Immunogenicity, safety and dual DIVA-like character of a recombinant candidate vaccine against neosporosis in cattle.

Neosporosis is the major infectious cause of abortion and reproductive losses in cattle worldwide; however, there are no available vaccines or drugs to control this disease. Recently, a dual (positive and negative) DIVA-like (Differentiation of Infected from Vaccinated Animals) vaccine was evaluated in a pregnant mouse model of neosporosis, showing promising immunogenic and protective results. The current report aimed to study the safety, the dose-dependent immunogenicity and the dual DIVA-like character of a recombinant subunit vaccine composed of the major surface antigen from Neospora caninum (rNcSAG1) and the carrier/adjuvant Heat shock protein 81.2 from Arabidopsis thaliana (rAtHsp81.2) in cattle. Healthy heifers were separated and assigned to experimental groups A-F and subcutaneously immunized with 2 doses of vaccine formulations 30 days apart as follows: A (n = 4): 50 µg rNcSAG1 + 150 µg rAtHsp81.2; B (n = 4): 200 µg rNcSAG1 + 600 µg rAtHsp81.2; C (n = 4): 500 µg rNcSAG1 + 1,500 µg rAtHsp81.2; D (n = 3): 150 µg rAtHsp81.2; E (n = 3):1,500 µg rAtHsp81.2, and F (n = 3) 2 ml of sterile PBS. The immunization of heifers with the different vaccine or adjuvant doses (groups A-E) was demonstrated to be safe and did not modify the mean value of the evaluated serum biomarkers of metabolic function (GOT/ASP, GPT/ALT, UREA, Glucose and total proteins). The kinetics and magnitude of the immune responses were dose-dependent. The higher dose of the vaccine formulation (group C) stimulated a broad and potent humoral and cellular immune response, characterized by an IgG1/IgG2 isotype profile and IFN-γ secretion. In addition, this was the first time that dual DIVA-like character of a vaccine against neosporosis was demonstrated, allowing us to differentiate vaccinated from infected heifers by two different DIVA compliant test approaches. These results encourage us to evaluate its protective efficacy in infected pregnant cattle in the future.

Safe plant Hsp90 adjuvants elicit an effective immune response against SARS-CoV2-derived RBD antigen.

To better understand the role of pHsp90 adjuvant in immune response modulation, we proposed the use of the Receptor Binding Domain (RBD) of the Spike protein of SARS-CoV2, the principal candidate in the design of subunit vaccines. We evaluated the humoral and cellular immune responses against RBD through the strategy "protein mixture" (Adjuvant + Antigen). The rRBD adjuvanted with rAtHsp81.2 group showed a higher increase of the anti-rRBD IgG1, while the rRBD adjuvanted with rNbHsp90.3 group showed a significant increase in anti-rRBD IgG2b/2a. These results were consistent with the cellular immune response analysis. Spleen cell cultures from rRBD + rNbHsp90.3-immunized mice showed significantly increased IFN-γ production. In contrast, spleen cell cultures from rRBD + rAtHsp81.2-immunized mice showed significantly increased IL-4 levels. Finally, vaccines adjuvanted with rNbHsp90.3 induced higher neutralizing antibody responses compared to those adjuvanted with rAtHsp81.2. To know whether both chaperones must form complexes to generate an effective immune response, we performed co-immunoprecipitation (co-IP) assays. The results indicated that the greater neutralizing capacity observed in the rRBD adjuvanted with rNbHsp90.3 group would be given by the rRBD-rNbHsp90.3 interaction rather than by the quality of the immune response triggered by the adjuvants. These results, together with our previous results, provide a comparative benchmark of these two novel and safe vaccine adjuvants for their capacity to stimulate immunity to a subunit vaccine, demonstrating the capacity of adjuvanted SARS-CoV2 subunit vaccines. Furthermore, these results revealed differences in the ability to modulate the immune response between these two pHsp90s, highlighting the importance of adjuvant selection for future rational vaccine and adjuvant design.

Dysfunction, oxidative stress markers, and cytokine expression in the placentae of mice experimentally infected with Neospora caninum.

Neosporosis is the major cause of abortion and reproductive failures in cattle, leading to significant economic losses. In this study, we evaluated the impact of Neospora caninum infection on oxidative stress (OS) markers and local cytokine mRNA expression at the placenta, as well as its effect on the progesterone (P4) serum levels and systemic cytokine profile in a pregnant mouse model. Infected pregnant mice (NC-1 group) showed increased percentages of fetal losses and IFN-γ serum levels, decreased serum progesterone, increased placental mRNA expression levels of both Th1-type (IFN-γ and TNF-α) and Th2-type (IL-4) cytokines, and inhibited expression of TGF-ß1 (Treg) compare to control dams (CONTROL group). In addition, lipid peroxidation and ROS were increased, whereas the antioxidant enzymes, superoxide dismutase (SOD), and catalase (CAT) activities were modified in the placentae of infected mice compared to control mice. These findings demonstrate that multiple factors, including placental OS, are involved in fetal losses associated with N. caninum infection in mice, thus OS contribution to the placental physiopathology of neosporosis in other hosts must not be ruled out.

Neosporosis in sheep: A systematic review and meta-analysis of global seroprevalence and related risk factors.

Neosporosis is recognized as the main cause of abortions in cattle worldwide and there is an increasing concern about its role in ovine reproductive losses; however, epidemiological studies regarding neosporosis in sheep are still limited. This meta-analysis aimed to estimate the global pooled seroprevalence and associated risk factors of ovine neosporosis. In the current report, a comprehensive strategy of search and data collection from 7 worldwide databases was performed. A final set of 73 studies (80 datasets) published from 2000 to 2021 were selected based on inclusion criteria, comprising data on 35,740 sheep (corresponding to 37,565 evaluated samples) from 30 countries worldwide. The global pooled seroprevalence of Neospora caninum infection in sheep estimated by the random-effects model was 13% (95% CI, 10-15) and showed high heterogeneity (Q = 5147.15, I2 = 98%, p< 0.001). Furthermore, by meta-analyses of subgroups it was demonstrated for the first time that seroprevalence significantly varied between continents (highest in Africa; 20%, 95% CI, 4-44), WHO regions (highest in African Region; 42%, 95% CI, 36-48), countries (highest in Colombia; 79%, 95% CI, 61-92%) and diagnostic methods (highest by IFAT; 17%, 95% CI, 12-23). Meta-regression indicated significant increasing trends in the prevalence of ovine neosporosis with decrease in geographical latitude (coefficient = -0.013; p<0.001), whereas longitude did not influence it (coefficient = -0.001; p=0.365). Regarding associated risk factors, older sheep were more likely to be infected with N. caninum than younger ones (OR 1.42; 95% CI 1.08-1.87), and sheep bred under intensive or semi-intensive systems resulted less susceptible to be seropositive than those bred under extensive system (OR 0.65; 95% CI 0.42-0.99 and OR 0.74; 95% CI 0.62-0.89, respectively). Conversely, no apparent association was found between seroprevalence and other variables, such as sex (OR 1.06; 95% CI 0.9-1.24), the presence of dogs on the farm (OR 1.15; 95% CI 0.63-2.12) or the presence of abortion (OR 1.80; 95% CI 0.87-3.74). In conclusion, the seroprevalence of ovine neosporosis is widely and heterogeneously distributed throughout the world, and it is negatively associated with increasing geographical latitude. In addition, age and extensive production system represent risk factors, which suggest that the horizontal transmission route is relevant for this host species. It is recommended to pay more attention to this disease and emphasize the global need for more indexed studies concerning the seroprevalence and risk factors of ovine neosporosis to better understand the epidemiology of this coccidian infection.

Oral Immunization With a Plant HSP90-SAG1 Fusion Protein Produced in Tobacco Elicits Strong Immune Responses and Reduces Cyst Number and Clinical Signs of Toxoplasmosis in Mice.

Plant 90kDa heat shock protein (HSP90) is a potent adjuvant that increases both humoral and cellular immune responses to diverse proteins and peptides. In this study, we explored whether Arabidopsis thaliana HSP90 (AtHsp81.2) can improve the immune effects of a Toxoplasma gondii surface antigen 1 (SAG1). We designed two constructs containing the sequence of mature antigen (SAG1m), from aa77 to aa322, and B- and T-cell antigenic epitope-containing SAG1HC, from aa221 to aa319 fused to AtHsp81.2 sequence. When comparing the transient expression in Nicotiana tabacum X-27-8 leaves, which overexpress the suppressor helper component protease HC-Pro-tobacco etch virus (TEV), to that in N. benthamiana leaves, co-agroinfiltrated with the suppressor p19, optimal conditions included 6-week-old N. benthamiana plants, 7-day time to harvest, Agrobacterium tumefaciens cultures with an OD600nm of 0.6 for binary vectors and LED lights. While AtHsp81.2-SAG1m fusion protein was undetectable by Western blot in any of the evaluated conditions, AtHsp81.2-SAG1HC was expressed as intact fusion protein, yielding up to 90µg/g of fresh weight. Besides, the AtHsp81.2-SAG1HC mRNA was strongly expressed compared to the endogenous Nicotiana tabacum elongation factor-alpha (NtEFα) gene, whereas the AtHsp81.2-SAG1m mRNA was almost undetectable. Finally, mice were orally immunized with AtHsp81.2-SAG1HC-infiltrated fresh leaves (plAtHsp81.2-SAG1HC group), recombinant AtHsp81.2-SAG1HC purified from infiltrated leaves (rAtHsp81.2-SAG1HC group), non-infiltrated fresh leaves (control group), or phosphate-buffered saline (PBS group). Serum samples from plAtHsp81.2-SAG1HC-immunized mice had significantly higher levels of IgGt, IgG2a, and IgG2b anti-SAG1HC antibodies than serum from rAtHsp81.2-SAG1HC, control, and PBS groups. The number of cysts per brain in the plAtHsp81.2-SAG1HC-immunized mice was significantly reduced, and the parasite load in brain tissue was also lower in this group compared with the remaining groups. In an immunoblot assay, plant-expressed AtHsp81.2-SAG1HC was shown to react with antibodies present in sera from T. gondii-infected people. Therefore, the plant expression of a T. gondii antigen fused to the non-pathogenic adjuvant and carrier plant HSP90 as formulations against T. gondii can improve the vaccine efficacy, and plant extract can be directly used for vaccination without the need to purify the protein, making this platform a suitable and powerful biotechnological system for immunogenic antigen expression against toxoplasmosis.

Use of Veterinary Vaccines for Livestock as a Strategy to Control Foodborne Parasitic Diseases.

Foodborne diseases (FBDs) are a major concern worldwide since they are associated with high mortality and morbidity in the human population. Among the causative agents of FBDs, Taenia solium, Echinococcus granulosus, Toxoplasma gondii, Cryptosporidium spp., and Trichinella spiralis are listed in the top global risk ranking of foodborne parasites. One common feature between them is that they affect domestic livestock, encompassing an enormous risk to global food production and human health from farm to fork, infecting animals, and people either directly or indirectly. Several approaches have been employed to control FBDs caused by parasites, including veterinary vaccines for livestock. Veterinary vaccines against foodborne parasites not only improve the animal health by controlling animal infections but also contribute to increase public health by controlling an important source of FBDs. In the present review, we discuss the advances in the development of veterinary vaccines for domestic livestock as a strategy to control foodborne parasitic diseases.

Heat Shock Proteins 90 kDa: Immunomodulators and Adjuvants in Vaccine Design Against Infectious Diseases.

Heat shock proteins 90 kDa (Hsp90s) were originally identified as stress-responsive proteins and described to participate in several homeostatic processes. Additionally, extracellular Hsp90s have the ability to bind to surface receptors and activate cellular functions related to immune response (cytokine secretion, cell maturation, and antigen presentation), making them very attractive to be studied as immunomodulators. In this context, Hsp90s are proposed as new adjuvants in the design of novel vaccine formulations that require the induction of a cell-mediated immune response to prevent infectious diseases. In this review, we summarized the adjuvant properties of Hsp90s when they are either alone, complexed, or fused to a peptide to add light to the knowledge of Hsp90s as carriers and adjuvants in the design of vaccines against infectious diseases. Besides, we also discuss the mechanisms by which Hsp90s activate and modulate professional antigen-presenting cells.

The potential of a DIVA-like recombinant vaccine composed by rNcSAG1 and rAtHsp81.2 against vertical transmission in a mouse model of congenital neosporosis.

Neospora caninum is the etiological agent of neosporosis, a worldwide infectious disease recognized as the major cause of abortions and reproductive failures in livestock, responsible for significant economic losses in cattle industries. Currently, there are not cost-effective control options for this pathology, and the development of a vaccine involving new and integrated approaches is highly recommended. In this study, we evaluated the immunogenic and protective efficacy, as well as the potential DIVA (Differentiation of Infected from Vaccinated Animals) character of a recombinant subunit vaccine composed by the major surface antigen from N. caninum (NcSAG1) and the carrier/adjuvant heat shock protein 81.2 from Arabidopsis thaliana (AtHsp81.2) in a mouse model of congenital neosporosis. BALB/c female mice were intraperitoneal (i.p.) immunized with a mixture of equimolar quantities of rNcSAG1 and rAtHSP81.2 or each protein alone (rNcSAG1 or rAtHsp81.2). The vaccine containing a mixture of rNcSAG1 and rAtHsp81.2 significantly enhanced the production of specific anti-rNcSAG1 total IgG (tIgG), IgG1 and IgG2a antibodies in immunized mice when compared to control groups (non-vaccinated and rAtHsp81.2 immunized mice) as well as to the group of mice immunized only with the antigen (rNcSAG1). In addition, partial protection against vertical transmission and improvement of the offspring survival time was observed in this group. On the other hand, rAtHsp81.2 induced the production of specific anti-rAtHsp81.2 tIgG, allowing us to differentiate vaccinated from infected mice. Despite further experiments have to be made in cattle to test the capability of this vaccine formulation to differentiate vaccinated from infected animals in the field, our results suggest that the formulation composed by rNcSAG1 and rAtHsp81.2 could serve as a basis for the development of a new vaccine approach against bovine neosporosis.

Heat treatment alleviates the growth and photosynthetic impairment of transplastomic plants expressing Leishmania infantum Hsp83-Toxoplasma gondii SAG1 fusion protein.

Previously, we showed that transplastomic tobacco plants expressing the LiHsp83-SAG1 fusion protein displayed a chlorotic phenotype and growth retardation, while plants expressing the SAG1 and GRA4 antigens alone did not. We conducted a comprehensive examination of the metabolic and photosynthetic parameters that could be affecting the normal growth of LiHsp83-SAG1 plants in order to understand the origin of these pleiotropic effects. These plants presented all photosynthetic pigments and parameters related to PSII efficiency significantly diminished. However, the expression of CHLI, RSSU and LHCa/b genes did not show significant differences between LiHsp83-SAG1 and control plants. Total protein, starch, and soluble sugar contents were also greatly reduced in LiHsp83-SAG1 plants. Since Hsp90 s are constitutively expressed at much higher concentrations at high temperatures, we tested if the fitness of LiHsp83-SAG1 over-expressing LiHsp83 would improve after heat treatment. LiHsp83-SAG1 plants showed an important alleviation of their phenotype and an evident recovery of the PSII function. As far as we know, this is the first report where it is demonstrated that a transplastomic line performs much better at higher temperatures. Finally, we detected that LiHsp83-SAG1 protein could be binding to key photosynthesis-related proteins at 37 °C. Our results suggest that the excess of this molecular chaperone could benefit the plant in a possible heat shock and prevent the expected denaturation of proteins. However, the LiHsp83-SAG1 protein content was weakly decreased in heat-treated plants. Therefore, we cannot rule out that the alleviation observed at 37 °C may be partially due to a reduction of the levels of the recombinant protein.

Plant Serine Protease Inhibitors: Biotechnology Application in Agriculture and Molecular Farming.

The serine protease inhibitors (SPIs) are widely distributed in living organisms like bacteria, fungi, plants, and humans. The main function of SPIs as protease enzymes is to regulate the proteolytic activity. In plants, most of the studies of SPIs have been focused on their physiological role. The initial studies carried out in plants showed that SPIs participate in the regulation of endogenous proteolytic processes, as the regulation of proteases in seeds. Besides, it was observed that SPIs also participate in the regulation of cell death during plant development and senescence. On the other hand, plant SPIs have an important role in plant defense against pests and phytopathogenic microorganisms. In the last 20 years, several transgenic plants over-expressing SPIs have been produced and tested in order to achieve the increase of the resistance against pathogenic insects. Finally, in molecular farming, SPIs have been employed to minimize the proteolysis of recombinant proteins expressed in plants. The present review discusses the potential biotechnological applications of plant SPIs in the agriculture field.

Plant Hsp90 is a novel adjuvant that elicits a strong humoral and cellular immune response against B- and T-cell epitopes of a Toxoplasma gondii SAG1 peptide.

BACKGROUND: The 90-kDa heat-shock protein (Hsp90) from Nicotiana benthamiana (NbHsp90.3) is a promising adjuvant, especially for those vaccines that require a T cell-mediated immune response. Toxoplasma gondii SAG1 is considered one of the most important antigens for the development of effective subunit vaccines. Some epitopes located in the SAG1 C-terminus region have showed a strong humoral and cellular immune response. In the present study, we aimed to assess the efficacy of NbHsp90.3 as carrier/adjuvant of SAG1-derived peptide (SAG1HC) in a T. gondii infection murine model. METHODS: In the present study, C57BL/6 mice were intraperitoneal immunized with the NbHsp90.3-SAG1HC fusion protein (NbHsp90.3-SAG1HC group), mature SAG1 (SAG1m group), NbHsp90.3 (NbHsp90.3 group) or PBS buffer 1× (PBS group). The levels of IgG antibodies and the cytokine profile were determined by ELISA. Two weeks after the last immunization, all mice were orally challenged with 20 cysts of T. gondii Me49 strain and the number of brain cysts was determined. In addition, both humoral and cellular immune responses were also evaluated during the acute and chronic phase of T. gondii infection by ELISA. RESULTS: The characterization of the immune response generated after vaccination with NbHsp90.3 as an adjuvant showed that NbHsp90.3-SAG1HC-immunized mice produced antibodies that were able to recognize not only rSAG1m but also the native SAG1 present in the total lysate antigen extract (SAG1TLA) from T. gondii tachyzoites, while control groups did not. Furthermore, anti-rSAG1m IgG2a/2b antibodies were significantly induced. In addition, only the spleen cell cultures from NbHsp90.3-SAG1HC-immunized mice showed a significantly increased production of IFN-γ. During the chronic phase of T. gondii infection, the antibodies generated by the infection were unable to detect the recombinant protein, but they did react with TLA extract. In addition, splenocytes from all groups showed a high production of IFN-γ when stimulated with rGRA4, but only those from NbHsp90.3-SAG1HC group stimulated with rSAG1m showed high production of IFN-γ. Finally, NbHsp90.3-SAG1HC-immunized mice exhibited a significant reduction in the cyst load (56%) against T. gondii infection. CONCLUSIONS: We demonstrated that NbHsp90.3 enhances the humoral and cell-mediated immune response through a Th1 type cytokine production. Mice vaccinated with NbHsp90.3-SAG1HC exhibited a partial protection against T. gondii infection and it was correlated with the induction of memory immune response. We developed and validated a vaccine formulation which, to our knowledge, for the first time includes the NbHsp90.3 protein covalently fused to a peptide from T. gondii SAG1 protein that contains T- and B-cell epitopes.

Promising Plant-Derived Adjuvants in the Development of Coccidial Vaccines.

Coccidial parasites cause medical and veterinary diseases worldwide, frequently leading to severe illness and important economic losses. At present, drugs, chemotherapeutics and prophylactic vaccines are still missing for most of the coccidial infections. Moreover, the development and administration of drugs and chemotherapeutics against these diseases would not be adequate in livestock, since they may generate unacceptable residues in milk and meat that would avoid their commercialization. In this scenario, prophylactic vaccines emerge as the most suitable approach. Subunit vaccines have proven to be biologically safe and economically viable, allowing researchers to choose among the best antigens against each pathogen. However, they are generally poorly immunogenic and require the addition of adjuvant compounds to the vaccine formulation. During the last decades, research involving plant immunomodulatory compounds has become an important field of study based on their potential pharmaceutical applications. Some plant molecules such as saponins, polysaccharides, lectins and heat shock proteins are being explored as candidates for adjuvant/carriers formulations. Moreover, plant-derived immune stimulatory compounds open the possibility to attain the main goal in adjuvant research: a safe and non-toxic adjuvant capable of strongly boosting and directing immune responses that could be incorporated into different vaccine formulations, including mucosal vaccines. Here, we review the immunomodulatory properties of several plant molecules and discuss their application and future perspective as adjuvants in the development of vaccines against coccidial infections.

Characterization of a novel Kazal-type serine proteinase inhibitor of Arabidopsis thaliana.

Many different types of serine proteinase inhibitors have been involved in several kinds of plant physiological processes, including defense mechanisms against phytopathogens. Kazal-type serine proteinase inhibitors, which are included in the serine proteinase inhibitor family, are present in several organisms. These proteins play a regulatory role in processes that involve serine proteinases like trypsin, chymotrypsin, thrombin, elastase and/or subtilisin. In the present work, we characterized two putative Kazal-type serine proteinase inhibitors from Arabidopsis thaliana, which have a single putative Kazal-type domain. The expression of these inhibitors is transiently induced in response to leaf infection by Botrytis cinerea, suggesting that they play some role in defense against pathogens. We also evaluated the inhibitory specificity of one of the Kazal-type serine proteinase inhibitors, which resulted to be induced during the local response to B. cinerea infection. The recombinant Kazal-type serine proteinase inhibitor displayed high specificity for elastase and subtilisin, but low specificity for trypsin, suggesting differences in its selectivity. In addition, this inhibitor exhibited a strong antifungal activity inhibiting the germination rate of B. cinerea conidia in vitro. Due to the important role of proteinase inhibitors in plant protection against pathogens and pests, the information about Kazal-type proteinase inhibitors described in the present work could contribute to improving current methods for plant protection against pathogens.

The fusion of Toxoplasma gondii SAG1 vaccine candidate to Leishmania infantum heat shock protein 83-kDa improves expression levels in tobacco chloroplasts.

Chloroplast transformation technology has emerged as an alternative platform offering many advantages over nuclear transformation. SAG1 is the main surface antigen of the intracellular parasite Toxoplasma gondii and a promising candidate to produce an anti-T. gondii vaccine. The aim of this study was to investigate the expression of SAG1 using chloroplast transformation technology in tobacco plants. In order to improve expression in transplastomic plants, we also expressed the 90-kDa heat shock protein of Leishmania infantum (LiHsp83) as a carrier for the SAG1 antigen. SAG1 protein accumulation in transplastomic plants was approximately 0.1-0.2 µg per gram of fresh weight (FW). Fusion of SAG1 to LiHsp83 significantly increased the level of SAG1 accumulation in tobacco chloroplasts (by up to 500-fold). We also evaluated the functionality of the chLiHsp83-SAG1. Three human seropositive samples reacted with SAG1 expressed in transplastomic chLiHsp83-SAG1 plants. Oral immunization with chLiHsp83-SAG1 elicited a significant reduction of the cyst burden that correlated with an increase of SAG1-specific antibodies. We propose the fusion of foreign proteins to LiHsp83 as a novel strategy to increase the expression level of the recombinant proteins using chloroplast transformation technology, thus addressing one of the current challenges for this approach in antigen protein production.

Plant heat shock protein 90 as carrier-adjuvant for immunization against a reporter antigen.

Here, we evaluated the modulation of the immune response induced by Hsp90 of Nicotiana benthamiana (NbHsp90.3) against the Maltose Binding Protein (MBP) as a reporter antigen. Equimolar quantities of recombinant proteins were administered in mice as follows: MBP alone (MBP group), a mixture of MBP and rNbHsp90.3 (MBP+rNbHsp90.3 group) and the fusion of MBP to rNbHsp90.3 (MBP-rNbHsp90.3 group). The covalent linkage between NbHsp90.3 and MBP to bring a fusion protein was essential to induce the strong specific antibody response with predominance of IgG2a. Eighty-four days after the first immunization, splenocyte proliferation from MBP-rNbHsp90.3-immunized mice was consistently higher than that from MBP and MBP+rNbHsp90.3 groups. In addition, splenocytes from MBP-rNbHsp90.3 immunized mice produced higher levels of IFN-γ than controls. Finally, both formulations with rNbHsp90.3 significantly enhanced the MHC class I expression levels, but only rNbHsp90.3 covalent bound to MBP induced a specific cellular immune response against MBP measured as increased percentage of CD8(+) T cells. Taken together, these results suggest that plant HSP90s could be incorporated as adjuvants in vaccines that require the generation of a Th1 response along with a CD8 cytotoxic cell response to confer immunity.

A chloroplast-derived Toxoplasma gondii GRA4 antigen used as an oral vaccine protects against toxoplasmosis in mice.

The parasitic protozoan Toxoplasma gondii, the causal agent of toxoplasmosis, can infect most mammals and birds. In human medicine, T. gondii can cause complications in pregnant women and immunodeficient individuals, while in veterinary medicine, T. gondii infection has economic importance due to abortion and neonatal loss in livestock. Thus, the development of an effective anti-Toxoplasma vaccine would be of great value. In this study, we analysed the expression of T. gondii GRA4 antigen by chloroplast transformation (chlGRA4) in tobacco plants and evaluated the humoral and cellular responses and the grade of protection after oral administration of chlGRA4 in a murine model. The Western blot analysis revealed a specific 34-kDa band mainly present in the insoluble fractions. The chlGRA4 accumulation levels were approximately 6 µg/g of fresh weight (equivalent to 0.2% of total protein). Oral immunization with chlGRA4 resulted in a decrease of 59% in the brain cyst load of mice compared to control mice. ChlGRA4 immunization elicited both a mucosal immune response characterized by the production of specific IgA, and IFN-γ, IL-4 and IL-10 secretion by mesenteric lymph node cells, and a systemic response in terms of GRA4-specific serum antibodies and secretion of IFN-γ, IL-4 and IL-10 by splenocytes. Our results indicate that oral administration of chlGRA4 promotes the elicitation of both mucosal and systemic balanced Th1/Th2 responses that control Toxoplasma infection, reducing parasite loads.

Systemic inflammation, cellular influx and up-regulation of ovarian VCAM-1 expression in a mouse model of polycystic ovary syndrome (PCOS).

PCOS, a major cause of anovulatory sterility, is associated with obesity, insulin resistance and chronic inflammation. New evidence suggests that the immune system aggravates the clinical features of PCOS. Our aim was to study the immune, metabolic and endocrine features of a mouse model of PCOS elicited by androgenisation using dehydroepiandrosterone (DHEA). We observed a significant weight gain and insulin resistance in DHEA-androgenised mice, coupled with the formation of ovarian follicular cysts. DHEA up-regulated the expression of vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 in the granulosa cell layer of the majority of cysts, and VCAM-1 expression in the theca cell layer of all follicles and cysts. The expression of these markers was low in control tissue. Peritoneal cells from PCOS-mice showed enhanced production of inflammatory cytokines, suggesting an association between chronic inflammation and PCOS. In addition, DHEA-androgenisation induced the activation of CD4(+) cells both in vivo and in vitro, and their expression of the respective ligands for VCAM-1 and ICAM-1, VLA-4 and LFA-1, as assessed in vitro. CD4(+) cells were present in androgenised ovaries, especially in the granulosa cell layer of cysts with high VCAM-1 expression. Herein, we present novel evidence that the immune system is activated systemically and locally in a mouse model for PCOS. We propose that VCAM-1 is involved in aggravating PCOS symptoms by promoting leukocyte recruitment to the ovaries and perpetuating local inflammation. These findings offer novel therapeutic opportunities for PCOS, such as blockage of VCAM-1 expression.

Alterations of folliculogenesis in women with polycystic ovary syndrome.

The objective of the present study was to examine some factors involved in follicular development of women with polycystic ovary syndrome (PCOS). Women with PCOS showed increased levels of serum luteinizing hormone (LH) but decreased follicular production of progesterone and estradiol by pre-ovulatory follicles. The mRNA expression corresponding to steroidogenic acute regulatory protein (StAR), and 20alpha-hydroxysteroid dehydrogenase (20α-HSD) was increased, while that corresponding to cytochrome P450 aromatase (P450arom) was decreased in PCOS follicles as compared to controls. No changes in the mRNA expression for 3beta-hydroxysteroid dehydrogenase 2 (3ß-HSD2), cytochrome P450 side-chain cleavage (P450scc), cytochrome P450 17 alpha hydroxylase/lyase (P450c17), cyclooxygenase 2 (COX2), and transcription factors (GATA-4 and GATA-6) were found. We conclude that despite the hyper-luteinized environment of PCOS follicles, these follicles produce lower levels of progesterone and estradiol, and that this is characterized by increased degradation of progesterone and decreased estradiol synthesis. Our data demonstrate that the synthesis of prostaglandin F2α (PGF2α) may be affected in PCOS-follicles and that the transcription factors GATA-4 and GATA-6 are present in PCOS-follicles but they are not involved in the abnormal transcription observed in the steroidogenic enzymes.

Hyperandrogenism alters intraovarian parameters during early folliculogenesis in mice.

This study aimed to investigate how hyperandrogenism affects early folliculogenesis. Hyperandrogenism was induced in prepuberal female BALB/c mice by daily s.c. injection of dehydroepiandrosterone (60 mg/kg body weight in 0.1 ml sesame oil) for 10 consecutive days. Although hyperandrogenism increased the growth rate of primary follicles, it also increased ovarian oxidative stress (evaluated by the increase in lipid peroxidation, the decrease in superoxide dismutase activity and the fact that glutathione content was not modified). By using the annexin V/cytometry assay it was found that the excess of androgens decreased viable ovarian cells and increased early apoptotic ones. The increased lipid peroxidation induced enhanced ovarian prostaglandin E production. In addition, hyperandrogenism increased the number of T lymphocytes that infiltrate ovarian tissue and modified their phenotype (decreased CD4+ or helper and increased the suppressor/cytotoxic CD8+). The excess of androgens decreased the ovarian expression of the long isoform of leptin receptor (Ob-Rb, the only isoform expressed in the ovarian tissue) when compared with controls. All these alterations increased serum concentrations of oestradiol, a pro-apoptotic agent. It is concluded that the excess of androgens impairs early follicular development by modulating some endocrine and immune parameters that are either directly or indirectly related to follicular atresia.