Isolation of human intrahepatic leukocytes for phenotypic and functional characterization by flow cytometry

With the growing appreciation of tissue-resident immunity, studying tissue-specific immune cells contributing to both homeostasis and disease is imperative. Here, we provide a protocol for the isolation of human intrahepatic leukocytes (IHL) maximizing viability, purity, and yield. Our protocol is scalable by tissue weight, allowing for reproducible and efficient IHL liberation suitable for functional characterization, cell isolation, and profiling by flow (or mass) cytometry. Furthermore, we provide a "guide" to determine an expected IHL yield per gram of tissue processed. For complete details on the use and execution of this protocol, please refer to Stegmann et al. (2016), Pallett et al. (2017), Easom et al. (2018), Swadling et al. (2020), Pallett et al. (2020), and Zakeri et al. (2022)

‘I can die today, I can die tomorrow’: lay perceptions of sickle cell disease in Kumasi, Ghana at a point of transition

Objective. To describe the lay meanings of sickle cell disease (SCD) in the Ashanti region of Ghana

X-Linked G6PD Deficiency Protects Hemizygous Males but Not Heterozygous Females against Severe Malaria

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) is important in the control of oxidant stress in erythrocytes, the host cells for Plasmodium falciparum. Mutations in this enzyme produce X-linked deficiency states associated with protection against malaria, notably in Africa where the A− form of G6PD deficiency is widespread. Some reports have proposed that heterozygous females with mosaic populations of normal and deficient erythrocytes (due to random X chromosome inactivation) have malaria resistance similar to or greater than hemizygous males with populations of uniformly deficient erythrocytes. These proposals are paradoxical, and they are not consistent with currently hypothesized mechanisms of protection. METHODS AND FINDINGS: We conducted large case-control studies of the A− form of G6PD deficiency in cases of severe or uncomplicated malaria among two ethnic populations of rural Mali, West Africa, where malaria is hyperendemic. Our results indicate that the uniform state of G6PD deficiency in hemizygous male children conferred significant protection against severe, life-threatening malaria, and that it may have likewise protected homozygous female children. No such protection was evident from the mosaic state of G6PD deficiency in heterozygous females. We also found no significant differences in the parasite densities of males and females with differences in G6PD status. Pooled odds ratios from meta-analysis of our data and data from a previous study confirmed highly significant protection against severe malaria in hemizygous males but not in heterozygous females. Among the different forms of severe malaria, protection was principally evident against cerebral malaria, the most frequent form of life-threatening malaria in these studies. CONCLUSIONS: The A− form of G6PD deficiency in Africa is under strong natural selection from the preferential protection it provides to hemizygous males against life-threatening malaria. Little or no such protection is present among heterozygous females. Although these conclusions are consistent with data from at least one previous study, they have not heretofore been realized to our knowledge, and they therefore give fresh perspectives on malaria protection by G6PD deficiency as an X-linked trait

Storage rot of seed yam resulting from speargrass injuries

Postharvest rot due to injury is a major contributing factor to the declining quality of stored seed yams ( Dioscorea spp.). Among the several known injuries, the piercing effect of speargrass rhizomes has become a serious constraint for yam production in Ghana. The objective of this study was to assess injuries on seed yams resulting from piercing of speargrass rhizomes and their effects on postharvest rots in Ghana. Eighty farmer fields from Mem, Watro, Asanteboa and Abour in the Atebubu-Amantin Municipal in the Bono East Region of Ghana were screened for speargrass incidence and injury on harvested tubers, for laboratory analysis of pathogens in 2016 and 2017. The tubers were sorted into four categories of seed yam based on weight. Thirty seed yams each of two selected white yam cultivars (Dente and Kpamyo) with visible speargrass rhizome-pierced-tubers (VSRPT) and non-speargrass rhizome pierced healthy tubers (NSRPHT) were randomly selected and stored in a ban for weekly assessment of rot. The rotten tissues from the localised area of VPSRT were subjected to pathological investigations in the laboratory. The incidence of injury seemingly increased with increasing tuber weight. It was 0% for < 100 g samples and averagely 14% for > 1 kg samples, irrespective of cultivars and locations. Incidence of rot from NSRPHT sample was observed 5 weeks after storage (WAS) for both cultivars; and 2 WAS from the VSRPT sample and 40% higher than NSRPHT at 8 WAS. Eight and six known rot pathogens were isolated from the rotten tissues of VSRPT of Dente and Kpamyo, respectively. Injury from the piercing of speargrass rhizome significantly contributed to hastening of tuber rots; while tuber injury increased with increasing speargrass density. Appropriate management of speargrass is essential for commercial seed yam growers to reduce tuber damage which affects yam quality, storage and marketing.La pourriture post-r\ue9colte due \ue0 une d\ue9chirure est un facteur majeur contribuant \ue0 la baisse de la qualit\ue9 des ignames des semences stock\ue9es ( Dioscorea spp.). Parmi les nombreuses d\ue9chirures connues, l\u2019effet per\ue7ant des rhizomes de la gerbe d\u2019herbe est devenu une contrainte s\ue9rieuse pour la production d\u2019igname au Ghana. L\u2019objectif de cette \ue9tude \ue9tait d\u2019\ue9valuer les d\ue9chirures sur les ignames de semence r\ue9sultant du per\ue7age des rhizomes de gerbe d\u2019herbe et leurs effets sur les pourritures post-r\ue9colte au Ghana. Quatre-vingts champs d\u2019agriculteurs de Mem, Watro, Asanteboa et Abour dans la municipalit\ue9 d\u2019Atebubu-Amantin dans la region de l\u2018 Est de Bono au Ghana ont \ue9t\ue9 examin\ue9s pour d\ue9terminer l\u2019incidence et les dommages de la gerbe d\u2019herbe sur les tubercules r\ue9colt\ue9s, pour une analyse en laboratoire des agents pathog\ue8nes en 2016 et 2017. Les tubercules ont \ue9t\ue9 tri\ue9s en quatre cat\ue9gories d\u2019igname de semence en fonction du poids. Trente ignames de semence de chacun des deux cultivars s\ue9lectionn\ue9s d\u2019igname blanche (Dente et Kpamyo) avec des tubercules perc\ue9s de rhizome de gerbe d\u2019herbe (VSRPT) et des tubercules sains perc\ue9s de rhizome non- gerbe d\u2019herbe (NSRPHT) ont \ue9t\ue9 s\ue9lectionn\ue9s au hasard et stock\ue9s dans une interdiction pour une \ue9valuation hebdomadaire de la pourriture . Les tissus pourris de la zone localis\ue9e de VPSRT ont \ue9t\ue9 soumis \ue0 des investigations pathologiques en laboratoire. L\u2019incidence des d\ue9chirures a apparemment augment\ue9 avec l\u2019augmentation du poids des tubercules. Il \ue9tait de 0% pour les \ue9chantillons <100 g et de 14% en moyenne pour les \ue9chantillons > 1 kg, quels que soient les cultivars et les emplacements. L\u2019incidence de pourriture de l\u2019\ue9chantillon NSRPHT a \ue9t\ue9 observ\ue9e 5 semaines apr\ue8s stockage (WAS) pour les deux cultivars; et 2 WAS de l\u2019\ue9chantillon VSRPT et 40% plus \ue9lev\ue9s que NSRPHT \ue0 8 WAS. Huit et six agents pathog\ue8nes de la pourriture connus ont \ue9t\ue9 isol\ue9s respectivement dans les tissus pourris du VSRPT de Dente et de Kpamyo. Les d\ue9chirures caus\ue9es par le per\ue7age du rhizome de gerbe d\u2019herbe ont consid\ue9rablement contribu\ue9 \ue0 acc\ue9l\ue9rer la pourriture des tubercules; tandis que les dommages aux tubercules augmentaient avec l\u2019augmentation de la densit\ue9 de la gerbe d\u2019herbe. Une gestion appropri\ue9e de la groseille verte est essentielle pour les producteurs commerciaux d\u2019ignames de semence afin de r\ue9duire les dommages aux tubercules qui affectent la qualit\ue9, le stockage et la commercialisation des ignames

Simulation of a detoxifying organ function: Focus on hemodynamics modeling and convection‐reaction numerical simulation in microcirculatory networks

International audienceWhen modeling a detoxifying organ function, an important component is the impact of flow on the metabolism of a compound of interest carried by the blood. We here study the effects of red blood cells (such as the Fahraeus-Lindqvist effect and plasma skimming) on blood flow in typical microcirculatory components such as tubes, bifurcations and entire networks, with particular emphasis on the liver as important representative of detoxifying organs. In one of the plasma skimming models, under certain conditions, oscillations between states are found and analyzed in a methodical study to identify their causes and influencing parameters. The flow solution obtained is then used to define the velocity at which a compound would be transported. A convection-reaction equation is studied to simulate the transport of a compound in blood and its uptake by the surrounding cells. Different types of signal sharpness have to be handled depending on the application to address different temporal compound concentration profiles. To permit executing the studied models numerically stable and accurate, we here extend existing transport schemes to handle converging bifurcations, and more generally multi-furcations. We study the accuracy of different numerical schemes as well as the effect of reactions and of the network itself on the bolus shape. Even though this study is guided by applications in liver micro-architecture, the proposed methodology is general and can readily be applied to other capillary network geometries, hence to other organs or to bioengineered network designs

Detection of PIGO-Deficient Cells Using Proaerolysin: A Valuable Tool to Investigate Mechanisms of Mutagenesis in the DT40 Cell System

While isogenic DT40 cell lines deficient in DNA repair pathways are a great tool to understand the DNA damage response to genotoxic agents by a comparison of cell toxicity in mutants and parental DT40 cells, no convenient mutation assay for mutagens currently exists for this reverse-genetic system. Here we establish a proaerolysin (PA) selection-based mutation assay in DT40 cells to identify glycosylphosphatidylinositol (GPI)-anchor deficient cells. Using PA, we detected an increase in the number of PA-resistant DT40 cells exposed to MMS for 24 hours followed by a 5-day period of phenotype expression. GPI anchor synthesis is catalyzed by a series of phosphatidylinositol glycan complementation groups (PIGs). The PIG-O gene is on the sex chromosome (Chromosome Z) in chicken cells and is critical for GPI anchor synthesis at the intermediate step. Among all the mutations detected in the sequence levels observed in DT40 cells exposed to MMS at 100 µM, we identified that ∼55% of the mutations are located at A:T sites with a high frequency of A to T transversion mutations. In contrast, we observed no transition mutations out of 18 mutations. This novel assay for DT40 cells provides a valuable tool to investigate the mode of action of mutations caused by reactive agents using a series of isogenic mutant DT40 cells

Polymorphisms in genes of interleukin 12 and its receptors and their association with protection against severe malarial anaemia in children in western Kenya

Abstract Background: Malarial anaemia is characterized by destruction of malaria infected red blood cells and suppression of erythropoiesis. Interleukin 12 (IL12) significantly boosts erythropoietic responses in murine models of malarial anaemia and decreased IL12 levels are associated with severe malarial anaemia (SMA) in children. Based on the biological relevance of IL12 in malaria anaemia, the relationship between genetic polymorphisms of IL12 and its receptors and SMA was examined. Methods: Fifty-five tagging single nucleotide polymorphisms covering genes encoding two IL12 subunits, IL12A and IL12B, and its receptors, IL12RB1 and IL12RB2, were examined in a cohort of 913 children residing in Asembo Bay region of western Kenya. Results: An increasing copy number of minor variant (C) in IL12A (rs2243140) was significantly associated with a decreased risk of SMA (P = 0.006; risk ratio, 0.52 for carrying one copy of allele C and 0.28 for two copies). Individuals possessing two copies of a rare variant (C) in IL12RB1 (rs429774) also appeared to be strongly protective against SMA (P = 0.00005; risk ratio, 0.18). In addition, children homozygous for another rare allele (T) in IL12A (rs22431348) were associated with reduced risk of severe anaemia (SA) (P = 0.004; risk ratio, 0.69) and of severe anaemia with any parasitaemia (SAP) (P = 0.004; risk ratio, 0.66). In contrast, AG genotype for another variant in IL12RB1 (rs383483) was associated with susceptibility to high-density parasitaemia (HDP) (P = 0.003; risk ratio, 1.21). Conclusions: This study has shown strong associations between polymorphisms in the genes of IL12A and IL12RB1 and protection from SMA in Kenyan children, suggesting that human genetic variants of IL12 related genes may significantly contribute to the development of anaemia in malaria patients

A Role for Fetal Hemoglobin and Maternal Immune IgG in Infant Resistance to Plasmodium falciparum Malaria

In Africa, infant susceptibility to Plasmodium falciparum malaria increases substantially as fetal hemoglobin (HbF) and maternal immune IgG disappear from circulation. During the first few months of life, however, resistance to malaria is evidenced by extremely low parasitemias, the absence of fever, and the almost complete lack of severe disease. This resistance has previously been attributed in part to poor parasite growth in HbF-containing red blood cells (RBCs). A specific role for maternal immune IgG in infant resistance to malaria has been hypothesized but not yet identified.We found that P. falciparum parasites invade and develop normally in fetal (cord blood, CB) RBCs, which contain up to 95% HbF. However, these parasitized CB RBCs are impaired in their binding to human microvascular endothelial cells (MVECs), monocytes, and nonparasitized RBCs--cytoadherence interactions that have been implicated in the development of high parasite densities and the symptoms of malaria. Abnormal display of the parasite's cytoadherence antigen P. falciparum erythrocyte membrane protein-1 (PfEMP-1) on CB RBCs accounts for these findings and is reminiscent of that on HbC and HbS RBCs. IgG purified from the plasma of immune Malian adults almost completely abolishes the adherence of parasitized CB RBCs to MVECs.Our data suggest a model of malaria protection in which HbF and maternal IgG act cooperatively to impair the cytoadherence of parasitized RBCs in the first few months of life. In highly malarious areas of Africa, an infant's contemporaneous expression of HbC or HbS and development of an immune IgG repertoire may effectively reconstitute the waning protective effects of HbF and maternal immune IgG, thereby extending the malaria resistance of infancy into early childhood

Haptoglobin and Sickle Cell Polymorphisms and Risk of Active Trachoma in Gambian Children

BACKGROUND: Susceptibility and resistance to trachoma, the leading infectious cause of blindness, have been associated with a range of host genetic factors. In vitro studies of the causative organism, Chlamydia trachomatis, demonstrate that iron availability regulates its growth, suggesting that host genes involved in regulating iron status and/or availability may modulate the risk of trachoma. The objective was to investigate whether haptoglobin (Hp) haplotypes constructed from the functional polymorphism (Hp1/Hp2) plus the functional promoter SNPs -61A-C (rs5471) and -101C-G (rs5470), or sickle cell trait (HbAS, rs334) were associated with risk of active trachoma when stratified by age and sex, in rural Gambian children. METHODOLOGY AND PRINCIPAL FINDINGS: In two cross sectional surveys of children aged 6-78 months (n = 836), the prevalence of the clinical signs of active trachoma was 21.4%. Within boys, haplotype E (-101G, -61A, Hp1), containing the variant allele of the -101C-G promoter SNP, was associated with a two-fold increased risk of active trachoma (OR = 2.0 [1.17-3.44]). Within girls, an opposite association was non-significant (OR = 0.58 [0.32-1.04]; P = 0.07) and the interaction by sex was statistically significant (P = 0.001). There was no association between trachoma and HbAS. CONCLUSIONS: These data indicate that genetic variation in Hp may affect susceptibility to active trachoma differentially by sex in The Gambia