Investigation of Hydrogen Sulfide Gas as a Treatment against P. falciparum,Murine Cerebral Malaria,and the Importance of Thiolation State in the Development of Cerebral Malaria

Introduction

Cerebral malaria (CM) is a potentially fatal cerebrovascular disease of complex pathogenesis caused by Plasmodium falciparum. Hydrogen sulfide (HS) is a physiological gas, similar to nitric oxide and carbon monoxide, involved in cellular metabolism, vascular tension, inflammation, and cell death. HS treatment has shown promising results as a therapy for cardio- and neuro- pathology. This study investigates the effects of fast (NaHS) and slow (GYY4137) HS-releasing drugs on the growth and metabolism of P. falciparum and the development of P. berghei ANKA CM. Moreover, we investigate the role of free plasma thiols and cell surface thiols in the pathogenesis of CM.

Methods

P. falciparum was cultured in vitro with varying doses of HS releasing drugs compared with artesunate. Growth and metabolism were quantified. C57Bl/6 mice were infected with P. berghei ANKA and were treated with varying doses and regimes of HS-releasing drugs. Free plasma thiols and cell surface thiols were quantified in CM mice and age-matched healthy controls.

Results

HS-releasing drugs significantly and dose-dependently inhibited P. falciparum growth and metabolism. Treatment of CM did not affect P. berghei growth, or development of CM. Interestingly, CM was associated with lower free plasma thiols, reduced leukocyte+erythrocyte cell surface thiols (infection day 3), and markedly (5-fold) increased platelet cell surface thiols (infection day 7).

Conclusions

HS inhibits P. falciparum growth and metabolism in vitro. Reduction in free plasma thiols, cell surface thiols and a marked increase in platelet cell surface thiols are associated with development of CM. HS drugs were not effective in vivo against murine CM.     

Maintaining the immunological balance in parasitic infections: a role for TGF-beta?

Transforming growth factor beta (TGF-beta) is an important regulator of inflammation, being proinflammatory at low concentrations and anti-inflammatory at high concentrations. As such, TGF-beta might be important in maintaining the balance between control and clearance of infectious organisms on the one hand and prevention of immune-mediated pathology on the other. In this article, Fakhereldin Omer, J?rgen Kurtzhals and Eleanor Riley review the immunoregulatory properties of TGF-beta in the context of parasitic infections. Data from murine malaria infections suggest that TGF-beta modifies the severity of the disease, and a number of potential protective mechanisms are discussed. Evidence is accumulating that TGF-beta is important for the regulation of other host-parasite interactions and that parasites might directly influence TGF-beta-dependent pathways via the synthesis of TGF-beta or TGF-beta-receptor homologues.     

V beta profiles in African children with acute cerebral or uncomplicated malaria: very focused changes among a remarkable global stability

T cells are thought to play a critical role in cerebral malaria pathogenesis. However, available evidences are restricted to rodent models in which V beta specific T cell expansion has been associated with neurological syndrome suggesting involvement of superantigens or dominant antigens. Using flow cytometry, we studied the peripheral V beta T cell repertoire of Ghanaian children with cerebral malaria, uncomplicated malaria and asymptomatic control children, to look for either expansion or deletion of specific V beta associated with cerebral malaria. At admission, the general pattern of the repertoire of the patients was very similar, with no major distortion compared to the control group a part a significant increase of the frequency of the V beta 21.3 subset correlating with disease severity and attributed to the CD4 subset. During convalescence very limited fluctuations were observed including a significant decrease of the V beta 21.3 subset and increase of the V beta 20 subset, a subset not detected at admission. The remarkable stability of the V beta repertoire observed in acute malaria either cerebral or uncomplicated argues against the idea that cerebral malaria would result from a T cell-mediated inflammatory shock syndrome driven by some dominant super-antigenic activity(ies). The significance of the reproducible increase of the CD4+V beta 21.3T cell subset deserves further investigations.     

Bone marrow suppression and severe anaemia associated with persistent Plasmodium falciparum infection in African children with microscopically undetectable parasitaemia

In sub-Saharan Africa the highest overlap between malaria and HIV infections occurs in female adolescents. Yet control activities for these infections are directed to different target groups, using disparate channels. This reflects the lack of priority given to adolescents and the absence of an accepted framework for delivering health and health-related interventions to this high-risk group. In this paper it is argued that female adolescents require a continuum of care for malaria and HIV – prior to conception, during and after pregnancy and that this should be provided through adolescent services. The evidence for this conclusion is presented. A number of African countries are commencing to formulate and implement adolescent-friendly policies and services and disease control programs for malaria and HIV will need to locate their interventions within such programs to ensure widespread coverage of this important target group. Failure to prioritize adolescent health in this way will seriously limit the success of disease control programs for malaria and HIV prevention.     

Plasmodium falciparum variant surface antigen expression varies between isolates causing severe and nonsevere malaria and is modified by acquired immunity

In areas of endemic parasite transmission, protective immunity to Plasmodium falciparum malaria is acquired over several years with numerous disease episodes. Acquisition of Abs to parasite-encoded variant surface Ags (VSA) on the infected erythrocyte membrane is important in the development of immunity, as disease-causing parasites appear to be those not controlled by preexisting VSA-specific Abs. In this work we report that VSA expressed by parasites from young Ghanaian children with P. falciparum malaria were commonly and strongly recognized by plasma Abs from healthy children in the same area, whereas recognition of VSA expressed by parasites from older children was weaker and less frequent. Independent of this, parasites isolated from children with severe malaria (cerebral malaria and severe anemia) were better recognized by VSA-specific plasma Abs than parasites obtained from children with nonsevere disease. This was not due to a higher infection multiplicity in younger patients or in patients with severe disease. Our data suggest that acquisition of VSA-specific Ab responses gradually restricts the VSA repertoire that is compatible with parasite survival in the semi-immune host. This appears to limit the risk of severe disease by discriminating against the expression of VSA likely to cause life-threatening complications, such as cerebral malaria and severe anemia. Such VSA seem to be preferred by parasites infecting a nonimmune host, suggesting that VSA expression and switching are not random, and that the VSA expression pattern is modulated by immunity. This opens the possibility of developing morbidity-reducing vaccines targeting a limited subset of common and particularly virulent VSA.     

Insights into deregulated TNF and IL-10 production in malaria: implications for understanding severe malarial anaemia

ABSTRACT: BACKGROUND: Severe malarial anaemia (SMA) is a major life-threatening complication of paediatric malaria. Protracted production of pro-inflammatory cytokines promoting erythrophagocytosis and depressing erythropoiesis is thought to play an important role in SMA, which is characterized by a high TNF/IL-10 ratio. Whether this TNF/IL-10 imbalance results from an intrinsic incapacity of SMA patients to produce IL-10 or from an IL-10 unresponsiveness to infection is unknown. Monocytes and T cells are recognized as the main sources of TNF and IL-10 in vivo, but little is known about the activation status of those cells in SMA patients. METHODS: The IL-10 and TNF production capacity and the activation phenotype of monocytes and T cells were compared in samples collected from 332 Ghanaian children with non-overlapping SMA (n = 108), cerebral malaria (CM) (n = 144) or uncomplicated malaria (UM) (n = 80) syndromes. Activation status of monocytes and T cells was ascertained by measuring HLADR + and/or CD69+ surface expression by flow cytometry. The TNF and IL-10 production was assessed in a whole-blood assay after or not stimulation with lipopolysaccharide (LPS) or phytohaemaglutinin (PHA) used as surrogate of unspecific monocyte and T cell stimulant. The number of circulating pigmented monocytes was also determined. RESULTS: Monocytes and T cells from SMA and CM patients showed similar activation profiles with a comparable decreased HLA-DR expression on monocytes and increased frequency of CD69+ and HLA-DR + T cells. In contrast, the acute-phase IL-10 production was markedly decreased in SMA compared to CM (P = .003) and UM (P = .004). Although in SMA the IL- 10 response to LPS-stimulation was larger in amplitude than in CM (P = .0082), the absolute levels of IL-10 reached were lower (P = .013). Both the amplitude and levels of TNF produced in response to LPS-stimulation were larger in SMA than CM (P = .019). In response to PHA-stimulation, absolute levels of IL-10 produced in SMA were lower than in CM (P = .005) contrasting with TNF levels, which were higher (P = .01). CONCLUSIONS: These data reveal that SMA patients have the potential to mount efficient IL-10 responses and that the TNF/IL-10 imbalance may reflect a specific monocyte and T cell programming/polarization pattern in response to infection.     

Plasmodium berghei ANKA: erythropoietin activates neural stem cells in an experimental cerebral malaria model

Cerebral malaria (CM) causes substantial mortality and neurological sequelae in survivors, and no neuroprotective regimens are currently available for this condition. Erythropoietin (EPO) reduces neuropathology and improves survival in murine CM. Using the Plasmodium berghei model of CM, we investigated if EPO’s neuroprotective effects include activation of endogenous neural stem cells (NSC). By using immunohistochemical markers of different NSC maturation stages, we show that EPO increased the number of nestin⁺ cells in the dentate gyrus and in the sub-ventricular zone of the lateral ventricles, relative to control-treatment. 75% of the EPO-treated CM mice displayed migration as nestin⁺ NSC. The NSC showed differentiation towards a neural cell lineage as shown by PSA-NCAM binding and NSC maturation and lineage commitment was significantly affected by exogenous EPO and by CM in the sub ventricular zone. These results indicate a rapid, EPO-dependent activation of NSC during CM pathology.     

In-depth validation of acridine orange staining for flow cytometric parasite and reticulocyte enumeration in an experimental model using Plasmodium berghei

Flow cytometry is potentially an effective method for counting malaria parasites, but inconsistent results have hampered its routine use in rodent models. A published two-channel method using acridine orange offers clear discrimination between the infected and uninfected erythrocytes. However, preliminary studies showed concerns when dealing with Plasmodium berghei-infected blood samples with high numbers of reticulocytes.In hyperparasitemic or chronic P. berghei infection, enhanced erythropoietic activity results in high numbers of circulating immature reticulocytes. We show that even though the protocol offered good discrimination in newly infected animals, discrimination between infected erythrocytes and uninfected reticulocytes became difficult in animals with hyperparasitemia or chronic infections maintained with subcurative treatment. Discrimination was especially hampered by increased nucleic acid content in immature uninfected reticulocytes. Our data confirms that though flow cytometry is a promising analytical tool in malaria research, care should still be taken when analysing samples from anemic or chronically infected animals.