P. falciparum Modulates Erythroblast Cell Gene Expression in Signaling and Erythrocyte Production Pathways

Global, genomic responses of erythrocytes to infectious agents have been difficult to measure because these cells are e-nucleated. We have previously demonstrated that in vitro matured, nucleated erythroblast cells at the orthochromatic stage can be efficiently infected by the human malaria parasite Plasmodium falciparum. We now show that infection of orthochromatic cells induces change in 609 host genes. 592 of these transcripts are up-regulated and associated with metabolic and chaperone pathways unique to P. falciparum infection, as well as a wide range of signaling pathways that are also induced in related apicomplexan infections of mouse hepatocytes or human fibroblast cells. Our data additionally show that polychromatophilic cells, which precede the orthochromatic stage and are not infected when co-cultured with P. falciparum, up-regulate a small set of genes, at least two of which are associated with pathways of hematopoiesis and/or erythroid cell development. These data support the idea that P. falciparum affects erythropoiesis at multiple stages during erythroblast differentiation. Further P. falciparum may modulate gene expression in bystander erythroblasts and thus influence pathways of erythrocyte development. This study provides a benchmark of the host erythroblast cell response to infection by P. falciparum

Differential gene expression mediated by 15-hydroxyeicosatetraenoic acid in LPS-stimulated RAW 264.7 cells

<p>Abstract</p> <p>Background</p> <p>Given the immuno-modulatory activity of native haemozoin (Hz), the effects of constitutive Hz components on immune response are of interest. Recently, gene expression changes mediated by HNE and the synthetic analogue of Hz, beta-haematin (BH), were identified and implicated a significant role for lipid peroxidation products in Hz's activity. The study presented herein examines gene expression changes in response to 15(S)-hydroxyeicosatetraenoic acid (HETE) in a model macrophage cell line.</p> <p>Methods</p> <p>LPS-stimulated RAW 264.7 macrophage-like cells were treated with 40 μM 15(S)-HETE for 24 h, and microarray analysis was used to identify global gene expression alterations. Fold changes were calculated relative to LPS-stimulated cells and those genes altered at least 1.8-fold (<it>p </it>value ≤ 0.025) were considered to be differentially expressed. Expression levels of a subset of genes were assessed by qRT-PCR and used to confirm the microarray results.</p> <p>Results</p> <p>Network analysis revealed that altered genes were primarily associated with "lipid metabolism" and "small molecule biochemistry". While several genes associated with PPAR-gamma receptor-mediated signaling were differentially expressed, a number of genes indicated the activation of secondary signaling cascades. Genes related to cytoadherence (cell-cell and cell-matrix), leukocyte extravasation, and inflammatory response were also differentially regulated by treatment, supporting a potential role for 15(S)-HETE in malaria pathogenesis.</p> <p>Conclusion</p> <p>These results add insight and detail to 15-HETE's effects on gene expression in macrophage-like cells. Data indicate that while 15-HETE exerts biological activity and may participate in Hz-mediated immuno-modulation, the gene expression changes are modest relative to those altered by the lipid peroxidation product HNE.</p

Synthetic Plasmodium-Like Hemozoin Activates the Immune Response: A Morphology - Function Study

Increasing evidence points to an important role for hemozoin (HZ), the malaria pigment, in the immunopathology related to this infection. However, there is no consensus as to whether HZ exerts its immunostimulatory activity in absence of other parasite or host components. Contamination of native HZ preparations and the lack of a unified protocol to produce crystals that mimic those of Plasmodium HZ (PHZ) are major technical limitants when performing functional studies with HZ. In fact, the most commonly used methods generate a heterogeneous nanocrystalline material. Thus, it is likely that such aggregates do not resemble to PHZ and differ in their inflammatory properties. To address this issue, the present study was designed to establish whether synthetic HZ (sHZ) crystals produced by different methods vary in their morphology and in their ability to activate immune responses. We report a new method of HZ synthesis (the precise aqueous acid-catalyzed method) that yields homogeneous sHZ crystals (Plasmodium-like HZ) which are very similar to PHZ in their size and physicochemical properties. Importantly, these crystals are devoid of protein and DNA contamination. Of interest, structure-function studies revealed that the size and shape of the synthetic crystals influences their ability to activate inflammatory responses (e.g. nitric oxide, chemokine and cytokine mRNA) in vitro and in vivo. In summary, our data confirm that sHZ possesses immunostimulatory properties and underline the importance of verifying by electron microscopy both the morphology and homogeneity of the synthetic crystals to ensure that they closely resemble those of the parasite. Periodic quality control experiments and unification of the method of HZ synthesis are key steps to unravel the role of HZ in malaria immunopathology

Neutrophil Paralysis in Plasmodium vivax Malaria

Plasmodium vivax is responsible for approximately 60–80% of the malaria cases in the world, and contributes to significant social and economic instability in the developing countries of Latin America and Asia. The pathogenesis of P. vivax malaria is a consequence of host derived inflammatory mediators. Hence, a better understanding of the mechanisms involved in induction of systemic inflammation during P. vivax malaria is critical for the clinical management and prevention of severe disease. The innate immune receptors recognize Plasmodium sp. and initiate a broad spectrum of host defense mechanisms that mediate resistance to infection. However, the innate immune response is the classic “two-edged sword”, and clinical malaria is associated with high levels of circulating pro-inflammatory cytokines. Our findings show that both monocytes and neutrophils are highly activated during malaria. Monocytes produced high levels of IL-1β, IL-6 and TNF-α during acute malaria. On the other hand, neutrophils were a poor source of cytokines, but displayed an enhanced phagocytic activity and superoxide production. Unexpectedly, we noticed an impaired chemotaxis of neutrophils towards an IL-8 (CXCL8) gradient. We proposed that neutrophil paralysis is in part responsible for the enhanced susceptibility to bacterial infection observed in malaria patients

Naturally Occurring Triggers that Induce Apoptosis-Like Programmed Cell Death in Plasmodium berghei Ookinetes

Several protozoan parasites have been shown to undergo a form of programmed cell death that exhibits morphological features associated with metazoan apoptosis. These include the rodent malaria parasite, Plasmodium berghei. Malaria zygotes develop in the mosquito midgut lumen, forming motile ookinetes. Up to 50% of these exhibit phenotypic markers of apoptosis; as do those grown in culture. We hypothesised that naturally occurring signals induce many ookinetes to undergo apoptosis before midgut traversal. To determine whether nitric oxide and reactive oxygen species act as such triggers, ookinetes were cultured with donors of these molecules. Exposure to the nitric oxide donor SNP induced a significant increase in ookinetes with condensed nuclear chromatin, activated caspase-like molecules and translocation of phosphatidylserine that was dose and time related. Results from an assay that detects the potential-dependent accumulation of aggregates of JC-1 in mitochondria suggested that nitric oxide does not operate via loss of mitochondrial membrane potential. L-DOPA (reactive oxygen species donor) also caused apoptosis in a dose and time dependent manner. Removal of white blood cells significantly decreased ookinetes exhibiting a marker of apoptosis in vitro. Inhibition of the activity of nitric oxide synthase in the mosquito midgut epithelium using L-NAME significantly decreased the proportion of apoptotic ookinetes and increased the number of oocysts that developed. Introduction of a nitric oxide donor into the blood meal had no effect on mosquito longevity but did reduce prevalence and intensity of infection. Thus, nitric oxide and reactive oxygen species are triggers of apoptosis in Plasmodium ookinetes. They occur naturally in the mosquito midgut lumen, sourced from infected blood and mosquito tissue. Up regulation of mosquito nitric oxide synthase activity has potential as a transmission blocking strategy

Induction of oxidative stress in human aqueous and vitreous humors by Nd:YAG laser posterior capsulotomy

AIM: To evaluate whether the Q-switched Nd:YAG laser treatment applied in routine capsulotomy elicits oxidative stress in aqueous and vitreous humors. METHODS: Thirty-six patients who had to undergo a 25 gauge pars plana vitrectomy due to vitreoretinal disorders were enrolled, 15 of them underwent a Q-switched Nd:YAG laser capsulotomy 7d before vitrectomy due to posterior capsule opacification (PCO) (Nd:YAG laser group) while the remaining 21 patients were not laser treated before vitrectomy (no Nd:YAG laser group). Samples of the aqueous and vitreous humors were collected during vitrectomy from all patients for the assessment of oxidative parameters which were compared between the Nd:YAG laser group and no Nd:YAG laser group. Thiobarbituric acid reactive substances (TBARS), a product of membrane lipid peroxidation, nitrite levels, the antioxidative activities of SOD and catalase, the 4-HNE-protein conjugate formation, indicating structural modifications in proteins due to lipoperoxidation, were assessed in aqueous and vitreous samples. RESULTS: In the human vitreous humor TBARS levels are significantly higher in the Nd:YAG laser group compared to the no Nd:YAG laser group and importantly, there is a significant correlation between the TBARS levels and the total energy of Nd:YAG laser used during capsulotomy. Moreover the anti-oxidative activities of SOD and catalase were significantly decreased by Nd:YAG laser treatment, both in aqueous and vitreous humors. In accordance with the TBARS data and anti-oxidative enzyme activities, significantly higher levels of proteins were conjugated with the lipoperoxidation product 4-HNE in the aqueous and vitreous humors in the Nd:YAG laser-treated group in comparison to no Nd:YAG laser group. CONCLUSION: These data, clearly suggest that any change that Q-switched Nd:YAG photo disruption may cause in the aqueous and vitreous compartments, resulting in a higher level of oxidative damage might be of considerable clinical significance particularly by accelerating the aging of the anterior and posterior segments of the eye and by worsening the intraocular pressure, the uveal, the retinal (especially macular) pathologies

Transfer of 4-hydroxynonenal from parasitized to non-parasitized erythrocytes in rosettes. Proposed role in severe malaria anemia.

Severe anaemia is a life-threatening complication of falciparum malaria associated with loss of predominantly non-parasitized red blood cells (npRBCs). This poorly elucidated process might be influenced by (i) rosettes, i.e. npRBCs cytoadherent to haemozoin-containing parasitized RBCs (pRBCs) and (ii) generation in pRBCs of 4-hydroxynonenal (4-HNE) through haemozoin-catalysed lipid peroxidation. We explored whether close proximity in rosettes may facilitate 4-HNE transfer to npRBCs, which is likely to enhance their phagocytosis and contribute to malaria anaemia. Fluorescence microscopy and flow cytometry data indicated 4-HNE transfer to npRBCs in rosettes. Rosettes were formed by 64·8 ± 1·8% varO-expressing pRBCs, and 8·7 ± 1·1% npRBCs were positive for 4-HNE-protein-conjugates, while low-rosetting parasites generated only 2·4 ± 1·1% 4-HNE-conjugate-positive npRBCs. 4-HNE transfer decreased after blocking rosetting by monoclonal antibodies. A positive linear relationship between rosette frequency and 4-HNE-conjugates in npRBCs was found in 40 malaria patients, a first indication for a role of rosetting in npRBCs modifications in vivo. Children with severe malaria anaemia had significantly higher percentages of 4-HNE-conjugate-positive npRBCs compared to children with uncomplicated malaria. In conclusion, 4-HNE transfer from pRBCs to npRBCs in rosettes is suggested to play a role in the phagocytic removal of large numbers of npRBCs, the hallmark of severe malaria anaemia