In vivo multiphoton microscopy technique to reveal the physiology of the mouse placenta.

PROBLEM: Pregnancy is a challenge to the maternal immune system as it must defend the body against pathogens while at the same time develop immune tolerance against the fetus growing inside the uterus. Despite ex vivo techniques being used to understand these processes, in vivo techniques are missing. METHOD OF STUDY: To directly study these phenomena, we have developed a new microscope stage and surgical procedures for use in two-photon microscopy, for in vivo observation of the mouse placenta. RESULTS: These tools and surgical procedures demonstrate fetal and maternal blood flow inside the labyrinth zone of the placenta, as well as its three dimensional structure. It was also useful to identify Plasmodium chabaudi-infected red blood cells inside this labyrinth zone. CONCLUSION: We believe this technique will represent an important contribution for expanding the available knowledge concerning cell dynamics and interactions at the fetal-maternal interface.

Transient attenuated Foxp3 expression on CD4⁺ T cells treated with 7D4 mAb contributes to the control of parasite burden in DBA / 2 mice infected with lethal Plasmodium chabaudi chabaudi AS.

CD4(+) CD25(+) regulatory T (Treg) cells expressing Foxp3(+) play a critical role in maintaining immune homoeostasis and controlling excessive immune responses. However, controversy about the immunoregulatory role of Treg cells exists in malaria studies. Given the role of maintenance of Foxp3 expression in Treg cells' activities, we investigated whether anti-CD25 mAb (7D4 clone) treatment affects Foxp3 expression in CD4(+) T cells in DBA/2 mice infected with Plasmodium chabaudi chabaudi AS (P. c. chabaudi AS). We found that DBA/2 mice succumbed to P. c. chabaudi AS infection, which was accompanied by increased expression of Foxp3 in CD4(+) T cells at the peak parasitemia. In contrast, Foxp3 expression was impaired in CD25-depleted mice with 7D4 mAb treatment, leading to delayed parasitemia and extended survival of infected mice. Production of IFN-γ, TNF-α and IL-6, as well as NO was significantly enhanced in CD25-depleted mice. The majority of CD4(+) CTLA-4(+) cells expressed high levels of Foxp3 (Foxp3(hi) cells) in control mice with P. c. chabaudi AS infection. However, the number of CD4(+) Foxp3(hi) CTLA-4(+) cells was reduced in CD25-depleted mice. Together, these data suggest that CD4(+) Foxp3(hi) CTLA-4(+) cells may be involved in regulating the intensity of pro-inflammatory responses via CTLA-4.

Flow cytometry as a tool for analyzing changes in Plasmodium falciparum cell cycle following treatment with indol compounds.

Melatonin and its derivatives modulate the Plasmodium falciparum and Plasmodium chabaudi cell cycle. Flow cytometry was employed together with the nucleic acid dye YOYO-1 allowing precise discrimination between mono- and multinucleated forms of P. falciparum-infected red blood cell. The use of YOYO-1 permitted excellent discrimination between uninfected and infected red blood cells as well as between early and late parasite stages. Fluorescence intensities of schizont-stage parasites were about 10-fold greater than those of ring-trophozoite form parasites. Melatonin and related indolic compounds including serotonin, N-acetyl-serotonin and tryptamine induced an increase in the percentage of multinucleated forms compared to non-treated control cultures. YOYO-1 staining of infected erythrocyte and subsequent flow cytometry analysis provides a powerful tool in malaria research for screening of bioactive compounds.

Melatonin and IP3-induced Ca2+ release from intracellular stores in the malaria parasite Plasmodium falciparum within infected red blood cells.

IP(3)-dependent Ca(2+) signaling controls a myriad of cellular processes in higher eukaryotes and similar signaling pathways are evolutionarily conserved in Plasmodium, the intracellular parasite that causes malaria. We have reported that isolated, permeabilized Plasmodium chabaudi, releases Ca(2+) upon addition of exogenous IP(3). In the present study, we investigated whether the IP(3) signaling pathway operates in intact Plasmodium falciparum, the major disease-causing human malaria parasite. P. falciparum-infected red blood cells (RBCs) in the trophozoite stage were simultaneously loaded with the Ca(2+) indicator Fluo-4/AM and caged-IP(3). Photolytic release of IP(3) elicited a transient Ca(2+) increase in the cytosol of the intact parasite within the RBC. The intracellular Ca(2+) pools of the parasite were selectively discharged, using thapsigargin to deplete endoplasmic reticulum (ER) Ca(2+) and the antimalarial chloroquine to deplete Ca(2+) from acidocalcisomes. These data show that the ER is the major IP(3)-sensitive Ca(2+) store. Previous work has shown that the human host hormone melatonin regulates P. falciparum cell cycle via a Ca(2+)-dependent pathway. In the present study, we demonstrate that melatonin increases inositol-polyphosphate production in intact intraerythrocytic parasite. Moreover, the Ca(2+) responses to melatonin and uncaging of IP(3) were mutually exclusive in infected RBCs. Taken together these data provide evidence that melatonin activates PLC to generate IP(3) and open ER-localized IP(3)-sensitive Ca(2+) channels in P. falciparum. This receptor signaling pathway is likely to be involved in the regulation and synchronization of parasite cell cycle progression.

Cell cycle analysis of asexual stages of erythrocytic malaria parasites.

Intra-erythrocytic Plasmodium species can be stained with the DNA binding dye, Hoechst 33342, and the distribution of DNA content determined for parasite populations by flow cytometric measurement of fluorescence. Analysis of this distribution will determine the parasitaemia (percentage of erythrocytes infected), and the percentages of trophozoite infected red blood cells, polyparasitized (trophozoite) red blood cells, and schizont/segmenter infected red blood cells. This analysis is based on the hypothesis that the asexual parasites cycle with single G1 period, and effectively, a single S phase with no significant G2/M period except at schizogony when the genome DNA content is equivalent to 8 N or higher, dependent on the species. Data are presented to support this model.