Platelet transfusion in neonatal alloimmune thrombocytopenia.
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Adult↗
Biomedical subjects
David J Roberts
Publications and source records attributed to David J Roberts.
A small-scale serum-free liquid cell culture model of erythropoiesis to assess the effects of exogenous factors.
Anaemia is an important global health problem. Therefore, it is crucial to understand its pathophysiology in various genetic or infectious diseases where dyserythropoiesis is a key pathological feature. To this effect, reproducible and reliable models of erythropoiesis in vitro are much needed as investigative tools. We have developed a serum-free liquid culture model of erythropoiesis using human umbilical cord blood CD34(+) cells cultured in the cytokine combination, interleukin-3 (IL-3), IL-6, stem cell factor (SCF) and erythropoietin (Epo), over 14 days. We found that these culture conditions favored erythroid differentiation over the expansion of the more primitive erythroid precursors. With an initiating culture density of 5x10(4) cells per ml, the nucleated cell fold expansion increased from 7.9+/-3.9 (range 4.5 to 11.1) after 4 days to 2990.2+/-1936.1 (range 626.6 to 6912.0) after 14 days in culture. Day-14 burst-forming unit-erythroid (BFU-E) frequencies peaked at day 4 (24.0+/-8.9%), with a marked decrease in BFU-E burst size as the cultures progressed. Time-course immunophenotypical profiles were characteristically erythroid with a decrease in CD34 expression (from 96.8+/-3.0% at day 0 to 0.8+/-0.8% at day 14), and a concomitant increase in the expression of erythroid-specific markers, CD36, glycophorin A (GpA) and CD71 (from 14.8+/-5.0%, 1.7+/-1.0% and 37.9+/-18.0% to 93.0+/-7.0%, 82.1+/-14.0% and 95.7+/-3.0%, respectively). Morphological studies revealed the presence of normoblasts with the complete absence of reticulocytes and mature erythrocytes after 14 days in culture. Once the culture conditions were optimized, we scaled down our culture model from 24-well plate (large-scale) to 96-well plate cultures (small-scale). We found that the small-scale cultures compared favorably with their large-scale counterpart in terms of erythroid progenitor cell proliferation and differentiation, particularly at low CD34(+) initiating cell doses. By using tumor necrosis factor-alpha (TNF-alpha), a known inhibitor of erythropoiesis, we validated our model system and showed a dose-dependent inhibition of erythroid differentiation with TNF-alpha in our cultures. Therefore, our results demonstrate a small-scale serum-free liquid culture model of erythropoiesis that is comparable with and complements our well-defined large-scale model. Our system would prove useful for screening the effects of exogenous factors on erythropoiesis in vitro.
Cell Culture Techniques↗
Lactate levels in severe malarial anaemia are associated with haemozoin-containing neutrophils and low levels of IL-12.
BACKGROUND: Hyperlactataemia is often associated with a poor outcome in severe malaria in African children. To unravel the complex pathophysiology of this condition the relationship between plasma lactate levels, parasite density, pro- and anti-inflammatory cytokines, and haemozoin-containing leucocytes was studied in children with severe falciparum malarial anaemia. METHODS: Twenty-six children with a primary diagnosis of severe malarial anaemia with any asexual Plasmodium falciparum parasite density and Hb < 5 g/dL were studied and the association of plasma lactate levels and haemozoin-containing leucocytes, parasite density, pro- and anti-inflammatory cytokines was measured. The same associations were measured in non-severe malaria controls (N = 60). RESULTS: Parasite density was associated with lactate levels on admission (r = 0.56, P < 0.005). Moreover, haemozoin-containing neutrophils and IL-12 were strongly associated with plasma lactate levels, independently of parasite density (r = 0.60, P = 0.003 and r = -0.46, P = 0.02, respectively). These associations were not found in controls with uncomplicated malarial anaemia. CONCLUSION: These data suggest that blood stage parasites, haemozoin and low levels of IL-12 may be associated with the development of hyperlactataemia in severe malarial anaemia.
Anemia↗
Suppression of erythropoiesis in malarial anemia is associated with hemozoin in vitro and in vivo.
Malarial anemia is a global public health problem and is characterized by a low reticulocyte response in the presence of life-threatening hemolysis. Although cytokines, in particular tumor necrosis factor-alpha (TNF-alpha), can suppress erythropoiesis, the grossly abnormal bone marrow morphology indicates that other factors may contribute to ineffective erythropoiesis. We hypothesized that the cytotoxic hemozoin (Hz) residues from digested hemoglobin (Hb) significantly contribute to abnormal erythropoiesis. Here, we show that not only isolated Hz, but also delipidated Hz, inhibits erythroid development in vitro in the absence of TNF-alpha. However, when added to cultures, TNF-alpha synergizes with Hz to inhibit erythropoiesis. Furthermore, we show that, in children with malarial anemia, the proportion of circulating monocytes containing Hz is associated with anemia (P < .001) and reticulocyte suppression (P = .009), and that this is independent of the level of circulating cytokines, including TNF-alpha. Plasma Hz is also associated with anemia (P < .001) and reticulocyte suppression (P = .02). Finally, histologic examination of the bone marrow of children who have died from malaria shows that pigmented erythroid and myeloid precursors are associated with the degree of abnormal erythroid development. Taken together, these observations provide compelling evidence for inhibition of erythropoiesis by Hz.
Anemia↗
Thrombocytopenia in falciparum malaria is associated with high concentrations of IL-10.
Over the last decade, a number of observations have suggested that platelets may play a role in the pathophysiology of severe malaria. However, somewhat paradoxically, thrombocytopenia is not associated clearly with outcome. We studied the relationship between thrombocytopenia and cytokines in Kenyan children with severe malaria and showed that thrombocytopenia (platelet count < 150 x 10(9)/L) strongly correlates with high levels of interleukin (IL)-10. Several studies have shown that high levels of IL-10 are associated with a favorable outcome in severe malaria. Taken together, these data suggest why thrombocytopenia has a complex relationship with severe disease and suggest one mechanism whereby IL-10 may modify the outcome of severe disease.
Child↗
Plasmodium falciparum antigenic variation: relationships between in vivo selection, acquired antibody response, and disease severity.
BACKGROUND: Variant surface antigens (VSA) on Plasmodium falciparum-infected erythrocytes are potentially important targets of immunity to malaria. We previously identified a VSA phenotype--VSA with a high frequency of antibody recognition (VSA(FoRH))--that is associated with young host age and severe malaria. We hypothesized that VSA(FoRH) are positively selected by host molecules such as intercellular adhesion molecule 1 (ICAM1) and CD36 and dominate in the absence of an effective immune response. Here, we assessed, in 115 Kenyan children, the potential role played by in vivo selection pressures in either favoring or selecting against VSA(FoRH) among parasites that cause malaria. METHODS: We tested for associations between VSA(FoRH) and (1) the repertoire of VSA antibodies carried by children at the time of acute malaria and (2) polymorphisms in ICAM1 (K29M) and CD36 (T188G) that could potentially reduce the positive selection of VSA(FoRH). RESULTS: An expected negative association between VSA antibody repertoire and VSA(FoRH) was observed in children with nonsevere malaria. However, this association did not extend to children with severe malaria, many of whom apparently had well-developed VSA antibody responses despite being infected by parasites expressing VSA(FoRH). There was no evidence for involvement of CD36 or ICAM1 in positive selection of VSA(FoRH). On the contrary, a weak positive association between carriage of the CD36 (T188G) allele and VSA(FoRH) was observed in children with severe malaria. CONCLUSION: The association between the VSA(FoRH) parasite phenotype and severe malaria cannot be explained simply in terms of the total repertoire of VSA antibodies carried at the time of acute disease.
Amino Acid Substitution↗
An immune basis for malaria protection by the sickle cell trait.
BACKGROUND: Malaria resistance by the sickle cell trait (genotype HbAS) has served as the prime example of genetic selection for over half a century. Nevertheless, the mechanism of this resistance remains the subject of considerable debate. While it probably involves innate factors such as the reduced ability of Plasmodium falciparum parasites to grow and multiply in HbAS erythrocytes, recent observations suggest that it might also involve the accelerated acquisition of malaria-specific immunity. METHODS AND FINDINGS: We studied the age-specific protection afforded by HbAS against clinical malaria in children living on the coast of Kenya. We found that protection increased with age from only 20% in the first 2 y of life to a maximum of 56% by the age of 10 y, returning thereafter to 30% in participants greater than 10 y old. CONCLUSIONS: Our observations suggest that malaria protection by HbAS involves the enhancement of not only innate but also of acquired immunity to the parasite. A better understanding of the underlying mechanisms might yield important insights into both these processes.
Age Factors↗
Antihistamines and driving ability: evidence from on-the-road driving studies during normal traffic.
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Animals↗
Mechanisms of inverse agonist action at D2 dopamine receptors.
Mechanisms of inverse agonist action at the D2(short) dopamine receptor have been examined. Discrimination of G-protein-coupled and -uncoupled forms of the receptor by inverse agonists was examined in competition ligand-binding studies versus the agonist [3H]NPA at a concentration labelling both G-protein-coupled and -uncoupled receptors. Competition of inverse agonists versus [3H]NPA gave data that were fitted best by a two-binding site model in the absence of GTP but by a one-binding site model in the presence of GTP. K(i) values were derived from the competition data for binding of the inverse agonists to G-protein-uncoupled and -coupled receptors. K(coupled) and K(uncoupled) were statistically different for the set of compounds tested (ANOVA) but the individual values were different in a post hoc test only for (+)-butaclamol. These observations were supported by simulations of these competition experiments according to the extended ternary complex model. Inverse agonist efficacy of the ligands was assessed from their ability to reduce agonist-independent [35S]GTP gamma S binding to varying degrees in concentration-response curves. Inverse agonism by (+)-butaclamol and spiperone occurred at higher potency when GDP was added to assays, whereas the potency of (-)-sulpiride was unaffected. These data show that some inverse agonists ((+)-butaclamol, spiperone) achieve inverse agonism by stabilising the uncoupled form of the receptor at the expense of the coupled form. For other compounds tested, we were unable to define the mechanism.
Animals↗
Neonatal ergot poisoning: a persistent iatrogenic illness.
Ergot toxicity in the newborn usually manifests itself as respiratory depression, cyanosis, oliguria, and seizures. Death is usually caused by respiratory failure. A limited number of neonatal cases have been reported worldwide, and almost all cases involved confusion of maternal methylergonovine with neonatal vitamin K. Previous case reports provided little information regarding the effectiveness and dosing of antidotal therapy, especially sodium nitroprusside. A full-term male infant was inadvertently given methylergonovine instead of naloxone at birth. Several hours later, he required intubation for respiratory failure. Peripheral perfusion, ventilation, and renal function improved rapidly with nitroprusside infusion, and he was extubated on the third hospital day. Even asymptomatic newborns should be transferred to a neonatal intensive care unit for close observation after methylergonovine administration because toxicity can be life threatening. Rapid recognition of the therapeutic error, ventilatory support, and prompt administration of sodium nitroprusside should lead to a good outcome.
Diagnosis, Differential↗
Fatal Plasmodium falciparum malaria causes specific patterns of splenic architectural disorganization.
The spleen is critical for host defense against pathogens, including Plasmodium falciparum. It has a dual role, not only removing aged or antigenically altered erythrocytes from the blood but also as the major lymphoid organ for blood-borne or systemic infections. The human malaria parasite P. falciparum replicates within erythrocytes during asexual blood stages and causes repeated infections that can be associated with severe disease. In spite of the crucial role of the spleen in the innate and acquired immune response to malaria, there is little information on the pathology of the spleen in human malaria. We performed a histological and quantitative immunohistochemical study of spleen sections from Vietnamese adults dying from severe falciparum malaria and compared the findings with the findings for spleen sections from control patients and patients dying from systemic bacterial sepsis. Here we report that the white pulp in the spleens of patients dying from malaria showed a marked architectural disorganization. We observed a marked dissolution of the marginal zones with relative loss of B cells. Furthermore, we found strong HLA-DR expression on sinusoidal lining cells but downregulation on cordal macrophages. P. falciparum infection results in alterations in splenic leukocytes, many of which are not seen in sepsis.
Adult↗
G protein activation by G protein coupled receptors: ternary complex formation or catalyzed reaction?
G protein coupled receptors catalyze the GDP/GTP exchange on G proteins, thereby activating them. The ternary complex model, designed to describe agonist binding in the absence of GTP, is often extended to G protein activation. This is logically unsatisfactory as the ternary complex does not accumulate when G proteins are activated by GTP. Extended models taking into account nucleotide binding exist, but fail to explain catalytic G protein activation. This review puts forward an enzymatic model of G protein activation and compares its predictions with the ternary complex model and with observed receptor phenomenon. This alternative model does not merely provide a new set of formulae but leads to a new philosophical outlook and more readily accommodates experimental observations. The ternary complex model implies that, HRG being responsible for efficient G protein activation, it should be as stable as possible. In contrast, the enzyme model suggests that although a limited stabilization of HRG facilitates GDP release, HRG should not be "too stable" as this might trap the G protein in an inactive state and actually hinder G protein activation. The two models also differ completely in the definition of the receptor "active state": the ternary complex model implies that the active state corresponds to a single active receptor conformation (HRG); in contrast, the catalytic model predicts that the active receptor state is mobile, switching smoothly through various conformations with high and low affinities for agonists (HR, HRG, HRGGDP, HRGGTP, etc.).
Animals↗
Mechanisms of agonist action at D2 dopamine receptors.
In this study, we investigated the biochemical mechanisms of agonist action at the G protein-coupled D2 dopamine receptor expressed in Chinese hamster ovary cells. Stimulation of guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding by full and partial agonists was determined at different concentrations of [35S]GTPgammaS (0.1 and 10 nM) and in the presence of different concentrations of GDP. At both concentrations of [35S]GTPgammaS, increasing GDP decreased the [35S]GTPgammaS binding observed with maximally stimulating concentrations of agonist, with partial agonists exhibiting greater sensitivity to the effects of GDP than full agonists. The relative efficacy of partial agonists was greater at the lower GDP concentrations. Concentration-response experiments were performed for a range of agonists at the two [35S]GTPgammaS concentrations and with different concentrations of GDP. At 0.1 nM [35S]GTPgammaS, the potency of both full and partial agonists was dependent on the GDP concentration in the assays. At 10 nM [35S]GTPgammaS, the potency of full agonists exhibited a greater dependence on the GDP concentration, whereas the potency of partial agonists was virtually independent of GDP. We concluded that at the lower [35S]GTPgammaS concentration, the rate-determining step in G protein activation is the binding of [35S]GTPgammaS to the G protein. At the higher [35S]GTPgammaS concentration, for full agonists, [35S]GTPgammaS binding remains the slowest step, whereas for partial agonists, another (GDP-independent) step, probably ternary complex breakdown, becomes rate-determining.
Animals↗
Investigation of the mechanism of agonist and inverse agonist action at D2 dopamine receptors.
This study investigated, for the D2 dopamine receptor, the relation between the ability of agonists and inverse agonists to stabilise different states of the receptor and their relative efficacies. Ki values for agonists were determined in competition versus the binding of the antagonist [3H]spiperone. Competition data were fitted best by a two-binding site model (with the exception of bromocriptine, for which a one-binding site model provided the best fit) and agonist affinities for the higher (Kh) (G protein-coupled) and lower affinity (Kl) (G protein-uncoupled) sites determined. Ki values for agonists were also determined in competition versus the binding of the agonist [3H]N-propylnorapomorphine (NPA) to provide a second estimate of Kh. Maximal agonist effects (Emax) and their potencies (EC50) were determined from concentration-response curves for agonist stimulation of guanosine-5'-O-(3-[32S]thiotriphosphate) ([35S]GTPgammaS) binding. The ability of agonists to stabilise the G protein-coupled state of the receptor (Kl/Kh determined from ligand-binding assays) did not correlate with either of two measures of relative efficacy (relative Emax, Kl/EC50) of agonists determined in [35S]GTPgammaS-binding assays, when the data for all of the compounds tested were analysed. For a subset of compounds, however, there was a relation between Kl/Kh and Emax. Competition-binding data versus [3H]spiperone and [3H]NPA for a range of inverse agonists were fitted best by a one-binding site model. Ki values for the inverse agonists tested were slightly lower in competition versus [3H]NPA compared to [3H]spiperone. These data do not provide support for the idea that inverse agonists act by binding preferentially to the ground state of the receptor.
Animals↗
Platelet-induced autoagglutination of Plasmodium falciparum-infected red blood cells and disease severity in Thailand.
The relationship of the platelet-mediated autoagglutination of Plasmodium falciparum-infected red blood cells (IRBCs) to disease severity was investigated in 182 Thai patients with falciparum malaria; it was evident in 43% of uncomplicated malaria (n=63), 41% of severe malaria (n=104), and 100% of cerebral malaria (n=15; P=.001) isolates. The median (range) number of IRBCs in agglutinates per 1000 IRBCs was significantly higher in cerebral malaria (6 [3-42]) than in severe (0 [0-52]) and uncomplicated (0 [0-24]) malaria (P=.01). In multivariate analyses, high parasitemia and cerebral malaria were associated independently with parasite agglutination.
Adult↗
GPVI levels in platelets: relationship to platelet function at high shear.
We have investigated the density of the collagen receptors glycoprotein VI (GPVI) and alpha 2 beta 1 on human platelets and their relationship to polymorphisms within the GPVI gene. GPVI levels varied 1.5-fold and showed a weak correlation (r = 0.35) with the levels of alpha 2 beta 1, which varied 3-fold. GPVI genotype had a significant effect on receptor levels with carriers of the proline 219 allele (approximately 22% of the population) having 10% lower GPVI levels than the more common serine homozygotes. GPVI and alpha 2 beta 1 levels were found to be significantly decreased on platelets from patients with myeloproliferative disorders (MPDs). In both the MPD and the control group, GPVI levels were found not to affect platelet function under high shear in whole blood. Similarly murine platelets that express up to 5-fold lower levels of GPVI showed no significant difference than controls in thrombus formation on a high-density collagen-coated surface. However platelets lacking the GPVI/Fc receptor gamma-chain (FcR gamma-chain) complex or a functional FcR gamma-chain (immunoreceptor tyrosine-based activation motif [ITAM] point mutant) exhibited severely abrogated thrombus formation at 800 s-1 and 1500 s-1. These results demonstrate that GPVI levels are tightly controlled and play a critical role in thrombus formation on collagen; nevertheless, a range of receptor densities can support platelet function under high shear.
Alleles↗
Inhibition of T cell function during malaria: implications for immunology and vaccinology.
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Animals↗
Chronic ethanol administration and withdrawal decreases 5-HT1A mRNA, but not 5-HT4 expression in the rat hippocampus.
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Animals↗