Murine Model for Preclinical Studies of Var2CSA-Mediated Pathology Associated with Malaria in Pregnancy

Plasmodium falciparum infection during pregnancy leads to abortions, stillbirth, low birth weight, and maternal mortality. Infected erythrocytes (IEs) accumulate in the placenta by adhering to chondroitin sulfate A (CSA) via var2CSA protein exposed on the P. falciparum IE membrane. Plasmodium berghei IE infection in pregnant BALB/c mice is a model for severe placental malaria (PM). Here, we describe a transgenic P. berghei parasite expressing the full-length var2CSA extracellular region (domains DBL1X to DBL6ε) fused to a P. berghei exported protein (EMAP1) and characterize a var2CSA-based mouse model of PM. BALB/c mice were infected at midgestation with different doses of P. berghei-var2CSA (P. berghei-VAR) or P. berghei wild-type IEs. Infection with 10(4) P. berghei-VAR IEs induced a higher incidence of stillbirth and lower fetal weight than P. berghei At doses of 10(5) and 10(6) IEs, P. berghei-VAR-infected mice showed increased maternal mortality during pregnancy and fetal loss, respectively. Parasite loads in infected placentas were similar between parasite lines despite differences in maternal outcomes. Fetal weight loss normalized for parasitemia was higher in P. berghei-VAR-infected mice than in P. berghei-infected mice. In vitro assays showed that higher numbers of P. berghei-VAR IEs than P. berghei IEs adhered to placental tissue. Immunization of mice with P. berghei-VAR elicited IgG antibodies reactive to DBL1-6 recombinant protein, indicating that the topology of immunogenic epitopes is maintained between DBL1-6-EMAP1 on P. berghei-VAR and recombinant DBL1-6 (recDBL1-6). Our data suggested that impairments in pregnancy caused by P. berghei-VAR infection were attributable to var2CSA expression. This model provides a tool for preclinical evaluation of protection against PM induced by approaches that target var2CSA.

Cellular and molecular interactions between the apicomplexan parasites Plasmodium and Theileria and their host cells

Apicomplexan parasites of the genera Theileria and Plasmodium have complicated life cycles including infection of a vertebrate intermediate host and an arthropod definitive host. As the Plasmodium parasite progresses through its life cycle, it enters a number of different cell types, both in its mammalian and mosquito hosts. The fate of these cells varies greatly, as do the parasite and host molecules involved in parasite-host interactions. In mammals, Plasmodium parasites infect hepatocytes and erythrocytes whereas Theileria infects ruminant leukocytes and erythrocytes. Survival of Plasmodium-infected hepatocytes and Theileria-infected leukocytes depends on parasite-mediated inhibition of host cell apoptosis but only Theileria-infected cells exhibit a fully transformed phenotype. As the development of both parasites progresses towards the merozoite stage, the parasites no longer promote the survival of the host cell and the infected cell is finally destroyed to release merozoites. In this review we describe similarities and differences of parasite-host cell interactions in Plasmodium-infected hepatocytes and Theileria-infected leukocytes and compare the observed phenotypes to other parasite stages interacting with host cells.

Mice deficient in interleukin-4 (IL-4) or IL-4 receptor alpha have higher resistance to sporozoite infection with Plasmodium berghei (ANKA) than do naive wild-type mice

BALB/c interleukin-4 (IL-4(-/-)) or IL-4 receptor-alpha (IL-4ralpha(-/-)) knockout (KO) mice were used to assess the roles of the IL-4 and IL-13 pathways during infections with the blood or liver stages of plasmodium in murine malaria. Intraperitoneal infection with the blood-stage erythrocytes of Plasmodium berghei (ANKA) resulted in 100% mortality within 24 days in BALB/c mice, as well as in the mutant mouse strains. However, when infected intravenously with the sporozoite liver stage, 60 to 80% of IL-4(-/-) and IL-4ralpha(-/-) mice survived, whereas all BALB/c mice succumbed with high parasitemia. Compared to infected BALB/c controls, the surviving KO mice showed increased NK cell numbers and expression of inducible nitric oxide synthase (iNOS) in the liver and were able to eliminate parasites early during infection. In vivo blockade of NO resulted in 100% mortality of sporozoite-infected KO mice. In vivo depletion of NK cells also resulted in 80 to 100% mortality, with a significant reduction in gamma interferon (IFN-gamma) production in the liver. These results suggest that IFN-gamma-producing NK cells are critical in host resistance against the sporozoite liver stage by inducing NO production, an effective killing effector molecule against Plasmodium. The absence of IL-4-mediated functions increases the protective innate immune mechanism identified above, which results in immunity against P. berghei infection in these mice, with no major role for IL-13.

Plasmodium induces swelling-activated ClC-2 anion channels in the host erythrocyte

Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum depends on delivery of nutrients. Moreover, infection challenges cell volume constancy of the host erythrocyte requiring enhanced activity of cell volume regulatory mechanisms. Patch clamp recording demonstrated inwardly and outwardly rectifying anion channels in infected but not in control erythrocytes. The molecular identity of those channels remained elusive. We show here for one channel type that voltage dependence, cell volume sensitivity, and activation by oxidation are identical to ClC-2. Moreover, Western blots and FACS analysis showed protein and functional ClC-2 expression in human erythrocytes and erythrocytes from wild type (Clcn2(+/+)) but not from Clcn2(-/-) mice. Finally, patch clamp recording revealed activation of volume-sensitive inwardly rectifying channels in Plasmodium berghei-infected Clcn2(+/+) but not Clcn2(-/-) erythrocytes. Erythrocytes from infected mice of both genotypes differed in cell volume and inhibition of ClC-2 by ZnCl(2) (1 mm) induced an increase of cell volume only in parasitized Clcn2(+/+) erythrocytes. Lack of ClC-2 did not inhibit P. berghei development in vivo nor substantially affect the mortality of infected mice. In conclusion, activation of host ClC-2 channels participates in the altered permeability of Plasmodium-infected erythrocytes but is not required for intraerythrocytic parasite survival.

The proteasome inhibitor MLN-273 blocks exoerythrocytic and erythrocytic development of Plasmodium parasites.

Protein degradation is regulated during the cell cycle of all eukaryotic cells and is mediated by the ubiquitin-proteasome pathway. Potent and specific peptide-derived inhibitors of the 20S proteasome have been developed recently as anti-cancer agents, based on their ability to induce apoptosis in rapidly dividing cells. Here, we tested a novel small molecule dipeptidyl boronic acid proteasome inhibitor, named MLN-273 on blood and liver stages of Plasmodium species, both of which undergo active replication, probably requiring extensive proteasome activity. The inhibitor blocked Plasmodium falciparum erythrocytic development at an early ring stage as well as P. berghei exoerythrocytic progression to schizonts. Importantly, neither uninfected erythrocytes nor hepatocytes were affected by the drug. MLN-273 caused an overall reduction in protein degradation in P. falciparum, as demonstrated by immunoblots using anti-ubiquitin antibodies to label ubiquitin-tagged protein conjugates. This led us to conclude that the target of the drug was the parasite proteasome. The fact that proteasome inhibitors are presently used as anti-cancer drugs in humans forms a solid basis for further development and makes them potentially attractive drugs also for malaria chemotherapy.

Manipulation of host hepatocytes by the malaria parasite for delivery into liver sinusoids.

The merozoite stage of the malaria parasite that infects erythrocytes and causes the symptoms of the disease is initially formed inside host hepatocytes. However, the mechanism by which hepatic merozoites reach blood vessels (sinusoids) in the liver and escape the host immune system before invading erythrocytes remains unknown. Here, we show that parasites induce the death and the detachment of their host hepatocytes, followed by the budding of parasite-filled vesicles (merosomes) into the sinusoid lumen. Parasites simultaneously inhibit the exposure of phosphatidylserine on the outer leaflet of host plasma membranes, which act as "eat me" signals to phagocytes. Thus, the hepatocyte-derived merosomes appear to ensure both the migration of parasites into the bloodstream and their protection from host immunity.

Lung Structure and the Intrinsic Challenges of Gas Exchange

Structural and functional complexities of the mammalian lung evolved to meet a unique set of challenges, namely, the provision of efficient delivery of inspired air to all lung units within a confined thoracic space, to build a large gas exchange surface associated with minimal barrier thickness and a microvascular network to accommodate the entire right ventricular cardiac output while withstanding cyclic mechanical stresses that increase several folds from rest to exercise. Intricate regulatory mechanisms at every level ensure that the dynamic capacities of ventilation, perfusion, diffusion, and chemical binding to hemoglobin are commensurate with usual metabolic demands and periodic extreme needs for activity and survival. This article reviews the structural design of mammalian and human lung, its functional challenges, limitations, and potential for adaptation. We discuss (i) the evolutionary origin of alveolar lungs and its advantages and compromises, (ii) structural determinants of alveolar gas exchange, including architecture of conducting bronchovascular trees that converge in gas exchange units, (iii) the challenges of matching ventilation, perfusion, and diffusion and tissue-erythrocyte and thoracopulmonary interactions. The notion of erythrocytes as an integral component of the gas exchanger is emphasized. We further discuss the signals, sources, and limits of structural plasticity of the lung in alveolar hypoxia and following a loss of lung units, and the promise and caveats of interventions aimed at augmenting endogenous adaptive responses. Our objective is to understand how individual components are matched at multiple levels to optimize organ function in the face of physiological demands or pathological constraints. © 2016 American Physiological Society. Compr Physiol 6:827-895, 2016.

Angiotensin II facilitates autoregulation in the perfused mouse kidney: An optimized in vitro model for assessment of renal vascular and tubular function

Background and Aims: We have optimized the isolated perfused mouse kidney (IPMK) model for studying renal vascular and tubular function in vitro using 24-28 g C57BL6J mice; the wild type controls for many transgenic mice. Methods and Results: Buffer composition was optimized for bovine serum albumin concentration (BSA). The effect of adding erythrocytes on renal function and morphology was assessed. Autoregulation was investigated during stepped increases in perfusion pressure. Perfusion for 60 min at 90-110 mmHg with Krebs bicarbonate buffer containing 5.5% BSA, and amino acids produced functional parameters within the in vivo range. Erythrocytes increased renal vascular resistance (3.8 +/- 0.2 vs 2.4 +/- 0.1 mL/min.mmHg, P < 0.05), enhanced sodium reabsorption (FENa = 0.3 +/- 0.08 vs 1.5 +/- 0.7%, P < 0.05), produced equivalent glomerular filtration rates (GFR; 364 +/- 38 vs 400 +/- 9 muL/min per gkw) and reduced distal tubular cell injury in the inner stripe (5.8 +/- 1.7 vs 23.7 +/- 3.1%, P < 0.001) compared to cell free perfusion. The IPMK was responsive to vasoconstrictor (angiotensin II, EC50 100 pM) and vasodilator (methacholine, EC50 75 nM) mediators and showed partial autoregulation of perfusate flow under control conditions over 65-85 mmHg; autoregulatory index (ARI) of 0.66 +/- 0.11. Angiotensin II (100 pM) extended this range (to 65-120 mmHg) and enhanced efficiency (ARI 0.21 +/- 0.02, P < 0.05). Angiotensin II facilitation was antagonized by methacholine (ARI 0.76 +/- 0.08) and papaverine (ARI 0.91 +/- 0.13). Conclusion: The IPMK model is useful for studying renal physiology and pathophysiology without systemic neurohormonal influences.

A pore-forming haemolysin from the hookworm, Ancylostoma caninum

Hookworms feed on blood, but the mechanism by which they lyse ingested erythrocytes is unknown. Here we show that Ancylostoma caninum, the common dog hookworm, expresses a detergent soluble, haemolytic factor. Activity was identified in both adult and larval stages, was heat-stable and unaffected by the addition of protease inhibitors, metal ions, chelators and reducing agents. Trypsin ablated lysis indicating that the haemolysin is a protein. A closely migrating doublet of hookworm proteins with apparent molecular weights of 60-65 kDa bound to the erythrocyte membrane after lysis of cells using both unlabeled and biotinylated detergent-solubilised hookworm extracts. In addition, separation of detergent-soluble parasite extracts using strong cation-exchange chromatography, resulted in purification of 60-65 kDa proteins with trypsin-sensitive haemolytic activity. Erythrocytes lysed with particulate, buffer-insoluble worm extracts were observed using scanning electron microscopy and appeared as red cell ghosts with approximately 100 nm diameter pores formed in the cell membranes. Red blood cell ghosts remained visible indicating that lysis was likely caused by pore formation and followed by osmotic disruption of the cell. (C) 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Capped acyclic permutants of the circular protein kalata B1

The cyclotides are a family of head-to-tail cyclized peptides that display exceptionally high stability and a range of biological activities. Acyclic permutants that contain a break in the circular backbone have been reported to be devoid of the haemolytic activity of the prototypic cyclotide kalata B1, but the potential role of the charges at the introduced termini in this loss of membraneolytic activity has not been fully determined. In this study, acyclic permutants of kalata B1 with capped N- and G termini were synthesized and found to adopt a native fold. These variants were observed to cause no measurable lysis of erythrocytes, strengthening the connection between backbone cyclization and haemolytic activity. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Cyclosporine A-induced changes to erythrocyte redox balance is time course-dependent

Cyclosporine A-treated transplant recipients develop pronounced cardiovascular disease and have increased oxidative stress and altered antioxidant capacity in erythrocytes and plasma. These experiments investigated the time-course of cyclosporine A-induced changes to redox balance in plasma and erythrocytes. Rats were randomly assigned to either a control or cyclosporine A-treated group. Treatment animals received 25 mg/kg of cyclosporine A via intraperitoneal injection for either 7 days or a single dose. Control rats were injected with the same volume of the vehicle. Three hours after the final injections, plasma was analysed for total antioxidant status, a-tocopherol, malondialdehyde, and creatinine. Erythrocytes were analysed for reduced glutathione (GSH), alpha-tocopherol, methaemoglobin, malondialdehyde, and the activities of superoxide dismutase, catalase, GSH peroxidase, and glucose-6-phosphate dehydrogenase (G6PD). Cyclosporine A administration for 7 days resulted in a significant increase (P < 0.05) in plasma malondialdehyde, methaemoglobin, and superoxide dismutase and catalase activities. There was a significant decrease (P < 0.05) in erythrocyte GSH concentration and G6PD activity in cyclosporine A animals. There were no significant differences (P > 0.05) between groups following a single dose of cyclosporine A in any of the measures. In summary, cyclosporine A alters erythrocyte redox balance after 7 days administration, but not after a single dose.

Cyclosporine A induced changes to plasma and erythrocyte antioxidant defences

Organ transplant recipients develop pronounced cardiovascular disease, and decreased antioxidant capacity in plasma and erythrocytes is associated with the pathogenesis of this disease. These experiments tested the hypothesis that the immunosuppressant cyclosporine A (CsA) alters erythrocyte redox balance and reduces plasma antioxidant capacity. Female Sprague-Dawley rats were randomly assigned to a control or CsA treated group. Treatment animals received 25 mg/kg/day of CsA via intraperitoneal injection for 18 days. Control rats were injected with the same volume of the vehicle. Three hours after the final CsA injection, rats were exsanguinated and plasma analysed for total antioxidant status (TAS), alpha-tocopherol, malondialdehyde (MDA), and creatinine. Erythrocytes were analysed for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glucose-6-phosphate dehydrogenase (G6PD) activities, alpha-tocopherol, and MDA. CsA administration resulted in a significant (P < 0.05) decrease in plasma TAS and significant increases (P < 0.05) in plasma creatinine and MDA. Erythrocyte CAT was significantly (P < 0.05) increased in CsA treated rats compared to controls. There were no significant differences (P > 0.05) in erythrocyte SOD, GPX, G6PD, alpha-tocopherol or MDA between groups. In summary, CsA alters erythrocyte antioxidant defence and decreases plasma total antioxidant capacity.

Parasitic isopods (Gnathia sp.) reduce haematocrit in captive blackeye thicklip (Labridae) on the Great Barrier Reef

Captive Hemigymnus melapterus exposed to large numbers of cultured juvenile parasitic isopods (Gnathia sp.) had significantly lower haematocrit (median 27-62% +/- 5-83% inter-quartile range) than uninfected, control fish (median 32-73% +/- 4-90%). This study is the first to show that juvenile Gnathia sp. reduce total blood volume in H. melapterus. The low haematocrit in infected fish was most likely due to plasma replacing erythrocytes lost as a result of isopods feeding on fish blood. (c) 2005 The Fisheries Society of the British Isles.

alpha-tocopherol and alpha-lipoic acid enhance the erythrocyte antioxidant defence in cyclosporine A-treated rats

The aim of this study was to determine the effects of dietary antioxidant supplementation with alpha-tocopherol and alpha-lipoic acid on cyclosporine A (cyclosporine)-induced alterations to erythrocyte and plasma redox balance. Rats were randomly assigned to either control, antioxidant (alpha-tocopherol 1000 IU/kg diet and alpha-lipoic acid 1.6 g/kg diet), cyclosporine (25 mg/kg/day), or cyclosporine + antioxidant treatments. Cyclosporine was administered for 7 days after an 8 week feeding period. Plasma was analysed for alpha-tocopherol, total antioxidant capacity, malondialdehyde, and creatinine. Erythrocytes were analysed for glutathione, methaemoglobin, superoxide dismutase, catalase, glutathione peroxidase, glucose-6-phosphate dehydrogenase, alpha-tocopherol and malondialdehye. Cyclosporine administration caused a significant decrease in superoxide dismutase activity (P < 0.05 control versus cyclosporine) and this was improved by antioxidant supplementation (P < 0.05 cyclosporine versus cyclosporine + antioxidant; P < 0.05 control versus cyclosporine + antioxidant). Animals receiving cyclosporine and antioxidants showed significantly increased (P < 0.05) catalase activity compared to both groups not receiving cyclosporine. Cyclosporine administration induced significant increases in plasma malondialdehyde and creatinine concentration (P < 0.05 control versus cyclosporine). Antioxidant supplementation prevented the cyclosporine induced increase in plasma creatinine (P < 0.05 cyclosporine versus cyclosporine + antioxidant; P > 0.05 control versus cyclosporine + antioxidant), however, supplementation did not alter the cyclosporine induced increase in plasma malondialdehyde concentration (P > 0.05 cyclosporine versus cyclosporine + antioxidant). Antioxidant supplementation resulted in significant increases (P < 0.05) in plasma and erythrocyte alpha-tocopherol in both of the supplemented groups compared to non-supplemented groups. In conclusion, dietary supplementation with alpha-tocopherol and alpha-lipoic acid enhanced the erythrocyte antioxidant defence and reduced nephrotoxicity in cyclosporine treated animals.

Antioxidant supplementation enhances erythrocyte antioxidant status and attenuates cyclosporine-induced vascular dysfunction

The aim of this study was to determine the effects of dietary antioxidant supplementation with a-tocopherol and a-lipoic acid on cyclosporine-induced alterations to erythrocyte and plasma redox balance, and cyclosporine-induced endothelial and smooth muscle dysfunction. Rats were randomly assigned to either control, antioxidant, cyclosporine or cyclosporine + antioxidant treatments. Cyclosporine A was administered for 10 days after an 8-week feeding period. Plasma was analyzed for alpha-tocopherol, total antioxidant capacity, malondialdehyde and creatinine. Erythrocytes were analyzed for glutathione, methemoglobin, superoxide dismutase, catalase, glutathione peroxidase, glucose-6-phosphate dehydrogenase, alpha-tocopherol and malondialdehye. Vascular endothelial and smooth muscle function was determined in vitro. Antioxidant supplementation resulted in significant increases in erythrocyte a-tocopherol concentration and glutathione peroxidase activity in both of the antioxidant-supplemented groups. Cyclosporine administration caused significant decreases in glutathione concentration, methemoglobin concentration and superoxide dismutase activity. Antioxidant supplementation attenuated the cyclosporine-induced decrease in superoxide dismutase activity. Cyclosporine therapy impaired both endothelium-independent and -dependent relaxation of the thoracic aorta, and this was attenuated by antioxidant supplementation. In summary, dietary supplementation with alpha-tocopherol and alpha-lipoic acid attenuated the cyclosporine-induced decrease in erythrocyte superoxide dismutase activity and attenuated cyclosporine-induced vascular dysfunction.