MSC-Derived Extracellular Vesicles: New Emergency Treatment to Limit the Development of Radiation-Induced Hematopoietic Syndrome?

Nuclear accidents or acts of terrorism involving radioactive sources might lead to mass casualties irradiation. The hematopoietic system is one of the most critical and radiation-sensitive tissues because the limited life span of blood cells requires the continuous division of hematopoietic stem cells (HSCs) into the bone marrow. The radiation-induced hematopoietic syndrome, RI-HS, is an impairment of the hematopoiesis that will result in pancytopenia of various degrees. In fact, treatment with granulocyte-colony stimulating factor (G-CSF) is considered as a valuable adjunct to treatment controls in some irradiated patients. Nevertheless, these overexposed patients with bone marrow suppression have minimal medullary territories that do not allow complete recovery of hematopoiesis but lead to significant immunoreactivity following allogeneic hematopoietic stem cell transplantation (HSCT). The high morbidity and mortality of these overexposed patients is a reminder of the lack of effective treatment for hematopoietic syndrome. During the last 20 y, a therapeutic approach for mesenchymal stem cells (MSC) has been proposed for the management of accidentally irradiated victims. Many preclinical animal studies have shown that MSC, mainly by their secretory activity, in particular extracellular vesicles (EVs), contribute to the control of inflammation and promote regeneration of tissues by accelerating angiogenesis and re-epithelialization processes. Therefore, we investigated the potential effect of EVs on the reduction of early bone marrow ionization toxicity, early anti-apoptotic therapy, and vascular protection in the RI-HS model. The main purpose is to propose an innovative treatment of non-patient-specific RI-HS emergency treatment in order to limit allogeneic HSC.

300-OR: Individualized Hemoglobin A1C: A New Glycemic Marker Determined Using Continuous Glucose Monitoring

Glycation of hemoglobin (Hb) measured by A1C depends on red blood cell (RBC) lifespan, thus may misrepresent intracellular glucose exposure in other tissues, and may explain observed dissociations between A1C and microvascular complications. A kinetic model that accounts for rates of RBC turnover and intracellular glucose transport was evaluated for A1C individualization using continuous glucose monitoring data (CGM) and laboratory A1C from 58 individuals with diabetes (type 1/type 2 = 5/53, mean age 58 years (range 27 to 77, M/F=38/20). A1C was performed at 0, 3, and 6 months with concurrent CGM for 6 months, having a minimum of 80% coverage and no gaps &amp;gt;36 hours. The adjustment required to calculate individualized A1C is expressed as follows: A1C/(A1C+(1-A1C)*KTR/KT), where KT is the Hb and RBC turnover rate, and KTR is the reference Hb and RBC turnover rate. The mean (±SD) rate of Hb and RBC turnover was 1.18±0.20%/day as depicted in the Figure, showing the individualized A1C calculation. These data suggest that laboratory A1C does not only underestimate cellular glucose exposure in some patients but can also overestimate average glucose levels making the individual prone to hypoglycemia through unnecessary escalation of antidiabetic therapies. Quantitative individualization of A1C derived from A1C and CGM readings has the potential to more accurately assess intracellular glycemic exposure. Disclosure R.M. Bergenstal: Research Support; Self; Abbott, Boehringer Ingelheim Pharmaceuticals, Inc., Dexcom, Inc., Glooko, Inc., Hygieia, JDRF, Johnson &amp; Johnson Medical Devices Companies, Lilly Diabetes, Medtronic MiniMed, Inc., Merck &amp; Co., Inc., National Institute of Diabetes and Digestive and Kidney Diseases, Novo Nordisk Inc., Roche Diabetes Care, Sanofi. Other Relationship; Self; Abbott, Helmsley Charitable Trust, Hygieia, Lilly Diabetes, Novo Nordisk Inc., Onduo LLC, Sanofi, UnitedHealth Group Inc. T. Dunn: Employee; Self; Abbott Laboratories. Y. Xu: Employee; Self; Abbott Laboratories. R. Ajjan: Consultant; Self; Abbott. Research Support; Self; Abbott, LifeScan, Inc. Funding Abbott Diabetes Care

Allium cepa Linn. (Liliaceae) (red onion) bulb aqueous extract increases membrane stability of red blood cells and ameliorates oxidative stress in diabetes

The aim of this study was to assess the effects of Allium cepa aqueous extract on red blood cell membrane stability and oxidative stress in streptozotocin-induced diabetic Wistar rats. Various alterations of red blood cell (RBC) membrane appear both in diabetes mellitus and during the physiological aging processes. Diabetes mellitus decreases the life span of red blood cells therefore, it may change the membrane stability by acting through its effect on the aging process. Fresh A. cepa bulbs were mashed and macerated in distilled water for 48 h. The freeze dried filtrate (413.7 g) was used for the experiment. Male rats (120–170 g) were made diabetic by a single intraperitoneal injection of streptozotocin (60 mg/kg). Animals with fasting blood sugar (FBS) of 10 mmol/l on day 3 post challenge were selected, grouped, and treated with aqueous extract of A. cepa for 21 days at doses of 50, 150, 300 mg/kg and glibenclamide (2 mg/kg). A negative (challenged and untreated) and a normal control (unchallenged and untreated) groups were included. On day 21, blood samples were collected through the retrobulbar plexus of the medial cantus of the eye for osmotic fragility and in vivo oxidative stress markers analysis. The results showing showed an increase in percent stabilization (through increasing median corpuscular fragility, MCF) with increasing dose of the extract treatment. It also showed an increase in serum catalase (CAT), glutathione (GSH), and superoxide dismutase (SOD) which were significant (p < 0.05) at 150 and 300 mg/kg when compared with the negative control. Malondialdehyde (MDA) was also significantly reduced in these groups with increasing dose of the extract. This study suggests that increase in osmotic fragility of red blood cells attributed to the disturbance in ionic motion through their membrane could be stabilized with aqueous extract of A. cepa. Also, the extract ameliorates oxidation caused by reactive oxygen species and other free radicals produced in diabetic states.

Decrease of α-Chains in β-Thalassemia

In the pathophysiology of β-thalassemia, globin chain imbalance plays a central role in predicting red blood cell (RBC) life span and disease severity. Strategies to improve globin chain imbalance are therefore a legitimate target in the management of this incurable genetic disorder. Classical gene addition with the available retroviral vectors can alter one of the two variables while combined reduction of achains could provide a more potent therapeutic effect. We developed foamy virus (FV) vectors for the production of β-globin and vectors targeting the a-globin transcript using the shRNA technology. Using FVderived vectors, we expressed human anti-a-globin short hairpin RNAs, off a potent PolIII promoter (H1); of the 4 different shRNAs tested, α-globin mRNA reduction varied from 6.3 to 54% of the control CD34+ cells. Similarly, vectors developed for the mouse α-globin, resulted in a significant reduction (range 15-28% of the control) of aglobin in erythroid colonies of Lin- cells. To assay vector performance in vivo, we investigated the hematological parameters in thal3+/- mice transpalnted with FV-transduced Lin- cells, transduced with anti-alpha-globin shRNA. Despite low chimerism and low vector copy numbers (&lt;0.5 per cell), we observed a 10% reduction in red cell distribution width, a marker for distorted erythropoiesis. We finally developed a combination FV vector expressing β-globin off a HS40 enhancer and anti-α-globin shRNA and tested its performance in human CD34+ cells from a thalassemic patient. Globin chain imbalance was ameliorated from a β/α ratio of 0.12 to the level of 0.5, clearly indicating a therapeutic benefit. Overall, shRNA control of α-globin excess is a feasible target but requires improvements since the RNAi effect is tough to predict and should ideally be combined with controllable elements.

Autoimmune Thrombozytopenie, Neutropenie und Hämolyse

Autoantibodies reduce the life span of platelets, granulocytes, and red blood cells. This may result in thrombocytopenia with bleeding, in neutropenia with infection, and in anemia, respectively. Immune-hemocytopenias can manifest as primary disease without another cause, or they are associated with other underlying morbidities such as autoimmune diseases, lymphoproliferative diseases, immune defects, or viral infections. Diagnosis is confirmed by laboratory tests showing autoantibodies against the respective blood cells. Indication for treatment is the clinical manifestation of symptoms: bleeding in autoimmune thrombocytopenia, infections in neutropenia, and symptomatic anemia, respectively. Especially in case of thrombocytopenia patients should not be treated because of abnormal laboratory values, only. To date steroids are the basic treatment in autoimmune thrombocytopenia and hemolytic anemia, while prophylactic antibiotics are the main treatment in autoimmune neutropenia. Growth factors like the new thrombopoietin receptor agnonists in autoimmune thrombocytopenia or G-CSF in autoimmune neutropenia, and anti-CD20 antibodies are new options for treatment.

Telomeres in the haemopoietic system.

The limited life span of most blood cells requires the continuous production of cells, which in adults exceeds 10(12) cells/day. This impressive production of cells (approximately 4 x 10(16) cells over a lifetime) is achieved by the proliferation and differentiation of committed progenitor cells, which themselves are derived from a population of pluripotent stem cells with self-renewal potential. Paradoxically, the large majority of stem cells in adult bone marrow are quiescent cells. One possibility is that stem cells, like other somatic cells, have only a limited replicative potential (< 100 divisions). This hypothesis is supported by two key observations and the consideration that, in theory, 55 divisions can yield 4 x 10(16) cells. First, it was shown that 'candidate' stem cells purified from fetal and adult tissue showed dramatic functional differences in turn-over time and the ability to produce cells with stem cell properties, Second, these functional differences were found to correlate with a measurable loss of telomere repeats despite the presence of low but readily detectable levels of telomerase in all purified cell fractions. In order to address questions about the role of telomeres in normal and malignant haemopoiesis, we developed a quantitative fluorescence in situ hybridization technique. Here we review the characteristics of this novel tool to assess the number of telomere repeats at the end of individual chromosomes and provide an overview of recent observations.

Hematological Changes in Cold-Acclimated Xenopus laevis.

While hematopoiesis in individuals is strictly regulated for maintenance of homeostasis, it has been reported that the numbers of peripheral blood cells are modulated in response to environmental temperature in vertebrates including hamsters, rats, squirrels, dogs, and bullfrogs. To date, the physiological and molecular mechanisms have not been elucidated yet. Amphibians are poikilothermic vertebrates exposed to various fluctuations of environmental conditions; therefore they need to exert their capacities to acclimate to such changes. Additionally numerous studies have demonstrated a measurable metabolic reduction in the metabolic rate of cold-acclimated frogs. We examined hematological changes in response to environmental temperature in an aquatic amphibian, Xenopus laevis. Frogs initially maintained at 25°C were acclimated to 10°C, and hematological changes were observed. The numbers of erythrocytes, leukocytes and thrombocytes gradually reduced as the transient phase by 4 weeks, and subsequently reached the steady-state that sustained for more than 4 months. Whereas the reduction in the numbers of white blood cells and thrombocytes were moderate, the number of erythrocytes and the level of hemoglobin at nadir were remarkably low (approximately 40% of the initial values). It is known that oxygen levels may reduce in ice-cold water, and cold-acclimated animals therefore tolerate prolonged severe hypoxia; nevertheless cold-acclimated Xenopus exhibited severe erythrocytopenia. In addition, morphological change of peripheral blood cells and hematopoietic tissues (liver, spleen, kidney, and bone marrow) were examined. There were no remarkable cellular changes in cellular size and shape. However, increased numbers of mature erythrocytes were observed in the bone marrow of the steady state cold-acclimated Xenopus, while mature erythrocytes were not found in the bone marrow in Xenopus at 25 °C. This cold-temperature-induced pancytopenia was reversible when the temperature was put back to 25°C, as all of blood cell counts returned to the normal levels within 4 weeks in a reverse fashion as the transient phase of cold-acclimation. During the recovery phase, immature erythrocytes that were scarcely existed in the normal peripheral blood appeared in the circulation, suggesting that erythrocytes were newly produced at 25 °C after prolonged exposure to cold temperature. The possible explanations for the reduction in the numbers of circulating peripheral blood cells might be due to a number of various reasons such as reduced productions of hamatopoietic progenitors and/or related cytokines, alternation in the storage capacities and/or the life span of blood cells, and systemic suspension of normal activities. To compare the lifespan of erythrocytes between normal and cold-acclimated Xenopus, erythrocytes were covalently labeled with biotin. The surviving biotinylated erythrocytes in the circulation were quantitatively detected as avidin-biotin complex by microscopy and flowcytometry. Furthermore expression levels of several genes responsible for the hematopoietic regulation were comparatively examined. The cold-acclimated Xenopus model developed here may allow for a valuable approach aiming at exploring undiscovered systems in hematopoietic regulation.

Increased mechanical fragility and intravascular lysis of erythrocytes in cats fed a propylene glycol-containing diet

The mechanism responsible for the decreased red blood cell (RBC) lifespan associated with feeding propylene glycol (PG)-containing diets was investigated to understand better how Heinz body-contained RBC are destroyed. Three cats were fed a diet containing 12% PG for 14 days and three other cats served as control. The experimental group developed reticulocytosis and increased Heinz body numbers. Red blood cell membrane immunoglobulih G (IgG) concentration and phagocytosis of RBC by peritoneal macrophages were lower in the PG group compared to the control group suggesting that neither IgG nor non-IgG-mediated phagocytosis was responsible for the RBC destruction. Osmotic fragility, rate of RBC proteolysis and mild mechanical fragility test results were not statistically different from controls. However, when RBC from cats fed PG were exposed to severe mechanical stress, their fragility were increased 2.2–2.8 times. Additionally, haptoglobin concentrations were decreased in the PG group. These data suggest that intravascular lysis may be involved in the pathogenesis of PG-induced RBC destruction.

Morphohistochemical changes of the blood cells in the hibernating frog (Rana esculenta L.)

Some morphological features (degree of nuclear segmentation in neutrophils and eosinophils), histochemical patterns (DHFR, LDH, G6PDH) and, in addition, the nucleic acid distribution (DNA, RNA with acridine orange) in the peripheral blood cells of Rana esculenta, during the hibernation phase were investigated. All the observed parameters varied significantly in the frog during hibernation in comparison with the active period. The most evident changes were an increase in the nuclear segmentation of the neutrophils and a lower activity of the histochemically demonstrable DHFR and LDH, probably due to cell ageing. These findings suggest that, in hibernating frogs, there is an increase in the life span of the peripheral blood cells as a consequence of a reduced metabolic activity and a slowing of haematopoiesis.

Erythropoiesis and Erythrocyte Age Distribution in Hemodialysis Patients Undergoing Erythropoietin Therapy

Renal anemia is caused in part by a reduced life span of red blood cells (RBCs) and by reduced erythropoietin biosynthesis in the damaged kidney. The RBC age can be determined by density gradient centrifugation and estimation of cell-age-dependent enzyme activities, as aspartate aminotransferase. The RBC age distribution influences the median density (D50) of RBCs and the blood rheology in coherence with the hematocrit. In our study, the median density was determined by Percoll density gradient centrifugation in 18 healthy subjects (D50 = 1.0674 +/- 0.0016 g/ml) and in 14 hemodialysis patients (D50 = 1.0674 +/- 0.0016 g/ml in the course of recombinant human erythropoietin (rhEPO) therapy. During the first 4 weeks of therapy, a strong rejuvenation of RBCs was observed whereby the D50 reached a minimum after 2 weeks (D50 = 1.0655 +/- 0.0022 g/ml; p less than 0.05 vs. value before therapy) and a steady state after 4 weeks (D50 = 1.0658 +/- 0.0013 g/ml; p less than 0.1 vs. value before therapy). In 5 of the patients with elevated plasma parathyroid hormone (i-PTH) concentrations greater than 10 pmol/l, a significantly (p less than 0.05) reduced amount of younger RBCs (D50 = 1.0675 +/- 0.0016 g/ml) was observed in the first 2 weeks of rhEPO therapy as compared to patients with i-PTH less than 10 pmol/l (D50 = 1.0677 +/- 0.0019 g/ml). Thus, erythropoiesis in the early phase of rhEPO therapy is strongly influenced by elevated plasma i-PTH concentrations. Therefore, a gradual increase in rhEPO doses is preferable before therapy at elevated doses with an uncontrolled increase in RBC amount.

Red Blood Cell Density Distribution in Uremic Patients on Acetate and Bicarbonate Hemodialysis

Renal anemia is the result of reduced erythropoietin (EPO) biosynthesis in the diseased kidney and also in part the result of a reduced life span of red blood cells (RBCs). An increase in density and a decrease in enzyme equipment (aspartate aminotransferase; GOT) of RBCs reflect cell age. In the following study, the density distribution (median density D50; determined by Percoll density gradients) and GOT activities of RBCs were measured in patients on acetate (HDA; n = 15) and bicarbonate (HDB; n = 51) hemodialysis. Hemoglobin (Hb) concentrations were: in the HDB group, 9.1 +/- 3.4 g/dl; in the HDA group, 6.2 +/- 1.2 g/dl, and, in a control (C) group of healthy persons, 14.0 +/- 1.5 g/dl. 14 HDB patients with severe anemia received EPO therapy during 1 year. D50 were found as follows: group C, 1.0674 +/- 0.0016 g/ml; HDB, 1.0674 +/- 0.0015 g/ml, and HDA, 1.0660 +/- 0.0012 g/ml (HDA vs. group C: p less than 0.05; HDA vs. HDB: p less than 0.05. D50 were elevated in the subgroups of HDA and HDB patients with severe anemia (Hb less than 8 g/dl). During activated erythropoiesis by EPO therapy, D50 decreased from 1.06739 +/- 0.0015 to 1.0656 +/- 0.0014 g/ml. The GOT activities in RBCs demonstrated a rejuvenation of the RBC population in the HDB group (6.4 +/- 2.5 U/g Hb) and HDA group (5.9 +/- 3.1 U/g Hb) compared to group C (3.9 +/- 1.3 U/g Hb).(ABSTRACT TRUNCATED AT 250 WORDS)

Microcythemia from anemic hypoxia and normal erythropoiesis in the newt.

Specimens of the newt, Triturus cristatus carnifex (Laurenti), rendered totally anemic, restore erythron by cyclic waves of erythropoietic activity that alternate with intervals of stasis. Hemolysis is obtained by administering 25 mg/liter of acetylphenylhydrazine in the breeding water for 36 h. The first cycle of erythropoietic activity produces microcytes, which have completely differentiated by 8 weeks after treatment. However, if the animals are raised in a hyperbaric chamber at a pressure of 1.5 atmospheres, in order to compensate for hypoxia, normocytes are produced. In both cases the hematocrit and hematic concentration of hemoglobin reach analogous values, so microcythemia appears to be the only effect of hypoxia. The hemoglobin, hematocrit values, and normocyte counts in hyperbaric animals are about one-half those of the controls newts. These data, together with those on the life span of red blood cells (RBC) and time span between two successive erythropoietic cycles (2 months and 1 month, respectively), indicate that the newts normally keep only two sets (one new, one old) of RBC in circulation, whose approximate parameters can be defined as RBC count: 60,000/mm3, hematocrit: 17%, and hemoglobin: 5.4 g/100 ml.

Hematopoiesis in the Human Spleen

For a few months in utero the normal human spleen is hematopoietic. By the close of the fifth fetal month, the function has moved permanently into the marrow. Except for some lymphocytes and plasma cells, which continue to be contributed from the white pulp of the spleen and other lymph tissues, the production of blood cells—monocytes, granulocytes, erythrocytes, and megakaryocytes—remains entirely in the marrow. The basis of this obligatory behavior probably resides in the quality of the marrow's unique sinusoidal microcirculation, which provides an essential environment. 1 In adult small mammals, the spleen remains a normal hematopoietic organ. In the mouse, for example, all available space in the marrow cavities contains what we would call, in the human condition, hypercellular marrow; there is no fat. In these tiny animals, the proportion of marrow cavity to solid bone is small and the life span of blood cells is relatively short (requiring

Biophysical methods for the assay of the life span of red blood cells.

Biophysical methods for the assay of the life span of red blood cells.