An AI-based imaging flow cytometry approach to study erythrophagocytosis.

Erythrophagocytosis is a process consisting of recognition, engulfment and digestion by phagocytes of antibody-coated or damaged erythrocytes. Understanding the dynamics that are behind erythrophagocytosis is fundamental to comprehend this cellular process under specific circumstances. Several techniques have been used to study phagocytosis. Among these, an interesting approach is the use of Imaging Flow Cytometry (IFC) to distinguish internalization and binding of cells or particles. However, this method requires laborious analysis. Here, we introduce a novel approach to analyze the phagocytosis process by combining Artificial Intelligence (AI) with IFC. Our study demonstrates that this approach is highly suitable to study erythrophagocytosis, categorizing internalized, bound and non-bound erythrocytes. Validation experiments showed that our pipeline performs with high accuracy and reproducibility.

Hemolysis in the spleen drives erythrocyte turnover.

Red pulp macrophages (RPMs) of the spleen mediate turnover of billions of senescent erythrocytes per day. However, the molecular mechanisms involved in sequestration of senescent erythrocytes, their recognition, and their subsequent degradation by RPMs remain unclear. In this study, we provide evidence that the splenic environment is of substantial importance in facilitating erythrocyte turnover through induction of hemolysis. Upon isolating human spleen RPMs, we noted a substantial lack of macrophages that were in the process of phagocytosing intact erythrocytes. Detailed characterization of erythrocyte and macrophage subpopulations from human spleen tissue led to the identification of erythrocytes that are devoid of hemoglobin, so-called erythrocyte ghosts. By using in vivo imaging and transfusion experiments, we further confirmed that senescent erythrocytes that are retained in the spleen are subject to hemolysis. In addition, we showed that erythrocyte adhesion molecules, which are specifically activated on aged erythrocytes, cause senescent erythrocytes to interact with extracellular matrix proteins that are exposed within the splenic architecture. Such adhesion molecule-driven retention of senescent erythrocytes under low shear conditions was found to result in steady shrinkage of the cell and ultimately resulted in hemolysis. In contrast to intact senescent erythrocytes, the remnant erythrocyte ghost shells were prone to recognition and breakdown by RPMs. These data identify hemolysis as a key event in the turnover of senescent erythrocytes, which alters our current understanding of how erythrocyte degradation is regulated.

Regulatory T cell features in chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in any of the genes encoding the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system, responsible for the production of reactive oxygen species (ROS). CGD is marked by invasive bacterial and fungal infections and by autoinflammation/autoimmunity, of which the exact pathophysiology remains elusive. Contributing factors include decreased neutrophil apoptosis, impaired apoptotic neutrophil clearance, increased proinflammatory protein expression and reduced ROS-mediated inflammasome dampening. We have explored a fundamentally different potential mechanism: it has been reported that macrophage-mediated induction of regulatory T cells (Tregs ) depends on ROS production. We have investigated whether numerical or functional deficiencies exist in Tregs of CGD patients. As the prevalence of autoinflammation/autoimmunity differs between CGD subtypes, we have also investigated Tregs from gp91phox -, p47phox - and p40phox -deficient CGD patients separately. Results show that Treg numbers and suppressive capacities are not different in CGD patients compared to healthy controls, with the exception that in gp91phox -deficiency effector Treg (eTreg ) numbers are decreased. Expression of Treg markers CD25, inducible T cell co-stimulator (ICOS), Helios, cytotoxic T lymphocyte antigen 4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor receptor (GITR) did not provide any clue for differences in Treg functionality or activation state. No correlation was seen between eTreg numbers and patients' clinical phenotype. To conclude, the only difference between Tregs from CGD patients and healthy controls is a decrease in circulating eTregs in gp91phox -deficiency. In terms of autoinflammation/autoimmunity, this group is the most affected. However, upon culture, patient-derived Tregs showed a normal phenotype and normal functional suppressor activity. No other findings pointed towards a role for Tregs in CGD-related autoinflammation/autoimmunity.

Non-polar lipids accumulate during storage of transfusion products and do not contribute to the onset of transfusion-related acute lung injury.

BACKGROUND AND OBJECTIVES: The accumulation of non-polar lipids arachidonic acid, 5-hydroxyeicosatetraenoic acid (HETE), 12-HETE and 15-HETE during storage of transfusion products may play a role in the onset of transfusion-related acute lung injury (TRALI), a syndrome of respiratory distress after transfusion. MATERIALS AND METHODS: We investigated non-polar lipid accumulation in red blood cells (RBCs) stored for 42 days, plasma stored for 7 days at either 4 or 20°C and platelet (PLT) transfusion products stored for 7 days. Furthermore, we investigated whether transfusion of RBCs with increased levels of non-polar lipids induces TRALI in a 'two-hit' human volunteer model. All products were produced following Dutch Blood Bank protocols and are according to European standards. Non-polar lipids were measured with high-performance liquid chromotography followed by mass spectrometry. RESULTS: All non-polar lipids increased in RBCs after 21 days of storage compared to baseline. The non-polar lipid concentration in plasma increased significantly, and the increase was even more pronounced in products stored at 20°C. In platelets, baseline levels of 5-HETE and 15-HETE were higher than in RBCs or plasma. However, the non-polar lipids did not change significantly during storage of PLT products. Infusion of RBCs with increased levels of non-polar lipids did not induce TRALI in LPS-primed human volunteers. CONCLUSION: We conclude that non-polar lipids accumulate in RBC and plasma transfusion products and that accumulation is temperature dependent. Accumulation of non-polar lipids does not appear to explain the onset of TRALI (Dutch Trial Register - NTR4455).

Susceptibility to lethal cerebral malaria is regulated by epistatic interaction between chromosome 4 (Berr6) and chromosome 1 (Berr7) loci in mice.

In humans, cerebral malaria is a rare but often lethal complication of infection with Plasmodium parasites, the occurrence of which is influenced by complex genetic factors of the host. We used a mouse model of experimental cerebral malaria (ECM) with Plasmodium berghei ANKA to study genetic factors regulating appearance of neurological symptoms and associated lethality. In a genome-wide screen of N-ethyl-N-nitrosourea-mutagenized mice derived from C57BL/6J (B6) and 129S1/SvImJ (129) mouse strains, we detected a strong interaction between the genetic backgrounds of these strains, which modulates ECM resistance. We have mapped a major gene locus to central chromosome 4 (log of the odds (LOD) 6.7; 79.6-97.3 Mb), which we designate Berr8. [corrected]. B6 alleles at Berr6 are associated with resistance, and are inherited in a co-dominant fashion. In mice heterozygous for Berr6 B6/129 alleles, resistance to ECM is strongly modulated by a second locus, Berr7, that maps to the proximal portion of chromosome 1 (LOD 4.03; 41.4 Mb). 129 alleles at Berr7 are associated with ECM resistance in a dosage-dependent fashion. Results are discussed in view of the possible role of this two-locus system in susceptibility to unrelated inflammatory conditions in mice and humans.

International point prevalence study of Intensive Care Unit transfusion practices-Pilot study in the Netherlands.

BACKGROUND: Anaemia and coagulopathy are common issues in critically ill patients. Transfusion can be lifesaving, however, is associated with potential life threatening adverse events. As an international transfusion guideline for this specific patient population is lacking, we hypothesize that a high heterogeneity in transfusion practices exists. In this pilot-study we assessed transfusion practice in a university hospital in the Netherlands and tested the feasibility of this protocol for an international multi-centre study. METHODS: A prospective single centre cohort study was conducted. For seven days all consecutive non-readmitted patients to the adult Intensive Care Unit (ICU) were included and followed for 28 days. Patients were prospectively followed until ICU discharge or up to day 28. Patient outcome data was collected at day 28. Workload for this study protocol was scored in hours and missing data. RESULTS: In total, 48 patients were included, needed in total three hours patient to include and collect all data, with 1.6% missing data showing the feasibility of the data acquisition. Six (12.5%) patients received red blood cells (RBCs), three patients (6.3%) received platelet concentrates, and two (4.2%) patients received plasma units. In total eight (16.7%) patients were transfused with one or more blood products. Median pre- and post-transfusion haemoglobin (Hb) levels were 7.6 (6.7-7.7) g/dL and 8.1 (7.6-8.7) g/dL, respectively. CONCLUSION: In this pilot-study we proved the feasibility of our protocol and observed in this small population a restrictive transfusion practice for all blood products.

Differential interaction between DARC and SDF-1 on erythrocytes and their precursors.

The Duffy Antigen Receptor for Chemokines (DARC) is expressed on erythrocytes and on endothelium of postcapillary venules and splenic sinusoids. Absence of DARC on erythrocytes, but not on endothelium, is referred to as the Duffy negative phenotype and is associated with neutropenia. Here we provide evidence that stromal cell-derived factor 1 (SDF-1), the chemokine that restricts neutrophil precursors to the bone marrow, binds to erythrocyte progenitors in a DARC-dependent manner. Furthermore, we show that SDF-1 binding to DARC is dependent on the conformation of DARC, which gradually changes during erythroid development, resulting in the absence of SDF-1 binding to mature erythrocytes. However, SDF-1 binding to erythrocytes was found to be inducible by pre-treating erythrocytes with IL-8 or with antibodies recognizing specific epitopes on DARC. Taken together, these novel findings identify DARC on erythrocyte precursors as a receptor for SDF-1, which may be of interest in beginning to understand the development of neutropenia in situations where DARC expression is limited.

Glycophorin-C sialylation regulates Lu/BCAM adhesive capacity during erythrocyte aging.

Lutheran/basal cell adhesion molecule (Lu/BCAM) is a transmembrane adhesion molecule expressed by erythrocytes and endothelial cells that can interact with the extracellular matrix protein laminin-α5. In sickle cell disease, Lu/BCAM is thought to contribute to adhesion of sickle erythrocytes to the vascular wall, especially during vaso-occlusive crises. On healthy erythrocytes however, its function is unclear. Here we report that Lu/BCAM is activated during erythrocyte aging. We show that Lu/BCAM-mediated binding to laminin-α5 is restricted by interacting, in cis, with glycophorin-C-derived sialic acid residues. Following loss of sialic acid during erythrocyte aging, Lu/BCAM is released from glycophorin-C and allowed to interact with sialic acid residues on laminin-α5. Decreased glycophorin-C sialylation, as observed in individuals lacking exon 3 of glycophorin-C, the so-called Gerbich phenotype, was found to correlate with increased Lu/BCAM-dependent binding to laminin-α5. In addition, we identified the sialic acid-binding site within the third immunoglobulin-like domain within Lu/BCAM that accounts for the interaction with glycophorin-C and laminin-α5. Last, we present evidence that neuraminidase-expressing pathogens, such as Streptococcus pneumoniae, can similarly induce Lu/BCAM-mediated binding to laminin-α5, by cleaving terminal sialic acid residues from the erythrocyte membrane. These results shed new light on the mechanisms contributing to increased adhesiveness of erythrocytes at the end of their lifespan, possibly facilitating their clearance. Furthermore, this work may contribute to understanding the pathology induced by neuraminidase-positive bacteria, because they are especially harmful to patients suffering from sickle cell disease and are associated with the occurrence of vaso-occlusive crises.

Therapeutic use of transferrin to modulate anemia and conditions of iron toxicity.

As the main iron transporter, transferrin delivers iron to target tissues like the bone marrow for erythropoiesis. Also, by binding free iron, transferrin prevents formation of reactive oxygen species. Transferrin deficiency due to congenital hypotransferrinemia is characterized by anemia as well as oxidative stress related to toxic free iron. Transferrin supplementation may be beneficial in two ways. First, transferrin can correct anemia by modulating the amount of iron that is available for erythropoiesis. This is obvious for patients that suffer from hypotransferrinemia, but may also have beneficial effects for ß-thalassemia patients. Second, under conditions of iron overload, transferrin reduces oxidative stress by binding free iron in the circulation and in tissues. Hereby, transferrin protects the host against the reactive oxygen species that can be formed as a consequence of free iron. This beneficial effect is shown in hematological patients undergoing chemotherapy and stem cell transplantation. Transferrin may also be beneficial in lung injury, ischemia-reperfusion injury and hypomyelination. This review summarizes the preclinical and clinical data on the efficacy of exogenous transferrin administration to modulate certain forms of anemia and to prevent the toxic effects of free iron. Thereby, we show that transferrin has promising therapeutic potential in a wide variety of conditions.

Effect and maintenance of the SLIMMER diabetes prevention lifestyle intervention in Dutch primary healthcare: a randomised controlled trial.

BACKGROUND/OBJECTIVES: To assess the effectiveness of the SLIMMER combined dietary and physical activity lifestyle intervention on clinical and metabolic risk factors, dietary intake, physical activity, and quality of life after 12 months, and to investigate whether effects sustained six months after the active intervention period ended. SUBJECTS/METHODS: SLIMMER was a randomised controlled intervention, implemented in Dutch primary healthcare. In total, 316 subjects aged 40-70 years with increased risk of type 2 diabetes were randomly allocated to the intervention group (10-month dietary and physical activity programme) or the control group (usual healthcare). All subjects underwent an oral glucose tolerance test and physical examination, and filled in questionnaires. Identical examinations were performed at baseline and after 12 and 18 months. Primary outcome was fasting insulin. RESULTS: The intervention group showed significantly greater improvements in anthropometry and glucose metabolism. After 12 and 18 months, differences between intervention and control group were -2.7 kg (95% confidence interval (CI): -3.7; -1.7) and -2.5 kg (95% CI: -3.6; -1.4) for weight, and -12.1 pmol l-1 (95% CI: -19.6; -4.6) and -8.0 pmol l-1 (95% CI: -14.7; -0.53) for fasting insulin. Furthermore, dietary intake, physical activity, and quality of life improved significantly more in the intervention group than in the control group. CONCLUSIONS: The Dutch SLIMMER lifestyle intervention is effective in the short and long term in improving clinical and metabolic risk factors, dietary intake, physical activity, and quality of life in subjects at high risk of diabetes.

Accelerated clearance of human red blood cells in a rat transfusion model.

BACKGROUND: Animal models are valuable in transfusion research. Use of human red blood cells (RBCs) in animal models facilitates extrapolation of the impact of storage conditions to the human condition but may be hampered by the use of cross species. METHODS: Investigation of clearance and posttransfusion recovery in a rat model using fresh and stored human RBCs. RESULTS: Directly following transfusion, human RBCs could be detected in the circulation of all recipients, with higher recovery rates for stored RBCs than for fresh RBCs. After 24 h following transfusion, no donor RBCs could be detected in the circulation, but donor RBCs could be detected in all organs of all recipients. CONCLUSION: The use of human donor RBCs in a rat transfusion model resulted in clearance from cells from the circulation. Donor cells were found in different organs of the recipients. Rat transfusion models are thus not appropriate to study the efficacy of human RBC transfusion.

[Low serum vitamin B 12 level and vitamin B 12 deficiency in the elderly. When should supplement be provided?]. / Een lage serum vitamine B12-spiegel en vitamine B12-deficiëntie bij ouderen. Wanneer suppleren?

Low serum vitamin B12 levels and vitamin B12 deficiencies are frequently found in the elderly. The full syndrome of a vitamin B12 deficiency is rather simple to diagnose. The large applicability of the vitamin B12 assay also gives rise to many probably incomplete features. Low/low-normal vitamin B12 levels in screening procedures raise uncertainty whether this finding represents deficiency and should be followed by supplementation. In this paper the occurrence of low serum levels of vitamin B12 are discussed. To assess the clinical relevance of low/low-normal outcomes supplementary diagnostic procedures will be needed. Within this scope we illustrate the d.o.s.-test and the MMA-assay. Especially the latter will probably provide more answers to the treatment question. Systematic research is needed to clarify this issue. Meanwhile supplementation of all low and low-normal outcomes of the B12-assay seems the best answer.

Calcium signalling is altered in myeloid cells with a deficiency in NADPH oxidase activity.

The relation of O(2.-)-production and Ca2+ homeostasis was investigated in PLB-985 cell lines and neutrophilic granulocytes from peripheral blood. In differentiated wild-type PLB-985 cells, a high level of O(2.-)-production was associated with a significant decrease in the membrane potential and the inhibition of capacitative Ca2+ entry. These correlations were not observed in gp91phox -/- cells or in cells transfected with a non-functional mutant of gp91phox (Thr341Lys). Membrane depolarization and inhibition of Ca2+ entry reappeared in cells transfected with wild-type gp91phox. These experiments demonstrate that inhibition of Ca2+ entry depends on the presence of a functional NADPH oxidase. The Ca2+ signal induced by stimulation of chemotactic receptors also showed remarkable differences: [Ca2+]ic in the sustained phase was higher in gp91phox-/- than in wild-type cells. Alteration of the Ca2+ signal was reproduced by treating peripheral blood neutrophils with the NADPH oxidase inhibitor diphenylene-iodonium. It is concluded that the deficiency in O(2.-)-production is accompanied by significant alterations of Ca2+ homeostasis in myeloid cells.