Non-nucleated Cells Research Articles

Sodium-23 NMR relaxation times in nucleated red blood cells and suspensions of nuclei

The relaxation behavior of intracellular 23Na in suspensions of chicken erythrocytes and of their nuclei was investigated. The transverse magnetization was found to decay biexponentially. The average relaxation rates for the nucleated chicken erythrocytes are considerably shorter than the average relaxation rates obtained for dog and human nonnucleated red blood cells. Of particular significance is the twofold decrease in the short component of T2. Calculations based on the measured 23Na NMR relaxation rates in suspensions of nuclei indicate that most of the difference between the relaxation rates in the mammalian as compared to the chicken erythrocytes, can be accounted for by the contribution of the nuclei in the latter.

Changes in alpha-globin gene expression in mice of two alpha-globin haplotypes during development

Adult alpha-globin in mice is synthesized in large amounts during development, first in the primitive, nucleated erythrocytes of yolk sac origin and later in the definitive, nonnucleated erythrocytes that differentiate in the fetal liver, spleen, and bone marrow. Isoelectric focusing analysis of hemoglobins of mice with the Hbag2 and Hbac haplotypes shows that the ratios of alpha chain 1 to chain 5m and alpha chain 1 to chain 4 in adult hemoglobins from Hbag2 and Hbac mice, respectively, change between day 11.5 and day 16.5 of gestation in nucleated red cells, while no change occurs in nonnucleated red cells. The percentage ratios of the two different alpha-globin chains are different in Hbag2 and Hbac mice for EII, EIII, and adult hemoglobin. In nucleated red cells of yolk sac origin, differences and changes in alpha-globin ratios are a composite of changing globin gene transcription and posttranslational competitive affinities among globins to form embryonic and adult hemoglobin tetramers.

IgM Autoantibodies against Two Cellular Antigens Always Appear in Acute Epstein-Barr Virus Infection

In the course of infectious mononucleosis, IgM antibodies are formed against 2 proteins present in nucleated and non-nucleated vertebrate cells. Antibodies were found in sera of all patients suffering from acute Epstein-Barr virus infection. In 40% of the cases these antibodies are monoclonal. Persons with former Epstein-Barr virus infection were negative. The antibodies against the 2 proteins were first detected in Raji cells with an IgM-specific immunofluorescence test. The proteins were demonstrated in extracts of different cells and tissues by immunoblot technique. The molecular weight of the proteins measured in SDS polyacrylamide gel electrophoresis was 26 kd and 29 kd, respectively. Their presence in the cells does not depend on the presence of the Epstein-Barr virus genome. The relevance of the new findings concerning diagnostics as well as pathogenetic aspects remains to be established.

The requirements for triggering of lysis by cytolytic T lymphocyte clones. II. Cyclosporin A inhibits TCR-mediated exocytosis by only selectively inhibits TCR-mediated lytic activity by cloned CTL.

TCR-mediated granule exocytosis, as measured by the release of serine esterase activity, has been implicated in the lytic process of Ag-specific CTL. Exocytosis appears to be the mechanism of release of other lysis-relevant molecules including cytotoxic lymphokines and proteins that have the capacity to induce membrane lesions as measured by the hemolysis of non-nucleated SRBC. In the studies presented here, we assessed the contribution of exocytosis and lymphokine production in CTL lysis of nucleated and non-nucleated target cells by using a panel of murine CTL clones. Ag-mediated activation of cytolysis, lymphokine production, and exocytosis could be mimicked by mAb against the TCR/CD3 complex, or by stimulation with the combination of PMA + calcium ionophore, which appear to bypass the TCR (neither PMA nor calcium ionophore alone induced these functions efficiently in our CD8+ CTL clones). Although lysis, IFN-gamma production and exocytosis of N-alpha-benzyloxycarbonyl-L-lysin esterase (BLTE) activity were induced by either stimulus, we were able to identify distinct activation requirements for each of these functions. We found that lymphokine production, exocytosis, and cytolysis could be selectively inhibited. Cycloheximide inhibited IFN-gamma production, but did not inhibit exocytosis of BLTE activity or cytolysis. In addition we showed that cyclosporine A (CsA) profoundly inhibited IFN-gamma production as well as exocytosis induced by stimulation through the Ag receptor or by PMA + calcium ionophore. In contrast, CsA had little or no effect on lysis of nucleated target cells that bear the relevant Ag. These findings indicate that our CTL clones can lyse target cells by a mechanism independent of exocytosis or (de novo) lymphokine production. To directly assess the capacity of our CTL clones to lyse target cells without inducing nuclear damage we developed a system of coating non-nucleated SRBC with anti-CD3 mAb for use as stimuli and as targets for lysis. We found that our cloned CTL were indeed activated to produce IFN-gamma by SRBC that were coated with anti-CD3 mAb, and, furthermore, they were able to lyse the SRBC in a short term cytolytic assay. Thus our CD8+ CTL are capable of lysing certain target cells by a mechanism independent of DNA degradation, presumably by inducing a membrane lesion. In addition, CsA did inhibit lysis of the non-nucleated SRBC targets as well as exocytosis of BLTE activity.(ABSTRACT TRUNCATED AT 400 WORDS)

A new method to improve the discrimination between lymphocytes and contaminating erythrocytes in flow cytometric analytical techniques

A method is described for the removal of contaminating erythrocytes from immunofluorescence labelled lymphocyte preparations and allowing accurate gating on the lymphocyte population during flow cytometric analysis. The technique involves an improved erythrolytic fixative combined with the DNA stain propidium iodide permitting non-nucleated cells (i.e., red blood cells) to be gated out during analysis. The erythrolytic fixative was shown not to be detrimental to the major T cell markers detected using indirect immunofluorescence. This method will improve the analysis of blood lymphocytes and their subsets in mononuclear cell preparations and more particularly in samples of body fluids and tissue washings or extracts which may contain variable and often large numbers of erythrocytes.

Metabolism in lizards: low lactate turnover and advantages of heterothermy.

The bobtail lizard (Tiliqua rugosa) encounters environmental temperatures that range from 7 to 40 degrees C. We have studied the effects of temperature on the whole-body turnover of glucose, lactate, and fatty acid. At 35 degrees C turnover rates of glucose and fatty acid account for the majority of O2 uptake and reflect the low metabolic rate of the bobtail compared with that of a mammal of equivalent mass. Lactate turnover at rest is very low. The low rates of lactate production correlate with a lack, or relative lack, of those tissues that normally produce most of the lactate in a resting mammal, such as nonnucleated red blood cells, renal medulla, and smooth muscle associated with blood vessels. Q10 (change in rate with a 10 degrees C change in temperature) values for turnover rates of glucose and fatty acid increase as temperature decreases. It is concluded that there is an inverse temperature effect that minimizes fuel usage during torpor.

Ion flux and Na+,K+-ATPase activity of erythrocytes and leucocytes in thyroid disease.

In hyperthyroidism, erythrocytes show decreased Na+,K+-ATPase activity, decreased [3H]ouabain binding capacity (an index of the number of sodium pumps) and decreased active sodium and potassium flux rates, with a high intracellular sodium concentration. As erythrocytes are non-nucleated and atypical cells, we have studied electrolyte status in thyroid disease using mixed leucocytes as well; the results obtained differed from those in erythrocytes. When compared with findings in healthy subjects, leucocyte Na+,K+-ATPase activity, [3H]-ouabain binding capacity, total and active rubidium (used instead of potassium) influx were all significantly increased in untreated hyperthyroidism and decreased in untreated hypothyroidism. In hyperthyroidism, there was also a decrease in plasma potassium, an increase in sodium efflux rate and efflux rate constant, but no significant changes in cell sodium and potassium concentrations. All these changes returned to normal in successfully treated patients. There was a significant correlation between these abnormalities of electrolyte status and thyroid disease status (as serum thyroid stimulating hormone and free thyroxine).

Lysis of nonnucleated red blood cells by cytotoxic T lymphocytes.

To establish the role of direct membrane damage in cytotoxic T lymphocytes (CTL)-mediated lysis we investigated whether CTL would be capable of lysing nonnucleated target cells. By antibody-mediated targeting we show that CTL are indeed capable of lysing sheep and human red blood cells.

Expression of the two adult β-globin genes in mouse yolk sac and fetal liver erythrocytes

The two adult β-globin genes (β1 s2major and β2 sminor) of the Hbb s2 haplotype are differentially expressed during development. Centrifugal elutriation was used to separate the two populations of erythrocytes present in developing fetuses. Hemoglobin analysis showed that the larger, nucleated erythrocytes (yolk sac-derived) have relatively larger amounts of β-sminor hemoglobin than do smaller, nonnucleated cells (fetal liver-, spleen-, and bone marrow-derived) at the same stage of development.

Blood Platelets Contain Factors Involved in Both Normal and Neoplastic Growth

Human blood platelets are small (2-5 ixm diameter) nonnucleated cells produced by controlled fragmentation of multinucleated megakaryocytes resident in the bone marrow (Weiss, 1975). Their role in hemostasis has been recognized for 100 years (Baumgartner and Muggli, 1976): by aggregating together, with or without fibrin, they can form a barrier which pre? vents the flow of blood out of a damaged vessel. Platelets also participate in activa? tion of the clotting cascade which ultimately results in the formation of fibrin from fibrinogen. In addition to this role in hemostasis, there is increasing evidence that platelets also contain several polypeptide growth factors; that these growth fac? tors are released from the platelet specifically at sites of tissue damage; and that they function there to promote the pro? cesses of cell proliferation and synthesis of structural protein which are needed to repair the damage. The best characterized growth factors in platelets are: plateletderived growth factor (PDGF), epidermal growth factor (EGF), and transforming growth factor (TGF). Each of these factors is probably involved in both neoplastic and normal cell proliferation.

Effect of oestrogen on Pasteurella pneumotropica in rat vagina.

The effects of ovarian hormones on the vaginal population of Pasteurella pneumotropica in rats were investigated. Specified-pathogen-free adult female Wistar-Imamichi rats with a 4 day oestrous cycle were inoculated with P. pneumotropica in the vagina. Cyclic changes in the vaginal population of P. pneumotropica were not observed in ovariectomized rats and the bacterial population was at a similar level to that at normal dioestrus. Administration of oestrogen to ovariectomized rats caused an increase in the numbers of P. pneumotropica and total bacteria in the vagina nearly equal to that at oestrus in intact rats. The numbers of these organisms in the vagina of ovariectomized rats treated with progesterone did not change and were similar to those of control ovariectomized rats treated with sesame oil. Vaginal smears of ovariectomized rats treated with oestrogen were characterized by abundant cornified non-nucleated epithelial cells with many adherent Gram-negative coccobacilli and were similar to smears from intact rats at oestrus. These findings suggest that the proliferation of P. pneumotropica at oestrus in rat vagina may be primarily due to the environment provided by the degeneration of vaginal epithelial cells promoted by oestrogen secretion from the ovaries.

Histological observations of amniotic fluid cellular content in the ear of neonates and infants

Amniotic fluid cellular content (AFCC), especially keratinized, non-nucleated squamous epithelial cells and lanugo hair, have been detected in the fetal and neonatal ear. We wished to evaluate the incidence, amount and histological findings of AFCC in the neonatal and early infant period. Temporal bones (n = 63) from children aged 10 min to 70 days were examined. AFCC was discovered in 39 of 43 bones in neonates. In children of 31-70 days of age, AFCC was present in 11 of 20 bones. A measurement of the amount of AFCC was performed; the maximum was 11.47 mm3 with a mean of 2.40 mm3. Histological findings varied from the mere presence of AFCC to the formation of epithelialized granulation tissue. This granulation tissue was found in 19 of the bones. Theoretical clinical implications are considered for middle ear adhesions, otitis media and cholesteatoma.

Action de l’acide 13-cis-rétinoïque et du rétinoïde aromatique sur divers paramètres cutanés, chez le rat rendu hyperséborrhéique

Castrated rats submitted to androgenic stimulation present hyperseborrhea with an increase in foamy cells associated with an increase in cutaneous lipids and a change in the number of epidermal cell layers. 13-cis-retinoic acid and aromatic retinoid (Ro 10-9359) were given orally during 17 days (10 mg/kg) to castrated rats which had received a testosterone implant. Cyproterone acetate was chosen as the antiandrogenic drug; it was given at doses of 10 mg/kg i.m. once every other day. As opposed to cyproterone acetate, the two substances studied had no effect on the seminal vesicle and ventral prostate. 13-cis-retinoic acid and retinoic aromatic acid increased the total number of epidermal layers: In the case of 13-cis-retinoic acid, the increase involves especially the nonnucleated cell layers, whereas with retinoid aromatic acid it involves the nucleated cell layer. The number of sebaceous glands is not changed by either products, on the other hand, the number of foamy cells is considerably decreased and the weight of cutaneous lipids is decreased as well.

Proliferation of Pasteurella pneumotropica at oestrus in the vagina of rats.

Using a colony of Wistar-Imamichi rats contaminated with P. pneumotropica, the vaginal microflora was qualitatively and quantitatively investigated by swabbing. P. pneumotropica was the most dominant organism in the majority of rats examined. The population of P. pneumotropica and indigenous bacteria increased significantly higher at oestrus than in other oestrous stages. By the vaginal flushing technique changes in the population of P. pneumotropica and total bacteria, and changes in vaginal cell type and bacterial counts adhering to vaginal epithelial cells were consecutively investigated. The populations of P. pneumotropica and total bacteria were maximal at oestrus. The increase was correlated with an increase in cornified non-nucleated cells, with large numbers of adherent Gram-negative coccobacilli. The findings indicate that the vagina is a suitable site for colonization by P. pneumotropica in adult female rats, and that proliferation of P. pneumotropica may be due to increased affinity of the organism for cornified non-nucleated cells.

Comparison of methyl- p-hydroxybenzimidate and 1,3,5-trichlorotriazine in producing sensitive target cells for use in the hemolytic spot assay

Two bifunctional reagents were shown to be useful in the coupling of immunogens to the surface of either nucleated or non-nucleated cells. The heterobifunctional reagent methyl- p-hydroxybenzimidate was used to couple aromatic amino-haptens to the surface of SRBC which resulted in a stable and sensitive target cell capable of detecting as little as 20 pg of purified anti-hapten antibody in the hemolytic spot assay. The multifunctional reagent 1,3,5-trichlorotriazine yielded similar results when coupling amino-haptens to the surface of SRBC for use in the hemolytic spot assay. This reagent was also used to couple protein to the surface of SRBC which were able to detect as low as 1 ng of purified anti-protein antibody in the hemolytic spot assay. The sensitized SRBC produced using either of these coupling reagents were shown to remain stable for several months giving reproducible results from one test to another. Lastly, 1,3,5-trichlorotriazine was used to couple the hapten 4-aminophthalate to the surface of nucleated cells with retention of >90% cell viability with continued growth and cellular division in culture.

Molecular and cell biologic aspects of erythropoiesis in long-term bone marrow cultures

In long-term marrow cultures, proliferation and differentiation of hemopoietic stem cells occurs for several months. Normally, only the most primitive erythroid progenitor cells are produced (the BFU-E). Following treatment with anemic mouse serum (AMS) or normal mouse serum plus erythropoietin, the BFU-E mature into CFU-E, which then go to produce mature nonnucleated red cells. This development is associated with the production of adult type hemoglobin. Furthermore, erythropoiesis and granulopoiesis occur in association with discrete cellular elements of the adherent cell layer in the long-term culture. Following treatment with AMS, erythropoiesis is enhanced while granulopoiesis is depressed, with no apparent competition at the stem cell or progenitor cell level.

Molecular and Cell Biologic Aspects of Erythropoiesis in Long-Term Bone Marrow Cultures

In long-term marrow cultures, proliferation and differentiation of hemopoietic stem cells occurs for several months. Normally, only the most primitive erythroid progenitor cells are produced (the BFU-E). Following treatment with anemic mouse serum (AMS) or normal mouse serum plus erythropoietin, the BFU-E mature into CFU-E, which then go to produce mature nonnucleated red cells. This development is associated with the production of adult type hemoglobin. Furthermore, erythropoiesis and granulopoiesis occur in association with discrete cellular elements of the adherent cell layer in the long-term culture. Following treatment with AMS, erythropoiesis is enhanced while granulopoiesis is depressed, with no apparent competition at the stem cell or progenitor cell level.

Visualization of actin polymerization and depolymerization cycles during polyamine-induced cytokinesis in living Amoeba proteus.

Microinjection of spermine induces cytokinesis of Amoeba proteus. Within 30--60 s after spermine injection cells form one, or less commonly, two cleavage furrows and within the following 4--10 min the constrictions are completed. The resulting nucleated cell parts show normal streaming and locomotion, whereas the non-nucleated cell parts remain stationary and later degenerate. The intracellular distribution of fully polymerization-competent fluorescently labelled muscle actin was followed by image intensification. Double injection experiments initially using labelled action and 30 min later spermine revealed a ring-like structure of enhanced fluorescence corresponding to the constricting cleavage furrow. Immediately after cleavage was completed, the ring disappeared. Electron microscopy of cells fixed during spermine-induced cytokinesis showed numerous well aligned actin and myosin filaments in the developing cleavage furrow. These filaments are a specialized manifestation of the cell cortex. The results demonstrate that cycles of actin and myosin polymerization and depolymerization and the parallel alignment of preexisting filaments (crosslinking) represent a basic mechanism in the generation of the motive force during cytokinesis.

Rat embryonic and foetal erythrocytes. High 2,3-bisphosphoglycerate and ATP and low oxygen affinity in vitro for nucleated embryonic cells.

Embryonic nucleated red cells of the rat have high ATP and 2,3-bisphosphoglycerate and relatively low oxygen affinity. During foetal life they are replaced by large non-nucleated red cells with high ATP, low bisphosphoglycerate and high oxygen affinity. After birth, small non-nucleated red cells with high bisphosphoglycerate and low oxygen affinity rapidly predominate.

An automated blood smear analysis system. Clinical experience and performance.

A detailed clinical evaluation of the diff3 system for examining peripheral blood smears was performed over a six-month period in a service hematology laboratory in a large teaching hospital. The diff3 automatically classifies 11 categories of nucleated cells; qualitatively evaluates erythrocyte size, shape, and color; estimates platelet and leukocyte count; and generates a quantitative estimate of platelet number. The analyzer contains a system for signaling abnormal or suspicious slides for technologist review. As many as 14 slides can be processed in sequence without operator attendance. Throughput averages 33 slides/hour in no review mode. The analyzer produced similar in-run and day-to-day precision when examining a wide variety of blood smears. Morphologic evaluation of nucleated and non-nucleated blood cells correlated with that of technologists. The diff3's normal range compared closely with that obtained by senior technologists examining conventional smears. Band counts, while somewhat higher, did not lead to judgement errors in left-shift direction. The diff3's sensitivity to recognizing abnormal smears was virtually identical to that of technologists with a false-positive level only slightly greater than theirs; diff3's false negatives were equal in frequency to those experienced by technologists.