COLONY FORMING CELLS IN THE LIVERS OF PRENATAL FLEXED (f/f) ANAEMIC MICE

The colony forming unit (c.f.u.) content of foetal livers of f/f and f /+ genotypes has been investigated. Although proportions of c.f.u. to nucleated liver cells do not vary with genotype, the total number of c.f.u. and erythroid cells per foetus is reduced in f/f embryos from 11 to 16 days of gestation. The differentiative capacity of f/f stem cells, measured by ⁵⁹Fe uptake into blood 7 days after transplantation of cells, was less than that of f /+ stem cells. An initially low growth rate of c.f.u. in early foetal liver and in young spleen colonies can explain several characteristics of flexed erythropoiesis.     

EXPRESSION OF CONGENITAL DEFECTS IN THE HAEMOPOIETIC MICRO-ENVIRONMENT: PRE-NATAL ERYTHROPOIESIS IN ANAEMIC 'STEEL'(SljSlj) MICE

The production of red cells by the livers of congenitally anaemic Sl^jSl^j foetal mice is reduced, so that the red cell concentration reaches only 0.4 × 10⁹/ml, compared to 2.3 × 10⁹/ml in normal littermates. The deficiency in haemoglobin concentration is not so severe (3 g/100 ml compared to 10 g/100 ml), because Sl^jSl^j red cells are macrocytic and normochromic. The number of erythroblasts in the livers of Sl^jSl^j foetuses is considerably less than in normal littermates, but the ratio of early erythroblasts to late erythroblasts is not altered. The cell cycles of Sl^jSl^j and normal early erythroblasts are similar, but the cycle time of Sl^jSl^j late erythroblasts is longer than that of the corresponding normal cells. Sl^jSl^j foetal liver cells have a restricted response to erythropoietin in vitro in terms of haem synthesis, whereas foetal liver cells of normal littermates are highly responsive until the 16th day of gestation. Mixtures of late 16 day Sl^jSl^j and normal foetal liver cells (both non-responsive alone) respond to erythropoietin in vitro.     

Characteristics of the pathways of erythroid cell differentiation in birds in anemia

A comparative study has been made of erythroid cell development pathways in the peripheral blood of pigeons during severe, moderate and weak forms of anaemia. Three modes of erythrocyte formation from bone marrow precursor are described: 1. A reserve erythropoiesis--the principal process during severe anaemia; the bone marrow precursors are basophylic erythroblasts which are reversibly blocked in phase G2 of the cell cycle; in results the rapid, increase of erythrocyte population above the normal level, although the cells have 25-30 per cent deficiency in haemoglobin content. 2) A mode of erythropoiesis, whose precursors are proliferating polychromatophylic erythroblasts; this is the principal mode of erythropoiesis at the moderate anaemia, leading to restoration of the normal quantity of erythrocytes with a normal haemoglobin content. 3) A mode of erythropoiesis with proliferating orthochromatic erythroblasts being precursors (which do not divide normally); this is the principal mode during the weak anaemia to result in a slow restoration of the number of erythrocytes with an excess in haemoglobin content. It is shown that regulation of the restoration processes during anaemia are characterized by a specific combination of cell proliferation and differentiation.     

Novel approaches to manipulating foetal cells in the maternal circulation for non-invasive prenatal diagnosis of the unborn child

Due to the risks to the foetus with invasive prenatal diagnosis, non-invasive prenatal diagnosis (NIPD) is gaining tremendous interest but no reliable method that can be widely used has been developed to date. Manipulation of foetal cells and foetal cell-free genetic material in the maternal blood are two promising approaches being researched. The manipulation of foetal cells in the maternal circulation is more popular as it can provide complete genetic information of the foetus particularly the diagnosis of aneuploidies. However, the foetal cell numbers in the maternal circulation are small and their enrichment and ex vivo culture remain two major challenges for NIPD. Primitive foetal erythroblasts (pFEs) have been considered as a good potential candidate for early first trimester NIPD but their nature, properties and manipulation to provide adequate cell numbers remain a challenging task and several approaches need to be meticulously evaluated. In this review we describe the current status of NIPD and suggest some novel approaches in manipulating pFEs for future clinical application of NIPD. These novel approaches include (1) understanding the pFE enucleation process, (2) enriching pFE numbers by individual pick-up of pFEs from maternal blood using micromanipulation and microdroplet culture, (3) expansion of pFEs using mitogens and (4) decondensation of the pFE nucleus with histone deacetylase (HDAC) inhibitors followed by reprogramming using gene delivery protocols with/without small reprogramming molecules to improve reprogrammed pFE proliferation rates for successful NIPD.     

Critical periods for foetal mortality in gilts identified by analysing the length distribution of mummified foetuses and frequency of non-fresh stillborn piglets

The objective of this study was to investigate the timing of foetal mortality in gilts of a segregating F2 cross of Large White and Meishan pigs on the basis of the length distribution of mummified foetuses and the frequency of non-fresh stillborn piglets in order to establish whether critical periods for foetal mortality exist. All expelled conceptuses and placentae of 192 farrowing gilts with a normal health status were meticulously investigated to recover all mummified foetuses. The length of each mummified foetus was measured. The predicted number of foetuses present per gilt at the early foetal stage of gestation was calculated as the sum of numbers of mummified foetuses and non-fresh stillborn, fresh stillborn and liveborn piglets. Foetal loss was calculated as the sum of mummified foetuses and non-fresh stillborn piglets. The average foetal mortality rate per gilt was 8.7%. In total 162 mummified foetuses were found (average 0.84 per litter), ranging in length from 0.4 to 33.0 cm. This indicates a range in foetal age at death of approximately 35-100 days. Although mummified foetuses of all lengths within the above mentioned range were found, relatively many had a length of less than 4 cm or of 10-21 cm. The total number of non-fresh stillborn piglets (i.e. late foetal deaths) was 58 (average 0.30 per litter). It can be concluded that foetal mortality occurred in these gilts throughout the period from day 35 to term, with relatively high incidences at the early foetal stage (days 35-40), shortly after mid-pregnancy (days 55-75) and after approximately day 100 of gestation. These three periods coincide with reported periods of change in porcine placental growth.     

Intraclutch variation in avian eggshell pigmentation: the anaemia hypothesis

Many passerine species lay eggs that are speckled with dark protoporphyrin pigmentation. Because protoporphyrin is mainly derived from the blood, we here formulate and test a new hypothesis that links an increase in anaemia along the laying sequence to within-clutch variation in egg pigmentation. More intense pigmentation is expected if pigments accumulate during enhanced red blood cell production in response to anaemia. Reduced pigmentation is expected if pigments are derived from the degradation of red blood cells that circulate in smaller numbers due to blood loss. To test this hypothesis, we manipulated anaemia in great tit (Parus major) females by infesting the nests with hen fleas (Ceratophyllus gallinae) prior to egg laying. Polychromatophil (i.e., immature red blood cells) percentage, as a measure of blood cell production, was positively correlated with parasite load confirming that female great tits experienced stronger anaemia when infested with haematophagous parasites during egg laying. We found a positive relationship between spot darkness and laying order that weakened under high parasite load. This result suggests that anaemia in females due to blood-sucking parasites led to diminished protoporphyrin from disintegrated red blood cells and hence a decreased deposition of protoporphyrin. However, the overall increase in pigment darkness along the laying sequence suggests that pigments also accumulate by enhanced red blood cell production caused by anaemia due to egg production itself.     

The Identification of Foetal Antigens in Human Bone Marrow Cells

Using an anti-serum raised in rabbits against 12 week old human foetuses, foetal-specific materials were identified in bone marrow cells in a number of neoplastic and non-neoplastic conditions.
Particularly high levels were found in patients with pernicious anaemia and folic acid deficiency and to a lesser extent, iron deficiency. Several types of leukaemia also demonstrated high proportion of the cells staining positive with the anti-foetal serum described.
The importance of the expression of foetal substances by cells during both neoplastic and non-neoplastic abnormal states is discussed. Emphasis is also placed on the importance of maturation arrest and abnormalities being one of the conditions under which foetal-specific substances (also called foetal antigens), can be expressed.     

Source of cell injected is a critical factors for short and long engraftment in xeno-transplantation

Abstract. This study aims to investigate engraftment of human cord blood and foetal bone marrow stem cells after in utero transplantation via the intracoelomic route in the sheep. Here, we performed transplantation in 14 single and 1 twin sheep foetuses at 40–47 days of development, using a novel schedule for injection. (i) Single injection of CD34⁺ human cord blood stem cells via the coelomic route (from 10 to 50 × 10⁴) in seven single foetuses. (ii) Single injection of CD34⁺ foetal bone marrow stem cells via the intracoelomic route with further numbers of cells (20 × 10⁵ and 8 × 10⁵, respectively) in three single and in one twin foetuses. (iii) Double fractioned injection (20–30 × 10⁶) via the coelomic route and 20 × 10⁶ postnatally, intravenously, shortly after birth of CD3-depleted cord blood stem cells in four single foetuses. In the first group, three single foetuses showed human/sheep chimaerism at 1, 8 and 14 months after birth. In the second group, the twin foetuses showed human/sheep chimaerism at 1 month after birth. In the third group, only two out of four single foetuses that underwent transplantation showed chimaerism at 1 month. While foetal bone marrow stem cells showed good short-term engraftment (1 month after birth), cord blood stem cells were able to persist longer in the ovine recipients (at 1, 8 and 14 months after birth).     

Primitive erythropoiesis in early chick embryogenesis. II. Correlation between hemoglobin synthesis and the mitotic history

Primitive erythroblasts in the circulating blood of the chick embryo continue to divide while synthesizing hemoglobin (Hb). Hb measurements on successive generations of erythroblasts show that there is a progressive increase in the Hb content of both interphase and metaphase cells. Furthermore, for any given embryo the Hb content of metaphase cells is always significantly greater than that of interphase cells. The distribution of Hb values for metaphase cells suggests that there are six Hb classes corresponding to the number of cell cycles in the proliferative phase. The location of erythroblasts in the cell cycle was determined by combining Feulgen cytophotometry with thymidine radioautography on the same cells. Measurements of the Hb content for erythroblasts in different compartments of the cell cycle (G1, S, G2, and M) show a progressive increase through the cycle. Thus, the amount of Hb per cell is a function of the number of cell divisions since the initiation of Hb synthesis and, to a lesser degree, the stage of the cell cycle. Earlier generations of erythroblasts synthesize Hb at a faster rate than the terminal generation. Several models have been proposed to explain these findings.     

The effect of single and concomitant pathogen infections on condition and fecundity of the wild rabbit (Oryctolagus cuniculus)

We assessed the effect of two pathogens (myxoma virus and Eimeria stiedae) and five macroparasites (gastrointestinal helminth species) of the wild rabbit (Oryctolagus cuniculus) upon total host body mass and abdominal fat level. Additionally, we assessed the effects of these organisms on the number of foetuses in adult females during the peak breeding period. Both mass of abdominal fat and total body mass of the rabbit were negatively associated with myxoma virus infection and increasing helminth species richness. Total body mass was also negatively associated with the protozoan parasite E. steidae. No relationship was found between any of the parasites/pathogens and the number of foetuses in adult females, although only relatively small sample sizes were available for this section of the analysis. Increasing host body mass was positively associated with number of foetuses and we propose that mass reduction caused by the pathogen and parasite species could also have the consequence of reducing foetal number.     

Shiga toxin of enterohaemorrhagic Escherichia coli directly injures developing human erythrocytes

Haemolytic anaemia is one of the characteristics of life‐threatening extraintestinal complications in humans during infection with enterohaemorrhagic Escherichia coli (EHEC). Shiga toxins (Stxs) of EHEC preferentially damage microvascular endothelial cells of the kidney and the brain, whereby occluded small blood vessels may elicit anaemia through mechanical erythrocyte disruption. Here we show for the first time that Stx2a, the major virulence factor of EHEC, is also capable of direct targeting developing human erythrocytes. We employed an ex vivo erythropoiesis model using mobilized CD34⁺ haematopoietic stem/progenitor cells from human blood and monitored expression of Stx receptors and Stx2a‐mediated cellular injury of developing erythrocytes. CD34⁺ haematopoietic stem/progenitor cells were negative for Stx2a receptors and resistant towards the toxin. Expression of Stx2a‐binding glycosphingolipids and toxin sensitivity was apparent immediately after initiation of erythropoietic differentiation, peaked for basophilic and polychromatic erythroblast stages and declined during maturation into orthochromatic erythroblasts and reticulocytes, which became highly refractory to Stx2a. The observed Stx‐mediated toxicity towards erythroblasts during the course of erythropoiesis might contribute, although speculative at this stage of research, to the anaemia caused by Stx‐producing pathogens.     

Malarial anaemia: mechanisms and implications of insufficient erythropoiesis during blood-stage malaria

It has been proposed that the basis of severe malarial anaemia, a major cause of morbidity and mortality in endemic areas, is multifactorial. Inappropriately low reticulocytosis is observed in malaria patients suggesting that insufficient erythropoiesis is a major factor. Clinical studies provide conflicting data concerning the production of adequate levels of erythropoietin (EPO) during malaria. Plasmodium chabaudi AS causes non-lethal infection in resistant C57BL/6 mice, and lethal infection in susceptible A/J mice. In P. chabaudi AS infected C57BL/6 and A/J mice, which experience varying degrees of severity of anaemia, kidney EPO production is appropriate to the severity of anaemia and is regulated by haematocrit level. Neutralisation of endogenous EPO during infection leads to lethal anaemia while timely administration of exogenous EPO rescues mice although reticulocytosis is suppressed in proportion to the parasitemia level. Characterisation of alterations in splenic erythroid compartments in naive and P. chabaudi AS infected A/J mice revealed that infection, with or without EPO treatment, leads to sub-optimal increases in TER119+ erythroblasts compared to EPO-treated naive mice. A lower percentage of TER119+ erythroblasts in infected mice undergo terminal differentiation to become mature haemoglobin-producing cells. Furthermore, there is a shift in transferrin receptor (CD71) expression from TER119+ cells to a non-erythroid population. Deficiencies in the number and maturation of TER119+ erythroblasts during infection coincide with blunted proliferation to EPO stimulation in vitro by splenocytes, although a high frequency express EPO receptor (EPOR). Together, these data suggest that during malaria, EPO-induced proliferation of early EPOR+ erythroid progenitors is suppressed, leading to sub-optimal generation of TER119+ erythroblasts. Moreover, a shift in CD71 expression may result in impaired terminal maturation of erythroblasts. Thus, suppressed proliferation, differentiation, and maturation of erythroid precursors in association with inadequate reticulocytosis may be the basis of insufficient erythropoiesis during malaria.     

Predicting iron and folate deficiency anaemias from standard blood testing: the mechanism and implications for clinical medicine and public health in developing countries

Background  

Developing countries have high prevalence of diseases, but facilities to diagnose and treat them are limited. We must use available resources in ways not needed where there are sophisticated equipment and trained staff. Anaemia is common; iron deficiency affects health and productivity; folate deficiency in pregnant women causes foetal abnormalities. Few developing countries can measure serum folate or ferritin, but standard automated blood analyses are widely available and can help predict folate and iron deficiency. The RDW-CV% (coefficient of variation of the red cell width) measures the variability in the size of red blood cells (RBC) in routine automated analysis of blood cells, but is seldom reported. Levels of RDW-CV% and haemoglobin (Hb) can predict iron deficiency anaemia.     

Abnormalities of gonadotrophs in the adenohypophysis of infertile male PD rats

Male PD strain rats are sterile in the homozygous condition (pd/pd) due to abnormal spermatogenesis detectable at around nine weeks of age. Previous studies have indicated electron microscopic abnormalities in the Sertoli cells of pd/pd males at three and 12 weeks of age. Since spermatogenesis and Sertoli cell function depend on gonadotropins (luteinizing and follicle-stimulating hormones [LH and FSH]) and testosterone, production and/or secretion of these hormones might be altered in pd/pd males. The aim of the study reported here was to investigate the hormonal status of pd/pd males at three, six, and nine weeks of age. Although alteration was not evident in the LH-immunoreactive cells, FSH-immunoreactive cells in pd/pd males were small in size with scant cytoplasm and were reduced in number and area (73 and 51% of phenotypically normal pd/+ males, respectively) at three weeks of age, although serum FSH concentration was similar to that in pd/+ males. At six and nine weeks of age, percentages of the areas occupied by LH- and FSH-immunoreactive cells in pd/pd males were higher than those in pd/+ males. Serum FSH concentration in pd/pd males was significantly high at nine weeks of age, although a difference in serum LH and testosterone concentration was not evident. These results suggest that FSH production in pd/pd males is decreased at three weeks of age. This might be associated with the Sertoli cell abnormalities and subsequent abnormal spermatogenesis seen in adult life.     

Flow cytometric DNA analysis of nurse shark, Ginglymostoma cirratum (Bonaterre) and clearnose skate, Raja eglanteria (Bosc) peripheral red blood cells

Flow cytometric DNA analysis was used to determine the DNA content of red blood cells from Ginglymostoma cirratum (Bonaterre) and Raja eglanteria (Bosc). The DNA content/nucleus was calculated to be 7.6 and 7.1 pg/nucleus, respectively. Peripheral red blood cells obtained both from G. cirratum and from R. eglanteria were demonstrated to be actively cell cycling. Percentages of red blood cells in the phases of the cell cycle ( G ₀/ G ₁, S , and G ₂/ M ) not only varied considerably between different fishes but also were quite variable in weekly samples obtained from individual animals.     

Litter-size-dependent intrauterine growth restriction in sheep

Regulation of foetal development in sheep depends on interactions between the intrinsic capacity of the foetus for growth and the maternal environment. Lambs born in multi-foetus litters have relatively small placentae with fewer cotelydons, and lower birth weights. Litter-size-dependent intrauterine growth restriction (IUGR) is evident at mid gestation when metabolic needs of the conceptus are moderate, and overnutrition of ewes with multiple foetuses does not promote growth of their foetuses to the size of singletons. Those observations suggest that placental and conceptus growth in multi-foetus pregnancies is reprogrammed at mid gestation by an as yet undefined mechanism to attenuate foetal growth. This may protect the foetus from severe nutritional insult during late gestation, when its daily growth rate is at a maximum. In that way, lambs born in large litters with relatively lower birth weights may not experience the long-term physiological insults that can be observed in small lambs born to undernourished ewes.     

Splenic haematopoiesis in primary (idiopathic) osteomyelofibrosis: immunohistochemical and morphometric evaluation of proliferative activity of erythro- and endoreduplicative capacity of megakaryopoiesis (PCNA- and Ki-67 staining)

Using monoclonal antibodies against proliferating cell nuclear antigen or PCNA (PC10) and the Ki-67 antigen (MIB1), an immunohistological and morphometric study was performed on routinely processed splenic tissue from ten patients with primary (idiopathic) osteomyelofibrosis (OMF). To determine the proliferation capacity of erythroid precursors and the endoreduplicative activity of megakaryocytes, corresponding antibodies (Ret40f and CD61) were applied in combination with the cell-cycle markers (sequential double-immunostaining). Morphometric analysis revealed no significant differences in PCNA or Ki-67 reactivity in either cell lineages. In comparison with previous studies on normal bone marrow, in splenic tissue showing myeloid metaplasia, the numbers of PCNA-labelled proerythroblasts, erythroblasts and megakaryocytes were conspicuously increased. Considering the ineffective erythropoiesis in OMF, there seemed to be a disproportional enhancement in PCNA and Ki-67 immunostaining of the red cell lineage. Similarly, the small size of megakaryocytes in advanced, OMF-associated myeloid metaplasia was in keeping with an impairment of endoreduplicative activity. In addition to various other contributory factors, anaemia in OMF may be partially caused by secondary folate (haematinic) deficiency. From experimental studies this defect is known to cause an abnormal arrest in the S-phase of the cell-cycle, comparable to that characterising pernicious anaemia. As a sequel of this pathomechanism, an undue overexpression of PCNA and Ki-67 has to be assumed, that is not necessarily associated with DNA synthesis or cell cycling. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG-Th 390/1-3)     

Fetal erythroblast isolation up to purity from cord blood and their culture in vitro

BACKGROUND: Erythroblasts have been the most encouraging candidate cell type for noninvasive prenatal genetic investigation. We previously showed that human erythroblasts can be recovered from bone marrow and blood bank buffy coats by a physical cell separation. In the present study, we modified our previous methodology, taking into account the peculiar behavior of erythroblasts in response to modifications of pH and osmolality of the separation medium. METHODS: Twenty to forty milliters of cord blood were initially centrifuged on Ficoll/diatrizoate (1.085 g/ml). The interphase cells were further separated on a continuous density gradient (1.040-1.085 g/ml). Two different gradients were initially compared: the first was iso-osmolar and neutral, whereas the second also contained an ionic strength gradient and a pH gradient (triple gradient). A subsequent monocyte depletion was performed by using magnetic microbeads coated with anti-CD14 monoclonal antibody (mAb), and erythroblasts were purified by sedimentation velocity. Purified cells were investigated by analyses with fluorescence-activated cell sorting (FACS) and fluorescence in situ hybridization (FISH) and immunocytochemistry with mAb against fetal hemoglobin and were cultured in vitro. RESULTS: When nucleated cells were spun on an iso-osmolar and neutral continuous density gradient, two separated bands of nucleated red blood cells (NRBCs) were obtained: a light fraction banding at 1.062 g/ml and an heavy fraction banding at 1.078 g/ml. Conversely, when cells were spun in the triple gradient, NRBCs were shifted to the low-density region. Monocyte depletion by immunomagnetic microbeads and velocity sedimentation provided a pure erythroblast population. FACS and FISH analyses and immunocytochemistry substantiated the purity of the isolated cell fraction, which was successfully cultured in vitro. CONCLUSIONS: We have shown that fetal erythroblasts can be purified up to homogeneity from cord blood, but further refinements of the isolation procedure are necessary before the same results can be obtained from maternal peripheral blood.     

Morphological and biochemical characterization of mitochondria in Torpedo red blood cells

A study is presented on the morphology and respiratory functions of mitochondria from Torpedo marmorata red blood cells. In vivo staining of red blood cells and transmission electron microscopy showed the existence of a considerable number of vital and orthodox mitochondria which decreased from young erythroblasts to mature erythrocytes from 60-50 to 30-20 per cell. In erythrocytes mitochondria exhibited a canonical, functional respiratory chain. The content and activity of cytochromes in erythrocytes were, however, significantly lower as compared to mammalian tissues.