A Mitochondria-Penetrating Peptide Exerts Potent Anti-Plasmodium Activity and Localizes at Parasites' Mitochondria.

Mitochondria are considered a novel drug target as they play a key role in energy production and programmed cell death of eukaryotic cells. The mitochondria of malaria parasites differ from those of their vertebrate hosts, contributing to the drug selectivity and the development of antimalarial drugs. (Fxr)3, a mitochondria-penetrating peptide or MPP, entered malaria-infected red cells without disrupting the membrane and subsequently killed the blood stage of P. falciparum parasites. The effects were more potent on the late stages than on the younger stages. Confocal microscopy showed that the (Fxr)3 intensely localized at the parasite mitochondria. (Fxr)3 highly affected both the lab-strain, chloroquine-resistant K1, and freshly isolated malaria parasites. (Fxr)3 (1 ng/mL to 10 µg/mL) was rarely toxic towards various mammalian cells, i.e., mouse fibroblasts (L929), human leukocytes and erythrocytes. At a thousand times higher concentration (100 µg/mL) than that of the antimalarial activity, cytotoxicity and hemolytic activity of (Fxr)3 were observed. Compared with the known antimalarial drug, atovaquone, (Fxr)3 exhibited more rapid killing activity. This is the first report on antimalarial activity of (Fxr)3, showing localization at parasites' mitochondria.

A novel, rationally designed, hybrid antimicrobial peptide, inspired by cathelicidin and aurein, exhibits membrane-active mechanisms against Pseudomonas aeruginosa.

Antimicrobial peptides (AMPs) are promising alternatives to classical antibiotics for the treatment of drug-resistant infections. Due to their versatility and unlimited sequence space, AMPs can be rationally designed by modulating physicochemical determinants to favor desired biological parameters and turned into novel therapeutics. In this study, we utilized key structural and physicochemical parameters, in combination with rational engineering, to design novel short α-helical hybrid peptides inspired by the well-known natural peptides, cathelicidin and aurein. By comparing homologous sequences and abstracting the conserved residue type, sequence templates of cathelicidin (P0) and aurein (A0) were obtained. Two peptide derivatives, P7 and A3, were generated by amino acid substitution based on their residue composition and distribution. In order to enhance antimicrobial activity, a hybrid analog of P7A3 was designed. The results demonstrated that P7A3 had higher antibacterial activity than the parental peptides with unexpectedly high hemolytic activity. Strikingly, C-terminal truncation of hybrid peptides containing only the α-helical segment (PA-18) and shorter derivatives confer potent antimicrobial activity with reduced hemolytic activity in a length-dependent manner. Among all, PA-13, showed remarkable broad-spectrum antibacterial activity, especially against Pseudomonas aeruginosa with no toxicity. PA-13 maintained antimicrobial activity in the presence of physiological salts and displayed rapid binding and penetration activity which resulted in membrane depolarization and permeabilization. Moreover, PA-13 showed an anti-inflammatory response via lipopolysaccharide (LPS) neutralization with dose-dependent, inhibiting, LPS-mediated Toll-like receptor activation. This study revealed the therapeutic potency of a novel hybrid peptide, and supports the use of rational design in development of new antibacterial agents.

A novel in vitro model reveals distinctive modulatory roles of Plasmodium falciparum and Plasmodium vivax on naïve cell-mediated immunity.

BACKGROUND: To date, human peripheral blood mononuclear cells (PBMCs) have been used mainly in immune stimulation assays and the interpretation of data can be influenced by the previous immunological history of donors and cross reactivity with other infectious agents. Resolving these limitations requires an alternative in vitro model to uncover the primary response profiles. METHODS: A novel in vitro model of mononuclear cells (MNCs) generated from haematopoietic stem cells (HSCs) was developed and these cells were then co-cultured with various antigens from Plasmodium falciparum and Plasmodium vivax to investigate the response of naïve immune cells to malaria antigens by flow cytometry. RESULTS: In vitro stimulation of naïve lymphocytes showed that CD4+ and CD8+ T lymphocytes were significantly reduced (P < 0.01) by exposure to lysates of infected erythrocytes or intact erythrocytes infected with P. falciparum. The depletion was associated with the expression of CD95 (Fas receptor) on the surface of T lymphocytes. Maturation of T lymphocytes was affected differently, showing elevated CD3+CD4+CD8+ and CD3+CD4-CD8- T lymphocytes after stimulation with cell lysates of P. falciparum and P. vivax, respectively. In addition, antigen presenting monocytes and dendritic cells derived from haematopoietic stem cells showed impaired HLA-DR expression as a consequence of exposure to different species of malaria parasites. CONCLUSION: These results suggest that naïve mononuclear cells differentiated in vitro from HSCs could provide a valid model for the assessment of immunity. P. falciparum and P. vivax malaria parasites could modulate various populations of immune cells starting from newly differentiated mononuclear cells.

Enhanced Sensitivity for Detection of Plasmodium falciparum gametocytes by magnetic nanoparticles combined with enzyme substrate system.

The highly sensitive and specific detection of Pfg377 gene of Plasmodium falciparum gametocyte using Magnetic Nanoparticles PCR Enzyme-Linked Gene Assay (MELGA) was successfully developed. The MELGA included amplification of the Pfg377 gene by polymerase chain reaction (PCR) using magnetic nanoparticles (MNPs)-conjugated forward primer and biotinylated reverse primer, followed by post-analytical process using horseradish peroxidase (HRP)-conjugated streptavidin (SA). The complexes of MELGA product were incubated with the peroxidase substrate and hydrogen peroxide to produce the signal for colorimetric measurement. Altogether, the MELGA technique provided a highly sensitive and specific detection at 1 P. falciparum gametocyte/µL, which was more efficient than that of microscopic examination and rapid diagnostic tests (RDTs). Additionally, the MELGA could detect target gene at femtogram level, which was greater sensitive than the conventional PCR, nested PCR and loop-mediated isothermal amplification (LAMP). The MELGA technique could become a novel and practical method that overcome limitation of traditional gametocyte detection.

Optimized high gradient magnetic separation for isolation of Plasmodium-infected red blood cells.

BACKGROUND: Highly purified infected red blood cells (irbc), or highly synchronized parasite cultures, are regularly required in malaria research. Conventional isolation and synchronization rely on density and osmotic fragility of irbc, respectively. High gradient magnetic separation (HGMS) offers an alternative based on intrinsic magnetic properties of irbc, avoiding exposure to chemicals and osmotic stress. Successful HGMS concentration in malaria research was previously reported using polymer coated columns, while HGMS depletion has not been described yet. This study presents a new approach to both HGMS concentration and depletion in malaria research, rendering polymer coating unnecessary. METHODS: A dipole magnet generating a strong homogenous field was custom assembled. Polypropylene syringes were fitted with one-way stopcocks and filled with stainless steel wool. Rbc from Plasmodium falciparum cultures were resuspended in density and viscosity optimized HGMS buffers and HGMS processed. Purification and depletion results were analysed by flow cytometer and light microscopy. Viability was evaluated by calculating the infection rate after re-culturing of isolates. RESULTS: In HGMS concentration, purity of irbc isolates from asynchronous cultures consistently ranged from 94.8% to 98.4% (mean 95.7%). With further optimization, over 90% of isolated irbc contained segmented schizonts. Processing time was less than 45 min. Reinfection rates ranged from 21.0% to 56.4%. In HGMS depletion, results were comparable to treatment with sorbitol, as demonstrated by essentially identical development of cultures. CONCLUSION: The novel HGMS concentration procedure achieves high purities of segmented stage irbc from standard asynchronous cultures, and is the first HGMS depletion alternative to sorbitol lysis. It represents a simple and highly efficient alternative to conventional irbc concentration and synchronization methods.

The effect of mimicking febrile temperature and drug stress on malarial development.

BACKGROUND: Malaria remains one of the most important tropical diseases of human with 1-2 million deaths annually especially caused by P. falciparum. During malarial life cycle, they exposed to many environmentally stresses including wide temperature fluctuation and pharmacological active molecules. These trigger malarial evolutionarily adaptive responses. The effect of febrile temperature on malarial growth, development and drug susceptibility by mimicking patient in treatment failure before and after drug uptake was examined. METHODS: Sensitivities of P. falciparum to antimalarial drug (chloroquine, mefloquine, quinine and artesunate) were investigated based on the incorporation of [3H] hypoxanthine into parasite nucleic acids or radioisotopic technique. The number of parasites was examined under microscope following Giemsa staining and the parasite development at the end of each phase was counted and comparison of parasite number was made. The proteome was separated, blotted and hybridized with anti-Hsp70s primary antibody. The hybridized proteins were separately digested with trypsin and identified by MALDI-TOF peptide mass fingerprint. RESULTS: The results show that febrile temperature is capable of markedly inhibiting the growth of field isolate P. falciparum but not to K1 and 3D7 standard strains. K1 and 3D7 grown under heat shock developed greater and the reinfection rate was increased up to 2-folds when compared to that of non-heat shock group. The IC50 value of K1 toward chloroquine, mefloquine and quinine under heat shock was higher than that of K1 under non-heat shock which is opposite to that of 3D7. Heat shock caused death in field isolated parasite. It was also found that the febrile temperature coped with chloroquine uptake had no effect to the development, drug sensitivity and the parasite number of K1 strain. In the opposite way, heat shock and chloroquine shows extremely effect toward 3D7 and field isolate PF91 as shown by higher number of dead parasites compared to that of control group. After culture under high temperature with artesunate, the total parasite number of all strains including K1, 3D7 and PF91 was extremely decreased and the parasite was not found at the end. Additionally, the expression of pfHsp70s was found in all strains and conditions as shown in 120 kDa hybridized band. However, the proteome extracted from K1 grown under heat shock with chloroquine, anti-pfHsp70 interacted with additional three bands identified by MALDI-TOF as elongation factor-1alpha (83 kDa), pfHsp86 (60 kDa) and phosphoethanolamine N-methyltransferase (43 kDa). CONCLUSION: In conclusion, febrile temperature was capable of markedly inhibiting the growth of field isolate P. falciparum while the development, reinfection rate and drug (chloroquine, mefloquine and quinine) resistant level of standard strain K1 was enhanced. However, the febrile temperature coped with chloroquine had no effect to the development, drug sensitivity and the parasite number of K1 strain. In the opposite way, heat shock and chloroquine showed extremely effect toward 3D7 and field isolate PF91 as shown by some died parasites. Heat shock protein 70 (pfHSP70) of strain K1 under heat shock with chloroquine might involved in many pathways in order to sustain the parasite.

Short-term in vitro culture of field isolates of Plasmodium vivax using umbilical cord blood.

Plasmodium vivax research has been hampered by the lack of technology for culturing this parasite. Culturing P. vivax is difficult because the parasite selectively invades reticulocytes. Here we describe a modified procedure to establish and maintain short-term cultures of freshly collected P. vivax parasites using reticulocyte-enriched cord blood. Using this method, parasites could be cultured for a month. Manipulation of the culture allowed procurement of synchronized stages of the parasite. This short-term culture method can be easily adapted to study various aspects of the parasite biology.

Natural human humoral response to salivary gland proteins of Anopheles mosquitoes in Thailand.

During blood feeding, arthropod vectors inject saliva into vertebrate hosts. The saliva is biochemically complex and pharmacologically active, and may play an important role in pathogen transmission. To examine whether mosquito saliva could elicit humoral immune response in humans under natural conditions, we have collected sera from malaria patients, healthy villagers, and people from a non-malarious region in Thailand. Here we have demonstrated that anti-Anopheles salivary protein antibodies occurred predominantly in patients with acute Plasmodium falciparum or P. vivax malaria, whereas people from a non-malarious area had no such antibodies. Besides, antibody levels against mosquito salivary proteins in malaria patients were highly variable, which may be related to the levels of mosquito exposure. Despite variability, patients' sera with high IgG titers consistently detected several proteins in Anopheles dirus salivary gland protein extracts. Immunohistochemical staining of Anopheles salivary glands with human sera showed that the salivary gland-specific IgGs reacted strongly with the median lobe. Comparison using Anopheles and Aedes salivary proteins suggests that the anti-salivary protein antibodies detected in malaria patients were Anopheles-specific, consistent with the major malaria vector status of An. dirus in this area.