Raman spectroscopy of lymphocytes from patients with the Epstein-Barr virus infection.

In this study, Raman spectroscopy is applied to trace lymphocytes activation following contact with the Epstein-Barr virus (EBV) of the herpesvirus family. The biomarker of cell activation is found to be the 520 cm-1 band, indicating formation of immunoglobulins. The blood samples are obtained from patients diagnosed with infectious mononucleosis and treated at the University Hospital in Kraków. The lymphocytes' Raman spectra are collected using a mapping technique, exciting samples with a 514.5 nm line of Ar + laser. Measurements are performed on the 1st, 4th, 6th, 12th and 30th day of hospitalization, until the patient has recovered. The highest intensity of the immunoglobulin marker is observed on the 4th day of hospitalization, while the results of the blood count of patients show the greatest increase in the number of lymphocytes at the beginning of hospitalization. No activated lymphocytes were observed in the blood of healthy volunteers. Some information is provided by the evaluation of B-cell activation by estimating the activated areas in the cells, which are determined by the presence of the Ig marker. The 900 cm-1 band and band around 1450 cm-1 are also analyzed as markers of the presence of the latent membrane protein, LMP2A (and 2B), of the EBV viral protein. The anomalous degree of depolarization observed in B-cells in the course of EBV infection appears to be due to the influence of a virus protein, disrupting BCR signal transduction.

Course of Plasmodium infection studied using 2D-COS on human erythrocytes.

BACKGROUND: The threat of malaria is still present in the world. Recognizing the type of parasite is important in determining a treatment plan. The golden routine involves microscopic diagnostics of Giemsa-stained thin blood smears, however, alternative methods are also constantly being sought, in order to gain an additional insight into the course of the disease. Spectroscopic methods, e.g., Raman spectroscopy, are becoming increasingly popular, due to the non-destructive nature of these techniques. METHODS: The study included patients hospitalized for malaria caused by Plasmodium falciparum or Plasmodium vivax, in the Department of Infectious Diseases at the University Hospital in Krakow, Poland, as well as healthy volunteers. The aim of this study was to assess the possibility of using Raman spectroscopy and 2D correlation (2D-COS) spectroscopy in understanding the structural changes in erythrocytes depending on the type of attacking parasite. EPR spectroscopy and two-trace two-dimensional (2T2D) correlation was also used to examine the specificity of paramagnetic centres found in the infected human blood. RESULTS: Two-dimensional (2D) correlation spectroscopy facilitates the identification of the hidden relationship, allowing for the discrimination of Raman spectra obtained during the course of disease in human red blood cells, infected by P. falciparum or P. vivax. Synchronous cross-peaks indicate the processes taking place inside the erythrocyte during the export of the parasite protein towards the cell membrane. In contrast, moieties that generate asynchronous 2D cross-peaks are characteristic of the respective ligand-receptor domains. These changes observed during the course of the infection, have different dynamics for P. falciparum and P. vivax, as indicated by the asynchronous correlation cross-peaks. Two-trace two-dimensional (2T2D) spectroscopy, applied to EPR spectra of blood at the beginning of the infection, showed differences between P. falciparum and P. vivax. CONCLUSIONS: A unique feature of 2D-COS is the ability to discriminate the collected Raman and EPR spectra. The changes observed during the course of a malaria infection have different dynamics for P. falciparum and P. vivax, indicated by the reverse sequence of events. For each type of parasite, a specific recycling process for iron was observed in the infected blood.

Blood-stage antiplasmodial activity and oocyst formation-blockage of metallo copper-cinchonine complex.

In the fight against malaria, the key is early treatment with antimalarial chemotherapy, such as artemisinin-based combination treatments (ACTs). However, Plasmodium has acquired multidrug resistance, including the emergence of P. falciparum strains with resistance to ACT. The development of novel antimalarial molecules, that are capable of interfering in the asexual and sexual blood stages, is important to slow down the transmission in endemic areas. In this work, we studied the ability of the mettalo copper-cinchonine complex to interfere in the sexual and asexual stages of Plasmodium. The tested compound in the in vitro assay was a cinchonine derivative, named CinCu (Bis[Cinchoninium Tetrachlorocuprate(II)]trihydrate). Its biological functions were assessed by antiplasmodial activity in vitro against chloroquine-resistant P. falciparum W2 strain. The mice model of P. berghei ANKA infection was used to analyze the antimalarial activity of CinCu and chloroquine and their acute toxicity. The oocyst formation-blocking assay was performed by experimental infection of Anopheles aquasalis with P. vivax infected blood, which was treated with different concentrations of CinCu, cinchonine, and primaquine. We found that CinCu was able to suppress as high as 81.58% of parasitemia in vitro, being considered a molecule with high antiplasmodial activity and low toxicity. The in vivo analysis showed that CinCu suppressed parasitemia at 34% up to 87.19%, being a partially active molecule against the blood-stage forms of P. berghei ANKA, without inducing severe clinical signs in the treated groups. The transmission-blocking assay revealed that both cinchonine and primaquine were able to reduce the infection intensity of P. vivax in A. aquasalis, leading to a decrease in the number of oocysts recovered from the mosquitoes' midgut. Regarding the effect of CinCu, the copper-complex was not able to induce inhibition of P. vivax infection; however, it was able to induce an important reduction in the intensity of oocyst formation by about 2.4 times. It is plausible that the metallo-compound also be able to interfere with the differentiation of parasite stages and/or ookinete-secreted chitinase into the peritrophic matrix of mosquitoes, promoting a reduction in the number of oocysts formed. Taken together, the results suggest that this compound is promising as a prototype for the development of new antimalarial drugs. Furthermore, our study can draw a new pathway for repositioning already-known antimalarial drugs by editing their chemical structure to improve the antimalarial activity against the asexual and sexual stages of the parasite.