Role In Pathogenesis Of Malaria Research Articles

Solution-State Structure of a Long-Loop G-Quadruplex Formed Within Promoters of Plasmodium falciparum B var Genes.

We report the high-resolution NMR solution-state structure of an intramolecular G-quadruplex with a diagonal loop of ten nucleotides. The G-quadruplex is formed by a 34-nt DNA sequence, d[CAG3T2A2G3TATA2CT3AG4T2AG3T2], named UpsB-Q-1. This sequence is found within promoters of the var genes of Plasmodium falciparum, which play a key role in malaria pathogenesis and evasion of the immune system. The [3+1]-hybrid G-quadruplex formed under physiologically relevant conditions exhibits a unique equilibrium between two structures, both stabilized by base stacking and non-canonical hydrogen bonding. Unique equilibrium of the two closely related 3D structures originates from a North-South repuckering of deoxyribose moiety of residue T27 in the lateral loop. Besides the 12 guanines involved in three G-quartets, most residues in loop regions are involved in interactions at both G-quartet-loop interfaces.

Immunofluorescence study of cytoskeleton in endothelial cells induced with malaria sera

BackgroundEndothelial cells (ECs) play a major role in malaria pathogenesis, as a point of direct contact of parasitized red blood cells to the blood vessel wall. The study of cytoskeleton structures of ECs, whose main functions are to maintain shape and provide strength to the EC membrane is important in determining the severe sequelae of Plasmodium falciparum malaria. The work investigated the cytoskeletal changes (microfilaments-actin, microtubules-tubulin and intermediate filaments-vimentin) in ECs induced by malaria sera (Plasmodium vivax, uncomplicated P. falciparum and complicated P. falciparum), in relation to the levels of pro-inflammatory cytokines.MethodsMorphology and fluorescence intensity of EC cytoskeleton stimulated with malaria sera were evaluated using immunofluorescence technique. Levels of tumour necrosis factor (TNF) and interferon (IFN)-gamma (γ) were determined using enzyme-linked immunosorbent assay (ELISA). Control experimental groups included ECs incubated with media alone and non-malaria patient sera. Experimental groups consisted of ECs incubated with malaria sera from P. vivax, uncomplicated P. falciparum and complicated P. falciparum. Morphological scores of cytoskeletal alterations and fluorescence intensity were compared across each experiment group, and correlated with TNF and IFN-γ.ResultsThe four morphological changes of cytoskeleton included (1) shrinkage of cytoskeleton and ECs with cortical condensation, (2) appearance of eccentric nuclei, (3) presence of “spiking pattern” of cytoskeleton and EC membrane, and (4) fragmentation and discontinuity of cytoskeleton and ECs. Significant damages were noted in actin filaments compared to tubulin and vimentin filaments in ECs stimulated with sera from complicated P. falciparum malaria. Morphological damages to cytoskeleton was positively correlated with fluorescence intensity and the levels of TNF and IFN-γ.ConclusionsECs stimulated with sera from complicated P. falciparum malaria showed cytoskeletal alterations and increased in fluorescence intensity, which was associated with high levels of TNF and IFN-γ. Cytoskeletal changes of ECs incubated with complicated P. falciparum malaria sera can lead to EC junctional alteration and permeability changes, which is mediated through apoptotic pathway. The findings can serve as a basis to explore measures to strengthen EC cytoskeleton and alleviate severe malaria complications such as pulmonary oedema and cerebral malaria. In addition, immunofluorescence intensity of cytoskeleton could be investigated as potential prognostic indicator for malaria severity.

Plasmodium Falciparum Rifins: Role in malaria pathogenesis

Malaria kills an estimated 600,000 people each year, especially children under five years who reside in sub-Saharan Africa. Malaria fatalities are associated with severe forms such as cerebral malaria, acute respiratory failure, severe anaemia, renal failure, hypoglycaemia, and pulmonary oedema. Although the underlying pathogenic mechanisms in immune responses and parasite immune evasion, cytoadherence of parasitized red blood cells, and rosetting are enumerated, the mechanisms are not fully understood. P. falciparum parasite-derived surface protein, repetitive interspersed family (RIFIN) genes are involved in rosetting, blocking microcirculation, and playing a role in malaria pathogenesis; it is unclear which RIFIN family genes are involved in the various pathogenic mechanisms in malaria. RIFINs are the extensive malaria family genes expressed throughout the malaria parasite stages, indicating their diverse roles. Malaria pathogenesis occurs in erythrocyte-stage infection, and the expression of RIFINs at this phase could play a diverse role in the various pathogenic mechanisms. They are involved in major phenomena such as cytoadherence, merozoite evasion, and immune evasion. RIFINs aid in the immune evasion of P. falciparum through various molecular interactions by binding to the inhibitory receptors LAIR1, LILRB1, and LILRB2. RIFINs in severe forms of malaria (such as cerebral malaria and severe anaemia) require a considerable understanding to target and control malaria severity and mortality. RIFINs are implicated in severe malaria and are discussed together with other variant surface antigens such as STEVORS or PfEMP1 in the specific pathophysiology of malaria. This review details the role of RIFINs in the various malaria pathophysiological mechanisms underlying severe malaria and mortality

Role of MyD88-Adaptor-Like (MAL) Gene Polymorphism rs8177374 and Cytokine (IFN-γ, TNF-α, IL-10, TGF-β) Levels in Diverse Malaria Manifestations upon P. falciparum and P. vivax Infections.

This study investigated the role of genetic variant rs8177374 in MAL/TIRAP gene in mediating the cytokine levels of IFN-γ, TNF-α, IL-10, and TGF-β in malaria patients due to Plasmodium falciparum or P. vivax infection. The study included human blood samples collected from patients with malaria (n = 228) and healthy controls (n = 226). P. falciparum and P. vivax groups were established based on the causative species of Plasmodium. Malaria samples were divided into mild and severe malaria groups based on the symptoms that appeared in the patients, according to the WHO criteria. In a previous study, we genotyped rs8177374 via allele specific PCR strategy. In this study, cytokine levels were estimated in the blood plasma of rs8177374 genotype samples via Sandwich Enzyme Linked Immunosorbent Assay kits. Increased IFN-γ and TNF-α levels in presence of CC genotype indicates the role of CC genotype in both severe and mild malaria groups. Enhanced IL-10 levels in the CT genotype and mild malaria groups suggest a role of CT genotype and IL-10 in the mild clinical outcomes of malaria. The rs8177374 polymorphism in MAL/TIRAP plays an important role in malaria pathogenesis.

Association of reduced glutathione levels with Plasmodium falciparum and Plasmodium vivax malaria: a systematic review and meta-analysis

Reduced glutathione (GSH) is a crucial antioxidant with recognized roles in malaria pathogenesis and host response. Despite its importance, reports on the association of GSH with malaria are inconsistent. Therefore, this systematic review and meta-analysis investigated the differences in GSH levels in relation to Plasmodium infection. A comprehensive literature search of six electronic databases (Embase, MEDLINE, Ovid, PubMed, Scopus, and ProQuest) was conducted. Of the 2158 initially identified records, 18 met the eligibility criteria. The majority of studies reported a significant decrease in GSH levels in malaria patients compared with uninfected controls, and this was confirmed by meta-analysis (P < 0.01, Hedges g: − 1.47, 95% confidence interval [CI] − 2.48 to − 0.46, I2: 99.12%, 17 studies). Additionally, there was no significant difference in GSH levels between Plasmodium falciparum malaria and P. vivax malaria (P = 0.80, Hedges g: 0.11, 95% CI − 0.76 to 0.98, I2: 93.23%, three studies). Similarly, no significant variation was observed between symptomatic and asymptomatic malaria cases (P = 0.78, Hedges g: 0.06, 95% CI − 0.34 to 0.46, I2: 48.07%, two studies). In conclusion, although GSH levels appear to be generally lower in malaria patients, further detailed studies are necessary to fully elucidate this complex relationship.

Erythrocyte Indices in Asymptomatic Malaria Infected Pregnant Women

Background: Red blood cell changes are one of the most common complications in malaria and they play a very crucial role in malaria pathogenesis. Malaria infections are one of the common causes of maternal anaemia especially during pregnancy. The aim of this study was to determine red cell indices of pregnant women with asymptomatic malaria.&#x0D; Place and Duration of Study: Department of Haematology and Antenatal Unit both of Enugu State University of Science and Technology Teaching Hospital, between June and September 2022.&#x0D; Methodology: The study population consisted of 90 pregnant women (65 pregnant women positive to malaria parasite without symptoms and 25 pregnant women negative to malaria parasite) and 26 control non-pregnant women. For the whole study population, red cell indices which include hemoglobin (HGB), packed cell volume (PCV), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width standard deviation (RDW-SD), red cell distribution width coefficient of variation (RDW-CV) were measured by automated haematology analyzer.&#x0D; Results: In the asymptomatic malaria group (AMG), 21 (32.3%) had mild anaemia (HGB level 9.0-10.0 g/dl), 11 (16.9%) had moderate anaemia (HGB level 7.0-8.0 g/dl) and 2 (3.1%) had severe anaemia (HGB level &lt;7.0 g/dl). Also in AMG group, the RDW-SD was 54.22 +/- 11.45 fl, whereas in control group it was 48.75 +/- 10.24 fl (p=0.002). Again in the AMG group the MCHC of those that had two pluses was 318.03 +/- 16.31 g/l, whereas in those that had one plus, it was 309 +/- 20.17 g/l. The comparison between the first, second and third trimester showed significant decrease in HGB (7.63 +/- 1.36 vs 11.64 +/- 0.72 g/dl) and PCV (26.98 +/- 5.14 vs 36.20 +/- 2.19 %) in third trimester compared to first trimester (p= &lt;0.001, &lt;0.001). whereas RDW-CV (18.96 +/- 5.04 vs 15.00 +/- 2.64 %) and RDW-SD (59.04 +/- 15.19 vs 49.16+/- 7.00 fl) (p= 0.002, 0.003) significantly increased in third trimester compared to first trimester&#x0D; Conclusion: This study found anaemia in asymptomatic malaria infected pregnant women, significant decrease in haemoglobin and packed cell volume at third trimester, higher MCHC in those with two pluses of malaria and significant increase in red cell distribution width at third trimester.

Optimization of CIDR1α-PfEMP1 Gene Transformation in Eschericia coli BL21(DE3) by Heat-shock Method: Stages of Malaria Vaccine Development

Malaria is still a world health problem, especially in developing countries. Data from the World Malaria Report 2020, malaria cases in the world in 2019 reached 229 million cases with a total death of 409,000 people. The highest mortality occurred in patients under five, which reached 272,000 people. The high mortality rate of severe malaria worldwide needs multiple approaches including the malaria vaccine. Cysteine-rich Interdomain Regions 1α-Plasmodium falciparum Erythrocyte Membrane Protein 1 (CIDR1α-PfEMP1) is a candidate for malaria vaccine due to its important role in malaria pathogenesis. The development of CIDR1α-PfEMP1-based malaria vaccine is conducted by DNA recombinant technology where transformation is a pivotal step. The study aimed to optimize the transformation of CIDR1α-PfEMP1 into Escherichia coli BL21(DE3) using heat shock method to find out the highest transformation efficiency. The variables were duration of heat shock in 30, 50, and 70 secs and plating volume of 100 µL and 200 µL. The results showed that the highest transformation efficiency was 1.9 x 102 CFU/µg, it was achieved by 50 secs heat shock and 200 µL plating volume. This was an optimum transformation condition that can be used further for cloning of CIDR1α-PfEMP1 gene to develop a malaria vaccine.

Platelet Parameters and Their Correlation with Parasitemia Levels Among Malaria Infected Adult Patients at Jinella Health Center, Harar, Eastern Ethiopia: Comparative Cross-Sectional Study.

Malaria is a major public health problem with the highest morbidity and mortality in developing countries. Hematological changes play a great role in malaria pathogenesis through platelets and platelet parameters. However, the changes in platelet parameters are not clearly described in Ethiopia. Therefore, this study aimed to compare platelet parameters and their correlation with parasitemia among malaria-infected adult patients and healthy adults. An institutional-based comparative cross-sectional study was conducted involving 186 (93 malaria-infected patients and 93 healthy adults) study participants using a convenient sampling technique at Jinella health center, Harar, Eastern Ethiopia, from July 10-August 10, 2022. Five milliliters of venous blood were collected from each study participant, and platelet parameters were analyzed using a Unicel (DxH 800) automated hematologic analyzer. A drop of blood was taken from malaria-suspected patients for blood film preparation. Results between two groups were compared using the Mann-Whitney U-test. Spearman's rank correlation coefficient was used to evaluate the relationships between two continuous variables. A P-value of< 0.05 was considered statistically significant. Platelet, plateletcrit, and mean platelet volume of malaria-infected patients were significantly lower as compared with healthy adults (103 x103cells/μL vs 268 x103cells/μL, 0.13 fl vs 0.23 fl, and 9.6 fl vs 15.3 fl), respectively). Conversely, platelet distribution width and platelet large cell ratio were higher in malaria-infected patients than healthy adults (19.2% vs 15.3% and 0.35% vs 0.29%), respectively). Parasitemia levels had a moderately inverse correlation with platelet count (r= -0.419) and a weakly positive correlation with mean platelet volume (r=0.278). The platelet, plateletcrit, and mean platelet volume of malaria-infected patients were significantly lower as compared with healthy adults. Malaria parasitemia had a moderate inverse correlation with platelet count and a weak positive correlation with mean platelet volume. Thrombocytopenia and alteration of platelet parameters should be considered in malaria patients.

Structural and Biophysical Characterization of Plasmodium falciparum Bromodomain Protein 1

Epigenetic regulatory mechanisms are important for the growth and survival of eukaryotic organisms. Post‐translational modifications found on histone proteins in the nucleosome function as a dynamic cell signaling mechanism that controls many essential functions such as cell growth, DNA replication, and gene expression. Bromodomains are conserved protein interaction modules that specifically recognize acetylated lysine residues on histones. Among five Plasmodium species, P. falciparum is the major cause of malaria cases worldwide. According to a WHO report in 2019 there were 409,000 deaths reported worldwide and the most affected age group was of children under 5 years of age. Interestingly, the P. falciparum genome encodes eight bromodomain‐containing proteins, and bromodomain protein 1 (PfBDP1) has been shown to play an important role in malaria pathogenesis. The domain architecture of PfBDP1 includes a single C‐terminal bromodomain and several ankyrin repeats. PfBDP1 is thought to act by tethering a transcriptional activator complex to acetylated histone H3 to control genes required for parasite invasion. Additionally, PfBDP1 forms a complex with the bromodomain protein 2 (PfBDP2) to carry out its cellular functions. We hypothesized that the bromodomain is crucial for the function of PfBDP1 via recognition of acetylated lysine modifications. We carried out several histone binding assays to identify novel histone interactions of PfBDP1 bromodomain. We also utilized a combination of structural biology and biophysical approaches to identify amino acid residues critical for ligand coordination. The rise of drug‐resistance towards P. falciparum has created an urgent need to outline the molecular mechanisms driving pathogenesis in hopes of uncovering new potential drug targets. Importantly, the results from this study may improve the development of new therapeutics to treat malaria.

Role of Oxidative Stress on Malarial Anaemia: Significance of Oxidative Stress Index in Patients with Malarial Infection

Introduction: An alteration of oxidant and antioxidant levels is thought to be involved in the pathogenesis of malaria. Limited data is found on association between oxidant and antioxidant status and significance of oxidative stress index in patients with malarial infection and anaemia in south Indian population. Aim: To measure the levels of oxidants and antioxidants and oxidative stress index in patients with malaria and their association with anaemia. Materials and Methods: The present case-control study was conducted in Department of Biochemistry, Osmania General Hospital, Hyderabad, India. A total of 50 subjects of malaria aged 20-45 years were included as cases based on inclusion criteria and exclusion criteria. Control group comprised of 50 healthy age and gender matched subjects. Haemoglobin (Hb), serum Malondialdehyde (MDA), Superoxide Dismutase (SOD), Total Antioxidant Capacity (TAC) was estimated in both the groups by standard methods using Ultraviolet Spectrophotometer. Independent sample t-test was used to test for the difference in all biochemical parameters among study and control groups. The study of orrelation among the parameters was done by Pearson’s correlation. Results: Both the groups were age and gender matched (p=0.39). MDA was significantly elevated in cases as compared to controls (p&lt;0.0001). SOD, Ferric Reducing Ability of Plasma (FRAP), Hb were significantly lower in cases as compared to controls with p-value &lt;0.0001. In the present study, it was found that there was a significant negative correlation between Hb and oxidative stress index (MDA/FRAP=-0.480) with p-value=0.0001 in patients with malaria. Conclusion: The present study concluded that oxidative stress might be a cause of malarial anaemia. Alterations in oxidant and antioxidant levels might have a role in pathogenesis of malaria. As compared to individual markers, oxidative stress index was found to be significantly associated with malarial anaemia denoting its role as a better marker in these patients. Antimalarial therapy targeting the above factors might decrease the pathogenesis, morbidity and mortality in patients with malaria.

Skeleton binding protein-1-mediated parasite sequestration inhibits spontaneous resolution of malaria-associated acute respiratory distress syndrome.

Malaria is a hazardous disease caused by Plasmodium parasites and often results in lethal complications, including malaria-associated acute respiratory distress syndrome (MA-ARDS). Parasite sequestration in the microvasculature is often observed, but its role in malaria pathogenesis and complications is still incompletely understood. We used skeleton binding protein-1 (SBP-1) KO parasites to study the role of sequestration in experimental MA-ARDS. The sequestration-deficiency of these SBP-1 KO parasites was confirmed with bioluminescence imaging and by measuring parasite accumulation in the lungs with RT-qPCR. The SBP-1 KO parasites induced similar lung pathology in the early stage of experimental MA-ARDS compared to wildtype (WT) parasites. Strikingly, the lung pathology resolved subsequently in more than 60% of the SBP-1 KO infected mice, resulting in prolonged survival despite the continuous presence of the parasite. This spontaneous disease resolution was associated with decreased inflammatory cytokine expression measured by RT-qPCR and lower expression of cytotoxic markers in pathogenic CD8+ T cells in the lungs of SBP-1 KO infected mice. These data suggest that SBP-1-mediated parasite sequestration and subsequent high parasite load are not essential for the development of experimental MA-ARDS but inhibit the resolution of the disease.

The mTOR Pathway in Platelets Contributes to the Pathophysiology of Experimental Cerebral Malaria

Background: Cerebral malaria is a highly prevalent infectious disease in Sub-Saharan Africa caused by the Plasmodium parasite. The pathogenesis of cerebral malaria results from damaged vascular endothelium induced by parasite sequestration, inflammatory cytokine production and vascular leakage, which results in increased brain permeability and death. While maladaptive responses from immune cells are thought to contribute, growing evidence suggests a crucial role of platelets in malaria pathophysiology. The mammalian target of rapamycin (mTOR) pathway is critical in regulating outcomes in malaria. Previous studies have demonstrated an mTOR specific inhibitor, rapamycin, is protective in a mouse model of experimental cerebral malaria (ECM). However, if the mTOR pathway in platelets specifically contributes to the pathogenesis of malaria is unknown.Methods: Platelet-specific mTOR-deficient (mTOR plt-/-) mice and littermate controls were subjected to a well-established model of ECM, using Plasmodium berghei ANKA. In addition, platelets isolated from human malaria patients were examined for differential regulation of the mTOR pathway using RNA-seq.Results: Platelet RNA-seq and Ingenuity Pathway Analysis from patients infected with P. vivax demonstrated enrichment of mTOR-associated pathways in platelets, such as mTOR signaling and p70S6K signaling, indicating mTOR associated genes are upregulated in human platelets during malaria infection. In mice infected with P. berghei ANKA, the mTOR pathway was activated in bone marrow-megakaryocytes and platelets based on phosphorylation of mTOR and its downstream effector, 4E-BP1. As the mTOR pathway regulates protein translation in platelets, we examined de novo protein synthesis and observed increased protein translation in platelets isolated from mice infected with P. berghei ANKA compared to uninfected controls.To study the specific role of platelet mTOR during ECM pathogenesis, mTOR plt-/- mice and wild-type controls (mTOR plt+/+), were infected with P. berghei ANKA. Platelet deficient-mTOR mice had significantly (p=0.0336) prolonged survival compared to wild-type mice. Increased survival was independent of parasitemia, suggesting platelets did not alter parasite reproduction. While thrombocytopenia and anemia were similar in both genotypes, mTOR plt-/- mice had significantly reduced brain (p=0.0067) and lung (p<0.0001) vascular permeability during late-stage ECM. Interestingly, flow cytometric assessment of leukocyte recruitment to the brain demonstrated a 1.7-fold (p=0.0442) reduction in inflammatory monocytes in platelet-deficient mTOR mice. However, mTOR plt-/- mice had significantly (1.4-fold, p=0.007) more inflammatory monocytes in the blood. Interestingly, circulating platelet-monocytes aggregates were significantly less in mTOR plt-/- compared to mTOR plt+/+ (p=0.0433). Taken together, these results suggest that platelets assist in the recruitment of leukocytes to the brain vasculature during ECM, which is impaired when mTOR is ablated.Conclusions: Our data demonstrates that the mTOR pathway in platelets plays a significant role in malaria pathogenesis. Deletion of platelet mTOR reduces vascular permeability and prolongs survival during ECM. We hypothesize that altered platelet-inflammatory monocyte interactions drive this phenotype. DisclosuresRondina: Platelet Transcriptomics: Patents & Royalties; Acticor Biotech: Membership on an entity's Board of Directors or advisory committees; Platelet Biogenesis: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding.

Structural basis of LAIR1 targeting by polymorphic Plasmodium RIFINs

RIFIN, a large family of Plasmodium variant surface antigens, plays a crucial role in malaria pathogenesis by mediating immune suppression through activation of inhibitory receptors such as LAIR1, and antibodies with LAIR1 inserts have been identified that bind infected erythrocytes through RIFIN. However, details of RIFIN-mediated LAIR1 recognition and receptor activation have been unclear. Here, we use negative-stain EM to define the architecture of LAIR1-inserted antibodies and determine crystal structures of RIFIN-variable 2 (V2) domain in complex with a LAIR1 domain. These structures reveal the LAIR1-binding region of RIFIN to be hydrophobic and membrane-distal, to exhibit extensive structural diversity, and to interact with RIFIN-V2 in a one-to-one fashion. Through structural and sequence analysis of various LAIR1 constructs, we identify essential elements of RIFIN-binding on LAIR1. Furthermore, a structure-derived LAIR1-binding sequence signature ascertained >20 LAIR1-binding RIFINs, including some from P. falciparum field strains and Plasmodium species infecting gorillas and chimpanzees.

Inhibition of Activin A suppressed tumor necrosis factor-α secretion and improved histopathological conditions in malarial mice.

Malaria infection still remains as one of the most prominent parasitic diseases afflicting mankind in tropical and subtropical regions. The severity of malaria infection has often been associated to exuberant host immune inflammatory responses that could possibly lead to severe immunopathological conditions and subsequent death of host tissues. Activin A is a protein belonging to the transforming growth factor-beta (TGF-β) family that regulates multiple physiological processes and pathological-associated diseases. The biological roles of activin A have been associated with manipulation of inflammation-related processes and modulation of host immune responses. This implies that activin A protein could play a role in malaria pathogenesis since malaria infection has been closely linked to severe immune responses leading to death, However, the actual in vivo role of activin A in malaria infection remains elusive. Hence, this study was undertaken to investigate the involvement of activin A in malaria infection as well as to assess the modulating effects of activin A on the cytokine releases (TNF-α, IFN-γ and IL-10) and histopathological changes in major affected organs (kidney, liver, lung, brain and spleen) in malarial mice infected with Plasmodium berghei ANKA. Our results showed that the concentrations of plasma activin A were significantly increased in malarial mice throughout the study periods. Also. the systemic activin A level was positively correlated with malaria parasitemia. This indicates that activin A could play a role in malaria pathogenesis and malaria parasitemia development. Plasma TNF-α, IFN-γ and IL-10 cytokine levels were significantly increased in malarial mice at day-5 post infection, suggesting that these cytokines attributed to severe malaria pathogenesis. Histopathological features such as sequestration of parasitized red blood cells (pRBCs) and hemozoin formation were amongst the most common pathological conditions observed in tissues of major affected organs (kidney, liver, lung, brain and spleen) in malarial mice. Neutralization of activin A production via recombinant mouse activin RIIA Fc chimera (rmActivin RIIA Fc chimera) had significantly reduced the parasitemia levels in malarial mice. The release of TNF-α cytokine was significantly reduced as well as the sequestration of parasitized pRBCs and hemozoin formation in major affected organs in malarial mice were also alleviated following inhibition of activin A production. Overall, this preliminary study suggests that activin A could play an immune modulation role in malaria pathogenesis through modulation of TNF-α release that benefits host from severe pathological destructions provoked by intensified inflammatory responses. Further studies are warranted to elucidate the precise mechanism of immune modulation mediated by activin A and its associated immune-modulation mediators in regulating the inflammatory responses elicited during the course of malaria infection.

&lt;i&gt;In vivo&lt;/i&gt; antimalarial activity of methanol extracts and fractions of &lt;i&gt;Brachystegia eurycoma&lt;/i&gt; and &lt;i&gt;Mondia whitei&lt;/i&gt;on chloroquine-resistant &lt;i&gt;Plasmodium berghei&lt;/i&gt;

Malaria remains a major public health problem in the tropics. According to WHO estimates, sub-Saharan Africa continues to carry a disproportionately high share of global malaria burden. Oxidative stress plays significant role in malaria pathogenesis. Recently, there is increasing effort to develop more potent antimalarials from plant source. Brachystegia eurycoma and Mondia whitei are used locally for malaria treatment. Medicinal plants used in therapy quite often possess antioxidant activities as a result of its inherent phytoconstituents. The phytochemical composition of Brachystegia eurycoma and Mondia whitei were qualitatively examined. The antioxidant activities of the methanolic extracts of the leaves of two medicinal plants were determined using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) antioxidant assay. The Brine shrimp lethality assay (BSLA) and Lorke‘s acute toxicity study were used to estimate their toxicities; while Peter‘s 4-day chemosuppresive test was employed to evaluate their antimalarial activities. The flavonoid and saponins content were particularly high in both plants. Using ascorbic acid as reference (IC50 9.26 μg/mL), the antioxidant IC50 values of B. eurycoma and M. whitei were 11.14 μg/ mL and 19.81μg/mL respectively; the BSLA LC50 were 1.8 mg/mL and 1.2 mg/mL; acute toxicity LD50 were 5000 mg/kg and 4500 mg/kg. Brachystegia eurycoma showed stronger daily average antimalarial activity (62.0 %) than Mondia whitei (39.3%), the standard drug, chloroquine, was 85.4%. The chloroform fraction of Brachystegia eurycoma was the most active with (65%) daily average suppression. This suggests that the extracts of leaves of the plants have good antioxidant activities, are non-toxic and supports their antimalarial use in ethnomedicine.

Associations between IgG reactivity to Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens and Burkitt lymphoma in Ghana and Uganda case-control studies.

BackgroundEndemic Burkitt lymphoma (eBL) is an aggressive childhood B-cell lymphoma linked to Plasmodium falciparum (Pf) malaria in sub-Saharan Africa. We investigated antibody reactivity to several human receptor-binding domains of the Pf erythrocyte membrane protein 1 (PfEMP1) that play a key role in malaria pathogenesis and are targets of acquired immunity to malaria.MethodsSerum/plasma IgG antibody reactivity was measured to 22 Pf antigens, including 18 to PfEMP1 CIDR domains between cases and controls from two populations (149 eBL cases and 150 controls from Ghana and 194 eBL cases and 600 controls from Uganda). Adjusted odds ratios (aORs) for case-control associations were estimated by logistic regression.FindingsThere was stronger reactivity to the severe malaria associated CIDRα1 domains than other CIDR domains both in cases and controls. eBL cases reacted to fewer antigens than controls (Ghana: p = 0·001; Uganda: p = 0·03), with statistically significant lower ORs associated with reactivity to 13+ antigens in Ghana (aOR 0·39, 95% CI 0·24–0·63; pheterogeneity = 0·00011) and Uganda (aOR 0·60, 95% CI 0.41–0·88; pheterogeneity = 0·008). eBL was inversely associated with reactivity, coded as quartiles, to group A variant CIDRδ1 (ptrend = 0·035) in Ghana and group B CD36-binding variants CIDRα2·2 (ptrend = 0·006) and CIDRα2·4 (ptrend = 0·033) in Uganda, and positively associated with reactivity to SERA5 in Ghana (ptrend = 0·017) and Uganda (ptrend = 0·007) and group A CIDRα1·5 variant in Uganda only (ptrend = 0·034).InterpretationeBL cases reacted to fewer antigens than controls using samples from two populations, Ghana and Uganda. Attenuated humoral immunity to Pf EMP1 may contribute to susceptibility to low-grade malaria and eBL risk.FundingIntramural Research Program, National Cancer Institute and National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services.

Assessment of the Combined Effect of Epstein-Barr Virus and Plasmodium falciparum Infections on Endemic Burkitt Lymphoma Using a Multiplex Serological Approach.

Epstein–Barr virus (EBV) is a necessary cause of endemic Burkitt lymphoma (eBL), while the role of Plasmodium falciparum in eBL remains uncertain. This study aimed to generate new hypotheses on the interplay between both infections in the development of eBL by investigating the IgG and IgM profiles against several EBV and P. falciparum antigens. Serum samples collected in a childhood study in Malawi (2005–2006) from 442 HIV-seronegative children (271 eBL cases and 171 controls) between 1.4 and 15 years old were tested by quantitative suspension array technology against a newly developed multiplex panel combining 4 EBV antigens [Z Epstein–Barr replication activator protein (ZEBRA), early antigen-diffuse component (EA-D), EBV nuclear antigen 1, and viral capsid antigen p18 subunit (VCA-p18)] and 15 P. falciparum antigens selected for their immunogenicity, role in malaria pathogenesis, and presence in different parasite stages. Principal component analyses, multivariate logistic models, and elastic-net regressions were used. As expected, elevated levels of EBV IgG (especially against the lytic antigens ZEBRA, EA-D, and VCA-p18) were strongly associated with eBL [high vs low tertile odds ratio (OR) = 8.67, 95% confidence interval (CI) = 4.81–15.64]. Higher IgG responses to the merozoite surface protein 3 were observed in children with eBL compared with controls (OR = 1.29, 95% CI = 1.02–1.64), showing an additive interaction with EBV IgGs (OR = 10.6, 95% CI = 5.1–22.2, P = 0.05). Using elastic-net regression models, eBL serological profile was further characterized by lower IgM levels against P. falciparum preerythrocytic-stage antigen CelTOS and EBV lytic antigen VCA-p18 compared with controls. In a secondary analysis, abdominal Burkitt lymphoma had lower IgM to EBV and higher IgG to EA-D levels than cases with head involvement. Overall, this exploratory study confirmed the strong role of EBV in eBL and identified differential IgG and IgM patterns to erythrocytic vs preerythrocytic P. falciparum antigens that suggest a more persistent/chronic malaria exposure and a weaker IgM immune response in children with eBL compared with controls. Future studies should continue exploring how the malaria infection status and the immune response to P. falciparum interact with EBV infection in the development of eBL.

Microfluidic analysis of red blood cell deformability as a means to assess hemin-induced oxidative stress resulting from Plasmodium falciparum intraerythrocytic parasitism.

Hemolytic anemia is one of the hallmarks of malaria and leads to an increase in oxidized heme (hemin) within the plasma of infected individuals. While scavenger proteins sequester much of the circulating heme, it has been hypothesized that extracellular heme may play a central role in malaria pathogenesis. We have previously developed the multiplex fluidic plunger (MFP) device for the measurement of red blood cell (RBC) deformability. Here, we demonstrate that the measurement of changes in RBC deformability is a sensitive method for inferring heme-induced oxidative stress. We further show that extracellular hemin concentration correlates closely with changes in RBC deformability and we confirm that this biophysical change correlates with other indicators of cell stress. Finally, we show that reduced erythrocyte deformability corresponds with both erythrophagocytosis and RBC osmotic fragility. The MFP microfluidic device presents a simple and potentially inexpensive alternative to existing methods for measuring hemolytic cell stress that could ultimately be used to perform clinical assessment of disease progression in severe malaria.

Phage Display cDNA Screens Reveal a Specific Region of Plasmodium falciparum Glutamic Acid‐rich Protein that Binds to Human Red Blood Cells. A Functional Role in Malaria Pathogenesis

Severe malaria caused by Plasmodium falciparum is a serious disease that affects a large number of people regardless of the mode of treatment with currently existing antimalarial drugs. In 2015, the Centers for Disease Control and Prevention (CDC) estimated nearly 214 million cases of malaria infection worldwide resulting in ~400,000 deaths mainly in sub‐Saharan Africa. Infection of red blood cells (RBCs) by P. falciparum, the most lethal species among the human malaria parasites, leads to several complications such as cytoadherence, which involves binding of infected red blood cells (iRBCs) to endothelial receptors, and rosetting, which is the binding of iRBCs to uninfected RBCs. These features contribute to specific organ damage and exacerbate disease pathogenesis. To elucidate the biological mechanisms of P. falciparum infection in human RBCs, we are currently investigating the virulence phenomena including parasite invasion and rosetting. Using phage display technology, we previously identified several novel ligand‐receptor interactions at the blood stage of malaria infection. Here, we used two independent phage display cDNA library screens and identified P. falciparum glutamic acid‐rich protein (Pf‐GARP) as a novel parasite ligand that binds to the surface of human RBCs. Specifically, we screened phage display cDNA libraries constructed from P. falciparum FCR‐3 and 3D7 strains and identified several overlapping Pf‐GARP clones expressing peptides that bind to intact RBCs. Two phage clones encoding Pf‐GARP392–437 and Pf‐GARP356–552 revealed a specific binding region located nearly in the middle of full length Pf‐GARP1–673. Using bioinformatics tools, we chemically synthesized a codon‐optimized segment, Pf‐GARP370–444, located within the overlapping phage clones. The phage cDNA inserts were cloned into pET‐32b vector, and recombinant peptides were expressed in E. coli. Recombinant fusion proteins encoded by all three Pf‐GARP cDNA were confirmed to bind human RBCs in vitro. The codon‐optimized Pf‐GARP370–444 was cloned into the pRE4 vector, and expressed on the surface of Chinese hamster ovary (CHO) cells. The transfected CHO cells formed rosette‐like clusters in the presence of uninfected human RBCs in vitro. Importantly, direct biochemical interaction between purified Pf‐GARP370–444 protein and human RBCs was abolished upon treatment of RBCs with chymotrypsin but not with trypsin and neuraminidase. These results indicate that a centrally‐located segment of Pf‐GARP recognizes a specific chymotrypsin‐sensitive receptor(s) on the surface of host RBCs. Our findings suggest a functional role of Pf‐GARP in the rosetting and invasion phenomena with implications in inter‐membrane transport and vaccine development against malaria.Support or Funding InformationFunding source: NIH‐HL060961

The role of regulatory T cells during Plasmodium chabaudi chabaudi AS infection in BALB/c mice.

An inappropriate immune response to parasite infection is one of the primary drivers of malaria pathogenesis. Regulatory T cells (Tregs), an important subset of CD4(+) T cells, can maintain self-tolerance and prevent autoimmune diseases. However, there is little consensus about their role in malaria pathogenesis. In this study, we transiently depleted Tregs (CD25(+) T cells) using an anti-CD25 mAb (7D4 clone) at different time points following Plasmodium chabaudi chabaudi AS infection in BALB/c mice and investigated the effect of depletion of Tregs in this model. In control mice, Tregs proliferated significantly and their suppressive function was enhanced after infection. IL-10 was increased drastically during infection. Depletion of Tregs at various time points can lead to divergent outcomes. When Tregs were depleted prior to or during the early phase of infection, most mice survived and had a robust Th1 immune response. In contrast, when Tregs were depleted close to peak parasitemia, all mice died as a result of inflammation. Taken together, these data suggest that in P. c. chabaudi AS-infected BALB/c mice, Tregs inhibit the Th1 response and macrophage activation, leading to increased parasite load; however, they also control inflammation-mediated pathology by secreting high levels of IL-10.