Febrile temperatures induce cytoadherence of ring-stage Plasmodium falciparum-infected erythrocytes

In falciparum malaria, the malaria parasite induces changes at the infected red blood cell surface that lead to adherence to vascular endothelium and other red blood cells. As a result, the more mature stages of Plasmodium falciparum are sequestered in the microvasculature and cause vital organ dysfunction, whereas the ring stages circulate in the blood stream. Malaria is characterized by fever. We have studied the effect of febrile temperatures on the cytoadherence in vitro of P. falciparum-infected erythrocytes. Freshly obtained ring-stage-infected red blood cells from 10 patients with acute falciparum malaria did not adhere to the principle vascular adherence receptors CD36 or intercellular adhesion molecule-1 (ICAM-1). However, after a brief period of heating to 40 degrees C, all ring-infected red blood cells adhered to CD36, and some isolates adhered to ICAM-1, whereas controls incubated at 37 degrees C did not. Heating to 40 degrees C accelerated cytoadherence and doubled the maximum cytoadherence observed (P < 0.01). Erythrocytes infected by ring-stages of the ICAM-1 binding clone A4var also did not cytoadhere at 37 degrees C, but after heating to febrile temperatures bound to both CD36 and ICAM-1. Adherence of red blood cells infected with trophozoites was also increased considerably by brief heating. The factor responsible for heat induced adherence was shown to be the parasite derived variant surface protein PfEMP-1. RNA analysis showed that levels of var mRNA did not differ between heated and unheated ring-stage parasites. Thus fever-induced adherence appeared to involve increased trafficking of PfEMP-1 to the erythrocyte membrane. Fever induced cytoadherence is likely to have important pathological consequences and may explain both clinical deterioration with fever in severe malaria and the effects of antipyretics on parasite clearance.     

Impaired cytoadherence of Plasmodium falciparum-infected erythrocytes containing sickle hemoglobin

Sickle trait, the heterozygous state of normal hemoglobin A (HbA) and sickle hemoglobin S (HbS), confers protection against malaria in Africa. AS children infected with Plasmodium falciparum are less likely than AA children to suffer the symptoms or severe manifestations of malaria, and they often carry lower parasite densities than AA children. The mechanisms by which sickle trait might confer such malaria protection remain unclear. We have compared the cytoadherence properties of parasitized AS and AA erythrocytes, because it is by these properties that parasitized erythrocytes can sequester in postcapillary microvessels of critical tissues such as the brain and cause the life-threatening complications of malaria. Our results show that the binding of parasitized AS erythrocytes to microvascular endothelial cells and blood monocytes is significantly reduced relative to the binding of parasitized AA erythrocytes. Reduced binding correlates with the altered display of P. falciparum erythrocyte membrane protein-1 (PfEMP-1), the parasite's major cytoadherence ligand and virulence factor on the erythrocyte surface. These findings identify a mechanism of protection for HbS that has features in common with that of hemoglobin C (HbC). Coinherited hemoglobin polymorphisms and naturally acquired antibodies to PfEMP-1 may influence the degree of malaria protection in AS children by further weakening cytoadherence interactions.     

Combination antiretroviral drugs in PLGA nanoparticle for HIV-1

Background  

Combination antiretroviral (AR) therapy continues to be the mainstay for HIV treatment. However, antiretroviral drug nonadherence can lead to the development of resistance and treatment failure. We have designed nanoparticles (NP) that contain three AR drugs and characterized the size, shape, and surface charge. Additionally, we investigated the in vitro release of the AR drugs from the NP using peripheral blood mononuclear cells (PBMCs).     

Ultrastructural analysis of fresh Plasmodium falciparum-infected erythrocytes and their cytoadherence to human leukocytes.

Sixty fresh Plasmodium falciparum isolates obtained from Gambian children with mild or cerebral malaria were investigated by transmission electron microscopy for the expression of knob-like protrusions (K+) on the surface of the infected erythrocytes. More than six-hundred infected erythrocytes were analyzed. Knob-forming parasites were present in all 60 isolates. Although knobless parasites (K-) were found in 25 (42%) of the isolates, only 39 were K-, while 577 were K+. Nine of the 39 K- infected erythrocytes that were studied in greater detail appeared to be asexual parasites because they were either segmented or they lacked mitochondrial DNA-like filaments and cristae, which are abundant in immature gametocytes. No difference was observed in the relative frequency of K+K- infected erythrocytes in isolates from patients with mild or cerebral malaria. Binding of both knobby and knobless infected erythrocytes to autologous leukocytes including monocytes, neutrophils, lymphocytes and plasma cells was found in some of the primary in vitro cultures. By using P. falciparum laboratory strains of known phenotypes and leukocytes from healthy blood bank donors, it was established that this novel adherence phenomenon was related to that of cytoadherence to certain melanoma or endothelial cells. Cytoadherent infected erythrocytes that bind to leukocytes enhance antibody-independent phagocytosis and induce cellular aggregation, while non-cytoadherent or rosetting infected erythrocytes do not.(ABSTRACT TRUNCATED AT 250 WORDS)     

约氏疟原虫感染对小鼠红细胞膜蛋白组分及其与内皮细胞黏附力的影响

目的分析约氏疟原虫感染后小鼠红细胞膜蛋白及受染红细胞与内皮细胞黏附能力的变化,为进一步探索脑型疟的发生机制提供线索。方法提取约氏疟原虫感染的BALB/c小鼠红细胞膜蛋白,进行SDS-PAGE电泳,检测红细胞膜蛋白的变化。用内皮细胞株与小鼠红细胞共同培养,观察受染红细胞与内皮细胞黏附能力的改变。结果 SDS-PAGE显示约氏疟原虫感染后的小鼠红细胞膜蛋白中含有分子质量约137ku组分,53/54ku固有膜蛋白减少。与未感染对照组比较,受染红细胞对内皮细胞的黏附能力增强约3倍,差异有统计学意义(P<0.05)。结论疟原虫感染可改变宿主红细胞膜蛋白组成,增强受染红细胞与内皮细胞的黏附力。这些变化可能与脑型疟的发生有关。     

Multiple ligands for cytoadherence can be present simultaneously on the surface of Plasmodium falciparum-infected erythrocytes.

A major virulence factor of Plasmodium falciparum is the adherence of parasitized erythrocytes to the wall of postcapillary venules via a specific interaction between parasite-derived erythrocyte surface ligands and receptors on endothelial cells. To study this phenomenon in vitro, we selected a parasite population that expressed at least two different ligands and demonstrated that parasitized cells may coexpress ligands with specificity for multiple receptors. This selected parasite line had several antigenic and cytoadherence characteristics that were different from those of the parent line. Single parasitized erythrocytes were able to adhere to three distinct receptors via at least two separate ligands; a trypsin-sensitive molecule mediated cytoadherence to CD36 and intercellular adhesion molecule 1 and a trypsin-insensitive molecule(s) was responsible for adherence to a third receptor on the surface of melanoma cells. We present evidence that this newly discovered receptor for cytoadherence is an N-linked glycosaminoglycan, as treatment of melanoma cells with endoglycosidase H abolished cytoadherence. These observations emphasize the adaptability of P. falciparum and the complexity of the cytoadherence phenomenon.     

Effect of highly active antiretroviral therapy on cervicovaginal HIV-1 RNA

OBJECTIVES: To determine the frequency of cervicovaginal lavage and plasma HIV-1 RNA levels that are below detectable levels (< 400 copies/ml) among women on highly active antiretroviral therapy (HAART), non-HAART and on no therapy. To compare the effect of initiating HAART on the timing of HIV-1 RNA suppression in the blood plasma and genital tract among antiretroviral-na?ve women. METHODS: Data were obtained from 205 HIV-infected women with paired plasma and cervicovaginal lavage viral load measurements. Seven antiretroviral-na?ve women starting HAART had viral load measurements performed daily for one week, at 2 weeks and at 1 month after initiating therapy. Viral load quantification was carried out by nucleic acid sequence-based amplification assay. The lower limit of detection was 400 copies/ml. RESULTS: Plasma and cervicovaginal HIV-1 RNA was detectable in 71 and 26% of the women, respectively. Among women with plasma viral loads less than 400, 400-9999, and 10,000 copies/ml or over, genital tract HIV-1 RNA was detected in 3, 17 and 48%, respectively (P < 0.001). Fifty-one per cent of the women with CD4 cell counts of less than 200/mm3 had detectable cervicovaginal viral loads compared with 18% among women with CD4 cell counts of 200/mm3 or over (P < 0.001). Cervicovaginal HIV-1 RNA was less than 400 copies/ml in 85% of those on HAART, 69% of those on non-HAART and 69% of those on no therapy (P < 0.045). In seven antiretroviral-na?ve women initiating HAART, cervicovaginal HIV-1 RNA decreased by 0.7-2.1 log10 within 1-14 days of starting therapy. CONCLUSION: The cervicovaginal HIV-1 RNA level was positively correlated with plasma HIV-1 RNA and negatively with the CD4 cell count. The use of HAART was significantly associated with below-detectable levels of HIV-1 RNA in both plasma and the genital tract. HIV-1 RNA suppression in the genital tract may occur rapidly after initiating therapy.     

Parasite sequestration in Plasmodium falciparum malaria: spleen and antibody modulation of cytoadherence of infected erythrocytes.

Sequestration, the adherence of infected erythrocytes containing late developmental stages of the parasite (trophozoites and schizonts) to the endothelium of capillaries and venules, is characteristic of Plasmodium falciparum infections. We have studied two host factors, the spleen and antibody, that influence sequestration of P. falciparum in the squirrel monkey. Sequestration of trophozoite/schizont-infected erythrocytes that occurs in intact animals is reduced in splenectomized animals; in vitro, when infected blood is incubated with monolayers of human melanoma cells, trophozoite/schizont-infected erythrocytes from intact animals but not from splenectomized animals bind to the melanoma cells. The switch in cytoadherence characteristics of the infected erythrocytes from nonbinding to binding occurs with a cloned parasite. Immune serum can inhibit and reverse in vitro binding to melanoma cells of infected erythrocytes from intact animals. Similarly, antibody can reverse in vivo sequestration as shown by the appearance of trophozoite/schizont-infected erythrocytes in the peripheral blood of an intact animal after inoculation with immune serum. These results indicate that the spleen modulates the expression of parasite alterations of the infected erythrocyte membrane responsible for sequestration and suggest that the prevention and reversal of sequestration could be one of the effector mechanisms involved in antibody-mediated protection against P. falciparum malaria.     

First data on HIV-1 resistance mutations to antiretroviral drugs in Central African Republic

In a background of high genomic HIV-1 variability with a predominance of CRF11_cpx and CRF22_01A1, we have studied the emergence of resistance mutations in isolates from Central African patients at failure of d4T-AZT/3TC/NVP-EFV plus two at failure of a PI-including regimen; the resistance mutations observed are those which are expected on HIV-1 subtype B.     

Evolution of resistance to drugs in HIV-1-infected patients failing antiretroviral therapy

BACKGROUND AND OBJECTIVE: The optimal time for changing failing antiretroviral therapy (ART) is not known. It involves balancing the risk of exhausting future treatment options against the risk of developing increased drug resistance. The frequency with which new drug-resistance mutations (DRM) developed and their potential consequences in patients continuing unchanged treatment despite persistent viremia were assessed. DESIGN: A retrospective study of consecutive sequence samples from 106 patients at one institution with viral load (VL) of more than 400 copies/ml, with no change in ART for more than 2 months despite virologic failure. METHODS: Two consecutive pol sequences, CD4 cell counts and VL were analyzed to quantify the development of new DRM and to identify changes in immunologic and virologic parameters. Genotypic susceptibility scores (GSS) and viral drug susceptibilities were calculated by a computer program (HIVDB). Poisson log-linear regression models were used to predict the expected number of mutations at the second time point. RESULTS:: After a median of 14 months of continued ART, 75% (80 of 106) of patients acquired new DRM and were assigned a significantly lower GSS, potentially limiting the success of future ART. The development of new DRM was proportional to the time between the two sequences and inversely proportional to the number of DRM in the first sequence. However, the development of DRM was not associated with significant changes in CD4 or VL counts. CONCLUSIONS: Despite stable levels of CD4 and VL over time, maintaining a failing therapeutic regimen increases drug resistance and may limit future treatment options.     

CD47 regulates the phagocytic clearance and replication of the Plasmodium yoelii malaria parasite

Several Plasmodium species exhibit a strong age-based preference for the red blood cells (RBC) they infect, which in turn is a major determinant of disease severity and pathogenesis. The molecular basis underlying this age constraint on the use of RBC and its influence on parasite burden is poorly understood. CD47 is a marker of self on most cells, including RBC, which, in conjunction with signal regulatory protein alpha (expressed on macrophages), prevents the clearance of cells by the immune system. In this report, we have investigated the role of CD47 on the growth and survival of nonlethal Plasmodium yoelii 17XNL (PyNL) malaria in C57BL/6 mice. By using a quantitative biotin-labeling procedure and a GFP-expressing parasite, we demonstrate that PyNL parasites preferentially infect high levels of CD47 (CD47^hi)-expressing young RBC. Importantly, C57BL/6 CD47^−/− mice were highly resistant to PyNL infection and developed a 9.3-fold lower peak parasitemia than their wild-type (WT) counterparts. The enhanced resistance to malaria observed in CD47^−/− mice was associated with a higher percentage of splenic F4/80⁺ cells, and these cells had a higher percentage of phagocytized parasitized RBC than infected WT mice during the acute phase of infection, when parasitemia was rapidly rising. Furthermore, injection of CD47-neutralizing antibody caused a significant reduction in parasite burden in WT C57BL/6 mice. Together, these results strongly suggest that CD47^hi young RBC may provide a shield to the malaria parasite from clearance by the phagocytic cells, which may be an immune escape mechanism used by Plasmodium parasites that preferentially infect young RBC.Malaria, caused by Plasmodium parasites, remains a major cause of mortality and morbidity in the developing world. Among the four principal human Plasmodium species, Plasmodium falciparum is the most virulent, being responsible for more than 90% of malaria-associated deaths. Likewise, Plasmodium species that infect rodents and nonhuman primates also differ widely in their fulminant nature and in the mortality they cause (1–3). How different Plasmodium species have evolved to exhibit this wide array of virulence and disease severity remains one of the major unsolved questions in malaria biology and pathogenesis.One important factor that is associated with Plasmodium parasite burden and disease severity is the age constraint of the host red blood cells (RBC) they infect. The age-based preference for restricted invasion of RBC by the Plasmodium parasite is characterized as young RBC (reticulocyte), aged RBC (mature), or both young and aged RBC. Plasmodium species that preferentially infect and grow inside young RBC generally cause a low-grade, self-resolving infection that is rarely fatal (e.g., Plasmodium vivax and Plasmodium ovale), whereas those that infect both young and aged RBC cause more fulminant infection that can be fatal in the absence of immunity (e.g., P. falciparum) (1, 4–6). Thus, along with host genetic background and immune response, restriction for age-specific RBC invasion is a major determinant of the severity and outcome of malaria infection.Malaria parasites have evolved to use redundant receptors and pathways to invade the RBC. For example, sialic acid (7) and Duffy antigen (8) are the major RBC receptors for invasion of P. falciparum and P. vivax, respectively, although other receptors and invasion pathways are known to exist (9, 10). Although a redundancy in RBC receptor use would ensure successful invasion by mitigating the effects of polymorphism and immune targeting, the reasons behind the RBC age-based preference for invasion are not fully clear and remain a subject of debate.Survival of normal cells through the course of their life cycle is essential to maintain homeostasis, and aberrant cells (e.g., senescent or foreign antigen-expressing cells) are eliminated through a sophisticated programmed cell removal system that relies on the recognition of self and nonself determinants (11). CD47, a cell surface molecule in the Ig superfamily, is ubiquitously expressed on many cell types, including RBC, and is a marker of self to avoid early clearance by phagocytic cells through ligation of signal regulatory protein alpha (12). In contrast, altered expression or conformational changes in CD47 may lead to a molecular switch that triggers a phagocytic signal to remove aged or damaged cells (11). Recent studies have shown that the level of CD47 expression is higher in progenitor cells and declines as they undergo maturation and are subsequently aged (13). This age-dependent difference in CD47 expression shields young cells but allows clearance of aging and damaged cells from the system.CD47 is overexpressed in cancer cells (11, 14, 15), and the CD47–signal regulatory protein alpha interaction is considered a major pathway of immune evasion by tumor cells (15). Administration of anti-CD47 antibodies enabled the phagocytosis of tumor cells in vitro, reduced their growth, and prevented the metastasis of human patient tumor cells (14). In this article, using the murine Plasmodium yoelii nonlethal model, we provide quantitative evidence for age of RBC as the basis for the survival and growth of malaria parasites and provide supporting data that suggest that P. yoelii nonlethal parasites prefer to grow inside younger RBC, which allows them to evade immune clearance by phagocytic cells through a CD47-mediated process, and that CD47 modulates the clearance of malaria infection. To our knowledge, this is the first report that provides a molecular basis for the age-dependent preference for infection of RBC by a Plasmodium parasite and sheds light on its implications for the severity of malaria infection in a host.     

Effect of antiretroviral therapy on HIV-1 genetic evolution during acute infection

The rapid evolution of HIV-1 is a major obstacle to viral eradication. Early antiretroviral therapy (ART) during primary HIV-1 infection could limit viral diversity. Eighteen patients recently infected with HIV-1 were selected. Nine initiated ART soon after enrolment and nine remained untreated. Replication-competent (RC) viruses were quantified at baseline and after one year of follow-up. Viral diversity in the C2V5 envelope region was evaluated from plasma, peripheral blood mononuclear cells (PBMCs), and cell culture at both time points. The amount of RC virus in the treated group declined (median -5.42 infectious units per million [IUPM]) while it remained stable or increased in the untreated group (median +0.87 IUPM). At one year post infection, we observed a significant increase in diversity for the C2V5 (+0.150%) region, specifically in the hypervariable loops V4 (+0.73%) and V5 (+0.77%), in the untreated group. More importantly, viral diversity did not significantly increase in treated individuals during the first year post infection. Genetic diversity during primary infection remains low through the first year of infection. Early treatment could contribute to a decrease in RC viruses from PBMCs and to limitation of viral diversification in the viral reservoir. These findings may have relevance for the rational design of specific immunotherapeutic strategies.     

In vitro rosetting, cytoadherence, and microagglutination properties of Plasmodium falciparum-infected erythrocytes from Gambian and Tanzanian patients

To understand the molecular mechanisms that lead to sequestration of red blood cells infected with mature stages of Plasmodium falciparum and to examine the relevance of earlier studies on adherence properties of laboratory-derived P falciparum parasites to the natural parasite population, we analyzed Gambian and Tanzanian isolates for in vitro cytoadherence and antibody-mediated microagglutination. Eighteen cryopreserved isolates of ring-stage parasites were cultured for 20 to 30 hours in vitro, in the patients original erythrocytes, to the trophozoite and schizont stage. All parasites were positive in the microagglutination assay with at least one of four African hyperimmune sera. In a rosetting assay, only 2 of the 18 isolates were strongly positive (35% and 41% of parasitized erythrocytes with more than two uninfected cells bound). Thirteen isolates showed either intermediate (5% to 18%) or low (less than 5%) rosetting while three isolates did not form rosettes. Infected cell-binding of the different isolates to immobilized CD36 or thrombospondin, or C32 melanoma cells correlated with the percentage of mature parasites in the blood samples (r = .932 for CD36, r = .946 for thrombospondin, and r = .881 for C32 melanoma cells). There was a high correlation between binding to CD36 and thrombospondin (r = .982). The extent of infected cell rosetting with uninfected cells in these blood samples was not correlated with these other receptor properties. We also observed coexpression of rosetting and cytoadherence receptors on the same parasitized erythrocytes.     

Effect of HIV-1 antiretroviral prophylaxis on hepatic and hematological parameters of African infants

OBJECTIVE: To measure hepatic and hematological parameters among neonates randomized to receive ultra-short antiretroviral regimens. DESIGN: As part of an on-going clinical trial in Malawi, infants born to women who received (early presenters) or did not receive (late presenters) standard intrapartum nevirapine (NVP) dosing were randomized to receive orally either single dose NVP alone or NVP plus zidovudine (twice daily for 1 week). An additional group of untreated infants (born to HIV-uninfected women) was enrolled as a control. METHODS: Laboratory measurements were performed at birth and repeated at 6 weeks of age. Serum alanine aminotransferase (ALT) was measured on approximately 200 infants consecutively enrolled and randomized at the start of the trial. Complete blood count (CBC) was performed on approximately 800 infants at birth and 600 infants at 6 weeks of age. ALT and CBC were also determined on approximately 200 control infants. RESULTS: At birth there were no differences in ALT values between the groups of children. At 6 weeks of age, ALT levels were significantly higher among the treated groups compared with control group (geometric mean of 11.5 U/l for controls and 16.2-19.1 U/l for treated groups; P < 0.0001). Hematological parameters did not differ between groups at birth. At 6 weeks of age, levels of hemoglobin, hematocrit, granulocytes, and platelets were significantly (P < 0.0001) lower among antiviral drug-treated groups compared with controls. These changes were consistent with grade 1 (mild) toxicity, and were more noticeable among HIV-infected infants. CONCLUSIONS: Hepatic and hematologic abnormalities associated with short-term neonatal antiretrovirals among African children are minimal.     

Effect of antiretroviral agents on carbohydrate metabolism in HIV-1 infected pregnant women

BACKGROUND: Despite the correlation between the use of protease inhibitors (PI) and adverse metabolic glycemic events, no prospective study has examined these parameters in pregnant women who use these drugs. METHODS: A prospective study was conducted on 57 pregnant women to investigate the effect of antiretroviral drugs (ARV) on the carbohydrate metabolism during pregnancy. The women were divided into three groups: ZDV Group, 20 HIV-1 infected women taking ZDV; TT Group, 25 patients on triple antiretroviral treatment (ZDV + 3TC + NFV); and Control Group, 12 pregnant women. Blood samples were obtained during the first visit for the determination of fasting plasma glycemia, when the patients were also submitted to a 75 g oral glucose test (OGTT-75g). These procedures were performed four times along pregnancy. RESULTS: The median values of the area under the glycemia curve (AUC) determined over a period of 120 min between the 33rd and 38th week were 11 685 mg/dL for the Control Group, 13 477 mg/dL for the ZDV Group, and 13 650 mg/dL for the TT Group (p = 0.049). There was an increase in the AUC along pregnancy for all three groups studied, regardless of the treatment used, although this increase was significant only in the TT Group (p = 0.001). The antiretroviral agents had no deleterious effects on prematurity, low birth weight, intrauterine growth restriction rates, or on Apgar score. CONCLUSION: An association was detected between the use of PI and the development of carbohydrate intolerance during pregnancy. The antiretroviral agents had no deleterious effects on perinatal prognosis.     

clag9: A cytoadherence gene in Plasmodium falciparum essential for binding of parasitized erythrocytes to CD36

The propensity of isolates of the malaria parasite Plasmodium falciparum to delete a segment of chromosome 9 has provided positional information that has allowed us to identify a gene necessary for cytoadherence. It has been termed the cytoadherence-linked asexual gene (clag9). clag9 encodes at least nine exons and is expressed in blood stages. The hydrophobicity profile of the predicted CLAG9 protein identifies up to four transmembrane domains. We show here that targeted gene disruption of clag9 ablated cytoadherence to C32 melanoma cells and purified CD36. DNA-induced antibodies to the clag9 gene product reacted with a polypeptide of 220 kDa in the parental malaria clone but not in clones with a disrupted clag9 gene.