Immature Erythrocytes Research Articles

Histopathological changes and erythrocytic nuclear abnormalities in Iberian green frogs ( Rana perezi Seoane) from a uranium mine pond

In spite of their sensitivity to anthropogenic stressors, adults of Rana perezi Seoane were found inhabiting effluent ponds from a uranium mine. Due to the presence of such organisms in this environment, it becomes of paramount importance to assess the damages induced by local contamination on these aquatic vertebrates, in order to integrate this information on a site-specific risk assessment that is being carried out in the area. To attain this purpose an ethically and statistically acceptable number of green frogs were captured in the mine pond (M) and in a pristine river (VR), a few kilometres from the mine. Bioaccumulation of metals and histopathological alterations were evaluated in the liver, kidneys, spleen, lungs and testes of the animals. Simultaneously, blood samples were collected for the evaluation of genotoxic damage on erythrocytes. Animals captured in the M pond showed significantly increased levels of Be, Al, Mn, Fe and U in the liver, as well as Pb and U in the kidney. The liver was the main target organ for the bioaccumulation of Be, Al, Fe and U. However, renal histopathologies were more severe than those of liver. The main tissue alterations recorded in animals from the mine were: a slight increase in melanomacrophagic centers (MMC) in liver, lung and kidneys; dilatation of the renal tubules lumen associated with tubular necrosis. A significantly higher number of erythrocytic abnormalities (lobed, notched and kidney shaped nuclei and micronuclei) were recorded in frogs from M than in frogs from VR, along with a significantly lower frequency of immature erythrocytes. Both observations suggested that the removal of abnormal blood cells might be compromised.

Erythropoietin Lacking Hypoxia Responsive Element in 3′ Untranslated Region Stimulates Hepatic Erythropoiesis in Anemic Xenopus.

Recent studies on EPO-EPOR systems in non-mammalian vertebrate including frog and teleost fishes demonstrate extramedullary adult erythropoiesis instead of bone marrow. In adult Xenopus laevis (African clawed frog), an animal model of hepatic erythropoiesis should give an opportunity for understanding the microenvironment of vascular niche. Therefore, we investigate on erythrocyte development, gene expressions and gene regulations of EPO and EPOR molecules in adult Xenopus liver. In situ hybridization and immunostaining revealed that Xenopus EPOR (xlEPOR) expressing erythrocyte progenitors, which have low hemoglobin content, were localized among liver sinusoids. The maximum number of xlEPOR expressing cells was observed when peripheral RBC count reached a nadir after hemolytic anemia. Flowcytometric analysis of peripheral blood cells and dispersed liver cells using anti-xlEPOR antibodies also indicate that the xlEPOR+ cells in liver were increased as decreasing count of peripheral RBC. After a nadir of RBC count, xlEPOR+ immature nucleated erythrocytes were emerged in the circulation. The count of xlEPOR+ immature erythrocytes was gradually decreased as increasing count of xlEPOR− mature erythrocytes. Since mature erythrocytes are still nucleated in Xenopus, we used xlEPOR molecules as erythroid differentiation marker. Meanwhile real time RT-PCR analysis of Xenopus EPO (xlEPO) mRNA revealed that xlEpo gene expression was significantly induced in anemic liver compared to normal liver. These data suggest that xlEPO-xlEPOR signaling between erythrocyte progenitors and liver cells progress the proliferation and differentiation of erythrocyte progenitors, and the mobilization of immature erythrocytes into the circulation. Our previous study showed that the anemic serum of phenylhydrazine administrated Xenopus contains erythroid colony forming activity; however, there is no information about the relationship between anemia and hypoxia enough to stimulate erythropoiesis in adult Xenopus. In mammalian species, Epo gene expression is upregulated by binding of hypoxia inducible factor-1a (HIF-1a) and ARNT complex to hypoxia response element (HRE) located in 3′ enhancer region of Epo gene. In frog and fishes, EPO mRNAs are expressed even in normoxia condition. In fish Epo genes, consensus HRE sequence (ACGTG) were not found in 3′UTR, as well as the reporter assay failed to show Epo upregulation respond to hypoxia. Since any consensus HRE sequence was not found in 3′ UTR of xlEpo gene, we examined whether HIF-1a mediates xlEpo gene regulation. By western blot analysis of HIF-1a, we assessed whether HIF-1a is stabilized in anemia; meanwhile binding capacity of HIF-1a to 5′, 3′ UTRs and intron regions of xlEpo gene was analyzed by gel shift mobility assay. The findings in non-mammalian animal model demonstrate the basis of erythropoietic gene regulations, as well as molecular mechanism underlying adult extramedullary erythropoiesis.

Erythrocytic nuclear abnormalities in wild and caged fish ( Liza aurata) along an environmental mercury contamination gradient

Laranjo basin (Aveiro, Portugal) has been subjected to mercury contamination from a chlor-alkali plant, presenting a well-described mercury gradient. This study aims the assessment of mercury genotoxicity in this area by measuring erythrocytic nuclear abnormalities (ENA) frequency in the mullet Liza aurata, and its relation with total mercury concentration (Hg t) in blood. Wild fish were seasonally analysed, and, complementarily, fish were caged for 3 days at three locations differing on their distances to the mercury source. The results from Laranjo were compared with those from a reference area (S. Jacinto). Wild fish from Laranjo showed elevated ENA frequency in summer and autumn in concomitance with increased blood Hg t. Surprisingly, no ENA induction was found in winter, despite the highest blood Hg t, which may be explained by haematological dynamics alterations, as supported by a decreased immature erythrocytes frequency. Caged fish displayed ENA induction only at the closest site to the contamination source, also showing a correlation with blood Hg t.

Evidence for canonical beta-catenin signaling in neuroblastomas

Ghole, Saif A. MD; Kuscuoglu, Unsal PhD; Hu, Min MD, PhD; Jeong, Yeon Jun MD, PhD; Sylvester, Karl G. MD, FACS Author Information

Novel Erythrocyte Pits in the Small Tropical Ruminant, Lesser Mouse Deer

We examined unique erythrocyte pits of the peripheral blood and bone marrow in the lesser mouse deer, Tragulus javanicus, using scanning electron microscope (SEM) and transmission electron microscope (TEM). Under the SEM observation, the pit was observed as a hole on both mature erythrocytes of the peripheral blood and immature erythrocytes of the bone marrow. By the TEM, the mature erythrocytes had a vacuole, which showed complicated shape and occupied considerable space within the cytoplasm. The vacuole was communicated extracellularly by perforation, which corresponded to the hole on the cell surface. In the bone marrow, erythroblast and reticulocytes have a cytoplasmic vacuole. This abnormal feature of the erythrocytes is peculiar to the mouse deer, and not found in other tropical ruminants. Despite the disadvantage of volume loss from the small erythrocytes, the mouse deer were healthy and showed no signs of anaemia.

Evaluation of the in vitro and in vivo genotoxicity of magnolia bark extract

Magnolia bark extract (MBE) is an extract of the dried stem, root, or branch bark of magnolia trees that has been used historically in traditional Chinese and Japanese medicines, and more recently as a component of dietary supplements and cosmetic products. To study the genotoxic potential of MBE, a bacterial reverse mutation assay and an in vivo micronucleus test were conducted. Compositional analysis of the test substance revealed that MBE contains 94% magnolol and 1.5% honokiol. MBE exerted no mutagenic activity in various bacterial strains of Salmonella typhimurium and in Escherichia coli WP2 uvrA, either in the absence or presence of metabolic activation at all doses tested. In the micronucleus test, various doses of MBE did not affect the proportions of immature to total erythrocytes, nor did it increase the number of micronuclei in the immature erythrocytes of Swiss albino mice. The results of these studies demonstrate that MBE is not genotoxic under the conditions of the in vitro bacterial reverse mutation assay and the in vivo micronucleus test, and support the safety of MBE for dietary consumption.

Haematological characteristics of injured western ringtail possums (Pseudocheirus occidentalis)

Haematological data from 12 western ringtail possums with tissue trauma resulting from motor vehicle impact or dog bite wounds were analysed. The greatest concentration of neutrophils observed was 7.22 × 109/l. Morphological atypia of neutrophils was evident in four animals. Four animals had significant anaemia, and three animals exhibited increased erythropoiesis (6–17.2% polychromatophilic erythrocytes). The study illustrates that marked neutrophilia is unlikely to occur in response to trauma-induced tissue inflammation in this species. However, marked increases in immature erythrocytes may be observed in the peripheral blood in response to anaemia.

Behaviour of reticulocyte counts and immature reticulocyte fraction during a competitive season in élite athletes of four different sports

The role of reticulocytes (Ret) in sports medicine became important when the count of immature erythrocytes has been introduced in protocols used and officially approved for antidoping purposes. The use of modern automated analysers, which allow the easy count and the description of characteristics of reticulocytes, increased the possible use of these parameters in sports medicine. We studied the behaviour of Ret and immature reticulocyte fraction (IRF) in top-level athletes practising rugby, ski, soccer and cyclism, throughout a competitive season. We aimed at increasing the knowledge of physiological characteristics of these sportsmen and supplying valuable suggestions to trainers and sports physicians. We observed a stability of Ret counts, also during training and competitions, although some modifications, namely decrease during competitions periods in cyclists, and in rugby and soccer players, occurred. No significant correlation was found between Ret count and Hb in each sport discipline. IRF values tend to be high in athletes owing to continuous bone marrow stimulation linked to haemolysis, typical of sports activities. We confirm the validity of the use of Ret counts for antidoping purposes and also for evaluating health status and iron metabolism of sportsmen.

Some changes in the haematological parameters of rainbow trout (Oncorhynchus mykiss) exposed to three artificial photoperiod regimes

Haematological parameters are often used as health status and stress indicators in fish. However, information on the effects of artificial photoperiods on these parameters is scarce and ambiguous. The consequences of three different artificial photoperiod regimes [light/day 12 h:12 h (LD 12:12) for 150 days as controls; LD 14:10 and LD 24:0, both for an initial 60 days, and then LD 12:12 for the remaining 90 days] were evaluated in trout. In all fish the haemogram and the erythrocyte production index (EPI) were assessed at days 7, 14, 30 and 60 (phase 1) and at days 90, 120 and 150 (phase 2). At the end of phase 1, photoperiods LD 14:10 and 24:0 induced higher production of immature (late basophilic) erythrocytes (0.06–0.08 × 1012 cells/l, P < 0.05) and elevated EPI (young erythrocytes = 1.6 in LD 14:10, 2.25 in LD 24:0; late basophilic erythrocytes = 4.1–4.9 in LD 14:10, 3.3 in LD 24:0) than in controls (late basophilic erythrocytes = 0.02–0.04 × 1012 cells/l; EPI young erythrocytes = 0.2–1.0; EPI late basophilic erythrocytes = 1.4–2.7). During phase 2, only reduced numbers of lymphocytes and thrombocytes were observed at day 150 in the LD 14:00 group. An increase in the EPI (1.9) of control young erythrocytes was found at day 150.

In vivo erythrocyte micronucleus assay : III. Validation and regulatory acceptance of automated scoring and the use of rat peripheral blood reticulocytes, with discussion of non-hematopoietic target cells and a single dose-level limit test

The in vivo micronucleus assay working group of the International Workshop on Genotoxicity Testing (IWGT) discussed new aspects in the in vivo micronucleus (MN) test, including the regulatory acceptance of data derived from automated scoring, especially with regard to the use of flow cytometry, the suitability of rat peripheral blood reticulocytes to serve as the principal cell population for analysis, the establishment of in vivo MN assays in tissues other than bone marrow and blood (for example liver, skin, colon, germ cells), and the biological relevance of the single-dose-level test. Our group members agreed that flow cytometric systems to detect induction of micronucleated immature erythrocytes have advantages based on the presented data, e.g., they give good reproducibility compared to manual scoring, are rapid, and require only small quantities of peripheral blood. Flow cytometric analysis of peripheral blood reticulocytes has the potential to allow monitoring of chromosome damage in rodents and also other species as part of routine toxicology studies. It appears that it will be applicable to humans as well, although in this case the possible confounding effects of splenic activity will need to be considered closely. Also, the consensus of the group was that any system that meets the validation criteria recommended by the IWGT (2000) should be acceptable. A number of different flow cytometric-based micronucleus assays have been developed, but at the present time the validation data are most extensive for the flow cytometric method using anti-CD71 fluorescent staining especially in terms of inter-laboratory collaborative data. Whichever method is chosen, it is desirable that each laboratory should determine the minimum sample size required to ensure that scoring error is maintained below the level of animal-to-animal variation. In the second IWGT, the potential to use rat peripheral blood reticulocytes as target cells for the micronucleus assay was discussed, but a consensus regarding acceptability for regulatory purposes could not be reached at that time. Subsequent validation efforts, combined with accumulated published data, demonstrate that blood-derived reticulocytes from rats as well as mice are acceptable when young reticulocytes are analyzed under proper assay protocol and sample size. The working group reviewed the results of micronucleus assays using target cells/tissues other than hematopoietic cells. We also discussed the relevance of the liver micronucleus assay using young rats, and the importance of understanding the maturation of enzyme systems involved in the processes of metabolic activation in the liver of young rats. Although the consensus of the group was that the more information with regard to the metabolic capabilities of young rats would be useful, the published literature shows that young rats have sufficient metabolic capacity for the purposes of this assay. The use of young rats as a model for detecting MN induction in the liver offers a good alternative methodology to the use of partial hepatectomy or mitogenic stimulation. Additional data obtained from colon and skin MN models have been integrated into the data bases, enhancing confidence in the utility of these models. A fourth topic discussed by the working group was the regulatory acceptance of the single-dose-level assay. There was no consensus regarding the acceptability of a single dose level protocol when dose-limiting toxicity occurs. The use of a single dose level can lead to problems in data interpretation or to the loss of animals due to unexpected toxicity, making it necessary to repeat the study with additional doses. A limit test at a single dose level is currently accepted when toxicity is not dose-limiting.

Relationships between hemorheology, erythrocyte metabolism, and von willebrand factor in athletes and patients with peripheral arterial disease

The relationship between hemorheology, erythrocyte ATP and 2,3-diphosphoglycerate (2,3-DPG) concentrations, and von Willebrand factor antigen was studied in athletes and peripheral arterial disease patients. Lower blood viscosity, mainly due to a higher erythrocyte deformability, was found in athletes compared to control subjects. Higher 2,3-DPG/Ht levels in athletes were correlated with blood viscosity, erythrocyte deformability, the rigidity index, and erythrocyte suspension viscosity at low shear stress. It is suggested that these relationships might be determined by the predominance of immature erythrocytes in the blood circulation of the athletes. In the group of patients, a decrease in ATP/Ht was related to increased erythrocyte aggregation and a higher erythrocyte suspension viscosity. Moreover, the concentration of von Willebrand factor was positively correlated with the erythrocyte aggregation index, erythrocyte suspension viscosity, and plasma viscosity. The results show that alterations in erythrocyte and plasma rheology may be involved in the modification of the functional state of the vascular endothelium and the development of atherosclerosis.

Genotoxicity Studies on Sel-Plex®, a Standardized, Registered High-Selenium Yeast

Selenium, recognized as an essential nutrient for human health, is a component of proteins and enzymes required for various biological functions and is currently being used as a feed supplement for livestock in geographical areas that are naturally low in selenium. Selenium is structurally similar to sulfur, replacing the sulfur atom in stoichiometric amounts and thus functions through an association with proteins, termed selenoproteins. In geographic areas low in selenium, there is the potential for animals (including humans) to become selenium deficient and this potential deficiency can be remedied by consumption of exogenous selenium, including selenium-enriched yeast (Saccharomyces cerevisiae) that contains high levels of organic selenium (e.g., selenized yeast). A unique, standardized, registered high selenium food-grade baker's yeast (S. cerevisiae; Sel-Plex), was tested in the following battery of Genotoxicity assays; (1) a bacterial reverse mutation test (Ames test); (2) an in vitro mammalian chromosome aberration test; and (3) a mouse micronucleus test. Under the conditions of this assay, Sel-Plex showed no evidence of mutagenic activity in Salmonella typhimurium, in the bacterial reverse mutation test. Sel-Plex did not induce significant chromosomal aberrations in cultured human lymphocytes in the in vitro mammalian chromosome aberration test. Sel-Plex did not statistically increase the frequency or proportion of micronucleated immature erythrocytes in the mouse micronucleus test. Thus, from the studies presented here, the authors conclude that Sel-Plex is nongenotoxic.

Morphological, cytochemical, and ultrastructural observations on the blood cells of the reptile Tupinambis merianae (Squamata)

Tupinambis merianae is a lizard of the Brazilian fauna and belongs to the Squamata order. Blood cell data are scarce. Blood samples from six specimens of adult T. merianae were used to evaluate some hematological parameters. For structural analysis, 2 ml of blood was collected in the presence of EDTA. Part of the blood was used for preparing blood smears which were submitted to the methods of Leishman, Laur, and toluidine blue, and the cytochemical reactions of periodic acid-Schiff, sirius red, sudan black B, and ortho-toluidine-H2O2. The remainder was centrifuged and the leukocyte buffy coat was fixed in Karnovsky’s fluid for electron microscopy examination. The following blood cells were identified: mature and immature erythrocytes, spherical and elliptical thrombocytes, heterophilic granulocytes, eosinophils and basophils types I and II, lymphocytes, monocytes, and azurophilic cells. The more significant results obtained were: the presence of glycogen in the cytoplasm of the thrombocytes, heterophils, and basophils; the presence of basic polyaminoacid-rich proteins in the granules of heterophils and eosinophils and myeloperoxidase in the granules of the heterophils, eosinophils, and azurophilic cells; and sudanophilic small granules in the heterophils, eosinophils, and azurophilic cells. More detailed morphological aspects of the cells were observed by means of ultrastructural analysis.

Absence of Erythroblast Macrophage Protein (Emp) Leads to Failure of Erythroblast Nuclear Extrusion

In mammals, the functional unit for definitive erythropoiesis is the erythroblastic island, a multicellular structure composed of a central macrophage surrounded by developing erythroblasts. Erythroblast-macrophage interactions play a central role in the terminal maturation of erythroblasts, including enucleation. One possible mediator of this cell-cell interaction is the protein Emp (erythroblast macrophage protein). We used targeted gene inactivation to define the function of Emp during hematopoiesis. Emp null embryos die perinatally and show profound alterations in the hematopoietic system. A dramatic increase in the number of nucleated, immature erythrocytes is seen in the peripheral blood of Emp null fetuses. In the fetal liver virtually no erythroblastic islands are observed, and the number of F4/80-positive macrophages is substantially reduced. Those present lack cytoplasmic projections and are unable to interact with erythroblasts. Interestingly, wild type macrophages can bind Emp-deficient erythroblasts, but these erythroblasts do not extrude their nuclei, suggesting that Emp impacts enucleation in a cell autonomous fashion. Previous studies have implicated the actin cytoskeleton and its reorganization in both erythroblast enucleation as well as in macrophage development. We demonstrate that Emp associates with F-actin and that this interaction is important in the normal distribution of F-actin in both erythroblasts and macrophages. Thus, Emp appears to be required for erythroblast enucleation and in the development of the mature macrophages. The availability of an Emp null model provides a unique experimental system to study the enucleation process and to evaluate the function of macrophages in definitive erythropoiesis.

Do parasites matter? Assessing the fitness consequences of haemogregarine infection in snakes

Although much research in evolutionary ecology is based upon the premise that high levels of parasitism impair the host's functioning, the assumed link between parasitism and fitness has been assessed for relatively few kinds of animals. At our study site in tropical Australia, keelback snakes ( Tropidonophis mairii (Gray, 1841), Colubridae) are heavily infected with haemogregarine blood parasites: 90% of snakes that we tested carried the parasite, with the proportion of erythrocytes containing haemogregarines averaging 15% and ranging up to a remarkable 64%. Prevalence increased with snake body size, but intensity decreased with age. Unlike lizards studied previously, the snakes did not respond to haemogregarine infection by releasing immature erythrocytes into the circulation. In striking contrast to results from a recent study on a sympatric snake species, we did not find any empirical links between parasite numbers and several measures of host fitness (body condition, growth rate, feeding rate, antipredator behaviour, locomotor performance, reproductive status, reproductive output, and recapture rate). The association between this parasite and its host thus appears to be surprisingly benign, suggesting that host–parasite interactions sometimes may have only trivial consequences for host fitness in natural populations. Plausibly, host–parasite coevolution weakens or eliminates fitness costs of parasitism.

Haematological changes in the three Indian major carps, Catla catla (Hamilton), Labeo rohita (Hamilton) and Cirrhinus mrigala (Hamilton) exposed to acidic and alkaline water pH

The effect of water pH on certain haematological parameters was studied in the three species of Indian major carps, catla ( Catla catla), rohu ( Labeo rohita) and mrigal ( Cirrhinus mrigala) for 21 days by subjecting fingerlings of these fishes to acidic (pH 5.5, 6.5) and alkaline (pH 8.0, 8.5 and 9.0) water, control groups were maintained at neutral pH. A change in water pH either to acidic or alkaline conditions exerted stress in fish characterized by swelling of erythrocytes, production of immature erythrocytes, and reductions in the total erythrocyte counts, haemoglobin and serum protein content. There were also increases in total leukocyte counts and blood glucose. However, the degree of these responses varied among the three species. Rohu was found to be least affected followed by mrigal to the stress of altered water pH, while catla was the most vulnerable to pH changes.

Bone marrow immunosuppression in Salmonella-infected mice is prolonged following influenza virus infection

It has been shown previously that infection with diverse viruses induces alterations in bone marrow lineage-specific progenitor cells. As complications arising from secondary bacterial infections can adversely affect the host, we investigated whether virally induced hematological alterations could contribute to the enhanced illness observed in such cases. Mice were infected with influenza virus alone or influenza virus followed by a vaccine strain of Salmonella typhimurium. The effects on hematopoiesis were analyzed by fluorescein-activated cell sorting analysis and immunohistology. Systemic Salmonella typhimurium infection induces depletion of bone marrow erythroid and lymphoid cells. The depletion lasted longer in mice that had been previously infected with influenza virus, compared with mice that had been previously treated with allantoic fluid. Although an increase in splenic lymphoid cells was apparent in the spleens of Salmonella-infected mice, the majority of cells in the enlarged spleens were found to be both immature and mature erythrocytes. These results show that bone marrow progenitor cell depletion induced by bacterial infection is prolonged following a viral infection. It is possible that hematological alterations may contribute to the enhanced clinical illness observed in consecutive viral:bacterial infections.

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.

Serum Free Hemoglobin Concentrations in Healthy Individuals Are Related to Haptoglobin Type

To the Editor: Hemoglobin (Hb) is the major erythrocyte protein, and measurement of free Hb in serum can be used as a marker of hemolysis. Classically, cyanohemoglobin spectrometry and immunonephelometric methods have been used for assaying free Hb in serum (1)(2). Hb metabolism partly occurs in tissue macrophages, which can engulf senescent erythrocytes (extravascular hemolysis) or take up Hb released from ruptured erythrocytes (intravascular hemolysis) and immature erythrocytes in the bone marrow. Because of the oxidative and toxic properties of the iron-containing heme in Hb, Hb is complexed by the plasma protein haptoglobin (Hp). Hp is involved in promoting the clearance of plasma Hb because Hp strongly binds free Hb and is depleted during increased hemolysis. Hp is a positive …

Reproductive Parameters of Adult Birds and Morphophysiological Characteristics of Chicks in the Pied Flycatcher (Ficedula hypoleuca Pall.) in Technogenically Polluted Habitats

Effects of toxic emissions (heavy metals and sulfur dioxide) on reproductive parameters of adult pied flycatchers (Ficedula hypoleuca Pall.) and morphophysiological characteristics of their progeny have been studied in the vicinity of the Middle Ural Copper-Smelting Plant. In polluted areas, the fecundity of adult birds is lower, mortality among the progeny is higher, and changes in the physiological parameters of birds are observed. The effects of toxic load are better manifested in nestlings than in adult birds. They include an increase in the liver index, the reduction of hemoglobin concentration, and an increase in the proportion of immature erythrocytes in the peripheral blood. The proportion of anemic birds also increases. The weakening of nestlings facilitates their infestation by the larvae of parasitic flies. Changes in the differential blood count of nestlings reflect the combined impact of toxic pollutants and parasites.