scholarly journals Babesia duncani as a model organism to study the development, virulence and drug susceptibility of intraerythrocytic parasites in vitro and in vivo

2021 ◽  
Author(s):  
Choukri Mamoun ◽  
Anasuya C. Pal ◽  
Isaline Renard ◽  
Pallavi Singh ◽  
Pratap Vydyam ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Molecular Mechanisms ◽  
Babesia Microti ◽  
Dual Model ◽  
Ideal System ◽  
Human Babesiosis

Hematozoa are a subclass of protozoan parasites that invade and develop within vertebrate red blood cells to cause the pathological symptoms associated with diseases of both medical and veterinary importance such as malaria and babesiosis. A major limitation in the study of the most prominent hematozoa, Plasmodium spp, the causative agents of malaria, is the lack of a broadly accessible mouse model to evaluate parasite infection in vivo as is the case for P. falciparum or altogether the lack of an in vitro culture and mouse models as is the case for P. vivax, P. malariae and P. ovale. Similarly, no in vitro culture system exists for Babesia microti, the predominant agent of human babesiosis. In this study, we show that human red blood cells infected with the human pathogen Babesia duncani continuously propagated in culture, as well as merozoites purified from parasite cultures, can cause lethal infection in immunocompetent C3H/HeJ mice. Furthermore, highly reproducible parasitemia and survival outcomes were established using specific parasite loads and different mouse genetic backgrounds. Using the combined in culturein mouse (ICIM) model of B. duncani infection, we demonstrate that current recommended combination therapies for the treatment of human babesiosis, while synergistic in cell culture, have weak potency in vitro and failed to clear infection or prevent death in mice. Interestingly, using the ICIM model, we identified two new endochin-like quinolone prodrugs, ELQ-331 and ELQ468, that alone or in combination with atovaquone are highly efficacious against B. duncani and B. microti. The novelty, ease of use and scalability of the B. duncani ICIM dual model make it an ideal system to study intraerythrocytic parasitism by protozoa, unravel the molecular mechanisms underlying parasite virulence and pathogenesis, and accelerate the development of innovative therapeutic strategies that could be translated to unculturable parasites and important pathogens for which an animal model is lacking.

scholarly journals CD59 Deficiency Is Critical for C3 Binding on Red Blood Cells of Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) during Anti-C5 Treatment (eculizumab)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 401-401 ◽  
Author(s):  
MIchela Sica ◽  
Tommaso Rondelli ◽  
Patrizia Ricci ◽  
Maria De Angioletti ◽  
Antonio M Risitano ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Research Funding ◽  
Molecular Mechanisms ◽  
Alternative Pathway ◽  
Red Cells ◽  
Classical Pathway ◽  
Complement Alternative Pathway

Abstract C5-blockade with eculizumab prevents complement-mediated intravascular hemolysis in PNH patients and its clinical consequences. However, a distinct population of PNH red blood cells bound with C3 fragments appears in almost all treated patients. This C3 binding results in extravascular hemolysis that in some patients reduces the clinical benefit from eculizumab. In each PNH patients on eculizumab there are always two distinct populations of PNH red blood cells, one with (C3+) and one without (C3-) C3 binding. This phenomenon is somehow paradoxical since the glycosylphosphatidylinositol (GPI)-linked complement regulators, CD55 and CD59, are uniformly deficient on the surface of PNH red cells. To investigate this phenomenon, we have modeled in vitro the C3 binding in the context of C5 blockade by incubating red blood cells from PNH patients with AB0-matched sera from patients on eculizumab. Complement alternative pathway has been activated by mild acidification (in presence of Mg/EGTA to prevent the activation of complement classical pathway) and C3 binding has been assessed by flow cytometry at serial time points. In these experimental conditions a fraction of PNH red blood cells, similar to what happens in vivo, become promptly C3+ and its size increases with the time: from 9.4±2.7% after 5 minutes to 21.2±9.5% after 24 hours. The membrane defects of PNH cells suggested that the deficiency of CD55, which regulates the formation and accelerates the dissociation of C3 convertases, should be responsible for C3 binding to PNH red blood cells in presence of eculizumab (Parker CJ. Hematology Am Soc Hematol Educ Program. 2011;2011:21-29). In order to verify experimentally this hypothesis we have inactivated CD55 or CD59 on normal red blood cells by using blocking monoclonal antibodies (moAb - listed in the figure legend), and we have tested them in vitro upon activation of complement alternative pathway by mild acidification in presence or absence of C5 blockade. We found that CD55 inactivation on normal red blood cells results neither in hemolysis (without C5 blockade) nor in any C3 binding (with C5 blockade). As expected without C5 blockade CD59-inactivated normal red blood cells undergo hemolysis but, surprisingly, we found that in presence of C5 blockade they become bound with C3 fragments (Figure 1), just as it occurs in vivo in PNH patients on eculizumab. The simultaneous inactivation of both CD55 and CD59 further increased the level of C3 binding. Thus, at variance with the starting hypothesis, the deficiency of CD59, not that of CD55, plays the major role in C3 binding to PNH red cells of patients on eculizumab. Therapeutic C5 blockade in PNH patients has unmasked a novel function of CD59: in addition to prevent MAC formation, it plays a central role also in the regulation of C3 activation on cell surface through molecular mechanisms not elucidated yet. It remains to be established the physiological role, if any, of this novel function of CD59 and whether it play a role in determining the pleomorphic clinical features of the congenital CD59 deficiency. Finally, these findings may lead to investigate innovative approaches to reduce C3 binding and extravascular hemolysis in PNH patients on eculizumab and, in a broader context, to modulate complement activity. Figure 1 Figure 1. Disclosures Risitano: Novartis: Research Funding; Alexion Pharmaceuticals: Other: lecture fees, Research Funding; Rapharma: Research Funding; Alnylam: Research Funding.

Ultrastructural observations on the in vitro cytoadherence of Plasmodium falciparum infected red blood cells

1990 ◽  
Vol 48 (3) ◽  
pp. 914-915
Author(s):  
D.J.P. Ferguson ◽  
A.R. Berendt ◽  
J. Tansey ◽  
K. Marsh ◽  
C.I. Newbold
Keyword(s):  
Plasmodium Falciparum ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Umbilical Vein ◽  
Cell Types ◽  
Amelanotic Melanoma ◽  
Cytoplasmic Process ◽  
Umbilical Vein Endothelial Cells

In human malaria, the most serious clinical manifestation is cerebral malaria (CM) due to infection with Plasmodium falciparum. The pathology of CM is thought to relate to the fact that red blood cells containing mature forms of the parasite (PRBC) cytoadhere or sequester to post capillary venules of various tissues including the brain. This in vivo phenomenon has been studied in vitro by examining the cytoadherence of PRBCs to various cell types and purified proteins. To date, three Ijiost receptor molecules have been identified; CD36, ICAM-1 and thrombospondin. The specific changes in the PRBC membrane which mediate cytoadherence are less well understood, but they include the sub-membranous deposition of electron-dense material resulting in surface deformations called knobs. Knobs were thought to be essential for cytoadherence, lput recent work has shown that certain knob-negative (K-) lines can cytoadhere. In the present study, we have used electron microscopy to re-examine the interactions between K+ PRBCs and both C32 amelanotic melanoma cells and human umbilical vein endothelial cells (HUVEC).We confirm previous data demonstrating that C32 cells possess numerous microvilli which adhere to the PRBC, mainly via the knobs (Fig. 1). In contrast, the HUVEC were relatively smooth and the PRBCs appeared partially flattened onto the cell surface (Fig. 2). Furthermore, many of the PRBCs exhibited an invagination of the limiting membrane in the attachment zone, often containing a cytoplasmic process from the endothelial cell (Fig. 2).

scholarly journals Expression Profile of New Marker Genes Involved in Differentiation of Canine Adipose-Derived Stem Cells into Osteoblasts

2021 ◽  
Vol 22 (13) ◽  
pp. 6663
Author(s):  
Maurycy Jankowski ◽  
Mariusz Kaczmarek ◽  
Grzegorz Wąsiatycz ◽  
Claudia Dompe ◽  
Paul Mozdziak ◽  
...  
Keyword(s):  
Stem Cells ◽  
In Vitro Culture ◽  
Osteogenic Differentiation ◽  
Molecular Mechanisms ◽  
Marker Genes ◽  
P Value ◽  
Illumina Platform

Next-generation sequencing (RNAseq) analysis of gene expression changes during the long-term in vitro culture and osteogenic differentiation of ASCs remains to be important, as the analysis provides important clues toward employing stem cells as a therapeutic intervention. In this study, the cells were isolated from adipose tissue obtained during routine surgical procedures and subjected to 14-day in vitro culture and differentiation. The mRNA transcript levels were evaluated using the Illumina platform, resulting in the detection of 19,856 gene transcripts. The most differentially expressed genes (fold change >|2|, adjusted p value < 0.05), between day 1, day 14 and differentiated cell cultures were extracted and subjected to bioinformatical analysis based on the R programming language. The results of this study provide molecular insight into the processes that occur during long-term in vitro culture and osteogenic differentiation of ASCs, allowing the re-evaluation of the roles of some genes in MSC progression towards a range of lineages. The results improve the knowledge of the molecular mechanisms associated with long-term in vitro culture and differentiation of ASCs, as well as providing a point of reference for potential in vivo and clinical studies regarding these cells’ application in regenerative medicine.

scholarly journals Multimodal Diagnostics of Microrheologic Alterations in Blood of Coronary Heart Disease and Diabetic Patients

Diagnostics ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 76
Author(s):  
Anastasia Maslianitsyna ◽  
Petr Ermolinskiy ◽  
Andrei Lugovtsov ◽  
Alexandra Pigurenko ◽  
Maria Sasonko ◽  
...  
Keyword(s):  
Coronary Heart Disease ◽  
Heart Disease ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Optical Methods ◽  
Diabetic Patients ◽  
Aggregation Index ◽  
Significant Difference

Coronary heart disease (CHD) has serious implications for human health and needs to be diagnosed as early as possible. In this article in vivo and in vitro optical methods are used to study blood properties related to the aggregation of red blood cells in patients with CHD and comorbidities such as type 2 diabetes mellitus (T2DM). The results show not only a significant difference of the aggregation in patients compared to healthy people, but also a correspondence between in vivo and in vitro parameters. Red blood cells aggregate in CHD patients faster and more numerously; in particular the aggregation index increases by 20 ± 7%. The presence of T2DM also significantly elevates aggregation in CHD patients. This work demonstrates multimodal diagnostics and monitoring of patients with socially significant pathologies.

Clock gene-independent daily regulation of haemoglobin oxidation in red blood cells

2021 ◽  
Author(s):  
Andrew D. Beale ◽  
Priya Crosby ◽  
Utham K. Valekunja ◽  
Rachel S. Edgar ◽  
Johanna E. Chesham ◽  
...  
Keyword(s):  
Circadian Rhythms ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Human Subjects ◽  
Developmental Regulation ◽  
Physiological Role ◽  
Cell Types ◽  
The Body

AbstractCellular circadian rhythms confer daily temporal organisation upon behaviour and physiology that is fundamental to human health and disease. Rhythms are present in red blood cells (RBCs), the most abundant cell type in the body. Being naturally anucleate, RBC circadian rhythms share key elements of post-translational, but not transcriptional, regulation with other cell types. The physiological function and developmental regulation of RBC circadian rhythms is poorly understood, however, partly due to the small number of appropriate techniques available. Here, we extend the RBC circadian toolkit with a novel biochemical assay for haemoglobin oxidation status, termed “Bloody Blotting”. Our approach relies on a redox-sensitive covalent haem-haemoglobin linkage that forms during cell lysis. Formation of this linkage exhibits daily rhythms in vitro, which are unaffected by mutations that affect the timing of circadian rhythms in nucleated cells. In vivo, haemoglobin oxidation rhythms demonstrate daily variation in the oxygen-carrying and nitrite reductase capacity of the blood, and are seen in human subjects under controlled laboratory conditions as well as in freely-behaving humans. These results extend our molecular understanding of RBC circadian rhythms and suggest they serve an important physiological role in gas transport.

scholarly journals Antagonists of the system L neutral amino acid transporter (LAT) promote endothelial adhesivity of human red blood cells

2017 ◽  
Vol 117 (07) ◽  
pp. 1402-1411 ◽  
Author(s):  
Laura Beth Mann Dosier ◽  
Vikram J. Premkumar ◽  
Hongmei Zhu ◽  
Izzet Akosman ◽  
Michael F. Wempe ◽  
...  
Keyword(s):  
Amino Acid ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Amino Acid Transporter ◽  
Neutral Amino Acid ◽  
Neutral Amino Acid Transporter ◽  
System L

SummaryThe system L neutral amino acid transporter (LAT; LAT1, LAT2, LAT3, or LAT4) has multiple functions in human biology, including the cellular import of S-nitrosothiols (SNOs), biologically active derivatives of nitric oxide (NO). SNO formation by haemoglobin within red blood cells (RBC) has been studied, but the conduit whereby a SNO leaves the RBC remains unidentified. Here we hypothesised that SNO export by RBCs may also depend on LAT activity, and investigated the role of RBC LAT in modulating SNO-sensitive RBC-endothelial cell (EC) adhesion. We used multiple pharmacologic inhibitors of LAT in vitro and in vivo to test the role of LAT in SNO export from RBCs and in thereby modulating RBC-EC adhesion. Inhibition of human RBC LAT by type-1-specific or nonspecific LAT antagonists increased RBC-endothelial adhesivity in vitro, and LAT inhibitors tended to increase post-transfusion RBC sequestration in the lung and decreased oxygenation in vivo. A LAT1-specific inhibitor attenuated SNO export from RBCs, and we demonstrated LAT1 in RBC membranes and LAT1 mRNA in reticulocytes. The proadhesive effects of inhibiting LAT1 could be overcome by supplemental L-CSNO (S-nitroso-L-cysteine), but not D-CSNO or L-Cys, and suggest a basal anti-adhesive role for stereospecific intercellular SNO transport. This study reveals for the first time a novel role of LAT1 in the export of SNOs from RBCs to prevent their adhesion to ECs. The findings have implications for the mechanisms of intercellular SNO signalling, and for thrombosis, sickle cell disease, and post-storage RBC transfusion, when RBC adhesivity is increased.

scholarly journals Acetylsalicylic acid and morphology of red blood cells

2010 ◽  
Vol 53 (3) ◽  
pp. 575-582 ◽  
Author(s):  
Jacques Natan Grinapel Frydman ◽  
Adenilson de Souza da Fonseca ◽  
Vanessa Câmara da Rocha ◽  
Monica Oliveira Benarroz ◽  
Gabrielle de Souza Rocha ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Control Group ◽  
Blood Samples ◽  
Morphological Alterations ◽  
Blood Smears ◽  
In Vivo Treatment ◽  
Microscopy Data

This work evaluated the effect of in vitro and in vivo treatment with ASA on the morphology of the red blood cells. Blood samples or Wistar rats were treated with ASA for one hour. Blood samples or animals treated with saline were used as control group. Blood smears were prepared, fixed, stained and the qualitative and quantitative morphology of red blood cells were evaluated under optical microscopy. Data showed that the in vitro treatment for one hour with ASA at higher dose used significantly (p<0.05) modified the perimeter/area ratio of the red blood cells. No morphological alterations were obtained with the in vivo treatment. ASA use at highest doses could interfere on shape of red blood cells.

Decarboxylation of Radioactive DOPA by Erythrocytes in Schizophrenia

1971 ◽  
Vol 118 (545) ◽  
pp. 465-466 ◽  
Author(s):  
Ngo Tran ◽  
Marcel Laplante ◽  
Etienne Lebel
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Ionization Chamber ◽  
Present Experiment ◽  
Continuous Measurement ◽  
Human Tissues ◽  
Chamber Method ◽  
Human Red Blood Cells

The decarboxylation of 3, 4-dihydroxyphenyl-alanine (Dopa) to dopamine has been shown previously in animal and human tissues in both in vitro and in vivo studies (Sourkes, 1966; Vogel et al., 1970). However, very little information is available as to whether or not the decarboxylation of Dopa occurs in human red blood cells (RBC). In the present experiment we demonstrated this change in RBC from normals and from schizophrenics. An ionization chamber method was used for an instantaneous and continuous measurement of 14CO2 production from DL-dopa-carboxyl-14C by RBC in vitro.

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