Immunogens in Balamuthia mandrillaris: a proteomic exploration.

Balamuthia mandrillaris is the causative agent of granulomatous amoebic encephalitis, a rare and often fatal infection affecting the central nervous system. The amoeba is isolated from diverse environmental sources and can cause severe infections in both immunocompromised and immunocompetent individuals. Given the limited understanding of B. mandrillaris, our research aimed to explore its protein profile, identifying potential immunogens crucial for early granulomatous amoebic encephalitis diagnosis. Cultures of B. mandrillaris and other amoebas were grown under axenic conditions, and total amoebic extracts were obtained. Proteomic analyses, including two-dimensional electrophoresis and mass spectrometry, were performed. A 50-kDa band showed a robust recognition of antibodies from immunized BALB/c mice; peptides contained in this band were matched with elongation factor-1 alpha, which emerged as a putative key immunogen. Besides, lectin blotting revealed the presence of glycoproteins in B. mandrillaris, and confocal microscopy demonstrated the focal distribution of the 50-kDa band throughout trophozoites. Cumulatively, these observations suggest the participation of the 50-kDa band in adhesion and recognition mechanisms. Thus, these collective findings demonstrate some protein characteristics of B. mandrillaris, opening avenues for understanding its pathogenicity and developing diagnostic and therapeutic strategies.

Two MP2CL5 Antigen Vaccines from Naegleria fowleri Stimulate the Immune Response against Meningitis in the BALB/c Model.

Naegleria fowleri is an etiological agent that generates primary amoebic meningoencephalitis; unfortunately, no effective treatment or vaccine is available. The objective of this work was to determine the immunoprotective response of two vaccine antigens, as follows: (i) the polypeptide band of 19 kDa or (ii) a predicted immunogenic peptide from the membrane protein MP2CL5 (Smp145). Both antigens were administered intranasally in mice using cholera toxin (CT) as an adjuvant. The survival rate and immune response of immunized mice with both antigens and challenged with N. fowleri trophozoites were measured in the nose-associated lymphoid tissue (NALT) and nasal passages (NPs) by flow cytometry and enzyme-linked immunosorbent assay (ELISA). We also determined the immunolocalization of both antigens in N. fowleri trophozoites by confocal microscopy. Immunization with the polypeptide band of 19 kDa alone or coadministered with CT was able to confer 80% and 100% of protection, respectively. The immunization with both antigens (alone or coadministered with CT) showed an increase in T and B lymphocytes. In addition, there was an increase in the expression of integrin α4ß1 and IgA in the nasal cavity of protected mice, and the IgA, IgG, and IgM levels were increased in serum and nasal washes. The immunolocalization of both antigens in N. fowleri trophozoites was observed in the plasma membrane, specifically in pseudopod-like structures. The MP2CL5 antigens evaluated in this work were capable of conferring protection which would lead us to consider them as potential candidates for vaccines against meningitis caused by N. fowleri.

Role of FcγRIII in the nasal cavity of BALB/c mice in the primary amebic meningoencephalitis protection model.

Different mechanisms of the host immune response against the primary amebic meningoencephalitis (PAM) in the mouse protection model have been described. It has been proposed that antibodies opsonize Naegleria fowleri trophozoites; subsequently, the polymorphonuclear cells (PMNs) surround the trophozoites to avoid the infection. FcγRs activate signaling pathways of adapter proteins such as Syk and Hck on PMNs to promote different effector cell functions which are induced by the Fc portion of the antibody-antigen complexes. In this work, we analyzed the activation of PMNs, epithelial cells, and nasal passage cells via the expression of Syk and Hck genes. Our results showed an increment of the FcγRIII and IgG subclasses in the nasal cavity from immunized mice as well as Syk and Hck expression was increased, whereas in the in vitro assay, we observed that when the trophozoites of N. fowleri were opsonized with IgG anti-N. fowleri and interacted with PMN, the expression of Syk and Hck was also increased. We suggest that PMNs are activated via their FcγRIII, which leads to the elimination of the trophozoites in vitro, while in the nasal cavity, the adhesion and consequently infection are avoided.

Role of cathepsin B of Naegleria fowleri during primary amebic meningoencephalitis.

Naegleria fowleri causes primary amoebic meningoencephalitis in humans and experimental animals. It has been suggested that cysteine proteases of parasites play key roles in metabolism, nutrient uptake, host tissue invasion, and immune evasion. The aim of this work was to evaluate the presence, expression, and role of cathepsin B from N. fowleri in vitro and during PAM. Rabbit-specific polyclonal antibodies against cathepsin B were obtained from rabbit immunization with a synthetic peptide obtained by bioinformatic design. In addition, a probe was designed from mRNA for N. fowleri cathepsin B. Both protein and messenger were detected in fixed trophozoites, trophozoites interacted with polymorphonuclear and histological sections of infected mice. The main cathepsin B distribution was observed in cytoplasm or membrane mainly pseudopods and food-cups while messenger was in nucleus and cytoplasm. Surprisingly, both the messenger and enzyme were observed in extracellular medium. To determine cathepsin B release, we used trophozoites supernatant recovered from nasal passages or brain of infected mice. We observed the highest release in supernatant from recovered brain amoebae, and when we analyzed molecular weight of secreted proteins by immunoblot, we found 30 and 37 kDa bands which were highly immunogenic. Finally, role of cathepsin B during N. fowleri infection was determined; we preincubated trophozoites with E-64, pHMB or antibodies with which we obtained 60%, 100%, and 60% of survival, respectively, in infected mice. These results suggest that cathepsin B plays a role during pathogenesis caused by N. fowleri mainly in adhesion and contributes to nervous tissue damage.

Effect of soybean grain (Glycine max L.) supplementation on the production and fatty acid profile in milk of grazing cows in the dry tropics of Mexico.

Improving the lipid profile in milk of cows with the use of soybean grain (Glycine max L.) can be favored in the grazing systems in the dry tropics of Mexico. The objective was to evaluate the milk production, the chemical composition, and the fatty acids profile (FAs) of the milk of cows in continuous grazing and supplemented with and without ground roasted soybean in the dry tropics of Mexico. Ten cows randomly distributed in two equal groups were used. Daily during confinement for milking, the cows individually received the treatments on dry basis T0: supplement with 4.6 kg commercial concentrate® without soybean, T1: supplement with 3.7 kg commercial concentrate® with 380 g of soybean. During the 78 days of the experiment, milk production was measured in all cows, and samples were collected to determine the chemical composition and FAs profile. Milk production, protein, milk total fat, lactose, and non-fat solids did not vary with treatment (p >0.05). Linoleic acid content (C18: 2, cis, cis-∆9, ∆12) increased by 22% in milk fat of cows of the T1 (p ˂0.05). The sum of the mono- and polyunsaturated FAs 29.1%, the ratio of saturated-unsaturated FAs (1.65), and the atherogenicity index (1.71) also improved in the milk of cows supplemented with T1 (p ˂0.05). It was concluded that ground roasted soybean supplement in the diet of grazing dairy cows did not affect production and did improve the lipid profile in milk fat with favorable index to promote human health.

A 250-kDa glycoprotein of Naegleria fowleri induces protection and modifies the expression of α4ß1 and LFA-1 on T and B lymphocytes in mouse meningitis model.

The aims of this work were to evaluate the protective role of the 250-kDa polypeptide band of Naegleria fowleri. We designed an immunization strategy in Balb/c mice which were inoculated by i.n. route with an electrocuted 250-kDa polypeptide band of N. fowleri. We observed that the 250-kDa band induced 80% of protection, whereas the coadministration with Cholera Toxin induced 100% of protection. Moreover, high levels of IgA- and IgG-specific antibodies were detected by ELISA assay. We also analysed migration molecules (α4ß1 and LFA-1) on T and B lymphocytes in nose-associated lymphoid tissue (NALT), cervical lymph nodes (CN) and nasal passages (NP) by flow cytometry. We observed that the percentage of B cells (B220/α4ß1) and T cells (CD4/α4ß1) in NP were higher in all immunized groups compared with the other compartments analysed. Finally, we detected by immunohistochemistry ICAM-1 and V-CAM-1 in the nasal cavity. The immunization with the 250-kDa polypeptide band, protect mice against N. fowleri challenge and modifies migration molecules and their ligands.

Identification of differential protein recognition pattern between Naegleria fowleri and Naegleria lovaniensis.

Many pathogenicity factors are involved in the development of primary amoebic meningoencephalitis (PAM) caused by N fowleri. However, most of them are not exclusive for N fowleri and they have not even been described in other nonpathogenic Naegleria species. Therefore, the objective of this work was to identify differential proteins and protein pattern recognition between Naegleria fowleri and Naegleria lovaniensis using antibodies anti-N fowleri as strategy to find vaccine candidates against meningoencephalitis. Electrophoresis and Western blots conventional and 2-DE were performed for the identification of antigenic proteins, and these were analysed by the mass spectrometry technique. The results obtained in 2-DE gels and Western blot showed very notable differences in spot intensity between these two species, specifically those with relative molecular weight of 100, 75, 50 and 19 kDa. Some spots corresponding to these molecular weights were identified as actin fragment, myosin II, heat shock protein, membrane protein Mp2CL5 among others, with differences in theoretical post-translational modifications. In this work, we found differences in antigenic proteins between both species, proteins that could be used for a further development of vaccines against N fowleri infection.

Neutrophil extracellular traps and MPO in models of susceptibility and resistance against Entamoeba histolytica.

The main effector mechanisms of neutrophils are the release of neutrophil extracellular traps (NETs) and myeloperoxidase (MPO). In this work, we evaluated the role of NETs and the activity of MPO in the interactions of rodent neutrophils with amoebae and in amoebic liver abscess (ALA)-resistant and ALA-susceptible models. We showed with in vitro assays that mice produced greater amounts of NETs and MPO than did hamsters, and the elastase activity was high in both models. However, the inhibition of NETs and MPO promoted an increase in amoeba viability in the mice. The mouse ALAs showed a more profound presence of NETs and MPO than did the hamster ALAs. We concluded that both effector mechanisms were essential for the amoebic damage and could prevent the formation of ALAs in the resistant model.

Bioinformatics design and experimental validation of influenza A virus multi-epitopes that induce neutralizing antibodies.

Pandemics caused by influenza A virus (IAV) are responsible for the deaths of millions of humans around the world. One of these pandemics occurred in Mexico in 2009. Despite the impact of IAV on human health, there is no effective vaccine. Gene mutations and translocation of genome segments of different IAV subtypes infecting a single host cell make the development of a universal vaccine difficult. The design of immunogenic peptides using bioinformatics tools could be an interesting strategy to increase the success of vaccines. In this work, we used the predicted amino acid sequences of the neuraminidase (NA) and hemagglutinin (HA) proteins of different IAV subtypes to perform multiple alignments, epitope predictions, molecular dynamics simulations, and experimental validation. Peptide selection was based on the following criteria: promiscuity, protein surface exposure, and the degree of conservation among different medically relevant IAV strains. These peptides were tested using immunological assays to test their ability to induce production of antibodies against IAV. We immunized rabbits and mice and measured the levels of IgG and IgA antibodies in serum samples and nasal washes. Rabbit antibodies against the peptides P11 and P14 (both of which are hybrids of NA and HA) recognized HA from both group 1 (H1, H2, and H5) and group 2 (H3 and H7) IAV and also recognized the purified NA protein from the viral stock (influenza A Puerto Rico/916/34). IgG antibodies from rabbits immunized with P11 and P14 were capable of recognizing viral particles and inhibited virus hemagglutination. Additionally, intranasal immunization of mice with P11 and P14 induced specific IgG and IgA antibodies in serum and nasal mucosa, respectively. Interestingly, the IgG antibodies were found to have neutralizing capability. In conclusion, the peptides designed through in silico studies were validated in experimental assays.

Mouse neutrophils release extracellular traps in response to Naegleria fowleri.

Naegleria fowleri is a free-living amoeba, which is able to infect humans through the nasal mucosa causing a disease in the central nervous system known as primary amoebic meningoencephalitis (PAM). Polymorphonuclear cells (PMNs) play a critical role in the early phase of N fowleri infection. Recently, a new biological defence mechanism called neutrophil extracellular traps (NETs) has been attracting attention. These structures represent an important strategy to immobilize and kill invading microorganisms. In this work, we evaluate the capacity of N fowleri to induce the NETs release by PMNs cells in mice in vitro and in vivo. In vitro: Neutrophils from bone marrow were cocultured with N fowleri trophozoites. In vivo: we employed a mouse model of PAM. We evaluated DNA, histone and myeloperoxidase (MPO) and the formation of NETs by confocal microscopy. Our results showed N fowleri induce both NETs and MPO release by PMNs cells in mice after trophozoite exposure, which increased through time, in vitro and in vivo. These results demonstrate that NETs are somehow associated with the amoebas. We suggest PMNs release their traps trying to avoid N fowleri attachment at the apical side of the nasal epithelium.

Posterior Subdiaphragmatic Vagotomy Downmodulates the IgA Levels in the Small Intestine of BALB/c Mice.

OBJECTIVE: The posterior vagus nerve trunk innervates the entire small intestine, and elucidating its modulatory role in the IgA response was the aim of this study. METHODS: Two groups of six male BALB/c mice underwent sham or posterior subdiaphragmatic vagotomy and were euthanized on the 14th postoperative day; then, the small intestines were dissected. The intestinal fluid was harvested for antibody analysis by ELISA, and cell suspensions from Peyer's patches and lamina propria were prepared for cytofluorometric analysis of plasma cells and T lymphocytes. The CD4+ T cells were labeled for the intracellular IgA-producing interleukins (ILs)-4, -5, -6, and -10; transforming growth factor (TGF)-ß; and the inflammatory cytokines tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and IL-12. In the intestinal tissue samples, myeloperoxidase (MPO) visualization and the enzymatic activity were assessed by immunohistochemistry and ELISA, respectively. The data were analyzed by Student's t test, and the differences were considered significant at p < 0.05. RESULTS: In the vagotomy group, the IgA levels and the CD4+ T cells labeled with mediators that promote IgA secretion, including IL-4 (only at lamina propria), TNF-α, and IFN-γ, were decreased, whereas the lamina propria IgA+ plasma cells and MPO presence/activity were increased; changes in the IgM levels, IgM+ plasma cells, and CD4+ T cells labeled with TGF-ß, which have a role in class switch recombination, were not observed. CONCLUSION: The downmodulating impact of vagotomy on IgA levels may result from defective IgA secretion without affecting class switch recombination, whereas vagotomy evoked a proinflammatory response regarding MPO. These findings may reflect the role of the vagus nerve on the control of the IgA response in the small intestine.

Anti-pathogenic, antibiofilm, and technological properties of fermented food associated Staphylococcus succinus strain AAS2.

The present investigation was aimed to determine the anti-pathogenic, antibiofilm, and technological properties of fermented food associated Staphylococcus succinus strain AAS2. The anti-pathogenic attribute of cell-free neutralized supernatant (CFNS) of strain AAS2 was assessed against food-borne and enteric pathogens that revealed promising activity against Staphylococcus aureus and Enterobacter aerogenes with high arbitrary unit of 220.25 ± 3.3 and 170.2 ± 4.6 AU/mL, respectively. Furthermore, the antibiofilm and time-kill assay of CFNS of strain AAS2 depicted remarkable reduction in biofilm formation of indicator pathogens in a dose-dependent manner. Moreover, time-kill assay data revealed the drastic reduction in the viability (log cfu/mL) of S. aureus and E. aerogenes in the presence of varied minimum inhibitory concentration ranges of CFNS. The distinct technological properties of strain AAS2 were demonstrated using standard methodologies. Reported results estimated moderate level of exopolysaccharide (41.3 ± 0.6 mg/L) and lipase production (8.3 ± 0.3 mm), followed by remarkable autolytic (30.1 ± 1.2-43.1 ± 1.3%), catalase (13.82 ± 0.3 AU), and nitrate reductase (10.25 ± 0.3 mM nitrite/mg dry weight) activities under standard conditions. Most importantly, the strain cleared the specific in vitro safety assessment tests. The described anti-pathogenic and technological traits of strain AAS2 paved the way to utilize it in pharmaceutical as well as food processing industries as starter/adjunct culture.

Susceptibility of ruminal bacteria isolated from large and small ruminant to multiple conventional antibiotics.

The pivotal aim of the present context was to isolate diversified group of bacteria from the ruminants and to evaluate their antibiogram pattern against 22 antibiotics of 14 different classes. The bacterial isolates from small and large ruminant (sheep, cattle and calves) were isolated from the rumen based on various colonies morphology, and subjected for preliminary antibiotics susceptibility assay using disc diffusion method. The most sensitive isolates (based on zone of inhibition) were selected for determining the minimum inhibitory concentration (MIC) of each antibiotic ranging from 1 to 256 µg/mL. Results revealed the concentration dependent growth inhibitory property of antibiotics a species-specific process. The maximum tolerable concentration (MTC) of each antibiotic was further determined using disc diffusion method, and results exhibited that the tolerance nature of ruminal isolates to antibiotics is a species-specific mechanism. Based on the MIC and MTC values of antibiotics, amikacin, ciprofloxacin, and amoxicilline were observed to be the most potent antibiotics in terms of inhibiting the growth of ruminal isolates. In brief, the findings of the current study showed that despite the overexploitation of antibiotics as additives in the animal's feed, most of the ruminal isolates are sensitive to multiple conventional antibiotics tested. The growth inhibitory trait of antibiotics proves these antimicrobials a propitious agent against the pathogenesis of ruminal isolates in livestock.

Antibacterial activities of tannic acid against isolated ruminal bacteria from sheep.

This present study was conducted to evaluate the antimicrobial effects of tannic acid (TA) against isolated ruminal bacteria from adult sheep. Rumen samples were collected from two (2) adults sheep, and a total of nine (9) ruminal bacteria were isolated from the sample. The sensitivity of the ruminal bacteria isolates to 0.63, 1.25, 2.50, 5.00 and 10.00 mg TA/mL of growth medium was determined using clearance zone (CZ) of Kirby-Bauer disc diffusion susceptibility test. There was observable increase in the sensitivity of all bacterial isolates as the level of TA increases. Not all bacterial isolates have the capacity to tolerate more than 1.25 mg TA/mL. The result shows that only 20% of the bacterial isolates had the capacity to tolerate 0.63 and 1.25 mg of tannic acid per liter. This concentration of tannic acid would be equivalent to 2% tannin in the diet of ruminant. Our findings shows that increase in concentration of tannic acid completely inhibited the ruminal bacteria from the sheep rumen.

Cytotoxic effect of the immunotoxin constructed of the ribosome-inactivating protein curcin and the monoclonal antibody against Her2 receptor on tumor cells.

The toxicity of the curcin on cancer cells allows to consider this protein as the toxic component of an immunotoxin directed to Her2, which is associated with cancer. Reductive amination was proposed to conjugate curcin and an anti-Her2; the binding was tested using Polyacrylamide gel electrophoresis, western blot, and immunocytochemistry. The in vitro cytotoxicity of curcin and the immunotoxin was assessed on breast cancer cell lines SK-BR-3 (Her2(+)) and MDA-MB-231 (Her2(-)). IC50 values for curcin were 15.5 ± 8.3 and 18.6 ± 2.4 µg/mL, respectively, statistically equivalent (p < 0.05). While to the immunotoxin was 2.2 ± 0.08 for SK-BR-3 and 147.6 ± 2.5 µg/mL for MDA-MB-231. These values showed that the immunotoxin was seven times more toxic to the SK-BR-3 than curcin and eight times less toxic to the MDA-MB-231. The immunotoxin composed of curcin and an antibody against Her2 and constructed by reductive amination could be a therapeutic candidate against Her2(+) cancer.

17ß-Estradiol transcriptionally modulates Nlrp1 and Nlrp3 inflammasomes in gonadectomized rats with inflammation.

CONTEXT: It has been reported that 17ß-estradiol (E2) reduces the expression of inflammatory molecules, but there are no data that show the effect of E2 on the transcriptional regulation of innate immunity-related molecules and inflammasomes. OBJECTIVE: To study the effect of 17ß-estradiol (E2) on the transcriptional expression of the NLR family, pyrin domain containing 1 (Nlrp1) and (Nlrp3) inflammasomes, which are mediators of inflammation. MATERIALS AND METHODS: Inflammation was induced in adult female gonadectomized (Gdx) rats by intramuscular injection of complete Freund's adjuvant (CFA). Measurements were taken at different times after the treatment. Gene expression determinations were done by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: CFA-induced inflammation increased the transcription of Nlrp3, IL-1ß (p < 0.05), vascular cell adhesion molecule 1 (VCAM1), E-selectin and estrogen receptor 1 alpha (ERα) (p < 0.001) and decreased the transcription of Nlrp1, Caspase-1, IL-33, NFKB1, ICAM1, ICAM2, GCRα, GCRß, UCP3 and PGC1α (p < 0.001) compared to the control. The administration of E2 to the inflamed tissue significantly increased the expression of Nlrp1, NFKB1, ERα, UCP3, Caspase-1, E-selectin (p < 0.001), IL-18 and ERα (p < 0.05) and decreased IL-1ß and VCAM1 (p < 0.005) compared to the control. DISCUSSION AND CONCLUSION: CFA differentially modulates the transcription of inflammasome-related genes and the administration of E2 increases the expression of ERα and Nlrp1 together with NFKB1, a key molecule in the activation of the inflammasomes. Finally, an analysis using the web interface GeneMANIA revealed an interaction between several genes, indicating a functional correlation in this model.

Productive response of lambs fed Crescentia alata and Guazuma ulmifolia fruits in a tropical region of Mexico.

In vitro gas production with and without polyethylene glycol (PEG) of the fruits of Crescentia alata and Guazuma ulmifolia was evaluated, the degradation kinetics of lamb diets with added fruit of the tree was determined, and the ration intake and growth rate of lambs fed these diets were measured. Twenty-five entire male lambs of 23.5 ± 0.44 kg body weight were used and distributed in treatments: T0 (control without fruit); T1 and T2, 15 and 30 % of the fruit of C. alata; and T3 and T4, 15 and 30 % of the fruit of G. ulmifolia. Data variables chemical composition, fermentation kinetic, and digestibility in vitro were analyzed by a completely randomized design and data production response factorials design of five treatments by three evaluation periods. The total phenolic content (TP) (23.0 g/kg DM) was higher (P < 0.01) in the fruits of G. ulmifolia. The addition of PEG increased (P < 0.05) in vitro gas production (156.6 mL/g DM) in fruits of G. ulmifolia. In the fermentation kinetics, the total gas volume was higher (P < 0.01) at T0 (b = 293 mL/g DM), and the rate of degradation (c) but Lag time (t lag) was not different. In animal response, total dry matter intake was higher in lambs that received T4 (1.35 kg), and the daily weight gain and feed conversion did not differ (P > 0.05) among lambs receiving the treatments. Thirty percent G. ulmifolia fruit added in the diet increased dry matter intake and improved feed conversion but did not increase weight gain.

Prevalence of bovine subclinical mastitis, its etiology and diagnosis of antibiotic resistance of dairy farms in four municipalities of a tropical region of Mexico.

A region-wide survey was conducted in the tropical area of Tierra Caliente, State of Guerrero, Mexico to estimate the prevalence of subclinical bovine mastitis (SCM), distribution of mastitis pathogens, and in vitro antimicrobial susceptibility of different mastitis pathogens in dairy farms. In total, 1036 quarter milk samples were obtained from 259 cows at 87 different dairy farms. Collected quarter milk samples were submitted for California Mastitis Test (CMT), bacteriological examination, and testing for antimicrobial susceptibility. Overall prevalence of SCM in the studied area was 20.5 %. Prevalence in the different regions was as follows: 28 % in Arcelia municipality, 21 % in Tlalchapa municipality, 19.4 % in Pungarabato municipality, and 14.3 % in Finch Cutzamala municipality. Of all positive isolates, 97.5 % were Gram-negative bacteria. Moreover, of all positive isolates, 37.5 % were Proteus vulgaris, 25 % Salmonella spp., 12.5 % Enterobacter aerogenes, and 10 % Escherichia coli. Klebsiella pneumonia and E. coli were sensitive for netilmicin antimicrobial. However, E. coli was sensitive for pefloxacin and gentamicin with a sensitivity for pefloxacin for E. aerogenes, while Staphylococci were sensitive for gentamicin and dicloxacillin. It could be concluded that practices such as the implementation of mastitis control programs, improved milking hygiene together with an intramammary treatment with netilmicin, pefloxacin, and gentamicin antimicrobials should be considered for mastitis prevention in the study area of Tierra Caliente, in the tropical area of Guerrero, Mexico.

Intranasal coadministration of Cholera toxin with amoeba lysates modulates the secretion of IgA and IgG antibodies, production of cytokines and expression of pIgR in the nasal cavity of mice in the model of Naegleria fowleri meningoencephalitis.

The nasal mucosa is the first contact with antigens to induce IgA response. The role of this site has rarely been studied. We have shown than intranasal administration with Naegleria fowleri lysates plus Cholera toxin (CT) increased the protection (survival up to 100%) against N. fowleri infection in mice and apparently antibodies IgA and IgG together with polymorphonuclear (PMN) cells avoid the attachment of N. fowleri to apical side of the nasal epithelium. We also observed that nasal immunization resulted in the induction of antigen-specific IgG subclasses (IgG1 and IgG2a) in nasal washes at days 3 and 9 after the challenge and IgA and IgG in the nasal cavity, compared to healthy and infected mice. We found that immunization with both treatments, N. fowleri lysates plus CT or CT alone, increased the expression of the genes for alpha chain, its receptor (pIgR), and it also increased the expression of the corresponding proteins evidenced by the ∼65 and ∼74kDa bands, respectively. Since the production of pIgR, IgA and IgG antibodies, is up-regulated by some factors, we analyzed the expression of genes for IL-10, IL-6, IFN-γ, TNF-α and IL-1ß by using RT-PCR of nasal passages. Immunization resulted in an increased expression of IL-10, IL-6, and IFN-γ cytokines. We also aimed to examine the possible influences of immunization and challenge on the production of inflammatory cytokines (TNF-α and IL-1ß). We observed that the stimulus of immunization inhibits the production of TNF-α compared to the infected group where the infection without immunization causes an increase in it. Thus, it is possible that the coexistence of selected cytokines produced by our immunization model may provide a highly effective immunological environment for the production of IgA, IgG and pIgR as well as a strong activation of the PMN in mucosal effector tissue such as nasal passages.

Insecticidal activity of two organic extracts from Libidibia coriaria (Jacq.) Schltdl. fruits against Spodoptera frugiperda J. E. Smith.

This study describes the chemical characterisation of two extracts (ethyl acetate, EtOAc-E and hexanic, Hx-E) from Libidibia coriaria fruits and their insecticidal properties on Spodoptera frugiperda. The HPLC analysis in EtOAct-E revealed the presence of ellagic acid and phenolic compounds. The CG-MS analysis in Hx-E revealed the presence of Hexadecanoic acid, 11-Methylheptacosane, Dodecanoic acid and Nonacosane as major compounds. The application of both extracts was performed on the dorsal part of each larva via aspersion. The larval mortality, relative growth and emergence percentage of adults were evaluated. The Hx-E caused a 93.33% mortality at 100 mg/mL at 24 h post-application. A minor relative growth with both EtOAc-E (12.64 mg) and Hx-E (7.90 mg) was observed compared with their respective negative controls (methanol = 25.05 mg and tween20 = 24.53 mg). The lowest emergence percentage of adults with the Hx-E (25%) at 50 mg/mL was observed. Libidibia coriaria fruits exhibit insecticidal properties against S. frugiperda.