Search for antibodies against Trichinella in two synanthropic Procyonidae species from southeast Mexico: white-nosed coatis (Nasua narica) and raccoons (Procyon lotor).

Trichinella is a nematode that are spread by the consumption of parasitized meat. Carnivora, a mammalian order, serve as key hosts for this parasite. However, evidence of Trichinella in wildlife from the Neotropics is extremely scarce, with reports documenting its presence only for five carnivore species: two Felidae, one Otariidae and two Mustelidae. Other widely distributed species that are consumed as bushmeat, such as Procyonidae, have not been studied in this context. A long-term study was performed for antibodies against Trichinella in coatis (Nasua narica) and common raccoons (Procyon lotor) in southeastern Mexico. Between the summer of 2009 to the winter 2013, a total of 291 coati samples and 125 raccoon samples were collected from a tropical green area located within an urban zone. An Enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies against the excretory and secretory products of Trichinella spiralis muscle larva. ELISA-positive samples were further confirmed by Western Blot analysis. Results showed no evidence of antibodies during the first two years of study. However, in 2011, a sudden appearance of anti-Trichinella occurred. The seroprevalence reached its highest peak of 43% for coatis during winter 2013 and 53% for raccoons in summer 2013. This is the first study that provides evidence of Trichinella circulation within a neotropical procyonid community.

Tibolone Improves Locomotor Function in a Rat Model of Spinal Cord Injury by Modulating Apoptosis and Autophagy.

Spinal cord injury (SCI) harms patients' health and social and economic well-being. Unfortunately, fully effective therapeutic strategies have yet to be developed to treat this disease, affecting millions worldwide. Apoptosis and autophagy are critical cell death signaling pathways after SCI that should be targeted for early therapeutic interventions to mitigate their adverse effects and promote functional recovery. Tibolone (TIB) is a selective tissue estrogen activity regulator (STEAR) with neuroprotective properties demonstrated in some experimental models. This study aimed to investigate the effect of TIB on apoptotic cell death and autophagy after SCI and verify whether TIB promotes motor function recovery. A moderate contusion SCI was produced at thoracic level 9 (T9) in male Sprague Dawley rats. Subsequently, animals received a daily dose of TIB orally and were sacrificed at 1, 3, 14 or 30 days post-injury. Tissue samples were collected for morphometric and immunofluorescence analysis to identify tissue damage and the percentage of neurons at the injury site. Autophagic (Beclin-1, LC3-I/LC3-II, p62) and apoptotic (Caspase 3) markers were also analyzed via Western blot. Finally, motor function was assessed using the BBB scale. TIB administration significantly increased the amount of preserved tissue (p < 0.05), improved the recovery of motor function (p < 0.001) and modulated the expression of autophagy markers in a time-dependent manner while consistently inhibiting apoptosis (p < 0.05). Therefore, TIB could be a therapeutic alternative for the recovery of motor function after SCI.

Phagekines: Directed Evolution and Characterization of Functional Cytokines Displayed on Phages.

The current chapter focuses on the use of filamentous phages to display and modify biologically active cytokines, with special emphasis on directed evolution of novel variants showing improved receptor binding. Cytokines are essential protein mediators involved in cell-to-cell communication. Their functional importance and the complexity of their interactions with multichain receptors make cytokine engineering a promising tool for the discovery and optimization of therapeutic molecules. Protocols used at the laboratory are illustrated through examples of manipulation of interleukin-2 and interleukin-6, two members of the family of alpha-helix-bundle cytokines playing pivotal roles in immunity and inflammation.

Identification and immunogenic potential of glycosylphosphatidylinositol-anchored proteins in Paracoccidioides brasiliensis.

In fungal pathogens the cell wall plays an important role in host-pathogen interactions because its molecular components (e.g., polysaccharides and proteins) may trigger immune responses during infection. GPI-anchored proteins represent the main protein class in the fungal cell wall where they can perform several functions, such as cell wall remodeling and adhesion to host tissues. Genomic analysis has identified the complement of GPI-anchored proteins in many fungal pathogens, but the function has remained unknown for most of them. Here, we conducted an RNA expression analysis of GPI-anchored proteins of Paracoccidioides brasiliensis which causes paracoccidioidomycosis (PCM), an important human systemic mycosis endemic in Latin America. The expression of the GPI-anchored proteins was analyzed by quantitative PCR in both the mycelium and yeast forms. qPCR analysis revealed that the transcript levels of 22 of them were increased in hyphae and 10 in yeasts, respectively, while 14 did not show any significant difference in either form. Furthermore, we cloned 46 open reading frames and purified their corresponding GPI-anchored proteins in the budding yeast. Immunoblot and ELISA analysis of four purified GPI-anchored proteins revealed immune reactivity of these proteins against sera obtained from PCM patients. The information obtained in this study provides valuable information about the expression of many GPI-anchored proteins of unknown function. In addition, based on our immune analysis, some GPI-anchored proteins are expressed during infection and therefore, they might serve as good candidates for the development of new diagnostic methods.

Immune response dynamics of recent and chronic small ruminant lentivirus infection in the male reproductive system / Dinâmica da resposta imunológica da infecção recente e crônica por lentivírus de pequenos ruminantes no sistema reprodutor de machos

The objective of this study was to analyze the immune responses of bucks to small ruminant lentivirus (SRLV) with a focus on the reproductive system of males with recent and chronic infection. A total of 12 bucks were selected, six seronegative and six seropositive with chronic natural infection for more than 18 months (chronic infection group). After selecting the animals, the six seronegative males were intravenously inoculated with caprine arthritis-encephalitis virus (CAEV)-Co viral strain at a titer of 10-5,6 TCID50/mL. After viral inoculation, this group was called the recent infection group and was monitored weekly with the chronically infected group for 180 days with blood serum and seminal plasma Western Blot (WB) analysis. Of the animals with chronic SRLV infection, 18.94% (50/264) showed anti-SRLV antibodies in at least one of the samples, and 81.06% (214/264) were negative. Anti-SRLV antibodies were detected in 27.27% (36/132) of the blood serum samples from this group, while 10.60% (14/132) were reactive in the seminal plasma WB test. The animals inoculated with CAEV-Co became seropositive after the third week of viral inoculation. In this group, 31.06% (41/132) of seminal plasma samples had anti-SRLV antibodies, and of these, 70.73% (29/41) coincided with blood serum results. Of the remaining 29.27% (12/41), the seminal plasma sample of only three animals (RIA2, RIA3, and RIA5) had anti-SRLV antibodies. One of the animals with a recent infection presented anti-SRLV antibodies only in seminal plasma samples, possibly due to virus compartmentalization. Intermittent viral shedding was observed in both biological samples, regardless of the infection stage. The immune response in bucks with recent SRLV infection is more significant than in chronically infected animals. Regardless of the stage of infection, there is a fluctuation in antibody levels, therefore, this creates a risk of false-negative samples when performing the diagnosis.

O objetivo desse estudo foi analisar a resposta imunológica aos lentivírus de pequenos ruminantes (LVPR) com enfoque no sistema reprodutor de machos com infecção recente e crônica. Para isso, foram selecionados 12 reprodutores caprinos, sendo seis soronegativos e seis soropositivos com infecção natural crônica há mais de 18 meses (grupo com infecção crônica). Após seleção dos animais, os seis machos soronegativos foram inoculados com a cepa viral do vírus da artrite encefalite caprina (CAEV)-Co, título 10-5,6 TCID50/mL, por via intravenosa. A partir da inoculação viral este agrupamento passou a ser denominado de grupo com infecção recente e juntamente com o grupo com infecção crônica foram acompanhados, semanalmente por 180 dias, com análise dos testes de Western Blot (WB) no soro sanguíneo e plasma seminal. Nos animais com infecção crônica para LVPR, 18,94% (50/264) apresentaram anticorpos anti-LVPR em pelo menos uma das distintas amostras, e 81,06% (214/264) tiveram resultados negativos. Das amostras de soro sanguíneo do referido grupo, em 27,27% (36/132) detectou-se anticorpos anti-LVPR, enquanto que no plasma seminal 10,60% (14/132) foram reagentes no teste de WB. Nos animais inoculados com o CAEV-Co, ocorreu a soroconversão após a terceira semana da inoculação viral. Nesse grupo, 31,06% (41/132) das amostras de plasma seminal tiveram anticorpos anti-LVPR, sendo que dessas 41, 70,73% (29/41) coincidiram com resultado das amostras de soro sanguíneo. Nos 29,27% (12/41) restante, houve a detecção somente no plasma seminal e eram amostras provenientes de três animais (AIR2, AIR3 e AIR5). Em um dos animais com infecção recente, só foi identificado anticorpos anti-LVPR em amostras de plasma seminal, possivelmente em função da compartimentalização do vírus. Intermitência viral foi observada em ambas as amostras biológicas, independentemente do estágio de infecção. Conclui-se que a resposta imunológica em reprodutores com infecção recente LVPR é mais acentuada do que em animais cronicamente infectados. E, independentemente do estágio da infecção há uma flutuação nos níveis de anticorpos, sendo, portanto, um fator de risco, em virtude da existência de amostras falso-negativo ao realizar o diagnóstico.

Proteomic analysis of the excretory-secretory products from Strongyloides venezuelensis infective larvae: new insights for the immunodiagnosis of human strongyloidiasis.

Serodiagnosis of human strongyloidiasis is a practical alternative to parasitological methods due to its high sensitivity. However, cross-reactivity with other helminth infections limits its utility, and this problem is due to the use of homologous or heterologous somatic extracts of the parasite as an antigen source. Excretory-secretory (E/S) products from Strongyloides infective larvae can be used to improve the serodiagnosis. The combined use of western blot and proteomics became an interesting strategy to identify immunological markers for the serodiagnosis of strongyloidiasis. The present study describes the proteomic analysis of the antigenic components from E/S products of S. venezuelensis infective larvae that were recognized by IgG antibodies from patients with strongyloidiasis. Our results showed that IgG antibodies from patients with strongyloidiasis recognized between 15 and 16 antigenic bands in the E/S products from S. venezuelensis that were incubated in PBS or in RPMI culture medium, respectively. Overall, antigenic bands of low and high molecular weight were more specific than those of intermediate molecular weight, which were cross-reactive. A 36-kDa antigenic band was 93% sensitive and 100% specific (a probably arginine kinase of 37 kDa), while other antigenic bands were highly sensitive but low specific. Proteomic analysis revealed differences between the protein profiles from E/S-RPMI and E/S-PBS since only one-third of all proteins identified were common in both types of E/S products. Bioinformatic analysis showed that more than 50% of the proteins from E/S products are secreted within extracellular vesicles and only a small percentage of them are actually released by the classical secretory pathway. Several components from the E/S products were identified as plasminogen-binding proteins, probably used as an immune evasion mechanism. The data provided here provide valuable information to increase understanding of E/S products from S. venezuelensis infective larvae. This may help us to find new targets for the immunodiagnosis of human strongyloidiasis.

Prevalence of Toxoplasma gondii Measured by Western Blot, ELISA and DNA Analysis, by PCR, in Cats of Western Mexico.

Toxoplasma gondii is the causative agent of toxoplasmosis in humans and animals. The sexual reproductive cycle of Toxoplasma takes place in the small intestine of felines, the definitive hosts. In the final part of the sexual cycle, T. gondii forms oocysts in infected cats. Oocysts transferred via the faeces to the environment are highly infectious to both animals and humans. This study aimed to determine the prevalence and risk factors associated with T. gondii infection in cats from the metropolitan region of Guadalajara in western Mexico. Western blotting and ELISA for anti-Toxoplasma IgG antibodies was performed, and Toxoplasma DNA was identified using polymerase chain reaction. Prevalence of anti-T. gondii antibodies was 14.8% (44/297), and only 2/297 cases were positive for PCR. Cats older than one year were at an increased risk of infection (OR = 3.9, 95% CI 1.844-8.362). Sex, raw meat feeding, hunting habits, vaccination status, and body condition were not associated with positivity. The prevalence of T. gondii infection determined with Western blot in cats in the metropolitan area of Guadalajara, Jalisco, Mexico, was lower than that reported in previous studies.

Modelling diagnostics for Echinococcus granulosus surveillance in sheep using Latent Class Analysis: Argentina as a case study.

Echinococcus granulosus sensu lato is a globally prevalent zoonotic parasitic cestode leading to cystic echinococcosis (CE) in both humans and sheep with both medical and financial impacts, whose reduction requires the application of a One Health approach to its control. Regarding the animal health component of this approach, lack of accurate and practical diagnostics in livestock impedes the assessment of disease burden and the implementation and evaluation of control strategies. We use of a Bayesian Latent Class Analysis (LCA) model to estimate ovine CE prevalence in sheep samples from the Río Negro province of Argentina accounting for uncertainty in the diagnostics. We use model outputs to evaluate the performance of a novel recombinant B8/2 antigen B subunit (rEgAgB8/2) indirect enzyme-linked immunosorbent assay (ELISA) for detecting E. granulosus in sheep. Necropsy (as a partial gold standard), western blot (WB) and ELISA diagnostic data were collected from 79 sheep within two Río Negro slaughterhouses, and used to estimate individual infection status (assigned as a latent variable within the model). Using the model outputs, the performance of the novel ELISA at both individual and flock levels was evaluated, respectively, using a receiver operating characteristic (ROC) curve, and simulating a range of sample sizes and prevalence levels within hypothetical flocks. The estimated (mean) prevalence of ovine CE was 27.5% (95%Bayesian credible interval (95%BCI): 13.8%-58.9%) within the sample population. At the individual level, the ELISA had a mean sensitivity and specificity of 55% (95%BCI: 46%-68%) and 68% (95%BCI: 63%-92%), respectively, at an optimal optical density (OD) threshold of 0.378. At the flock level, the ELISA had an 80% probability of correctly classifying infection at an optimal cut-off threshold of 0.496. These results suggest that the novel ELISA could play a useful role as a flock-level diagnostic for CE surveillance in the region, supplementing surveillance activities in the human population and thus strengthening a One Health approach. Importantly, selection of ELISA cut-off threshold values must be tailored according to the epidemiological situation.

Natural latex serum: characterization and biocompatibility assessment using Galleria mellonella as an alternative in vivo model.

Natural latex serum (NLS) is one of the natural rubber latex fractions from Hevea brasiliensis tree, which is formed by centrifuged serum and is composed of proteins, acids, nucleotides, salts and carbohydrates. The proteins present in NLS have demonstrated several interesting biological properties, including angiogenic, healing, osteogenic, anti-inflammatory, antimicrobial, in addition to inducing neovascularization, bone formation and osseointegration. Thus, we proposed to characterize NLS by physicochemical techniques and to investigate the biocompatibility by toxicological assays and safety test in Galleria mellonella. Infrared spectrum showed vibrational bands characteristic of amide I, II and III that are linked to the protein content, which was confirmed by the High Performance Liquid Chromatography profile and by the Electrophoresis analysis. This material did not exhibit hemolytic (rate <0.5%) and cytotoxic effects (viability >70%) and was able to enhance the proliferation of fibroblasts (>600%) after 3 days. The pronounced proliferative effect observed in fibroblast cells can be explained by the presence of the fibroblast growth factor (FGF) like protein revealed by the Western blot test. Moreover, NLS did not provoke toxic effects (survival ∼ 80%) on the G. mellonella model, indicating that it is a biocompatible and safe material.

Proteomic analysis of the excretory-secretory products from Strongyloides venezuelensis infective larvae: new insights for the immunodiagnosis of human strongyloidiasis

Serodiagnosis of human strongyloidiasis is a practical alternative to parasitological methods due to its high sensitivity. However, cross-reactivity with other helminth infections limits its utility, and this problem is due to the use of homologous or heterologous somatic extracts of the parasite as an antigen source. Excretory-secretory (E/S) products from Strongyloides infective larvae can be used to improve the serodiagnosis. The combined use of western blot and proteomics became an interesting strategy to identify immunological markers for the serodiagnosis of strongyloidiasis. The present study describes the proteomic analysis of the antigenic components from E/S products of S. venezuelensis infective larvae that were recognized by IgG antibodies from patients with strongyloidiasis. Our results showed that IgG antibodies from patients with strongyloidiasis recognized between 15 and 16 antigenic bands in the E/S products from S. venezuelensis that were incubated in PBS or in RPMI culture medium, respectively. Overall, antigenic bands of low and high molecular weight were more specifc than those of intermediate molecular weight, which were cross-reactive. A 36-kDa antigenic band was 93% sensitive and 100% specifc (a probably arginine kinase of 37 kDa), while other antigenic bands were highly sensitive but low specifc. Proteomic analysis revealed diferences between the protein profles from E/S-RPMI and E/S-PBS since only one-third of all proteins identifed were common in both types of E/S products. Bioinformatic analysis showed that more than 50% of the proteins from E/S products are secreted within extracellular vesicles and only a small percentage of them are actually released by the classical secretory pathway. Several components from the E/S products were identifed as plasminogenbinding proteins, probably used as an immune evasion mechanism. The data provided here provide valuable information to increase understanding of E/S products from S. venezuelensis infective larvae. This may help us to find new targets for the immunodiagnosis of human strongyloidiasis.

A Novel Brighter Bioluminescent Fusion Protein Based on ZZ Domain and Amydetes vivianii Firefly Luciferase for Immunoassays.

Immunoassays are widely used for detection of antibodies against specific antigens in diagnosis, as well as in electrophoretic techniques such as Western Blotting. They usually rely on colorimetric, fluorescent or chemiluminescent methods for detection. Whereas the chemiluminescence methods are more sensitive and widely used, they usually suffer of fast luminescence decay. Here we constructed a novel bioluminescent fusion protein based on the N-terminal ZZ portion of protein A and the brighter green-blue emitting Amydetes vivianii firefly luciferase. In the presence of D-luciferin/ATP assay solution, the new fusion protein, displays higher bioluminescence activity, is very thermostable and produces a sustained emission (t1/2 > 30 min). In dot blots, we could successfully detect rabbit IgG against firefly luciferases, Limpet Haemocyanin, and SARS-CoV-2 Nucleoprotein (1-250 ng), as well as the antigen bound antibodies using either CCD imaging, and even photography using smartphones. Using CCD imaging, we could detect up to 100 pg of SARS-CoV-2 Nucleoprotein. Using this system, we could also successfully detect firefly luciferase and SARS-CoV-2 nucleoprotein in Western Blots (5-250 ng). Comparatively, the new fusion protein displays slightly higher and more sustained luminescent signal when compared to commercial HRP-labeled secondary antibodies, constituting a novel promising alternative for Western Blotting and immunoassays.

Near misdiagnosis of acute HIV-infection with ELISA-Western Blot scheme: Time for mindset change.

Some HIV-infection diagnostic guidelines and health care providers still rely on the ELISA-Western blot diagnostic algorithm. We present a near misdiagnosis case with discordant test results and a lack of proper counseling. We point out the need for an assertive update of health care providers on diagnostic HIV-tests.

Prevalencia de cisticercosis en porcinos de la provincia de Tambopata, Perú / Cysticercosis Prevalence in Pigs in the Tambopata Province, Perú

Resumen La cisticercosis es una de las principales enfermedades zoonóticas parasitarias que es causada por el establecimiento de la forma larvaria de Taenia solium. Esta enfermedad se desarrolla principalmente en cerdos que son criados en granjas sin tecnificación, donde el uso de la tecnología y las condiciones sanitarias son mínimas. Este tipo de crianza es muy usual, por lo que representa un riesgo de la salud pública. En ese sentido, se determinó la prevalencia de cisticercosis en porcinos de la provincia de Tambopata, donde fue evaluado un total de 98 porcinos. Se tomaron aproximadamente 5 ml de sangre de la vena cava en animales mayores de 6 meses y hembras que no estuviesen preñadas; posteriormente, se obtuvo el plasma para ser procesado mediante la prueba de enzyme-linked inmunoelectrotransfer bloot assay (EITB) o Western Blot. Se determinó que el 17 % de los cerdos evaluados dio positivo para cisticercosis; con respecto al sexo, se obtuvo una seroprevalencia de 5,21 % ± 0,82 % para machos y 11,45 % ± 1,93 % para hembras. Finalmente, se determinó una seroprevalencia de 10,41 % ± 1,75 % para animales jóvenes de 6 a 11 meses y 6,25 % ± 1,01 % para animales adultos de 12 meses a más. Estos resultados reflejan la importancia de la vigilancia y control de las enfermedades parasitarias en los animales de producción ya que pudo corroborarse que la cisticercosis porcina constituye un serio problema de salud pública.

Abstract Cysticercosis is one of the main zoonotic parasitic diseases caused by the larval settlement of Taenia solium. This disease develops mainly in pigs that are reared in non-technified farms where the use of technology and the sanitary conditions are poor. It is quite usual to rear pigs this way and, therefore, there is a public health risk. In this sense, the cysticercosis prevalence was determined among pigs in the Tambopata Province, including 98 animals in the evaluation. Approximately 5 ml of blood were taken from the vena cava in more than 6-month-old female pigs that were not pregnant. Next, the plasma was taken in order to be processed under an enzyme-linked inmunoelectrotransfer bloot assay (EITB) or western blot. It was found that 17% of pigs were positive to cysticercosis. Regarding the sex, the seroprevalence was 5.21% ± 0.82% in males and 11.45% ± 1.93% in females. Finally, the seroprevalence was determined at 10.41% ± 1.75% in young animals (6 to 11 months old) and 6.25% ± 1.01% in adult animals (12 months old and above). These results show how important it is to monitor and control the parasitic diseases in production animals as this study confirmed that porcine cysticercosis is a serious problem in public health.

Modulatory effects of photobiomodulation in the anterior cingulate cortex of diabetic rats.

Anterior Cingulate Cortex (ACC) has a crucial contribution to higher order pain processing. Photobiomodulation (PBM) has being used as integrative medicine for pain treatment and for a variety of nervous system disorders. This study evaluated the effects of PBM in the ACC of diabetic rats. Type 1 diabetes was induced by a single dose of streptozotocin (85 mg/Kg). A total of ten sessions of PBM (pulsed gallium-arsenide laser, 904 nm, 9500 Hz, 6.23 J/cm2) was applied to the rat peripheral nervous system. Glial fibrillary acidic protein (GFAP), mu-opioid receptor (MOR), glutamate receptor 1 (GluR1), and glutamic acid decarboxylase (GAD65/67) protein level expression were analyzed in the ACC of diabetic rats treated with PBM. Our data revealed that PBM decreased 79.5% of GFAP protein levels in the ACC of STZ rats. Moreover, STZ + PBM rats had protein levels of MOR increased 14.7% in the ACC. Interestingly, STZ + PBM rats had a decrease in 70.7% of GluR1 protein level in the ACC. Additionally, PBM decreased 45.5% of GAD65/67 protein levels in the ACC of STZ rats.

Reactividad de sueros de ratones inmunizados con vesículas de membrana externa derivadas de Salmonella entérica / Reactivity of sera from mice immunized with outer membrane vesicles derived from Salmonella enterica

La fiebre tifoidea causada por Salmonella Paratyphi A (fiebre paratifoidea) es indistinguible de la producida por Salmonella Typhi y el grado de incidencia ha aumentado en los últimos años, especialmente en el sudeste asiático. Por otro lado, la diarrea y otras complicaciones entéricas causadas por Salmonella Enteritidis y Salmonella Typhimurium continúan siendo un problema de salud grave, especialmente en países subdesarrollados. Las vacunas continúan siendo la forma más efectiva de prevenir estas enfermedades. Existen vacunas basadas en el polisacárido capsular de Salmonella Typhi que protegen contra la fiebre tifoidea; sin embargo, no hay vacunas efectivas licenciadas para uso en humanos que prevengan las enfermedades producidas por los serotipos de Salmonella no tifoideas. El desarrollo de una formulación con capacidad para proteger contra estas enfermedades sigue siendo un desafío para la comunidad científica. En este trabajo se evaluó, mediante Western blot, la reactividad de los sueros de ratones inmunizados por vía subcutánea con formulaciones basadas en vesículas de membrana externa derivadas de Salmonella Paratyphi A, Salmonella Enteritidis y Salmonella Typhimurium, contra los respectivos lisados celulares, para identificar la formulación que induce la mejor respuesta inmunológica cruzada. Los resultados obtenidos indicaron una alta reactividad de todos los sueros a los lisados, sin una diferencia aparente entre ellos. Sin lugar a dudas, se deberán realizar pruebas de inmunogenicidad seguidas de pruebas de retos cruzados para identificar un candidato vacunal. Estos resultados sugieren que las vesículas de membrana externa empleadas en este estudio están compuestas por antígenos posiblemente conservados en los tres serotipos de Salmonella y que pueden inducir una respuesta inmune de amplio espectro y protección cruzada(AU)

Typhoid fever caused by Salmonella Paratyphi A (paratyphoid fever) is indistinguishable from that caused by Salmonella Typhi and the degree of incidence has increased in recent years, especially in Southeast Asia. On the other hand, diarrhea and other enteric complications caused by Salmonella Enteritidis and Salmonella Typhimurium continue to be a serious health problem, especially in underdeveloped countries. Vaccines continue to be the most effective way to prevent these diseases. There are vaccines based on Salmonella Typhi capsular polysaccharide, which protects against typhoid fever; however, there are no effective vaccines licensed for use in humans to prevent disease caused by nontyphoidal Salmonella serotypes. Developing a formulation capable of protecting against these diseases remains a challenge for the scientific community. In this work, the reactivity of the sera of mice immunized subcutaneously with formulations based on Outer Membrane Vesicles (OMV) derived from Salmonella Paratyphi A, Salmonella Enteritidis and Salmonella Typhimurium, was evaluated by Western blot, against the respective cell lysates to identify the formulation that induces the best cross immune response. The results obtained indicated a high reactivity of all the sera to the lysates; without an apparent difference between them. Undoubtedly, immunogenicity tests followed by cross-challenge tests should be performed to identify a vaccine candidate. These results suggest that the OMV used in this study are composed of possibly conserved antigens in the three Salmonella serotypes and that they can induce a broad-spectrum immune response and cross protection(AU)

Seroprevalence of Toxoplasma gondii in free-range pigs in northern Peru.

Toxoplasma gondii is an important foodborne pathogen worldwide, with undercooked meat as the main source of human transmission. In this study, we determined the seroprevalence of T. gondii in free-range pigs from two adjacent villages in the Tumbes region of northern Peru, El Tutumo and Nuevo Progreso. We randomly selected 100 pig serum samples collected during a prior study and processed these using Western Blot to detect IgG anti-T. gondii antibodies. Results indicated a prevalence of 32% (32/100) to T. gondii in pigs. Free-ranging pigs from northern Peru represent a substantial risk for transmission of T. gondii to humans.

A freeze-and-thaw-induced fragment of the microtubule-associated protein tau in rat brain extracts: implications for the biochemical assessment of neurotoxicity.

Tau is a microtubule-associated protein (MAP) responsible for controlling the stabilization of microtubules in neurons. Tau function is regulated by phosphorylation. However, in some neurological diseases Tau becomes aberrantly hyperphosphorylated, which contributes to the pathogenesis of neurological diseases, known as tauopathies. Western blotting (WB) has been widely employed to determine Tau levels in neurological disease models. However, Tau quantification by WB should be interpreted with care, as this approach has been recognized as prone to produce artifactual results if not properly performed. In the present study, our goal was to evaluate the influence of a freeze-and-thaw cycle, a common procedure preceding WB, to the integrity of Tau in brain homogenates from rats, 3xTg-AD mice and human samples. Homogenates were prepared in ice-cold RIPA buffer supplemented with protease/phosphatase inhibitors. Immediately after centrifugation, an aliquot of the extracts was analyzed via WB to quantify total and phosphorylated Tau levels. The remaining aliquots of the same extracts were stored for at least 2 weeks at either -20 or -80°C and then subjected to WB. Extracts from rodent brains submitted to freeze-and-thaw presented a ∼25 kDa fragment immunoreactive to anti-Tau antibodies. An in-gel digestion followed by mass spectrometry (MS) analysis in excised bands revealed this ∼25 kDa species corresponds to a Tau fragment. Freeze-and-thaw-induced Tau proteolysis was detected even when extracts were stored at -80°C. This phenomenon was not observed in human samples at any storage condition tested. Based on these findings, we strongly recommend the use of fresh extracts of brain samples in molecular analysis of Tau levels in rodents.

Prevalencia de anticuerpos anti-hepatitis E de tipo IgG evaluados a través de tres ensayos comerciales / Prevalence of anti-Hepatitis E IgG analyzed in sera through three commercial kits

Resumen Introducción: El virus de la Hepatitis E (HVE) es de ácido ribonucleico desnudo, los genotipos 3 y 4 pueden presentarse como una zoonosis transmitida por agua o alimentos contaminados. En la zona del eje cafetero-Colombia, no se ha descrito la presencia de anticuerpos para este virus en la comunidad. Objetivo: Determinar la prevalencia de anticuerpos anti-HVE de tipo Inmuniglobulinas G (IgG) en muestras de suero de un laboratorio clínico del Eje Cafetero. Materiales y métodos: En un periodo de dos meses se analizaron 90 sueros de pacientes atendidos en un laboratorio clínico de la ciudad de Armenia, se utilizaron tres técnicas diferentes para la caracterización de los anticuerpos y se compararon sus resultados. Resultados: De los 90 sueros evaluados, la técnica de ELISA de anticuerpos totales ELISA IgG anti HVE Recom Well marca Mikrogen identificó 2 sueros positivos (2,2%), la Prueba ELISA IgG HVE versión ULTRA® marca Diapro evidenció una muestra equivoca (1,1%). La prueba western blot Recom line HVE marca Mikrogen detectó 4 muestras positivas (4,4%). Conclusiones: Se encontró una prevalencia de anticuerpos HVE IgG que oscila entre 0 y 4,4% dependiendo de la prueba comercial utilizada, evidenciando circulación del virus y un posible ciclo infecciosos en la región.

Abstract Introduction: Hepatitis E virus (HEV) is a nonenveloped, RNA virus. HEV genotypes 3 and 4 are considered zoonosis transmitted by contaminated water and/or food. The presence of antibodies against this virus have not been described in communities inhabiting the "Coffee Axis" region of Colombia. Objective: To determine the prevalence of anti-Hepatitis E IgG in serum samples analyzed in a clinical laboratory from the Colombian Coffee Axis. Materials and methods: 90 serum samples from patients treated at a clinical laboratory in the city of Armenia (Quindio) were analyzed and compared through three different methods that characterize antibodies. Results: The Mikrogen recomWell ELISA kit (IgG anti-HEV) identified two positive sera (2.2%). The Diapro HEV IgG ELISA (version ULTRA®) test registered a false positive sample (1.1%). The Mikrogen recom Line HVE western blot assay detected 4 positive samples (4.4%). Conclusions: Depending on the commercial kit used, the prevalence of anti-HEV IgG antibodies fluctuated between 0% to 4.4%, which demonstrates that the virus is circulating and that a possible infectious cycle in this region exists.

Selective Regional Isolation of Brain Microvessels.

The study of the regionalized function of the blood-brain barrier at the level of brain endothelial cells and pericytes is essential to understand the biological properties and molecular mechanisms regulating this biological barrier. The isolation of blood vessels from specific brain regions will allow to understand regional differences in susceptibility to pathological phenomena such as ischemia, traumatic brain injury, and neurodegenerative diseases, such as Alzheimer disease. Here, we propose an efficient and fast method to isolate brain endothelial cells and pericytes from a specific cerebral region. The isolated brain endothelial cells and pericytes are viable to perform conventional molecular and histological techniques such as Western blots, immunocytofluorescence, and scanning electron microscopy.

CDC6 is up-regulated and a poor prognostic signature in glioblastoma multiforme.

PURPOSE: Glioblastoma multiforme (GBM) represents the most common and the most malignant type of brain tumor. Cell division cycle 6 (CDC6), a gene associated with DNA replication initiation, has been proven to be associated with the prognosis of multiple tumors. In this study, we aim to explore the association between CDC6 expression and GBM carcinogenesis and prognosis. METHODS: CDC6 expression in normal cells and GBM cells was explored by analyzing TCGA dataset, as well as by RT-PCR and western blot methods. Survival analysis was performed by the Kaplan-Meier method. Multivariate Cox-regression analysis was adopted to estimate the independence of CDC6 as a GBM prognostic factor. RESULTS AND CONCLUSIONS: Elevated CDC6 levels in GBM tumor tissues compared with those in normal brain tissues were illustrated by analyzing the gene expression profiles from TCGA dataset, and confirmed by RT-PCR and western blot assays in GBM tumor and normal human astrocyte cell lines. Kaplan-Meier analysis indicated the negative influence of high CDC6 expression on GBM overall survival (OS) probability and days to progression (D2P) after initial treatment, but not on days to recurrence (D2R) after initial treatment. Multivariate Cox regression analysis showed CDC6 as an independent signature marker gene for GBM prognosis. In addition, the combination of CDC6 mRNA expression and CpG island methylator phenotype (CIMP) could sensitively predict 3-year OS and D2P. In conclusion, our study uncovered the role of CDC6 in GBM carcinogenesis and prognosis for the first time, which could shed new light on GBM diagnosis and treatment.