Functional and Immunologic Mapping of Domains of the Reticulocyte-Binding Protein Plasmodium vivax PvRBP2a.

We previously described a novel Plasmodium vivax invasion mechanism into human reticulocytes via the PvRBP2a-CD98 receptor-ligand pair. Using linear epitope mapping, we assessed the PvRBP2a epitopes involved in CD98 binding and recognized by antibodies from patients who were infected. We identified 2 epitope clusters mediating PvRBP2a-CD98 interaction. Cluster B (PvRBP2a431-448, TAALKEKGKLLANLYNKL) was the target of antibody responses in humans infected by P vivax. Peptides from each cluster were able to prevent live parasite invasion of human reticulocytes. These results provide new insights for development of a malaria blood-stage vaccine against P vivax.

The contribution of host cell-directed vs. parasite-directed immunity to the disease and dynamics of malaria infections.

Hosts defend themselves against pathogens by mounting an immune response. Fully understanding the immune response as a driver of host disease and pathogen evolution requires a quantitative account of its impact on parasite population dynamics. Here, we use a data-driven modeling approach to quantify the birth and death processes underlying the dynamics of infections of the rodent malaria parasite, Plasmodium chabaudi, and the red blood cells (RBCs) it targets. We decompose the immune response into 3 components, each with a distinct effect on parasite and RBC vital rates, and quantify the relative contribution of each component to host disease and parasite density. Our analysis suggests that these components are deployed in a coordinated fashion to realize distinct resource-directed defense strategies that complement the killing of parasitized cells. Early in the infection, the host deploys a strategy reminiscent of siege and scorched-earth tactics, in which it both destroys RBCs and restricts their supply. Late in the infection, a "juvenilization" strategy, in which turnover of RBCs is accelerated, allows the host to recover from anemia while holding parasite proliferation at bay. By quantifying the impact of immunity on both parasite fitness and host disease, we reveal that phenomena often interpreted as immunopathology may in fact be beneficial to the host. Finally, we show that, across mice, the components of the host response are consistently related to each other, even when infections take qualitatively different trajectories. This suggests the existence of simple rules that govern the immune system's deployment.

Neonatal CD71+ Erythroid Cells Do Not Modify Murine Sepsis Mortality.

Sepsis is a major cause of neonatal mortality and morbidity worldwide. A recent report suggested that murine neonatal host defense against infection could be compromised by immunosuppressive CD71(+) erythroid splenocytes. We examined the impact of CD71(+) erythroid splenocytes on murine neonatal mortality to endotoxin challenge or polymicrobial sepsis and characterized circulating CD71(+) erythroid (CD235a(+)) cells in human neonates. Adoptive transfer or an Ab-mediated reduction in neonatal CD71(+) erythroid splenocytes did not alter murine neonatal survival to endotoxin challenge or polymicrobial sepsis challenge. Ex vivo immunosuppression of stimulated adult CD11b(+) cells was not limited to neonatal splenocytes; it also occurred with adult and neonatal bone marrow. Animals treated with anti-CD71 Ab showed reduced splenic bacterial load following bacterial challenge compared with isotype-treated mice. However, adoptive transfer of enriched CD71(+) erythroid splenocytes to CD71(+)-reduced animals did not reduce bacterial clearance. Human CD71(+)CD235a(+) cells were common among cord blood mononuclear cells and were shown to be reticulocytes. In summary, a lack of effect on murine survival to polymicrobial sepsis following adoptive transfer or diminution of CD71(+) erythroid splenocytes under these experimental conditions suggests that the impact of these cells on neonatal infection risk and progression may be limited. An unanticipated immune priming effect of anti-CD71 Ab treatment, rather than a reduction in immunosuppressive CD71(+) erythroid splenocytes, was likely responsible for the reported enhanced bacterial clearance. In humans, the well-described rapid decrease in circulating reticulocytes after birth suggests that they may have a limited role in reducing inflammation secondary to microbial colonization.

Characterization of surface antigens of reticulated immature platelets.

Reticulated platelets (RPs) are immature platelets with high dense granules content and a residual amount of megakaryocyte-derived of mRNA. Increased level of RPs has been found to be an independent predictor of cardiovascular ischemic events, and has been associated with impaired response to various anti-platelet drugs. The study aimed to characterize and compare the surface antigenic properties of reticulated versus mature platelets. Platelets from healthy individuals and diabetic patients were tested at rest and after activation with adenosine diphosphate (ADP). For each patient, we calculated the proportion of RPs and mature platelets using flow cytometry analysis with thiazole orange staining (for RPs) and CD42b platelet-specific antibody. We also tested the surface expression of P-selectin and Annexin V, by double staining flow cytometry in RPs versus mature platelets. A total of 20 subjects were recruited (10 healthy individuals, 10 diabetics). Activation with ADP did not cause a significant change in the proportion of RPs. Following activation, RPs demonstrated a significant increase in the expression of both P-selectin and Annexin V, while mature platelets exhibited a non-significant increase in both markers. These findings were consistent in both healthy subjects and patients with diabetes. In conclusion, RPs have a significantly higher capacity to increase the expression of platelet activation markers compared with mature platelets.

Gene Models, Expression Repertoire, and Immune Response of Plasmodium vivax Reticulocyte Binding Proteins.

Members of the Plasmodium vivax reticulocyte binding protein (PvRBP) family are believed to mediate specific invasion of reticulocytes by P. vivax. In this study, we performed molecular characterization of genes encoding members of this protein family. Through cDNA sequencing, we constructed full-length gene models and verified genes that are protein coding and those that are pseudogenes. We also used quantitative PCR to measure their in vivo transcript abundances in clinical P. vivax isolates. Like genes encoding related invasion ligands of P. falciparum, Pvrbp expression levels vary broadly across different parasite isolates. Through antibody measurements, we found that host immune pressure may be the driving force behind the distinctly high diversity of one of the family members, PvRBP2c. Mild yet significant negative correlation was found between parasitemia and the PvRBP2b antibody level, suggesting that antibodies to the protein may interfere with invasion.

Naturally acquired antibodies specific for Plasmodium falciparum reticulocyte-binding protein homologue 5 inhibit parasite growth and predict protection from malaria.

BACKGROUND: Plasmodium falciparum reticulocyte-binding protein homologue 5 (PfRH5) is a blood-stage parasite protein essential for host erythrocyte invasion. PfRH5-specific antibodies raised in animals inhibit parasite growth in vitro, but the relevance of naturally acquired PfRH5-specific antibodies in humans is unclear. METHODS: We assessed pre-malaria season PfRH5-specific immunoglobulin G (IgG) levels in 357 Malian children and adults who were uninfected with Plasmodium. Subsequent P. falciparum infections were detected by polymerase chain reaction every 2 weeks and malaria episodes by weekly physical examination and self-referral for 7 months. The primary outcome was time between the first P. falciparum infection and the first febrile malaria episode. PfRH5-specific IgG was assayed for parasite growth-inhibitory activity. RESULTS: The presence of PfRH5-specific IgG at enrollment was associated with a longer time between the first blood-stage infection and the first malaria episode (PfRH5-seropositive median: 71 days, PfRH5-seronegative median: 18 days; P = .001). This association remained significant after adjustment for age and other factors associated with malaria risk/exposure (hazard ratio, .62; P = .02). Concentrated PfRH5-specific IgG purified from Malians inhibited P. falciparum growth in vitro. CONCLUSIONS: Naturally acquired PfRH5-specific IgG inhibits parasite growth in vitro and predicts protection from malaria. These findings strongly support efforts to develop PfRH5 as an urgently needed blood-stage malaria vaccine. CLINICAL TRIALS REGISTRATION: NCT01322581.

Reticulocyte-prone malaria parasites predominantly invade CD71hi immature cells: implications for the development of an in vitro culture for Plasmodium vivax.

BACKGROUND: The lack of a continuous in vitro culture system for blood stages of malarial parasites with a unique tropism for reticulocytes, such as Plasmodium vivax and the Plasmodium yoelii 17X reticulocyte-prone strain, hinders research in these organisms. The maturation of reticulocytes into erythrocytes is a complex process involving the selective removal of membrane proteins such as the transferrin receptor, CD71. In order to advance in the characterization of infected cells during experimental infections of BALB/c mice with P. yoelii 17X, CD71 expression in erythroid cells (TER119+) was assessed and in vitro culture of P. yoelii 17X was attempted by adding reticulocytes highly expressing CD71. METHODS: BALB/c mice were infected with P. yoelii 17X-GFP transgenic parasites and erythroid cells (TER119+) were analysed in blood, spleen and bone marrow cells. TER119, CD71 and GFP expression was assessed at different points post-infection by flow cytometry. Moreover, in vitro culture of P. yoelli 17X was attempted by adding red blood cells (RBCs) from mice with a pyruvate kinase deficiency, which contain high percentages of CD71hi cells in peripheral blood as compared to healthy animals. RESULTS: A predominance of erythroid cells lacking expression of CD71 (CD71-) was observed in peripheral blood and spleen in normal and infected animals up to ten days post-infection (pi). At ten days pi, however, a dramatic temporal switch to erythroid cells highly expressing CD71 (CD71hi) was observed in the spleen and at day 15 pi in peripheral blood of the infected cells. A distribution of erythroid cells expressing differently CD71 was noticed in the bone marrow. Yet, similar to peripheral blood and spleen, a predominance of CD71hi cells was observed at 15 days pi. Remarkably, CD71hi cells were the cells predominantly infected in these organs as well as in peripheral blood. Attempts were thus made to culture in vitro the P. yoelli 17X strain by adding RBCs from pyruvate kinase-deficient mice containing high percentages of CD71hi cells in peripheral blood. CONCLUSIONS: The parasite preference for immature cells that are rare in normal peripheral blood could have important implications for the development of an in vitro culture system for P. vivax.

Interlaboratory trial of the rat Pig-a mutation assay using an erythroid marker HIS49 antibody.

The peripheral blood Pig-a assay has shown promise as a tool for evaluating in vivo mutagenicity. In this study five laboratories participated in a collaborative trial that evaluated the transferability and reproducibility of a rat Pig-a assay that uses a HIS49 antibody reacts with an antigen found on erythrocytes and erythroid progenitors. In preliminary work, flow cytometry methods were established that enabled all laboratories to detect CD59-negative erythrocyte frequencies (Pig-a mutant frequencies) of <10×10(-6) in control rats. Four of the laboratories (the in-life labs) then treated male rats with a single oral dose of N-nitroso-N-ethylurea, 7,12-dimethylbenz[a]anthracene (DMBA), or 4-nitroquinoline-1-oxide (4NQO). Blood samples were collected up to 4 weeks after the treatments and analyzed by flow cytometry for the frequency of CD59-negative cells among total red blood cells (RBCs; RBC Pig-a assay). RBC Pig-a assays were conducted in the four in-life laboratories, plus a fifth laboratory that received blood samples from the other laboratories. In addition, three of the five laboratories performed a Pig-a assay on reticulocytes (RETs; PIGRET assay), using blood from the rats treated with DMBA and 4NQO. The four in-life laboratories detected consistent, time- and dose-related increases in RBC Pig-a mutant frequency (MF) for all three test articles. Furthermore, comparable results were obtained in the fifth laboratory that received blood samples from other laboratories. The three laboratories conducting the PIGRET assay also detected consistent, time- and dose-related increases in Pig-a MF, with the RET MFs increasing more rapidly with time than RBC MFs. These results indicate that rat Pig-a assays using a HIS49 antibody were transferable between laboratories and that data generated by the assays were reproducible. The findings also suggest that the PIGRET assay may detect the in vivo mutagenicity of test compounds earlier than the RBC Pig-a assay.

Innate immune activation during Salmonella infection initiates extramedullary erythropoiesis and splenomegaly.

Systemic Salmonella infection commonly induces prolonged splenomegaly in murine or human hosts. Although this increase in splenic cellularity is often assumed to be due to the recruitment and expansion of leukocytes, the actual cause of splenomegaly remains unclear. We monitored spleen cell populations during Salmonella infection and found that the most prominent increase is found in the erythroid compartment. At the peak of infection, the majority of spleen cells are immature CD71(-)Ter119(+) reticulocytes, indicating that massive erythropoiesis occurs in response to Salmonella infection. Indeed, this increase in RBC precursors corresponded with marked elevation of serum erythropoietin (EPO). Furthermore, the increase in RBC precursors and EPO production required innate immune signaling mediated by Myd88/TRIF. Neutralization of EPO substantially reduced the immature RBC population in the spleen and allowed a modest increase in host control of infection. These data indicate that early innate immunity to Salmonella initiates marked splenic erythropoiesis and may hinder bacterial clearance.

Reticulocyte and erythrocyte binding-like proteins function cooperatively in invasion of human erythrocytes by malaria parasites.

Plasmodium falciparum causes the most severe form of malaria in humans and invades erythrocytes using multiple ligand-receptor interactions. Two important protein families involved in erythrocyte binding are the erythrocyte binding-like (EBL) and the reticulocyte binding-like (RBL or P. falciparum Rh [PfRh]) proteins. We constructed P. falciparum lines lacking expression of EBL proteins by creating single and double knockouts of the corresponding genes for eba-175, eba-181, and eba-140 and show that the EBL and PfRh proteins function cooperatively, consistent with them playing a similar role in merozoite invasion. We provide evidence that PfRh and EBL proteins functionally interact, as loss of function of EBA-181 ablates the ability of PfRh2a/b protein antibodies to inhibit merozoite invasion. Additionally, loss of function of some ebl genes results in selection for increased transcription of the PfRh family. This provides a rational basis for considering PfRh and EBL proteins for use as a combination vaccine against P. falciparum. We immunized rabbits with combinations of PfRh and EBL proteins to test the ability of antibodies to block merozoite invasion in growth inhibition assays. A combination of EBA-175, PfRh2a/b, and PfRh4 recombinant proteins induced antibodies that potently blocked merozoite invasion. This validates the use of a combination of these ligands as a potential vaccine that would have broad activity against P. falciparum.

Antigenic structure of cell surfaces. An immunoferritin study of the occurrence and topography of H-2' theta, and TL alloantigens on mouse cells.

The representation of mouse alloantigens belonging to three systems, H-2, theta and TL, on the surface of cells from thymus, spleen, lymph nodes, and peritoneal cavity, was studied by electron microscopy with ferritin-labeled antibody. As expected from earlier serological data, TL was confined to thymocytes, theta was found on thymocytes and lymphocytes, and H-2 occurred to some extent on all cell types observed. On reticular cells, lymphocytes, plasma cells, and eosinophils, the majority of the cell surface was occupied by H-2; thymocytes had considerably less H-2, and erythrocytes and peritoneal macrophages least of all. In every instance the representation of antigen was discontinuous, the fraction of the cell surface covered being characteristic both of the antigen and of the type of cell. H-2 and theta provide a striking example of this; H-2 is present in far higher amounts on lymphocytes than on thymocytes, whereas the converse is true of theta. Within areas positive for H-2 or theta, protuberances of the surface membrane were often antigen-negative. A better definition of cell surface structure, gained from studies such as this, is necessary for further inquiry into how the cell surface is assembled, and into selective gene action in relation to cellular differentiation.

Plasmodium vivax in Hematopoietic Niches: Hidden and Dangerous.

Estimation of Plasmodium vivax biomass based on circulating biomarkers indicates the existence of a predominant biomass outside of the circulation that is not captured by peripheral parasitemia, in particular in patients with complicated outcomes. A series of recent studies have suggested that the hematopoietic niche of the bone marrow (BM) is a major reservoir for parasite replication and the development of transmission stages. However, significant knowledge gaps remain in our understanding of host-parasite interactions, pathophysiology, and the implications for treatment and diagnosis of such a reservoir. Here, we discuss the current status of this emerging research field in the context of P. vivax.

Sickle Cell Anemia Patients Display an Intricate Cellular and Serum Biomarker Network Highlighted by TCD4+CD69+ Lymphocytes, IL-17/MIP-1ß, IL-12/VEGF, and IL-10/IP-10 Axis.

BACKGROUND: Sickle cell anemia (SCA) is associated with a chronic proinflammatory state characterized by elevated leukocyte count, mortality from severe recurrent infections, and subsequent vasoocclusive complications with leukocyte adhesion to the endothelium and increased plasma levels of inflammatory cytokines. The immune system has a close connection with morbidity in SCA, but further studies are needed to uncover the involvement of innate and adaptive immunities in modulating the SCA physiopathology. We performed measurements of the frequency of innate and adaptive immunity cells, cytokines, chemokines, and growth factors and immunophenotyping of Toll-like receptor and adhesion molecule expression in the blood of SCA patients and healthy donors to evaluate the different profiles of these biomarkers, the relationship among them, and their correlation to laboratory records and death risk. Material and Methods. Immunophenotyping of cells, Toll-like receptors, and adhesion molecules were performed from peripheral blood samples of SCA patients and healthy donors by flow cytometry and cytokine/chemokine/growth factor measurement by the Luminex technique performed from the serum of the same subjects. RESULTS: Cells of adaptive immunity such as IL-12, IL-17, and IL-10 cytokines; IL-8, IP-10, MIP-1α, MIP-1ß, and RANTES chemokines; and VEGF, FGF-basic, and GM-CSF growth factors were higher in SCA patients than healthy donors regardless of any laboratorial and clinical condition. However, high death risk appears to have relevant biomarkers. CONCLUSION: In the SCA pathophysiology at steady state, there is a broad immunological biomarker crosstalk highlighted by TCD4+CD69+ lymphocytes, IL-12 and IL-17 inflammatory and IL-10 regulatory cytokines, MIP-1α, MIP-1ß, and IP-10 chemokines, and VEGF growth factor. High expression of TLR2 in monocytes and VLA-4 in TCD8+ lymphocytes and high levels of MIP-1ß and RANTES appear to be relevant in high death risk conditions. The high reticulocytosis and high death risk conditions present common correlations, and there seems to be a balance by the Th2 profile.

Scald Injury-Induced T Cell Dysfunction Can Be Mitigated by Gr1+ Cell Depletion and Blockage of CD47/CD172a Signaling.

Infection is a common and severe complication of burn injury: Sepsis accounts for 47% of postburn mortality. Burn-induced T cell suppression likely contributes to the increased infection susceptibility in burn patients. However, little is known about the kinetics of T cell dysfunction after burn and its underlying mechanisms. In this study, we show in a murine scald injury model that T cell activation of both CD4+ and CD8+ T cells as well as T cell cytokine production is suppressed acutely and persistently for at least 11 days after burn injury. Purified T cells from scald-injured mice exhibit normal T cell functions, indicating an extrinsically mediated defect. We further show that T cell dysfunction after burn appears to be cell-to-cell contact dependent and can be ameliorated by depletion of myeloid-derived suppressor cells. These cells expand after burn injury, particularly a subset expressing the checkpoint inhibitor CD172a, and infiltrate germinal centers. Expression of CD172a appears to be driven by ingestion of immature reticulocytes. Immature reticulocytes are drastically increased in the spleen of scald mice and may contribute to immunosuppression through more direct mechanisms as well. Overall, our study newly identifies two cell populations, myeloid-derived suppressor cells and immature reticulocytes, as well as the CD47/CD172a-signaling pathways as mediators of T cell suppressors after burn and thus opens up new research opportunities in the search for new therapies to combat increased infection susceptibility and the associated morbidity and mortality in burn victims.

Autosensitization of lymphocytes against thymus reticulum cells.

Lymph nodes of normal rats contain lymphocytes that can be induced in vitro to mediate a specific cellular immune reaction against reticulum cells derived from syngeneic adult thymus glands. It is likely that these lymphocytes had access to reticulum cell antigens during the period in which they developed immunocompetence within the thymus. This suggests that contact with self-antigens during development may not eliminate self-reactive lymphocytes. These findings are in conflict with the theory that natural tolerance to self-antigens depends upon elimination of lymphocyte clones.

Southeast Asian ovalocytosis is associated with increased expression of Duffy antigen receptor for chemokines (DARC).

The Duffy antigen receptor for chemokines (DARC or Fy glycoprotein) carries antigens that are important in blood transfusion and is the main receptor used by Plasmodium vivax to invade reticulocytes. Southeast Asian ovalocytosis (SAO) results from an alteration in RBC membrane protein band 3 and is thought to mitigate susceptibility to falciparum malaria. Expression of some RBC antigens is suppressed by SAO, and we hypothesized that SAO may also reduce Fy expression, potentiallyleading to reduced susceptibility to vivax malaria. Blood samples were collected from individuals living in the Madang Province of Papua New Guinea. Samples were assayed using a flow cytometry assay for expression of Fy on the surface of RBC and reticulocytes by measuring the attachment of a phycoerythrin-labeled Fy6 antibody. Reticulocytes were detected using thiazole orange. The presence of the SAO mutation was confirmed by PCR. There was a small (approximately 10%) but statistically significant (p=0.049, Mann-Whitney U test) increase in Fy expression on SAO RBC compared with RBC from individuals without this polymorphism: mean Fy expression (mean fluorescence intensity [MFI]) was 10.12 +/- 1.22 for SAO heterozygotes versus an MFI of 8.95 +/- 1.1 for individuals without SAO. For reticulocytes the MFI values were 27.61 +/- 19.12 for SAO heterozygotes and 16.47 +/- 3.81 for controls. SAO is associated with increased and not decreased Fy6 expression so that susceptibility to P. vivax infection is unlikely to be affected.

Autoimmune hemolytic anemia in HIV-infected patients: a hospital based study.

BACKGROUND: The prevalence of anemia in HIV/AIDS patients is high, with a multitude of possible etiologies; autoimmune hemolytic anemia (AIHA) in HIV/AIDS patients has been associated with a poor prognosis when treated with red cell transfusion. Our aim was to demonstrate the frequency of AIHA in a cohort of adult Nigerian HIV/AIDS patients and to see if the presence or not of AIHA is related to the severity of the disease with regards to the CD4 counts and the presence or absence of opportunistic infections. METHOD: Ninety- eight adult patients with HIV infection were screened for the presence of AIHA using the packed cell volume (PCV), direct antiglobulin test (DAT) and reticulocyte count (RC). RESULTS: The frequency of AIHA was 3.06%, 36.74% of our study population were anemic; 11.22% had a positive DAT. Mean RC was 2.22 +/- 0.90 for all the patients. There was no statistically significant difference in the PCV of patients that had positive and negative DAT. There was no correlation between the presence of AIHA, use of ART, presence of opportunistic infections or CD4 counts. CONCLUSION: We conclude that in spite of the low frequency of AIHA in HIV/AIDS patients, the fact that most patients will respond to standard treatment makes it imperative to screen HIV/AIDS patients with anemia for the presence of AIHA. Again since HIV/A IDS patients with AIHA may have a fatal reaction to red cell transfusion, we suggest that anemic patients with HIV/AIDS in non -emergency situations be screened for the presence of AIHA before receiving red cell transfusions when indicated.

Targeting a Reticulocyte Binding Protein and Duffy Binding Protein to Inhibit Reticulocyte Invasion by Plasmodium vivax.

Plasmodium vivax merozoite invasion is restricted to Duffy positive reticulocytes. Merozoite interaction with the Duffy antigen is mediated by the P. vivax Duffy binding protein (PvDBP). The receptor-binding domain of PvDBP maps to an N-terminal cysteine-rich region referred to as region II (PvDBPII). In addition, a family of P. vivax reticulocyte binding proteins (PvRBPs) mediates interactions with reticulocyte receptors. The receptor binding domain of P. vivax reticulocyte binding protein 1a (PvRBP1a) maps to a 30 kD region (PvRBP1a30). Antibodies raised against recombinant PvRBP1a30 and PvDBPII recognize the native P. vivax antigens and inhibit their binding to host receptors. Rabbit IgG purified from sera raised against PvRBP1a30 and PvDBPII were tested individually and in combination for inhibition of reticulocyte invasion by P. vivax field isolates. While anti-PvDBPII rabbit IgG inhibits invasion, anti-PvRBP1a30 rabbit IgG does not show significant invasion inhibitory activity. Combining antibodies against PvDBPII and PvRBP1a30 also does not increase invasion inhibitory activity. These studies suggest that although PvRBP1a mediates reticulocyte invasion by P. vivax merozoites, it may not be useful to include PvRBP1a30 in a blood stage vaccine for P. vivax malaria. In contrast, these studies validate PvDBPII as a promising blood stage vaccine candidate for P. vivax malaria.

A novel flow cytometric phagocytosis assay of malaria-infected erythrocytes.

Monocytes play a crucial role in controlling malaria infection. To facilitate our research into the development of antibody-mediated immunity against pregnancy-associated malaria we have established several novel malaria-specific flow cytometric phagocytosis assays based on ethidium bromide staining of DNA present in blood stage trophozoites. The first assay quantifies the ability of sera to opsonise trophozoites and promotes phagocytosis by the monocytic cell line THP1. This measures the levels of functional antibodies to the chosen strain of parasite. The second assay is a whole blood phagocytosis assay which measures the phagocytic ability of patient monocytes ex vivo. The third assay employs simultaneous labelling of trophozoites with ethidium bromide and erythrocytes with fluorescein isothiocyanate to compare phagocytosis of both non-infected and parasitised erythrocytes to assess possible bystander effects on uninfected erythrocytes. These assays have the advantage over other malaria phagocytosis assays in that they are rapid, simple and specific to malaria-infected cells and avoid potential bias associated with manual counting.

Suppressive effect of culture supernatant of erythrocytes and serum from dogs infected with Babesia gibsoni on the morphological maturation of canine reticulocytes in vitro.

The present study evaluated the effects of infected culture supernatant of erythrocytes, fractionation of culture supernatant and serum from dogs infected with Babesia gibsoni (B. gibsoni) on the maturation of canine reticulocytes in vitro. The SDS-PAGE demonstrated that significantly broader bands were generated by both the infected culture supernatant of erythrocytes and the serum from dogs chronically infected with B. gibsoni. The culture supernatant of erythrocytes infected with B. gibsoni strongly suppressed the maturation of reticulocytes. Prior studies showed that chronically infected serum had inhibitory effects on both the maturation of reticulocytes and the canine pyrimidine 5'-nucleotidase subclass I and purine-specific 5'-nucleotidase activity. In addition, serum free infected culture supernatant of erythrocytes had an inhibitory effect on the morphological maturation of reticulocytes. These results suggest that infected serum and culture supernatant of erythrocytes might accumulate excess proteins and/or metabolites as a result of the inhibited maturation of reticulocytes and decreased activity of erythrocyte 5'-nucleotidase. Furthermore, the fractions observed at >150 kDa- and 150-70 kDa- in the infected culture supernatant and serum retarded the maturation of canine reticulocytes in vitro. The results obtained from the in vitro examinations, in the present study, suggested that B. gibsoni itself and/or its metabolites might release certain proteins in the infected culture supernatant and serum from infected dogs and as a result delay morphological maturation of canine reticulocytes.