Variation of the 2D Pattern of Brain Proteins in Mice Infected with Taenia crassiceps ORF Strain.

Some parasites are known to influence brain proteins or induce changes in the functioning of the nervous system. In this study, our objective is to demonstrate how the two-dimensional gel technique is valuable for detecting differences in protein expression and providing detailed information on changes in the brain proteome during a parasitic infection. Subsequently, we seek to understand how the parasitic infection affects the protein composition in the brain and how this may be related to changes in brain function. By analyzing de novo-expressed proteins at 2, 4, and 8 weeks post-infection compared to the brains of the control mice, we observed that proteins expressed at 2 weeks are primarily associated with neuroprotection or the initial response of the mouse brain to the infection. At 8 weeks, parasitic infection can induce oxidative stress in the brain, potentially activating signaling pathways related to the response to cellular damage. Proteins expressed at 8 weeks exhibit a pattern indicating that, as the host fails to balance the Neuro-Immuno-Endocrine network of the organism, the brain begins to undergo an apoptotic process and consequently experiences brain damage.

2D immunoblots show differential response of mouse IgG and IgM antibodies to antigens of mammary carcinoma 4 T1 cells.

BACKGROUND: Immunosuppression in breast cancer has been reported in women and in the highly metastatic mouse mammary tumor model 4 T1. The immunosuppressive environment complicates the use of the humoral response against the tumor as an immunodiagnostic tool. IgM has not been used in immunodiagnostic in part because its antitumor responses, both innate and adaptive, have not been studied in function of time in breast cancer.We show a new approach to analyzing the mouse humoral immune response, and compare the evolution with time of IgG and IgM responses against the antigens of 4 T1 cells. METHODS: The study is based on 2-dimensional immunoblotting detection of antigens from 4 T1 cells by the IgG and IgM antibodies in the serum of female mice injected with 4 T1 cells. RESULTS: There was a high variability in the intra-and inter-mouse response. Variability in the IgM response was manifested as a pattern of spots that could become a multibinomial variable of 0 and 1, which could represent a signature of the immune response. Different numbers of spots was found in the IgG and IgM responses from week 1 to 5. On average, the IgM had more but the IgG response decrease with the time. The natural IgM at t = 0 responds stronger than w1; the adaptive response of both IgM and IgG were elicited where, with the former being stronger better than the latter. Antigens that are recognized by some female mice in the first week are also recognized by other female mice at time 0. Contamination of the natural IgM makes difficult use the adaptive IgM as a tool for immunodiagnostic. CONCLUSIONS: IgM and IgG response varied with the time and individuals. Spot variation in 2D pattern for the natural IgM could be expressed as a binomial signature, which opens up the way to correlate a particular pattern with resistance or susceptibility. This uncovers a battery of IgMs for each individual to confront cancer or infections. The possibility to differentiate between adaptive IgM antibodies from the natural IgM will allow investigation of the adaptive IgM for early immunodiagnosis.

Differential Protein Expression of Taenia crassiceps ORF Strain in the Murine Cysticercosis Model Using Resistant (C57BL/6) Mice.

A cysticercosis model of Taenia crassiceps ORF strain in susceptible BALB/c mice revealed a Th2 response after 4 weeks, allowing for the growth of the parasite, whereas resistant C57BL/6 mice developed a sustained Th1 response, limiting parasitic growth. However, little is known about how cysticerci respond to an immunological environment in resistant mice. Here, we show that the Th1 response, during infection in resistant C57BL/6 mice, lasted up to 8 weeks and kept parasitemia low. Proteomics analysis of parasites during this Th1 environment showed an average of 128 expressed proteins; we chose 15 proteins whose differential expression varied between 70 and 100%. A total of 11 proteins were identified that formed a group whose expression increased at 4 weeks and decreased at 8 weeks, and another group with proteins whose expression was high at 2 weeks and decreased at 8 weeks. These identified proteins participate in tissue repair, immunoregulation and parasite establishment. This suggests that T. crassiceps cysticerci in mice resistant under the Th1 environment express proteins that control damage and help to establish a parasite in the host. These proteins could be targets for drugs or vaccine development.

Proteomic analysis of IgM antigens from mammary tissue under pre- and post-cancer conditions using the MMTV-PyVT mouse model.

We analyzed the recognition of tumor antigens by IgM in transgenic MMTV-PyVT mice. PyVT female mice are a model of breast cancer that simulates its counterpart in humans. The PyVT model allows studying antigen recognition in two conditions: before and during tumor expression. We attempted to identify by sequence, the antigens recognized by IgM that are expressed or disappear in the membrane of breast transgenic tissue during the transition "No tumor-Tumor". 2D immunoblots were obtained of isolated membranes from the breast tissue in the fifth, sixth, and seventh week (transition point). Proteins recognized by IgM were sequenced in duplicate by MALDI-TOF. In the transition, we observed the disappearance of antigens in transgenic mice with respect to non-transgenic ones. We believe that in the diagnosis of cancer in its early stages, the expression of early antigens is as important as their early delocalization, with the latter having the advantage that, under normal conditions, we can know which proteins should be present at a given time. Therefore, we could consider that also the absence of antigens could be considered as a biomarker of cancer in progress.

Environmental parasitology and its impact on the host nueroimmunoendocrine network.

The communication between neuroendocrine and immune system maintains a bidirectional complex network. Both systems jointly act during a parasite infection to maintain homeostasis and to eliminate such pathogens. Parasites interfere with the synthesis, secretion, metabolism, action, and elimination of endogenous hormones, as well as with the immune system in the host. Here, we aim to address as how parasite colonization disrupts the normal homeostasis of endocrine organs of the host, likely due to the exacerbated immune response, or by the impact of the parasite directly affecting endocrine tissues.

Dehydroepiandrosterone Effect on Toxoplasma gondii: Molecular Mechanisms Associated to Parasite Death.

Toxoplasmosis is a zoonotic disease caused by the apicomplexa protozoan parasite Toxoplasma gondii. This disease is a health burden, mainly in pregnant women and immunocompromised individuals. Dehydroepiandrosterone (DHEA) has proved to be an important molecule that could drive resistance against a variety of infections, including intracellular parasites such as Plasmodium falciparum and Trypanozoma cruzi, among others. However, to date, the role of DHEA on T. gondii has not been explored. Here, we demonstrated for the first time the toxoplasmicidal effect of DHEA on extracellular tachyzoites. Ultrastructural analysis of treated parasites showed that DHEA alters the cytoskeleton structures, leading to the loss of the organelle structure and organization as well as the loss of the cellular shape. In vitro treatment with DHEA reduces the viability of extracellular tachyzoites and the passive invasion process. Two-dimensional (2D) SDS-PAGE analysis revealed that in the presence of the hormone, a progesterone receptor membrane component (PGRMC) with a cytochrome b5 family heme/steroid binding domain-containing protein was expressed, while the expression of proteins that are essential for motility and virulence was highly reduced. Finally, in vivo DHEA treatment induced a reduction of parasitic load in male, but not in female mice.

A novel model to culture cells from giant cell tumor of bone using three-dimensional (3D) polycaprolactone scaffold.

Two-dimensional (2D) culture of cells from giant cell tumor of bone (GCTB) is affected by loss of the multinucleated giant cells in subsequent passages. Therefore, there is limited time to study GCTB with all its histological components in 2D culture. Here, we explored the possibility of culturing GCTB cells on a polycaprolactone (PCL)-printed scaffold. We also evaluated the viability of the cultured cells and their adherence to the PCL scaffold at day 14 days using immunofluorescence analysis with calcein, vinculin, and phalloidin. Using the histological technique with hematoxylin and eosin staining, we observed all the histological components of GCTB in this 3D model. Immunohistochemical assays with cathepsin K, p63, and receptor activator of nuclear factor (NF)-κB ligand (RANKL) yielded positive results in this construct, which allowed us to confirm that the seeded cells maintained the expression of GCTB markers. Based on these findings, we concluded that the PCL scaffold is an efficient model to culture GCTB cells, and the cell viability and adherence to the scaffold can be preserved for up to 14 days. Moreover, this model can also be used in subsequent studies to assess in vitro cell-cell interactions and antineoplastic efficacy of certain agents to establish a treatment against GCTB.

Budding of Taenia crassiceps cysticerci in vitro is promoted by crowding in addition to hormonal, stress, and energy-related signals.

Taenia crassiceps cysticerci (cysts) reproduce by budding. The cysts' production of buds was measured in vitro to explore parasite and environmental-related factors involved in the extreme individual variation in parasite loads of inbred mice. Cysts were placed in in vitro culture for 10 days at initial parasite densities of 1, 5, 10 cysts/well in 1 ml of RPMI Medium 1640 without serum. Results showed that there is considerable intrinsic initial variation among inoculated cysts in their production of buds and that increasing parasite density (crowding) stimulates the overall production of buds and recruit into budding most of the cysts. Identical cultures were then subjected to various treatments such as heating and exposure to peroxide to induce stress, or to 17beta-estradiol, insulin, glucose, or insulin+glucose to supplement putatively limiting hormonal and energy resources. All treatments increased budding but the parasites' strong budding response to crowding alone overshadows the other treatments.

Progesterone induces scolex evagination of the human parasite Taenia solium: evolutionary implications to the host-parasite relationship.

Taenia solium cysticercosis is a health problem in underdeveloped and developed countries. Sex hormones are involved in cysticercosis prevalence in female and male pigs. Here, we evaluated the effects of progesterone and its antagonist RU486 on scolex evagination, which is the initial step in the development of the adult worm. Interestingly, progesterone increased T. solium scolex evagination and worm growth, in a concentration-independent pattern. Progesterone effects could be mediated by a novel T. solium progesterone receptor (TsPR), since RU486 inhibits both scolex evagination and worm development induced by progesterone. Using RT-PCR and western blot, sequences related to progesterone receptor were detected in the parasite. A phylogenetic analysis reveals that TsPR is highly related to fish and amphibian progesterone receptors, whereas it has a distant relation with birds and mammals. Conclusively, progesterone directly acts upon T. solium cysticerci, possibly through its binding to a progesterone receptor synthesized by the parasite.

A new MAP kinase protein involved in estradiol-stimulated reproduction of the helminth parasite Taenia crassiceps.

MAP kinases (MAPK) are involved in the regulation of cellular processes such as reproduction and growth. In parasites, the role of MAPK has been scarcely studied. Here, we describe the participation of an ERK-like protein in estrogen-dependent reproduction of the helminth parasite Taenia crassiceps. Our results show that 17beta-estradiol induces a concentration-dependent increase in the bud number of in vitro cultured cysticerci. If parasites are also incubated in presence of an ERK-inhibitor, the stimulatory effect of estrogen is blocked. The expression of ERK-like mRNA and its corresponding protein was detected in the parasite. The ERK-like protein was over-expressed by all treatments. Nevertheless, a strong induction of phosphorylation of this protein was observed only in response to 17beta-estradiol. Cross-contamination by host cells was discarded by flow cytometry analysis. Parasite cells expressing the ERK-like protein were exclusively located at the subtegument tissue by confocal microscopy. Finally, the ERK-like protein was separated by bidimensional electrophoresis and then sequenced, showing the conserved TEY activation motif, typical of all known ERK 1/2 proteins. Our results show that an ERK-like protein is involved in the molecular signalling during the interaction between the host and T. crassiceps, and may be considered as target for anti-helminth drugs design.

Protein expression profile of Taenia crassiceps cysticerci related to Th1- and Th2-type responses in the mouse cysticercosis model.

The intraperitoneal cysticercosis model with the Taenia crassiceps ORF strain in female BALB/cAnN mice has been widely used to study the immune response in cysticercosis. During early infection (2 weeks), the host develops a non-permissive Th1 response, whereas during late infection (8 weeks), molecules from the cysticerci induce a Th2 response that is permissive to parasite growth. The modulation of the Th2 response is induced by molecules excreted/secreted by the larval stage of the parasite. However, there is limited information regarding the response of cysticerci to the mouse immunological environment during infection. The proteomic profiles in T. crassiceps ORF cysticerci when faced with the mouse Th1 and Th2 responses were analyzed through two-dimensional gel electrophoresis (2DE), and the differential expression of proteins was evaluated. Thirteen proteins, whose differential expression varied between 70% and 100%, were selected randomly. Protein identification by MALDI-TOF MS and BLAST showed that the proteins were related to folding, signaling, enzymatic activities, cell-movement regulation, cell-cell interactions, motility, carbohydrate metabolism, detoxification, and redox regulation processes. Notably, some of the proteins can act as antigenic-protective molecules and elicit a weak Th1 response; however, most are involved in the avoidance of the immune system, which leads to a Th2 response, or apoptosis. The findings indicate the process by which T. crassiceps cysticerci responds based on the host environment and provides novel insights into the mechanism by which this facilitates its establishment and persistence in the mouse. Furthermore, these proteins could be used as targets for drug and vaccine development.

Protection induced by estradiol benzoate in the MPP+ rat model of Parkinson's disease is associated with the regulation of the inflammatory cytokine profile in the nigro striatum.

Previously, we have demonstrated that ß-estradiol-3-benzoate (EB) has a protective effect on the neurodegenerative experimental model of Parkinson's disease. The protective effect is through the induction of the expression of paraoxonase-2 (PON2) in the striatum. PON2 has proven to have antioxidant and anti-inflammatory activity, this protein has a beneficial effect in MPP+ model in rats decreasing the lipid peroxidation and the oxidative stress. Furthermore, the molecular effect and the pathway by which EB induces protection were not further pursued. This study shows the regulation by EB of the anti-inflammatory effect through the modulation of cytokines, antioxidant enzymes and PON2 in the rat striatum. Rats were gonadectomized and 30 days after were randomly assigned into four experimental groups; only vehicles (Control group); EB treatment (EB group); MPP+ injury (M group); EB plus MPP+ injured (EB/M group). EB treatment consisted of 100 µg of the drug administered every 48 h for 11 days. Results showed that EB (group EB/M) treatment decrease significantly (40%) the number of ipsilateral turns respect to the M group and prevents significantly the dopamine (DA) decreased induced by MPP+ (~75%). This results are correlate with a significant decrease in the level of lipid peroxidation (60%) of the EB/M group respect to the M group. The EB treatment showed protection against neurotoxicity induced with MPP+, this could be due to EB capacity to prevent the increase in the expression level of proinflammatory cytokines TNF-α, IL-1 and IL-6 induced by MPP+. While, TGF-ß1 and TGF-ß3 expression was reduced in the rats treated only with MPP+, in the rats of EB/M group the expression of both cytokines was increased. EB protective effect against MPP+ neurotoxicity is related to antioxidant effect of PON2, pro-inflammatory cytokines and GSHR but not to SOD2, catalase, GPX1 or GPX4.

Potential Novel Risk Factor for Breast Cancer: Toxocara canis Infection Increases Tumor Size Due to Modulation of the Tumor Immune Microenvironment.

Worldwide, breast cancer is the most important type of cancer in women with regard to incidence and prevalence. Several risk factors interact to increase the probability of breast cancer development. Biological environmental contaminants such as infectious agents play a significant role in tumor development, and helminths have been recognized as cancer enhancers or inducers due to their ability to regulate the host immune response. Toxocara canis is a zoonotic and cosmopolite nematode with immuno-regulatory abilities. T. canis infection has been related to T helper type-2 cell (Th2 or type 2) and regulatory responses. Type 2 and regulatory immune responses may favor the development of comorbidities that are usually controlled or eliminated through a type 1 response such as cancer. The aim of this study was to determine whether T. canis infection alters mammary tumor growth through modulation of the immune response. Infected mice developed larger tumors. Tumor immune cell milieu analysis revealed that infection reduced the proportions of CD8+ lymphocytes and increased the proportions of F4/80+ macrophages and CD19+ B cells. These changes were accompanied by a type 2 local response represented by increased amounts of IL-4 and VEGF and a regulatory microenvironment associated with higher IL-10 levels. Thus, this study demonstrates that T. canis infection enhances tumor development and suggests that this is through modulation of the tumor immune microenvironment.

M2 macrophage immunotherapy abolishes glucose intolerance by increasing IL-10 expression and AKT activation.

Aim: Glucose intolerance associates with M1/M2 macrophage unbalance. We thus wanted to examine the effect of M2 macrophage administration on mouse model of glucose intolerance. Materials & methods: C57BL/6 mice fed a high-fat diet (HFD) for 12 weeks and then received thrice 20 mg/kg streptozotocin (HFD-GI). Bone marrow-derived stem cells were collected from donor mice and differentiated/activated into M2 macrophages for intraperitoneal administration into HFD-GI mice. Results: M2 macrophage treatment abolished glucose intolerance independently of obesity. M2 macrophage administration increased IL-10 in visceral adipose tissue and serum, but showed no effect on serum insulin. While nitric oxide synthase-2 and arginase-1 remained unaltered, M2 macrophage treatment restored AKT phosphorylation in visceral adipose tissue. Conclusion: M2 macrophage treatment abolishes glucose intolerance by increasing IL-10 and phosphorylated AKT.

Trypanosoma cruzi SHSP16: Characterization of an alpha-crystallin small heat shock protein.

This report describes the characterization of a member of the alpha-crystallin small heat shock protein family in a trypanosomatid, which was isolated from the human pathogen Trypanosoma cruzi. One alpha-crystallin small heat shock protein gene was identified in a database search. The coding region is located in an open reading frame of 429bp encoding a protein of 142 amino acids. The amino acid sequence was deduced from the isolated gene. The protein has an alpha-crystallin domain characteristic of the alpha-crystallin small heat shock proteins and a molecular weight of 15.9kDa, so the protein was designated SHSP16. Analysis of the nucleotide sequences of four different T. cruzi strains showed two different sequences, which correspond to the two main T. cruzi genetic groups. Gene expression analysis by RT-PCR showed increased transcription of the gene after the parasite was exposed to heat stress. Recombinant SHSP16 showed molecular chaperone activity in vitro, because it inhibited the thermal aggregation of the mitochondrial malate dehydrogenase enzyme.

Neonatal Bisphenol A Exposure Affects the IgM Humoral Immune Response to 4T1 Breast Carcinoma Cells in Mice.

Bisphenol A (BPA) is an endocrine disruptor of estrogenic nature. During the early stages of development, any exposure to BPA can have long-term effects. In this work, we study the potential alterations to the humoral antitumor immune (IgM) response in adult life after a single neonatal exposure to BPA. Female syngeneic BALB/c mice were exposed to a single dose of BPA of 250 µg/kg. Once sexual maturity was reached, a breast tumor was induced. After 25 days, the serum was obtained, and the populations of B cells in the spleen and lymph nodes were analyzed by flow cytometry. The reactivity of IgM was evaluated by 2D immunoblots. No significant changes were found in the B cell populations in the peripheral lymph nodes and the spleen. The level of ERα expression was not significantly different. However, the IgM reactivity was affected. In individuals treated with BPA, a decrease in the number of IgMs that recognize tumor antigens was observed. The possibility that these antibodies are the high affinity products of the adaptive response is discussed. The recognition of IgG was also evaluated but a null recognition was found in the controls as in the individuals treated with the 4T1 cells.

Prolactin as immune cell regulator in Toxocara canis somatic larvae chronic infection.

Toxocariasis is a zoonotic disease produced by ingestion of larval Toxocara spp. eggs. Prolactin (PRL) has been considered to have an important role in Toxocara canis infection. Recent evidence has found that PRL directly can increase parasite growth and differentiation of T. canis The present study, evaluated the effect of high PRL levels on the immune system's response and parasites clearance in chronic infection. Our results showed that hyperprolactinemia did not affect the number of larvae recovered from several tissues in rats. Parasite-specific antibody production, showed no difference between the groups. Lung tissue presented eosinophilic granulomas typical of a chronic infection in all the experimental groups. Flow cytometry analysis was made in order to determine changes in the percentage of innate and adaptive immune cell subpopulations in the spleen, peripheric (PLN) and mesenteric (MLN) lymphatic nodes. The results showed a differential effect of PRL and infection on different immune compartments in the percent of total T cells, T helper cells, T cytotoxic cells, B cells, NK cells, and Tγδ cells. To our knowledge, for the first time it is demonstrated that PRL can have an immunomodulatory role during T. canis chronic infection in the murine host.

IL-6 KO mice develop experimental amoebic liver infection with eosinophilia.

Interleukin 6 (IL-6) is a multifunctional cytokine that regulates various aspects of the immune response, such as acute phase reaction and hematopoiesis, and is an important signal that coordinates activities of liver cells, macrophages, and lymphocytes. Amoebic liver lesions have been studied, usually in hamsters, due to the problem of abscess development in mice. We report here the development of an experimental amoebic liver abscess (ALA) model in mice deficient in IL-6. Axenically grown amoebae were injected directly into the livers of C57BL/6 wild type (WT) and IL-6 KO -/- mice; the abscesses produced were counted and the inflammatory process was examined on 5, 10, and 20 days postinfection. Our results showed that IL-6 KO -/- mice develop ALA, in contrast to the WT strain, which usually do not have signs of abscess or infection. Histological analysis of the abscesses showed extended inflammatory response, mainly mediated by eosinophils, which strongly infiltrate the abscess in IL-6 K -/- mice. The present results suggest that in mice, IL-6 could play a role in the resistance against ALA.

Recognition of tumor antigens in 4T1 cells by natural IgM from three strains of mice with different susceptibilities to spontaneous breast cancer.

The issue of antibody responses to tumors is potentially important to cancer immunologists. Early detection of cancer represents one of the most promising approaches to reduce the growing cancer burden. Natural immunoglobulin (Ig)M antibodies have been associated with the recognition and elimination of cancerous and precancerous cells. Using natural IgM antibodies, the present study identified a set of antigens in healthy mice from three different strains and examined whether the global patterns of antibodies are able to discriminate between a condition of more or less susceptibility to breast cancer. The current study performed two-dimensional (2D) immunoblotting to detect antigens from 4T1 cells using natural IgM from serum of healthy female mice from three different strains. The t-test was used to analyze the total number of spots. There were no significant differences in the numbers of antigens recognized in each strain. However, differences in patterns were observed on 2D immunoblots among the three strains. The reactivity patterns of natural IgM antibodies to particular antigens exhibited non-random clustering, which discriminated between strains with different susceptibilities to spontaneous breast cancer. The results demonstrated that the patterns of reactivity to defined subsets of antigens are able to provide information regarding differential diagnosis associated with breast cancer sensitivity. Therefore, it may be concluded that it is possible to segregate the IgM humoral immune response toward cancer antigens according to the genetic background of individuals. In addition, it is possible to identify the recognized antigens that allow grouping or discriminate between the different IgM antibodies expressed. The possible association between a particular antigen and cancer susceptibility requires further study, but the methodology exposed in the present study may identify potential candidates for this possible association.

ß-Estradiol-3-benzoate confers neuroprotection in Parkinson MPP+ rat model through inhibition of lipid peroxidation.

Estradiol (E2), in addition to its known hormone function, is a neuroactive steroid that has shown neuroprotective profile in several models of neurological diseases. The present study explores the antioxidant effect of ß-estradiol-3-benzoate (EB) on the neurotoxicity elicited by MPP+ in rat striatum. Male Wistar rats, that were gonadectomized 30days prior to EB, were given 100µgEB per rat every 48h for 11days and animals were infused with MPP+ via intrastriatal at day six after beginning EB treatment. EB treatment completely prevented the fall in dopamine caused by MPP+, such result was related with decreased lipid peroxidation, a marker of oxidative stress; diminished number of ipsilateral-to-lesion turns and increased signal of the dopamine-synthesizing enzyme Tyrosin Hydroxylase in substantia nigra. The protection elicited by EB was not related to Mn or Cu-Zn superoxide dismutase enzymatic activities or glutathione modulation since none of these parameters were influenced by EB at the times assayed. Whereas, increased expression of PON2 as a result of EB treatment was observed, this phenomenon could be one of the mechanism by which the steroid conferred protection to dopaminergic cells against MPP+ injury.