Adiponectin and glucose production in patients infected with Plasmodium falciparum

Infections are often complicated by an increase in glucose production due to stimulation of the secretion of glucose counter-regulatory hormones and cytokines. Adiponectin, a fat-derived hormone with insulin-sensitizing properties, could play a regulatory role in the degree of stimulation of glucose production by the infectious agent. Therefore, we investigated the possible correlation between glucose production and plasma adiponectin levels in 25 subjects: 7 patients with cerebral malaria, 6 with uncomplicated malaria, and 12 matched controls. Glucose production was significantly higher in patients with malaria compared to healthy controls (P < .001). Adiponectin levels were not different between the patients with malaria and the control group. However, patients with cerebral malaria had significantly higher values for adiponectin than the patients with uncomplicated malaria (P < .005). Glucose production and gluconeogenesis were positively correlated to plasma adiponectin in the patients (r = 0.835, P < .001 and r = 0.846, P < .001, respectively), whereas these correlations were absent in the controls (r = -0.329, NS and r = -0.028, NS, respectively). In conclusion, adiponectin levels were not different between patients with malaria and their matched controls. However, patients infected with Plasmodium falciparum who have higher glucose production also have higher adiponectin levels. In healthy subjects such a correlation was not found. As adiponectin is known to inhibit glucose production, stimulation of adiponectin secretion during infection could be intended to restrain the glucose production stimulating properties of hormones and cytokines secreted during infection.     

Plasmodium falciparum polypeptides associated with the infected erythrocyte plasma membrane.

Plasmodium falciparum proteins associated with plasma membranes of infected erythrocytes were identified by using three techniques: isolated plasma membranes from infected and uninfected erythrocytes were compared by gel electrophoresis and silver staining; isolated plasma membranes from cells metabolically labeled with [35S]methionine were assayed by gel electrophoresis; and uninfected and infected intact erythrocytes were surface-labeled by lactoperoxidase iodination, and the labeled polypeptides were compared by gel electrophoresis. The results from these experiments indicate that at least six parasite-derived polypeptides (Mr = greater than 240,000, 150,000, 55,000, 45,000, 35,000, and 20,000) are associated with the infected erythrocyte plasma membrane. At least four of these peptides (Mr = 55,000, 45,000, 35,000, and 20,000) may be exposed on the surface of the infected erythrocytes.     

Deficiency of Con A-induced suppressor cell activity in peripheral blood mononuclear cells from Thai adults naturally infected with Plasmodium falciparum and Plasmodium vivax

Con A-pretreated mononuclear (MNC) cells from Thai adults with naturally acquired P. falciparum or P. vivax malaria were significantly less effective in suppressing the responsiveness of autologous or normal allogeneic responder cells to mitogenic lectins or allogenic stimulator cells than pretreated cells from healthy donors. Serial studies of three patients demonstrated that reduced suppressor cell activity was present early in malaria infection but returned to normal soon after treatment. These studies demonstrate that the loss of T cells previously observed in patients with malaria, in part may functionally represent a loss of suppressor T cells.     

Human natural killer cells control Plasmodium falciparum infection by eliminating infected red blood cells

Immunodeficient mouse–human chimeras provide a powerful approach to study host-specific pathogens, such as Plasmodium falciparum that causes human malaria. Supplementation of immunodeficient mice with human RBCs supports infection by human Plasmodium parasites, but these mice lack the human immune system. By combining human RBC supplementation and humanized mice that are optimized for human immune cell reconstitution, we have developed RBC-supplemented, immune cell-optimized humanized (RICH) mice that support multiple cycles of P. falciparum infection. Depletion of human natural killer (NK) cells, but not macrophages, in RICH mice results in a significant increase in parasitemia. Further studies in vitro show that NK cells preferentially interact with infected RBCs (iRBCs), resulting in the activation of NK cells and the elimination of iRBCs in a contact-dependent manner. We show that the adhesion molecule lymphocyte-associated antigen 1 is required for NK cell interaction with and elimination of iRBCs. Development of RICH mice and validation of P. falciparum infection should facilitate the dissection of human immune responses to malaria parasite infection and the evaluation of therapeutics and vaccines.Malaria is caused by infection with parasites of the Plasmodium species which are transmitted by bites of infected Anopheles mosquitoes. Plasmodium species are highly host specific. making it difficult to model human parasite infection in laboratory animals. So far, most in vivo experimental studies of malaria have been carried out with mouse and rat Plasmodium strains in rodents. Differences in invasion and disease pathology between human and rodent parasite species have impeded the translation of findings from rodents into human. The lack of appropriate small animal models also has hampered the evaluation of new drugs and vaccines before clinical trials (1).To overcome this challenge, one approach is to supplement SCID mice with human RBCs. The resulting mice support a limited blood-stage P. falciparum infection (2–4). The need to inject large volumes of human RBCs repeatedly and to treat mice with anti-neutrophil antibody and highly toxic clodronate liposomes to suppress the rapid clearance of the injected human RBCs by macrophages in the recipient mice makes working with this system difficult. More recently NOD-SCID Il2rg^−/− (NSG) mice have been shown to support a more efficient P. falciparum infection without the treatment of clodronate liposomes or anti-neutrophil antibody (5). Furthermore, a recent report shows the development of liver-stage P. falciparum infection in immunocompromised and fumarylacetoacetate hydrolase-deficient (Fah^−/−, Rag2^−/−, Il2rg^−/−) (FRG) mice. Backcrossing of FRG mice to the NOD background and supplementing the resulting mice with human RBCs led to reproducible transition from liver-stage infection to blood-stage infection (6). Despite such progress, none of the existing mouse models of human parasite infection has a human immune system.The immune system plays a critical role in the control of parasite infection. Studies in mice using mouse Plasmodium strains have shown that mouse immune cells such as natural killer (NK) cells, T cells, dendritic cells, and B cells all contribute to antiparasitic immunity (7–10). Notably, depletion of NK cells in a mouse model of Plasmodium chabaudi infection results in more severe disease associated with higher parasitemia and mortality (11). In vitro, P. falciparum-infected human RBCs are shown to interact with human NK cells, leading to the induction of IFN-γ (12). Compared with P. falciparum schizonts, live infected RBCs (iRBCs) induce more rapid activation and more production of IFN-γ by NK cells (13). More recently, it has been shown that, in addition to IFN-γ, activated human NK cells also produce perforin and granzyme against P. falciparum-infected RBCs (14). However, because of the lack of appropriate models, little is known about the role of human NK cells in the control of P. falciparum infection in vivo. NK cells are cytolytic and can lyse virus-infected cells and tumor cells (15). However, whether NK cells also can eliminate parasite-infected RBCs directly has not been demonstrated comprehensively.In our study of humanized mice, we previously had developed a simple and effective method of enhancing human cell reconstitution by hydrodynamic expression of human cytokines. Expression of human IL-15 and Flt-3/Flk-2 ligand (Flt-3L) enhances the reconstitution of human NK cells, monocytes, and macrophages (16). In this study, we have constructed humanized mice that have an optimized human immune cell reconstitution as well as high levels of human RBCs through supplementation. We show that such humanized mice support an efficient infection by P. falciparum. Depletion of human NK cells, but not macrophages, in these mice results in a significant increase in parasitemia. Our additional studies in vitro show that NK cells interact preferentially with iRBCs and become activated, resulting in the elimination of iRBCs in a contact-dependent manner. We further show that the cell adhesion molecules lymphocyte-associated antigen 1 (LFA-1) and to some extent DNAX accessory molecule 1 (DNAM-1) mediate NK cell interaction with and elimination of iRBCs. Development of humanized mice with robust reconstitution of human immune cells and human RBCs and validation of the model for P. falciparum infection should facilitate the dissection of human immune responses to malaria parasite infection and the evaluation of therapeutics and vaccines.     

Chloroquine resistance of Plasmodium falciparum malaria in Ethiopia and Eritrea

We report on the first 2 years of operation of a new strategy for treatment for P. falciparum malaria patients who were not cured by a standard course of chloroquine. Any such patient who returned to a malaria treatment and detection post within 2 weeks was treated daily under supervision with chloroquine. Patients whose parasitaemia had not decreased below 25% of the initial level by day 3 or cleared completely by day 7 were given sulphadoxine/pyrimethamine (Fansidar). Of 39 824 patients treated initially with chloroquine, 4% returned to the malaria post within 2 weeks of treatment; 87% of these were chloroquine resistant and treated with Fansidar and 28% of the returning patients were RIII resistant. Resistance was associated with geographical area, initial parasite density and age. Earlier studies had shown resistance to be confined to border areas, but we found that it was highest in the centre of the region, notably in the lowlands of the Shewa and Arsi provinces, and lowest in the west. Although imported cases have been held responsible for the development of resistance in border areas, other factors are likely to be important in the middle of the region. The implications of these findings for a treatment policy of P. falciparum malaria in the region are discussed.     

恶性疟原虫氯喹敏感株与抗性株对青蒿素类药物体外敏感性的研究

目的 测定恶性疟原虫氯喹敏感株与抗性株对青蒿素类药物的体外敏感性. 方法 运用体外微量法与酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)测定青蒿琥酯、蒿甲醚及双氢青蒿素等3种青蒿素类抗疟药物对体外培养的恶性疟原虫氯喹敏感株与氯喹抗性株的体外敏感性,并比较两种方法测定的IC50值. 结果 体外微量法测定的3种药物对恶性疟原虫氯喹敏感株的IC50值依次为3.12 nmol/L、4.30 nmol/L、2.18 nmol/L,对恶性疟原虫氯喹抗性株的IC50值依次为4.31nmol/L、3.90 nmol/L、3.17 nmol/L;同时,将体外微量法与ELISA法所获的结果进行相关性分析,两种方法结果基本一致(r2=0.93,P＜0.001). 结论 恶性疟原虫氯喹抗性株对青蒿素类药物无明显的交叉抗性;ELISA法可用于恶性疟原虫对抗疟药物的体外敏感性检测.     

Levamisole inhibits sequestration of infected red blood cells in patients with falciparum malaria

BACKGROUND: Sequestration of infected red blood cells (iRBCs) in the microcirculation is central to the pathophysiology of falciparum malaria. It is caused by cytoadhesion of iRBCs to vascular endothelium, mediated through the binding of Plasmodium falciparum erythrocyte membrane protein-1 to several endothelial receptors. Binding to CD36, the major vascular receptor, is stabilized through dephosphorylation of CD36 by an alkaline phosphatase. This is inhibited by the alkaline phosphatase-inhibitor levamisole, resulting in decreased cytoadhesion. METHODS: Patients with uncomplicated falciparum malaria were randomized to receive either quinine treatment alone or treatment with a single 150-mg dose of levamisole as an adjunct to quinine. Peripheral blood parasitemia and parasite stage distribution were monitored closely over time. RESULTS: Compared with those in control subjects, peripheral blood parasitemias of mature P. falciparum parasites increased during the 24 h after levamisole administration (n=21; P=.006). The sequestration ratio (between observed and expected peripheral blood parasitemia) of early trophozoite and midtrophozoite parasites increased after levamisole treatment, with near complete prevention of early trophozoite sequestration and >65% prevention of midtrophozoite sequestration. CONCLUSION: These findings strongly suggest that levamisole decreases iRBC sequestration in falciparum malaria in vivo and should be considered as a potential adjunctive treatment for severe falciparum malaria. TRIAL REGISTRATION: Current Controlled Trials identifier: 15314870.     

Molecular Analysis of Chloroquine and Sulfadoxine-Pyrimethamine Resistance-Associated Alleles in Plasmodium falciparum Isolates from Nicaragua

Chloroquine (CQ) is used as a first-line therapy for the treatment of Plasmodium falciparum malaria in Nicaragua. We investigated the prevalence of molecular markers associated with CQ and sulfadoxine-pyrimethamine (SP) resistance in P. falciparum isolates obtained from the North Atlantic Autonomous Region of Nicaragua. Blood spots for this study were made available from a CQ and SP drug efficacy trial conducted in 2005 and also from a surveillance study performed in 2011. Polymorphisms in P. falciparum CQ resistance transporter, dihydrofolate reductase, and dihydropteroate synthase gene loci that are associated with resistance to CQ, pyrimethamine, and sulfadoxine, respectively, were detected by DNA sequencing. In the 2005 dataset, only 2 of 53 isolates had a CQ resistance allele (CVIET), 2 of 52 had a pyrimethamine resistance allele, and 1 of 49 had a sulfadoxine resistance allele. In the 2011 dataset, none of 45 isolates analyzed had CQ or SP resistance alleles.     

Multiple stiffening effects of nanoscale knobs on human red blood cells infected with Plasmodium falciparum malaria parasite

During its asexual development within the red blood cell (RBC), Plasmodium falciparum (Pf), the most virulent human malaria parasite, exports proteins that modify the host RBC membrane. The attendant increase in cell stiffness and cytoadherence leads to sequestration of infected RBCs in microvasculature, which enables the parasite to evade the spleen, and leads to organ dysfunction in severe cases of malaria. Despite progress in understanding malaria pathogenesis, the molecular mechanisms responsible for the dramatic loss of deformability of Pf-infected RBCs have remained elusive. By recourse to a coarse-grained (CG) model that captures the molecular structures of Pf-infected RBC membrane, here we show that nanoscale surface protrusions, known as “knobs,” introduce multiple stiffening mechanisms through composite strengthening, strain hardening, and knob density-dependent vertical coupling. On one hand, the knobs act as structural strengtheners for the spectrin network; on the other, the presence of knobs results in strain inhomogeneity in the spectrin network with elevated shear strain in the knob-free regions, which, given its strain-hardening property, effectively stiffens the network. From the trophozoite to the schizont stage that ensues within 24–48 h of parasite invasion into the RBC, the rise in the knob density results in the increased number of vertical constraints between the spectrin network and the lipid bilayer, which further stiffens the membrane. The shear moduli of Pf-infected RBCs predicted by the CG model at different stages of parasite maturation are in agreement with experimental results. In addition to providing a fundamental understanding of the stiffening mechanisms of Pf-infected RBCs, our simulation results suggest potential targets for antimalarial therapies.The most virulent human malaria parasite, Plasmodium falciparum (Pf), causes ∼700,000 deaths each year (1, 2). Following entry into red blood cells (RBCs), the parasite matures through the ring (0–24 h), trophozoite (24–36 h), and schizont stages (40–48 h). This intraerythrocyte maturation is accompanied by striking changes in the surface topography and membrane architecture of the infected RBC (3–5). A notable modification is the formation of nanoscale protrusions, commonly known as knobs, at the RBC surface during the second half (24–48 h) of the asexual cycle. These protrusions mainly comprise the knob-associated histidine-rich protein (KAHRP) and the membrane-embedded cytoadherence protein, Pf-erythrocyte membrane protein 1 (PfEMP1). KAHRP binds to the fourth repeat unit of the spectrin α-chain, to ankyrin, to spectrin–actin–protein 4.1 complexes, and to the cytoplasmic domain of PfEMP1 (6–9). These attachments enhance the vertical coupling between the lipid bilayer and the spectrin network. Another striking modification in the Pf-infected RBC membrane is the reorganization of the cytoskeletal network caused by parasite-induced actin remodeling (10). As a result of these molecular-level modifications, the Pf-infected RBC exhibits markedly increased stiffness [the shear modulus increases on average from ∼4−10 µN/m in normal/uninfected RBCs, to ∼40 µN/m at the trophozoite stage, and to as high as 90 µN/m at the schizont stage (11–13)] and cytoadherence to the vascular endothelium, which enable sequestration from circulation in vasculature, and evasion from the surveillance mechanisms of the spleen. Although in vitro experimental studies have revealed roles of particular parasite-encoded proteins in remodeling the host RBC (14–22), the mechanism by which Pf-infected RBCs gain dramatically increased stiffness has remained unclear. Indeed, uncertainty remains as to whether the loss of deformability arises from the structural reorganization of the host membrane components or from the deposition of parasite proteins. That is, it is not clear whether the stiffening is due to remodeling of the spectrin network, or to the formation of the knobs, or both. As experimental studies alone have heretofore not been able to determine the molecular details, numerical modeling, combined with a variety of experimental observations and measurements, offers an alternative approach to reveal the underlying mechanisms.We present here a coarse-grained (CG) molecular dynamics (MD) RBC membrane model to correlate structural modifications at the molecular ultrastructure level with the shear responses of the Pf-infected RBC membrane, focusing on the second half of the parasite’s intra-RBC asexual cycle (24–48 h), i.e., the trophozoite and schizont stages. The CG model is computationally efficient, and able to capture the molecular structures of the RBC membrane in both normal and infected states. CGMD simulations reveal that spectrin network remodeling accounts for a relatively small change in shear modulus. Instead, the knobs stiffen the membrane by multiple mechanisms, including composite strengthening, strain hardening, and knob density-dependent vertical coupling. Our findings provide molecular-level understanding of the stiffening mechanisms operating in Pf-infected RBCs and shed light on the pathogenesis of falciparum malaria.     

Multi-drug resistant Plasmodium falciparum malaria in Assam, India: timing of recurrence and anti-malarial drug concentrations in whole blood

The susceptibility of 23 cases of Plasmodium falciparum malaria from the Sonapur primary health center in the Kamrup district of Assam, India to different antimalarials was investigated using the 28-day World Health Organization in vivo test. Whole blood concentrations of chloroquine, sulfadoxine, and quinine were determined at different intervals and at the time of parasites recrudescence after completion of treatment with the respective drugs to confirm the status of drug sensitivity. A case of multi-drug resistant P. falciparum malaria was found where recrudescence occurred, despite standard oral treatment with chloroquine, sulfadoxine/pyrimethamine, and quinine sequentially. Whole blood concentrations of chloroquine, sulfadoxine, and quinine at the time of recrudescence were 0.35 microg/ml (day 7), 18 microg/ml (day 14), and 0.009 microg/ml (day 14), respectively. Therefore, monitoring of drug-resistant P. falciparum malaria and its proper treatment should be intensified to check the spread of multi-drug resistant strains in other parts of the country.     

Chloroquine resistance in Plasmodium falciparum and polymorphism of the CG2 gene

A distinct genotype (designated Dd2-type profile) consisting of 12 point mutations and 3 repetitive regions of the CG2 gene, a candidate gene for chloroquine resistance, has been associated with in vitro resistance in laboratory-adapted strains of Plasmodium falciparum. The DNA sequence of clinical isolates, characterized by in vitro and in vivo tests, was analyzed to evaluate whether the genotype corresponds to the phenotype in naturally occurring parasites. Eight of 11 chloroquine-resistant isolates had the Dd2 genotype. One resistant isolate (by in vitro assay) with a sensitive CG2 genotype was sensitive in vivo. Two resistant isolates and 6 sensitive isolates were multiple infections with mixed alleles. No typical CG2 genotype was found corresponding to the chloroquine-sensitive isolates. These results suggest a strong association between the drug-resistant and CG2 genotypes and support the hypothesis that the CG2 gene may be implicated in chloroquine resistance.     

恶性疟原虫对氯喹抗性及其逆转剂的研究进展

恶性疟原虫对氯喹抗性的出现和广泛传播迫使人类调整治疗疟疾的用药策略并寻找更加有效的新型抗疟药。然而,在一些贫困的疟疾流行区,氯喹仍被用于治疗恶性疟。了解氯喹抗性机制、探索逆转其抗性的方法,将使氯喹这一价廉高效的抗疟药继续发挥作用。抗性逆转剂的研究和发展为上述目标提供了线索,当与氯喹合用时它能够部分恢复氯喹对氯喹抗性株的作用。为此,本文对恶性疟原虫氯喹抗性机制及其逆转剂的研究进展作一综述。     

红细胞感染恶性疟原虫后抗氧化体系的变化

将体外培养的海南株恶性疟原虫感染宿主红细胞后,发现感染红细胞抗氧化体系中的超氧化物岐化酶和过氧化氢酶的活性明显降低,还原型谷胱甘肽的含量明显下降,过氧化氢显著增加,表明疟原虫感染可使宿主红细胞内抗氧化体系的抗氧化能力下降。     

Peripheral blood dendritic cells in children with acute Plasmodium falciparum malaria.

The importance of dendritic cells (DCs) for the initiation and regulation of immune responses not only to foreign organisms but also to the self has raised considerable interest in the qualitative and quantitative analysis of these cells in various human diseases. Plasmodium falciparum malaria is characterized by the poor induction of long-lasting protective immune responses. This study, therefore, investigated the percentage of peripheral blood DCs as lineage marker-negative and HLA-DR(+) or CD83(+) cells in healthy children and in children suffering from acute malaria in Kilifi, Kenya. Comparable percentages of CD83(+) DCs were found in peripheral blood of healthy children and children with malaria. However, the percentage of HLA-DR(+) peripheral blood DCs was significantly reduced in children with malaria. The results suggest that a proportion of peripheral blood DCs may be functionally impaired due to the low expression of HLA-DR on their surface.