Biosynthesized nanosilver as anti-oxidant,anti-apoptotic and anti-inflammatory agent against Plasmodium chabaudi infection in the mouse liver

In recent years, the use of plant-mediated nanoparticle synthesis to combat infectious diseases has become increasingly significant. Malaria is one of the world's most infectious diseases caused by Plasmodium species. The antioxidant, anti-apoptotic, and anti-inflammatory properties of nanosilver biosynthesized from Indigofera oblongifolia leaf extracts (NS) against Plasmodium chabaudi infection of the mouse liver were investigated in this research. Male mice were infected with P. chabaudi infected erythrocytes then treated with NS for 7 days. The parasitemia was suppressed by approximately 24, 28, 47 and 75% on days 4, 5, 6 and 7 postinfection, respectively after treatment of mice with NS. Also, NS was able to regulate the leucocytes count and the IL1β and TNF-α-mRNA expression in mice. Ns could increase the antioxidant activity in liver of mice and was able to regulate the apoptotic genes, Bcl2 and Casp3. We showed that NS has antioxidant, anti-apoptotic, and anti-inflammatory properties when it was used to treat the livers of mice infected with P. chabaudi.     

Indigofera oblongifolia leaf extract regulates spleen macrophage response during Plasmodium chabaudi infection

Malaria is a major health problem that still affects numerous countries. The current study aimed to identify the role of Indigofera oblongifolia leaf extract in regulating mouse spleen macrophages during the progression of Plasmodium chabaudi infection. Three doses of the leaf extract (100, 200, and 300 mg/kg) were administered to mice inoculated with P. chabaudi infected erythrocytes. The weight of the infected mice improved after the treatment with I. oblongifolia. The infection causes disorganization of macrophage distribution in the spleen. After the mice had been treated with the leaf extract, the macrophages appeared to be reorganized in the white and red pulp areas. In addition, the I. oblongifolia leaf extract (IOLE) significantly increased the total antioxidant capacity of the mice spleens infected with P. chabaudi. The phagocytic activity of spleen macrophages was increased in the infected group as indicated by the significant decrease in the number of fluorescent particles in the spleen sections. This number increased in the mice spleens after treatment with IOLE. Based on these results, it is suggested that IOLE regulate macrophage response of the spleen during the blood stage of malaria in mice.     

Mosquito mortality and the evolution of malaria virulence

Abstract Several laboratory studies of malaria parasites (Plasmodium sp.) and some field observations suggest that parasite virulence, defined as the harm a parasite causes to its vertebrate host, is positively correlated with transmission. Given this advantage, what limits the continual evolution of higher parasite virulence? One possibility is that while more virulent strains are more infectious, they are also more lethal to mosquitoes. In this study, we tested whether the virulence of the rodent malaria parasite P. chabaudi in the laboratory mouse was correlated with the fitness of mosquitoes it subsequently infected. Mice were infected with one of seven genetically distinct clones of P. chabaudi that differ in virulence. Weight loss and anemia in infected mice were monitored for 16–17 days before Anopheles stephensi mosquitoes were allowed to take a blood meal from them. Infection virulence in mice was positively correlated with transmission to mosquitoes (infection rate) and weakly associated with parasite burden (number of oocysts). Mosquito survival fell with increasing oocyst burden, but there was no overall statistically significant relationship between virulence in mice and mosquito mortality. Thus, there was no evidence that more virulent strains are more lethal to mosquitoes. Both vector survival and fecundity depended on parasite clone, and contrary to expectations, mosquitoes fed on infections more virulent to mice were more fecund. The strong parasite genetic effects associated with both fecundity and survival suggests that vector fitness could be an important selective agent shaping malaria population genetics and the evolution of phenotypes such as virulence in the vector.     

Double-drug development against antioxidant enzymes from Plasmodium falciparum

Abstract

New drugs against malaria are urgently and continuously needed. Plasmodium parasites are exposed to higher fluxes of reactive oxygen species and need high activities of intracellular antioxidant systems. A most important antioxidative system consists of (di)thiols which are recycled by disulfide reductases (DR), namely both glutathione reductases (GR) of the malarial parasite Plasmodium falciparum and man, and the thioredoxin reductase (TrxR) of P. falciparum. The aim of our interdisciplinary research is to substantiate DR inhibitors as antimalarial agents. Such compounds are active per se but, in addition, they can reverse thiol-based resistance against other drugs in parasites. Reversal of drug resistance by DR inhibitors is currently investigated for the commonly used antimalarial drug chloroquine (CQ). Our recent strategy is based on the synthesis of inhibitors of the glutathione reductases from parasite and host erythrocyte. With the expectation of a synergistic or additive effect, double-headed prodrugs were designed to be directed against two different and essential functions of the malarial parasite P. falciparum, namely glutathione regeneration and heme detoxification. The prodrugs were prepared by linking bioreversibly a GR inhibitor to a 4-aminoquinoline moiety which is known to concentrate in the acidic food vacuole of parasites. Drug-enzyme interaction was correlated with antiparasitic action in vitro on strains resistant towards CQ and in vivo in Plasmodium berghei-infected mice as well as absence of cytotoxicity towards human cells. Because TrxR of P. falciparum was recently shown to be responsible for the residual glutathione disulfide-reducing capacity observed after GR inhibition in P. falciparum, future development of antimalarial drug-candidates that act by perturbing the redox equilibrium of parasites is based on the design of new double-drugs based on TrxR inhibitors as potential antimalarial drug candidates.     

Haptoglobin and malaria

Abstract

Haptoglobin gene knockout mice and wild-type controls were infected with Plasmodium berghei ANKA or Plasmodium chabaudi. The peak parasitaemia and parasite burden were higher in Hp^-/- mice than in Hp^+/+ mice. The increase in spleen weight following malaria infection was smaller in Hp^-/- mice than in Hp^+/+ animals. The occurrence of cerebral malaria in P. berghei ANKA infection was not different in Hp gene knockout mice and their controls.     

Ameliorative role of Ziziphus spina-christi leaf extracts against hepatic injury induced by Plasmodium chabaudi infected erythrocytes

One of the most common deadliest parasitic diseases is Malaria. The biology and the pathogenesis of this fascinating parasite are not yet fully understood which make discovering effective alternative drugs a challenging task. Moreover, the emergence of resistant strains added an additional burden in the journey of malaria elimination. Traditional medicine used to be an alternative therapy choice owing to the presence of potent natural products. Ziziphus spina-christi (L.) considered being one of the common potent natural plant in gulf region and other nations. Therefore, this study designed to evaluate the ameliorative role of Z. spina-christi leaf extracts (ZSCLE) against Plasmodium chabaudi-induced hepatic injury. The study involved three groups were as follows; a vehicle control group, infected with 10⁶P. chabaudi-parasitized erythrocytes group and ZSCLE treated-infected mice with 10⁶P. chabaudi-parasitized erythrocytes group. The results showed a remarkable reduction of parasitemia level and notable reverse of the anemic picture among ZSCLE treated-infected mice. The effects of ZSCLE on the liver functions enzymes and on the histopathological pictures of liver were significant. It could be concluded that Z. spina-christi leaf extracts have a protective role against Plasmodium infection that also marked through significant restoration of hepatic oxidative markers.     

On the contribution of the rodent model Plasmodium chabaudi for understanding the genetics of drug resistance in malaria

Malaria is a devastating disease that still claims over half a million lives every year, mostly in sub–Saharan Africa. One of the main barriers to malaria control is the evolution and propagation of drug-resistant mutant parasites. Knowing the genes and respective mutations responsible for drug resistance facilitates the design of drugs with novel modes of action and allows predicting and monitoring drug resistance in natural parasite populations in real-time. The best way to identify these mutations is to experimentally evolve resistance to the drug in question and then comparing the genomes of the drug-resistant mutants to that of the sensitive progenitor parasites. This simple evolutive concept was the starting point for the development of a paradigm over the years, based on the use of the rodent malaria parasite Plasmodium chabaudi to unravel the genetics of drug resistance in malaria. It involves the use of a cloned parasite isolate (P. chabaudi AS) whose genome is well characterized, to artificially select resistance to given drugs through serial passages in mice under slowly increasing drug pressure. The end resulting parasites are cloned and the genetic mutations are then discovered through Linkage Group Selection, a technique conceived by Prof. Richard Carter and his group, and/or Whole Genome Sequencing. The precise role of these mutations can then be interrogated in malaria parasites of humans through allelic replacement experiments and/or genotype-phenotype association studies in natural parasite populations. Using this paradigm, all the mutations underlying resistance to the most important antimalarial drugs were identified, most of which were pioneering and later shown to also play a role in drug resistance in natural infections of human malaria parasites. This supports the use of P. chabaudi a fast-track predictive model to identify candidate genetic markers of resistance to present and future antimalarial drugs and improving our understanding of the biology of resistance.     

Wave expansion of CD34 progenitor cells in the spleen in rodent malaria

Defense against malaria depends upon amplification of the spleen structure and function for the clearance of parasitized red blood cells (pRBC). We studied the distribution and amount of CD34⁺ cells in the spleens of mice infected with rodent malaria. We sought to identify these cells in the spleen and determine their relationship to infection. C57BL/6J mice were infected with self-resolving, Plasmodium chabaudi CR, or one of the lethal rodent malaria strains, P. chabaudi AJ and P. berghei ANKA. We then recorded parasitemia, mortality, and the presence of CD34⁺ cells in spleen, as determined by immunohistochemistry and flow cytometry. In the non-lethal strain, the spleen structure was maintained during amplification, but disrupted in lethal models. The abundance of CD34⁺ cells increased in the red pulp on the 4th and 6th days p.i. in all models, and subsided on the 8th day p.i. Faint CD34⁺ staining on the 8th day p.i., was probably due to differentiation of committed cell lineages. In this work, increase of spleen CD34⁺ cells did not correlate with infection control.     

Detection and cartography of the fluorinated antimalarial drug mefloquine in normal and Plasmodium falciparum infected red blood cells by scanning ion microscopy and mass spectrometry

Summary— Due to the presence of fluorine atoms in its molecule, the antimalarial drug mefloquine (MQ) can be easily detected in normal and Plasmodium falciparum infected red blood cells (RBC) by scanning ion microscopy and mass spectrometry. The P falciparum infected RBC exhibited intense distribution of MQ inside the parasite. The main compartments of the parasite which accumulate the drug were the food vacuole and the cytoplasm. The correlation between fluorine (¹⁹F^?) and phosphorus (³¹P^?) as well as probes for the DNA synthesis (BrdU and IdU) emissions shows that the parasite nucleus is also accessible to the drug. This study demonstrates that SIMS technique on smear preparations is an efficient approach for the direct detection and cartography of fluorinated antimalarial drugs in normal and P falciparum infected RBC, without radioactive labelling.     

Classical CD11c dendritic cells, not plasmacytoid dendritic cells, induce T cell responses to Plasmodium chabaudi malaria

Dendritic cells play an important role in the development of immune responses in malaria, but the contribution of plasmacytoid dendritic cells (pDC) to CD4 T cell activation and immunopathology is unknown. We have investigated pDC in a Plasmodium chabaudi infection in mice. During infection, pDC increased in number and transiently up-regulated expression of Major Histocompatibility Complex class II and co-stimulatory molecules. However, in contrast to classical CD11c^high DC, pDC could not phagocytose parasites or process parasite proteins, to activate CD4 T cells. Activation of naïve pDC, but not CD11c^high DC, by infected red blood cells induced IFNα in vitro, which was dependent on the Toll-like receptor, TLR9. However, inactivation of TLR9 in knock-out mice had no effect on a P. chabaudi infection suggesting that TLR9 was not crucial for parasite elimination or pathology. Neither pDC nor IFNαβ were essential for parasite clearance as mice depleted of pDC or IFNαβ Receptor-knock-out mice could control infection. However, these mice lost significantly more weight than untreated or wild-type mice. We conclude that classical DC are the major antigen-presenting cells for CD4 T cells in this infection, but that pDC and IFNαβ may play minor roles in controlling the magnitude of acute stage pathology.     

Glutathione is involved in the antimalarial action of chloroquine and its modulation affects drug sensitivity of human and murine species of Plasmodium

Abstract

Ferriprotoporphyrin IX (FP) is released inside the food vacuole of the malaria parasite during the digestion of host cell hemoglobin. FP is detoxified by its biomineralization to hemozoin. This process is effectively inhibited by chloroquine (CQ) and amodiaquine (AQ). Undegraded FP accumulates in the membrane fraction and inhibits enzymes of infected cells in parallel with parasite killing. FP is demonstrably degraded by reduced glutathione (GSH) in a radical-mediated mechanism. This degradation is inhibited by CQ and AQ in a competitive manner, thus explaining the ability of increased GSH levels in Plasmodium falciparum-infected cells to increase resistance to CQ and vice versa, and to render Plasmodium berghei that were selected for CQ resistance in vivo sensitive to the CQ when glutathione synthesis is inhibited. Some over-the-counter drugs that are known to reduce GSH in body tissues when used in excess were found to enhance the antimalarial action of CQ and AQ in mice infected either with P. berghei or Plasmodium vinckei. In contrast, N-acetyl-cysteine which is expected to increase the cellular levels of GSH, antagonized the action of CQ. These results suggest that some over-the-counter drugs can be used in combination with some antimalarials to which the parasite has become resistant.     

Cysteamine, the natural metabolite of pantetheinase, shows specific activity against Plasmodium

In mice, loss of pantetheinase activity causes susceptibility to infection with Plasmodium chabaudi AS. Treatment of mice with the pantetheinase metabolite cysteamine reduces blood-stage replication of P. chabaudi and significantly increases survival. Similarly, a short exposure of Plasmodium to cysteamine ex vivo is sufficient to suppress parasite infectivity in vivo. This effect of cysteamine is specific and not observed with a related thiol (dimercaptosuccinic acid) or with the pantethine precursor of cysteamine. Also, cysteamine does not protect against infection with the parasite Trypanosoma cruzi or the fungal pathogen Candida albicans, suggesting cysteamine acts directly against the parasite and does not modulate host inflammatory response. Cysteamine exposure also blocks replication of P. falciparum in vitro; moreover, these treated parasites show higher levels of intact hemoglobin. This study highlights the in vivo action of cysteamine against Plasmodium and provides further evidence for the involvement of pantetheinase in host response to this infection.     

Beyond Schuh: early studies on the oxidation of LDL and other lipoproteins and its role in atherosclerosis

Abstract

The malaria parasite Plasmodium falciparum is still a major threat to human health in the non-industrialised world mainly due to the increasing incidence of drug resistance. Therefore, there is an urgent need to identify and validate new potential drug targets in the parasite's metabolism that are suitable for the design of new anti-malarial drugs. It is known that infection with P. falciparum leads to increased oxidative stress in red blood cells, implying that the parasite requires efficient antioxidant and redox systems to prevent damage caused by reactive oxygen species. In recent years, it has been shown that P. falciparum possess functional thioredoxin and glutathione systems. Using genetic and chemical tools, it was demonstrated that thioredoxin reductase, the first step of the thioredoxin redox cycle, and γ-glutamylcysteine synthetase (γ-GCS), the rate-limiting step of glutathione synthesis, are essential for parasite survival. Indeed, the mRNA levels of γ-GCS are elevated in parasites that are oxidatively stressed, indicating that glutathione plays an important antioxidant role in P. falciparum. In addition to this antioxidant function, glutathione is important for detoxification processes and is possibly involved in the development of resistance against drugs such as chloroquine.     

Plasmodium chabaudi: Antigenic variation during recrudescent parasitaemias in mice

The A/S strain of Plasmodium chabaudi at different times was twice mosquito passaged and cloned by limiting dilution. Large groups of NIH mice were infected with 10⁵ parasitized red cells of populations of parasites which were considered to be identical or very similar to the population forming the first erythrocytic parasitaemia seen in mice after mosquito transmission of the parasite. Most of the mice were killed immediately after the first patent parasitaemia had become subpatent and their sera pooled. The parasitaemias of surviving mice were followed until recrudescences appeared. The protective activity of the immune serum was then tested against the original infecting population and recrudescent populations by passive transfer tests in naive mice. Protection was measured as a delay in patent parasitaemia reaching 2% compared with normal serum recipients. The immune serum significantly delayed the 2% parasitaemia but in different experiments six out of seven recrudescent populations were found to be less sensitive to the effects of the immune serum than the original infecting population. The recrudescent populations retained their reduced or total insensitivity to the action of the immune serum after two blood passages and after eryopreservation. It appears, therefore, that P. chabaudi can undergo antigenic variation.     

Effect of gold nanoparticles on mice splenomegaly induced by schistosomiasis mansoni

Schistosomiasis is still one of the main parasitic diseases that affect human health in tropical regions. Whilst praziquantel (PZQ) is the main classic antischistosomal drug, the need for new drugs is still a must due to the low effectiveness of the drug on the schistosome young worms, and the evolving of PZQ resistant strains. Nanotechnology is one of the most important recent and current methods used to treat human diseases including parasitic ones. Therefore, the present study aimed to examine the curative role of gold nanoparticles (GNPs) on splenic tissue of mice infected with Schistosoma mansoni Sambon, 1907. High-resolution transmission electron microscopy was used for characterization of nanoparticles (NP). GNPs of 1 mg/kg mice body weight were inoculated into mice infected with S. mansoni. The parasite caused deteriorations in histological architecture of the spleen tissue, and splenomegaly. Additionally, the parasite induced a significant reduction in splenic tissue glutathione levels; however, the concentrations of nitric oxide and malondialdehyde were significantly increased. Treatment of mice with GNPs reduced the extent of histological impairment and oxidative stress in spleen tissue. Therefore, our results demonstrate the protective role of GNPs against splenic damage in mice infected with S. mansoni.     

Antimalarial activity of endoperoxide compound 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol

Plasmodium falciparum, the major causative parasite for the disease, has acquired resistance to most of the antimalarial drugs used today, presenting an immediate need for new antimalarial drugs. Here, we report the in vitro and in vivo antimalarial activities of 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol (N-251) against P. falciparum and Plasmodium berghei parasites. The N-251 showed high antimalarial potencies both in the in vitro and the in vivo tests (EC₅₀ 2.3 × 10⁻⁸ M; ED₅₀ 15 mg/kg (per oral)). The potencies were similar to that of artemisinin in vitro and greater than artemisinin's activity in vivo (p.o.). In addition, N-251 has little toxicity: a single oral administration at 2000 mg/kg to a rat gave no health problems to it. Administration of N-251 to mice bearing 1% of parasitemia (per oral 68 mg/kg, 3 times a day for 3 consecutive days) resulted in a dramatic decrease in the parasitemia: all the 5 mice given N-251 were cured without any recurrence, with no diarrhea or weight loss occurring in the 60 days of experiment. N-251 deserves more extensive clinical evaluation, desirably including future trials in the human.     

Plasmodium chabaudi: Adoptive transfer of immunity with different spleen cell populations and development of protective activity in the serum of lethally irradiated recipient mice

Groups of lethally X-irradiated NIH mice were injected with either glass wool-filtered (g.w.) immune spleen cells or nylon wool enriched immune T cells from syngeneic mice immune to Plasmodium chabaudi, or g.w. normal spleen cells. After cell recipients were infected with P. chabaudi the three groups reached similar mean peak parasitaemias on Day 11. In passive transfer tests serum obtained from mice sacrificed at this time gave little protection compared to normal serum. On Day 14 g.w. immune spleen cell recipients had subpatent infections and enriched immune T-cell recipients had a lower mean parasitaemia than g.w. normal spleen cell recipients. Serum obtained on Day 14 from g.w. immune spleen cell recipients gave better protection after passive transfer than sera from enriched immune T-cell or g.w. normal spleen cell recipients. Day 14 serum from enriched immune T-cell recipients, but not from g.w. normal spleen cell recipients, produced some initial protection after passive transfer. These results suggest that the transferred immune spleen cells contributed to the observed humoral immunity in lethally irradiated recipient mice.     

Double-drug development against antioxidant enzymes from Plasmodium falciparum

New drugs against malaria are urgently and continuously needed. Plasmodium parasites are exposed to higher fluxes of reactive oxygen species and need high activities of intracellular antioxidant systems. A most important antioxidative system consists of (di)thiols which are recycled by disulfide reductases (DR), namely both glutathione reductases (GR) of the malarial parasite Plasmodium falciparum and man, and the thioredoxin reductase (TrxR) of P. falciparum. The aim of our interdisciplinary research is to substantiate DR inhibitors as antimalarial agents. Such compounds are active per se but, in addition, they can reverse thiol-based resistance against other drugs in parasites. Reversal of drug resistance by DR inhibitors is currently investigated for the commonly used antimalarial drug chloroquine (CQ). Our recent strategy is based on the synthesis of inhibitors of the glutathione reductases from parasite and host erythrocyte. With the expectation of a synergistic or additive effect, double-headed prodrugs were designed to be directed against two different and essential functions of the malarial parasite P. falciparum, namely glutathione regeneration and heme detoxification. The prodrugs were prepared by linking bioreversibly a GR inhibitor to a 4-aminoquinoline moiety which is known to concentrate in the acidic food vacuole of parasites. Drug-enzyme interaction was correlated with antiparasitic action in vitro on strains resistant towards CQ and in vivo in Plasmodium berghei-infected mice as well as absence of cytotoxicity towards human cells. Because TrxR of P. falciparum was recently shown to be responsible for the residual glutathione disulfide-reducing capacity observed after GR inhibition in P. falciparum, future development of antimalarial drug-candidates that act by perturbing the redox equilibrium of parasites is based on the design of new double-drugs based on TrxR inhibitors as potential antimalarial drug candidates.     

Altered renal immune complexes deposition in female BWF1 lupus mice following Plasmodium chabaudi infection

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease that has a mysterious relationship with malaria infection. The current study was designated to compare between the effect of the live and the gamma irradiated Plasmodium chabaudi infection on BWF1 lupus murine model. A total of 30 female BWF1 mice were randomly divided into three groups (10 mice/group) as follows: group (I) lupus group (lupus non infected); group (II) live malaria infected group (lupus + live malaria infection); and group (III) irradiated malaria-infected group (lupus + gamma irradiated malaria infection). Live P. chabaudi infection was accompanied with a decrease in survival rate and food consumption in comparison to the control group of mice while gamma irradiated P. chabaudi -infection was unable to do this effect. Additionally, live P. chabaudi infection was accompanied with an increased level of proteinuria and increased rate of immune complexes deposition in kidney. Moreover, infection with live, but not gamma-irradiated P. chabaudi was accompanied with an increase in nitric oxide (NO), hydrogen peroxide (H₂O₂), and malondialdehyde (MDA) levels in plasma of lupus mice. The levels of both total cholesterol and triglycerides in plasma of lupus mice after live P. chabaudi infection were obviously decreased in comparison to the control group. On the other hand, gamma-irradiated P. chabaudi infection resembled the control group. Our data revealed that infection of lupus mice with live but not gamma-irradiated P. chabaudi has several histological and biochemical effects.