Martha Legorreta-Herrera

Pyrimethamine induces oxidative stress in Plasmodium yoelii 17XL-infected mice: a novel immunomodulatory mechanism of action for an old antimalarial drug?

Pyrimethamine is an antimalarial drug that has also been used successfully to treat autoimmune diseases such as lymphoproliferative syndrome. In this work, the effect of pyrimethamine (PYR) on the production of free radicals in malaria-infected mice was studied to better understand the drugs immunomodulatory properties. BALB/c and CBA/Ca mice were infected with Plasmodium yoelii 17XL. Seven days after infection, mice were treated with PYR or vehicle and sacrificed 24h later. Treatment with PYR increased superoxide dismutase and glutathione peroxidase activities in erythrocytes and the liver, augmented the levels of nitric oxide in the serum, and upregulated mRNA levels of superoxide dismutase, glutathione peroxidase, catalase, and iNOS in the spleen. In addition, PYR increased lipoperoxidation and protein carbonylation in infected mice. Our results indicate that P. yoelii 17XL reduces oxidative stress in infected cells, while PYR induces it, which is associated with increased parasite elimination. Thus, it is possible that oxidative stress generated by pyrimethamine is also involved in its immunomodulatory mechanism of action.

Helminth Parasites Alter Protection against Plasmodium Infection

More than one-third of the worlds population is infected with one or more helminthic parasites. Helminth infections are prevalent throughout tropical and subtropical regions where malaria pathogens are transmitted. Malaria is the most widespread and deadliest parasitic disease. The severity of the disease is strongly related to parasite density and the hosts immune responses. Furthermore, coinfections between both parasites occur frequently. However, little is known regarding how concomitant infection with helminths and Plasmodium affects the hosts immune response. Helminthic infections are frequently massive, chronic, and strong inductors of a Th2-type response. This implies that infection by such parasites could alter the hosts susceptibility to subsequent infections by Plasmodium. There are a number of reports on the interactions between helminths and Plasmodium; in some, the burden of Plasmodium parasites increased, but others reported a reduction in the parasite. This review focuses on explaining many of these discrepancies regarding helminth-Plasmodium coinfections in terms of the effects that helminths have on the immune system. In particular, it focuses on helminth-induced immunosuppression and the effects of cytokines controlling polarization toward the Th1 or Th2 arms of the immune response.

Pretreatment with Cry1Ac Protoxin Modulates the Immune Response, and Increases the Survival of Plasmodium-Infected CBA/Ca Mice

Malaria is a major global health problem that kills 1-2 million people each year. Despite exhaustive research, naturally acquired immunity is poorly understood. Cry1A proteins are potent immunogens with adjuvant properties and are able to induce strong cellular and humoral responses. In fact, it has been shown that administration of Cry1Ac protoxin alone or with amoebic lysates induces protection against the lethal infection caused by the protozoa Naegleria fowleri. In this work, we studied whether Cry1Ac is able to activate the innate immune response to induce protection against Plasmodium berghei ANKA (lethal) and P. chabaudi AS (nonlethal) parasites in CBA/Ca mice. Treatment with Cry1Ac induced protection against both Plasmodium species in terms of reduced parasitaemia, longer survival time, modulation of pro- and anti-inflammatory cytokines, and increased levels of specific antibodies against Plasmodium. Understanding how to boost innate immunity to Plasmodium infection should lead to immunologically based intervention strategies.

Immunomodulatory Role of Chloroquine and Pyrimethamine in Plasmodium yoelii 17XL Infected Mice

Chloroquine (CLQ) and Pyrimethamine (PYR) are used for the treatment of malaria and some autoimmune diseases; although their mechanism of action is only partially understood, their therapeutic effectiveness in the second case has been attributed to their ability to increase apoptosis of T lymphocytes. In view of the potential for immunomodulation during malaria chemotherapy, we investigated the effects of CLQ and PYR treatment on lymphocyte apoptosis and cytokine expression during infection with blood‐stage Plasmodium. This work shows that infection of BALB/c mice with Plasmodium yoelii 17XL (Py17XL) reduced apoptosis in spleen cells but when infected mice were treated with CLQ, apoptosis of B and T lymphocytes increased significantly via a Fas‐mRNA expression independent mechanism associated with downregulation of Bcl‐2 expression, whereas treatment with PYR increased apoptosis to a lesser extent and only in B lymphocytes. CLQ treatment of Py17XL infected mice upregulated tumour necrosis factor‐α mRNA expression, while PYR treatment increased interferon‐γ mRNA expression. In infected mice, treatment with CLQ downregulated expression of the anti‐inflammatory cytokines, interleukin‐10 and transforming growth factor‐β (TGF‐β), while PYR treatment upregulated TGF‐β. Thus, in addition to their anti‐malarial effects, both drugs modulate the immune response in malaria by increasing apoptosis and modulating the mRNA expression of cytokines involved in parasite elimination and regulation of inflammatory responses.

Analysis of a p53 mutation associated with cancer susceptibility for biochemistry and genetic laboratory courses

We have devised and implemented a module for an upper division undergraduate laboratory based on the amplification and analysis of a p53 polymorphism associated with cancer susceptibility. First, students collected a drop of peripheral blood cells using a sterile sting and then used FTA cards to extract the genomic DNA. The p53 region is then PCR amplified, and the PCR products are digested with the BstUI enzyme to detect the 72 codon polymorphism. Polyacrylamide gel electrophoresis is used to resolve the PCR products, and the results are statistically analyzed in the context of human population genetics. Blood samples in FTA cards were also collected from 50 women to detect the mutation in a wide range of ages and assess its relationship to familial cancer susceptibility. This module enables students to use materials and methods that are routinely used by scientific researchers to analyze polymorphisms. Therefore, it can be used for laboratory exercises in traditional biochemistry curricula as well as in the growing field of genomic science and education.

Sex hormones modulate the immune response to Plasmodium berghei ANKA in CBA/Ca mice

Susceptibility to malaria differs between females and males, and this sexual dimorphism may have important implications for the effects of vaccines and drugs. However, little is known about the mechanisms mediating these sexual differences. Because the main differences between sexes are dictated by sex hormones, we studied the effect of gonadal steroids on immune responses to malaria in CBA/Ca mice. We decreased sex hormones levels by gonadectomy and evaluated the splenic index and the cells involved in the immune response, including T cells (CD3+, CD4+, CD8+ and NK+), B cells and macrophages (Mac-3+) in the spleens of female and male mice infected with Plasmodium berghei ANKA. In addition, we measured antibody and cytokine levels in blood. Gonadectomy increased T+ and B+ splenic cells in both sexes but increased Mac-3+ cells only in male mice. By contrast, gonadectomy decreased the NK+ cell population only in male mice. In general, female mice developed higher antibody levels than males. Contrary to our expectations, gonadectomy increased the synthesis of IgG1, IgG2b, IgG3, and total IgG in female mice, indicating negative regulation of antibody production by female sex hormones. Gonadectomy increased the synthesis of tumour necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) only in female mice, suggesting that female sex hormones have anti-inflammatory properties. This work demonstrates that the levels of sex hormones affect the immune response and should be considered when designing malaria vaccines.

Exploring the antimalarial potential of whole Cymbopogon citratus plant therapy.

ETHNOPHARMACOLOGICAL RELEVANCE Cymbopogon citratus (lemon grass) has been used in traditional medicine as an herbal infusion to treat fever and malaria. Generally, whole plant extracts possess higher biological activity than purified compounds. However, the antimalarial activity of the whole C. citratus plant has not been experimentally tested. AIM OF THE STUDY To evaluate the antimalarial activity of an herbal infusion and the whole Cymbopogon citratus plant in two experimental models of malaria. MATERIAL AND METHODS The plant was dried for 10 days at room temperature and was then milled and passed through brass sieves to obtain a powder, which was administered to CBA/Ca mice with a patent Plasmodium chabaudi AS or P. berghei ANKA infection. We analysed the effects of two different doses (1600 and 3200mg/kg) compared with those of the herbal infusion and chloroquine, used as a positive control. We also assessed the prophylactic antimalarial activities of the whole C. citratus plant and the combination of the whole plant and chloroquine. RESULTS The C. citratus whole plant exhibited prolonged antimalarial activity against both P. chabaudi AS and P. berghei ANKA. The low dose of the whole C. citratus plant displayed higher antimalarial activity than the high dose against P. berghei ANKA. As a prophylactic treatment, the whole plant exhibited higher antimalarial activity than either the herbal infusion or chloroquine. In addition, the combination of the whole C. citratus plant and chloroquine displayed higher activity than chloroquine alone against P. berghei ANKA patent infection. CONCLUSIONS We demonstrated the antimalarial activity of the whole C. citratus plant in two experimental models. The whole C. citratus plant elicited higher anti-malarial activity than the herbal infusion or chloroquine when used as a prophylactic treatment. The antimalarial activity of the whole C. citratus plant supports continued efforts towards developing whole plant therapies for the management of malaria and other infectious diseases prevalent in resource-poor communities.

Gonadal Steroids Negatively Modulate Oxidative Stress in CBA/Ca Female Mice Infected with P. berghei ANKA

We decreased the level of gonadal steroids in female and male mice by gonadectomy. We infected these mice with P. berghei ANKA and observed the subsequent impact on the oxidative stress response. Intact females developed lower levels of parasitaemia and lost weight faster than intact males. Gonadectomised female mice displayed increased levels of parasitaemia, increased body mass, and increased anaemia compared with their male counterparts. In addition, gonadectomised females exhibited lower specific catalase, superoxide dismutase, and glutathione peroxidase activities in their blood and spleen tissues compared with gonadectomised males. To further study the oxidative stress response in P. berghei ANKA-infected gonadectomised mice, nitric oxide levels were assessed in the blood and spleen, and MDA levels were assessed in the spleen. Intact, sham-operated, and gonadectomised female mice exhibited higher levels of nitric oxide in the blood and spleen compared with male mice. MDA levels were higher in all of the female groups. Finally, gonadectomy significantly increased the oxidative stress levels in females but not in males. These data suggest that differential oxidative stress is influenced by oestrogens that may contribute to sexual dimorphism in malaria.

Amaranthus leucocarpus lectin recognizes a moesin-like O-glycoprotein and costimulates murine CD3-activated CD4(+) T cells.

The Galβ1,3GalNAcα1,O‐Ser/Thr specific lectin from Amaranthus leucocarpus (ALL) binds a ∼70 kDa glycoprotein on murine T cell surface. We show that in the absence of antigen presenting cells, murine CD4+ T cells activated by an anti‐CD3 antibody plus ALL enhanced cell proliferation similar to those cells activated via CD3/CD28 at 48 h of culture. Moreover, ALL induced the production of IL‐4, IL‐10, TNF‐alpha, and TGF‐beta in CD3‐activated cells. Proteomic assay using two‐dimensional electrophoresis and far‐Western blotting, ALL recognized two prominent proteins associated to the lipid raft microdomains in CD3/CD28‐activated CD4+ T cells. By mass spectrometry, the peptide fragments from ALL‐recognized proteins showed sequences with 33% homology to matricin (gi|347839 NCBInr) and 41% identity to an unnamed protein related to moesin (gi|74186081 NCBInr). Confocal microscopy analysis of CD3/CD28‐activated CD4+ T cells confirmed that staining by ALL colocalized with anti‐moesin FERM domain antibody along the plasma membrane and in the intercellular contact sites. Our findings suggest that a moesin‐like O‐glycoprotein is the ALL‐recognized molecule in lipid rats, which induces costimulatory signals on CD4+ T cells.

Detection of an ABCA1 variant associated with type 2 diabetes mellitus susceptibility for biochemistry and genetic laboratory courses

We selected diabetes mellitus for this laboratory exercise to provide students with an explicit model for scientific research concerning the association between the R230C polymorphism and susceptibility to type 2 diabetes mellitus, which is highly prevalent in the Mexican population. We used a collaborative project‐based learning to engage students to direct their own learning process. Students worked in small groups with the same learning goal to research, organize data, and present seminars to experimentally genotype the C230 variant and correctly interpret their results. At the conclusion of this laboratory exercise, the students were able to demonstrate a clear understanding of the relevant biological molecular principles to genotype the C230 variant, showed technical competency to carry out the experimental protocols with proficiency, and interpret their results using statistical analyses. The students discussed their understanding of the genetic technologies and the broader social and ethical implications of the research. A randomly selected team was trained to work as a “sentinel” to monitor their classmates and ensure the proper application of techniques. Moreover, the evaluation of this exercise is shared between the students and the instructors; the students evaluate their own work and the performance of their classmates. At the end of the course, the students complete a questionnaire to anonymously provide feedback and information regarding their perception of the learning outcomes. Overall, the student feedback was positive, indicating that the exercise was useful and that it would help to prepare the students for professional practice.