Quantification of Plasmodium falciparum HRP-2 as an alternative method to [3H]hypoxanthine incorporation to measure the parasite reduction ratio in vitro.

In the absence of a highly efficacious vaccine, chemotherapy remains the cornerstone to control malaria morbidity and mortality. The threat of the emergence of parasites resistant to artemisinin-based combination therapies highlights the need for new antimalarial drugs ideally with superior properties. The killing rate reflects the speed of action of antimalarial drugs, which can be measured in vitro through the parasite reduction ratio (PRR) assay to shortlist interesting candidates. As a standard, the in vitro PRR assay is performed by measuring [3H]hypoxanthine incorporation of Plasmodium falciparum. This methodology is restricted to specialised laboratories owing to the handling of radioactive material. In this work, we describe a sandwich enzyme-linked immunosorbent assay to detect P. falciparum histidine-rich protein 2 (HRP-2) as an alternative methodology to assess the PRR. We first validated the methodology with established antimalarial drugs (artesunate, chloroquine, pyrimethamine and atovaquone) by comparing our results with previous results of the [3H]hypoxanthine incorporation readout provided by an expert laboratory, and subsequently assessed the speed of action of four new antimalarial candidates (compound 22, chlorotonil A, boromycin and ivermectin). The HRP-2 PRR assay achieved comparable results to the [3H]hypoxanthine incorporation readout in terms of parasite growth rate over time, lag phase and parasite clearance time. In addition, parasite growth following drug exposure was quantified after 7, 14, 21 and 28 days of recovery time. In conclusion, the PRR assay based on HRP-2 is similar to [3H]hypoxanthine in determining a drug's parasite killing rate and can be widely used in all research laboratories.

Cattle Encephalon Glycoside and Ignotin Reduce Early Brain Injury and Cognitive Dysfunction after Subarachnoid Hemorrhage in Rats.

Subarachnoid hemorrhage (SAH) is a well-known hemorrhagic stroke with high rates of morbidity and mortality where patients frequently experience cognitive dysfunction. This study explores a potential treatment for cognitive dysfunction following SAH with the demonstration that multi-target drug cattle encephalon glycoside and ignotin (CEGI) can relieve cognitive dysfunction by decreasing hippocampal neuron apoptosis following SAH in rats. Experimentally, 110 male SD rats were separated at random into Sham (20), SAH + Vehicle (30), SAH + 4 ml/kg CEGI (30), and SAH + 1 ml/kg CEGI groups (30) and an endovascular perforation model was created to induce SAH. We discovered that the number of TUNEL-positive neurons in the hippocampus was markedly decreased in SAH + 4 ml/kg and SAH + 1 ml/kg CEGI groups compared to the SAH + Vehicle group. This finding was associated with an observed decrease in Bax/Bcl-2 ratio, cytochrome-c and PUMA expression, and the suppression of caspase-3 activation following SAH. In Morris water maze tests, the SAH + 4 ml/kg CEGI group demonstrated a decreased escape latency time and increase in time spent in the target quadrant as well as crossing times of platform region. These results indicate that high doses of CEGI can decrease hippocampal neuron apoptosis and relieve cognitive dysfunction in rats, suggesting that multitarget-drug CEGI exhibits a neuroprotective effect in SAH via the mitochondrial apoptosis pathway.

Neotrofin, a novel purine that induces NGF-dependent nociceptive nerve sprouting but not hyperalgesia in adult rat skin.

We report peripheral actions in rats of Neotrofin, a purine derivative of therapeutic interest. Systemic injections mimicked NGF in eliciting sprouting of nociceptive nerves without affecting their regeneration. The sprouting was prevented by anti-NGF treatment, implicating endogenous NGF. We detected no Neotrofin-induced increases in cutaneous NGF levels or in retrograde NGF transport. In contrast, both NGF and phosphorylation of trkA increased significantly in DRGs, with a marginal appearance of phosphorylated trkA in axons. We conclude that the DRG effects of Neotrofin are responsible for its induction of sprouting. Neotrofin also induced a striking phosphorylation of axonal erk 1 and 2, which was, however, unaffected by anti-NGF treatment. We suggest that this NGF-independent MAP kinase activation is involved in nonsprouting functions of Neotrofin such as neuroprotection. Unlike injected NGF, Neotrofin did not induce hyperalgesia, supporting its candidacy as a treatment for peripheral neuropathies like those induced by diabetes and anticancer chemotherapy.

2-Methoxyestradiol enhances reactive oxygen species formation and increases the efficacy of oxygen radical generating tumor treatment.

In an investigation of the antitumor effects of 2-methoxyestradiol (2-ME) in combination with other reactive oxygen generating treatments, 2-ME (0.5 microM) was found to completely inhibit cell proliferation of rat DS-sarcoma cells in vitro, with 71% of cells dying after exposure to 5 microM 2-ME. Concentration-dependent increases in ROS-formation, lipid peroxidation and mitochondrial changes were also observed, and an elevation in caspase-3 activity resulted in DNA fragmentation and apoptosis. Combination of 2-ME with hypoxanthine and xanthine oxidase enhanced in vitro cytotoxicity. In vivo, 2-ME caused a slight inhibition of tumor growth, with no tumors cured. Combination of 2-ME treatment with localized 44 degrees C hyperthermia, respiratory hyperoxia and xanthine oxidase caused a tumor growth delay with 51% of tumors cured. These results suggest that amplifying the levels of reactive oxygen species within tumor tissue with substances such as 2-ME may prove to be a promising strategy for adjuvant treatment of solid tumors.