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J.R. Cursino-Santos et al.
gen-carrying component and major protein of the RBC and is normally formed as a tetramer of two α-globins and two β-globins which constitute adult hemoglobin A (HbA). The major hemoglobinopathies result from muta- tions that either decrease the production of α- or β-glo- bins (in α- and β-thalassemia) or sickling of the erythro- cyte (in sickle HbS, HbC, and HbE diseases).8,9 Remarkably, small genetic variations confer dramatic lev- els of protection from malaria.10,11 HbS is the result of a single point mutation (Glu→Val) on the sixth codon of the β-globin gene. Homozygotes for hemoglobin S (HbSS) with two affected β chains develop sickle cell dis- ease (SCD), in which polymerized Hb causes RBC to sickle and occlude blood vessels, and results in high mor- bidity and mortality.12 Heterozygotes for sickle hemoglo- bin (HbAS) have sickle cell trait and are generally asymp- tomatic. Despite the obvious deleterious nature of HbSS, it is now widely accepted that the persistence of the sick- le mutation in human populations is due to the protec- tion from malaria afforded to heterozygous individu- als.13,14 Multiple divergent mechanisms have been put for- ward to explain this resistance to malaria, including enhanced macrophage uptake, impaired growth and maturation of parasite, and decreased deposition of par- asitized RBC in deep post capillary beds, but no single convincing explanation has yet been given.1,15,16
Babesiosis has long been recognized as a veterinary problem of great significance, but only in the last 50 years has it been recognized as an important pathogen in man.2 The four identified Babesia species that have so far been definitively confirmed to infect humans are B. microti,17 B. divergens,18 B. duncani,19,20 and B. venatorum.21-24 As sampling has become expansive and techniques have become more sensitive, there is evidence that more B. microti-like and B. divergens-like spp. are able to cause human infection (as reported in detail by Yabsley and Shock).25 However, the general life cycle within humans remains the same. Babesia parasites are intracellular obli- gates that target RBC, and the parasite's ability to first recognize and then invade host RBC is central to the dis- ease pathology. Besides its natural route of transmission via the infected tick, the parasite is also transmitted by transfusion of infected blood as its RBC host provides an optimum vehicle to facilitate its transmission. In fact, as the frequency of clinical cases has risen, there has been an associated increase in transfusion-transmitted Babesia (TTB), mainly reported for B. microti,26-28 making babesio- sis the most frequent transfusion-transmitted infection in the US. Patients with sickle cell anemia, especially those on chronic transfusion therapy, are at high risk for severe TTB.29,30 Whether the sickle red cells themselves are responsible for the increased susceptibility of these patients to TTB or whether this is due to other related factors, such as a compromised immune system, has not been investigated. In this paper, we focus on the ability of the Babesia parasite to invade, grow in and egress from sickle trait and sickle cell anemia erythrocytes. Use of in vitro invasion and development assays were developed in our laboratory,31 as our primary outcome provided a rare opportunity to systematically examine the cellular deter- minants of parasite development in the sickle cell anemia setting. These enabled a comparison between various components of the parasite life-cycle in RBC obtained from various hemoglobin genotypes, HbAA, HbAS and HbSS, and revealed altered parasite population progres-
sion, parasite maturation and egress phenotypes in the HbSS cells.
Methods
Ethics statement
Human blood from healthy volunteer donors was used to cul- ture B. divergens (Bd) in vitro. SCD patients' RBC were obtained from residual anticoagulated blood samples from same day collec- tions from patients with sickle cell anemia (hemoglobin genotype SS) who had not been transfused for at least three months prior to sample draw. Patients provided consent for use of de-identified blood for research purposes on a Montefiore Medical Center Institutional Review Board (IRB) approved protocol. HbAA RBC and sickle trait RBC were identified from New York Blood Center (NYBC) blood donors and confirmed through genotypic analysis. All blood was used within a few hours of drawing. The blood was de-identified and approved for use by the NYBC IRB. All blood donors gave informed written consent for use of their blood for research purposes.
B. divergens in vitro culture
B. divergens (Bd Rouen 1986 strain) were maintained in human
RBC as previously described.32,33 A+ RBC were collected in 10% CPD and washed 3X with RPMI 1640 medium for the complete plasma and white cell removal.
Free merozoites isolation
High concentration of viable free merozoites was isolated from unsynchronized cultures at high parasitemia (40%), as described previously.31,34
Assessment of invasion, development and egress in various red blood cells
Fresh cultures were seeded with purified merozoite suspension at 20% (v/v) of culture volume. To define time points to accurately estimate invasion in the different RBC (HbAA, HbAS; HbSS), invasion was assayed in the first set of samples at 5 minutes (min), 1 hour (h) or 6 h post invasion. At additional time points (24-72 h), samples were collected to assess the culture progression and sub- population dynamics from the perspective of parasite develop- ment and egress. Analysis was carried out at specific intervals of 24 h, 48 h and 72 h for the majority of cultures (6 cultures were monitored for 48 h). The culture size (parasitemia) and the para- site proliferation analysis were carried out by FACS (described below). Characterization of parasite morphology and develop- ment was performed by light microscopy. Cells were obtained from three replicate cultures for each RBC sample.
Light microscopy
Blood smears were fixed with methanol and stained with 20% Giemsa (Sigma-Aldrich, St. Louis, MO, USA) for the morphologi- cal analysis of parasites. A minimum of 2000 cells was scanned for assessment of changes in morphology using a Nikon Eclipse E 600 microscope.
Flow cytometry
The dual-color staining protocol was used to monitor the para- site cycle within the RBC over 72 h, as previously described31 with modifications.
Statistical analysis
Parasitemia was defined as the total number of infected RBC (iRBC) in every 100 RBC, not taking into consideration the num- ber of parasites seen in a given cell when measured by flow
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haematologica | 2019; 104(11)