Early appearance of functional plasma cells (CD138+CD98+) in non-immunized neonate mice.

Plasma cells (PCs) are terminally differentiated antibody-secreting cells, derived from activated B-lymphocytes in response to either T-independent or T-dependent antigens. The plasma cell population is scarce in circulation in non-immunized individuals. It is established that neonates are incapable of mounting an efficient immune response due to the immaturity of the immune system. However, this disadvantage is well overcome through the antibodies neonates receive from breastmilk. This implies that neonates will be only protected against antigens the mother had previously encountered. Thus, the child might be potentially susceptible to new antigens. This issue prompted us to seek for the presence of PCs in non-immunized neonate mice. We found a PC population identified as CD138+/CD98+ cells since day one after birth. These PCs were positive for Ki67 and expressed Blimp-1, B220, and CD19, which suggests the populations are plasmablasts and PCs with heterogeneous phenotype. These PCs were also determined to secrete antibodies, although mainly isotype IgM. Altogether, the results indicated that neonate PCs can produce antibodies against antigens they encounter in the first weeks of life, most likely coming from food, colonizing microbiota, or the environment.

Autodisplay of an avidin with biotin-binding activity on the surface of Escherichia coli.

OBJECTIVES: To display a recombinant avidin fused to the autotransporter ShdA to bind biotinylated molecules on the surface of Escherichia coli. RESULTS: Two chimeric protein constructs containing avidin fused to the autotransporter ShdA were expressed on the surface of Escherichia coli DH5α. One fusion protein contained 476 amino acids of the ShdA α and ß domains, whereas the second consisted of a 314 amino acid from α and truncated ß domains. Protein production was verified by SDS-PAGE using an antibody to the molecular FLAG-tag. The surface display of the avidin-shdA fusion protein was confirmed by confocal microscopy and flow cytometry analysis, and the biotin-binding activity was evaluated by fluorescence microscopy and flow cytometry using biotin-4-fluorescein and biotinylated-ovalbumin (OVA). CONCLUSIONS: Expression of a recombinant avidin with biotin-binding activity on the surface of E. coli was achieved using the autotransporter ShdA. This system is an alternative to bind biotinylated molecules to E. coli.

14-3-3 Proteins regulate Akt Thr308 phosphorylation in intestinal epithelial cells.

Akt activation has been associated with proliferation, differentiation, survival and death of epithelial cells. Phosphorylation of Thr308 of Akt by phosphoinositide-dependent kinase 1 (PDK1) is critical for optimal stimulation of its kinase activity. However, the mechanism(s) regulating this process remain elusive. Here, we report that 14-3-3 proteins control Akt Thr308 phosphorylation during intestinal inflammation. Mechanistically, we found that IFNγ and TNFα treatment induce degradation of the PDK1 inhibitor, 14-3-3η, in intestinal epithelial cells. This mechanism requires association of 14-3-3ζ with raptor in a process that triggers autophagy and leads to 14-3-3η degradation. Notably, inhibition of 14-3-3 function by the chemical inhibitor BV02 induces uncontrolled Akt activation, nuclear Akt accumulation and ultimately intestinal epithelial cell death. Our results suggest that 14-3-3 proteins control Akt activation and regulate its biological functions, thereby providing a new mechanistic link between cell survival and apoptosis of intestinal epithelial cells during inflammation.

CRTAM is negatively regulated by ZEB1 in T cells.

T cell activation leads to the induction of genes that are required for appropriate immune responses. This includes CRTAM (Class-I MHC-restricted T cell associated molecule), a protein that plays a key role in T cell development, proliferation, and generating cell polarity during activation. We previously characterized the CRTAM promoter and described how AP-1 family members are important for inducing CRTAM expression upon antigenic activation. Here, we show that CRTAM is a molecular target for ZEB1 (zinc finger E-box-binding protein), a homeodomain/Zn finger transcription factor. Overexpression of ZEB1 repressed CRTAM promoter activity, as well as endogenous CRTAM levels in human T cells. ZEB1-mediated transcriptional repression was abolished when E-box-like elements in the CRTAM promoter are mutated. In summary, ZEB1 functions as a transcriptional repressor for the CRTAM gene in both non-stimulated and stimulated T cells, thereby modulating adaptive immune responses.

Distribución de receptores de estrógenos beta en el ovario de ovejas prepúberes / Beta estrogen receptors distribution in the ovary of ewe lambs

Las hormonas esteroidales tienen un papel esencial en la fisiología reproductiva, actúan en el ovario estableciendo comunicación entre este y la glándula hipofisiaria y también en forma paracrina como reguladores locales. Se ha descrito expresión de receptores de estrógeno a nivel de ovarios fetales, neonatales y adulto, siendo necesario determinar cuales son los tipos celulares que expresan estos receptores. El ovario ovino es estereogénico activo desde la etapa fetal y por lo tanto los esteroides desempeñan un importante papel en el desarrollo gonadal. La regulación del crecimiento folicular está relacionado a varios factores por un lado la secreción de gonadotropinas por la hipófisis interviene durante el desarrollo folicular tardío, existiendo evidencias de una acción intra-ovárica directa de los estrógenos, en la regulación del crecimiento folicular temprano. Nuestro objetivo fue, evaluar la expresión y distribución de receptores de estrógeno b en las distintas poblaciones celulares del ovario de la oveja prepúber.

Steroid hormones play an essential role in reproductive physiology, acting in the ovary establishing communication between it and the pituitary gland as well as local paracrine regulators. Described estrogen receptor expression level fetal, neonatal and adult ovaries, which are necessary to determine the cell types that express these receptors. Sheep ovarian stereogenic is active from the fetal stage and therefore steroids play an important role in gonadal development. The regulation of follicle growth is related to several factors on the one hand the secretion of gonadotropins by the pituitary follicular development occurring during late, there was evidence of a direct intra-ovarian estrogen action in the early follicular growth regulation. Our objective was to evaluate the expression and distribution of estrogen receptor b in different cell populations of the ovary of prepubertal sheep.

Effects of Vitamin D Supplementation during the Induction and Progression of Osteoarthritis in a Rat Model.

Epidemiological studies correlate low levels of vitamin D with the osteoarthritis (OA) progression. Cytokines and metalloproteases play a major role in OA promoting the inflammation and degradation of the cartilage and can be induced through the Toll-like receptor (TLR) pathway. The aim of this study was to evaluate the protective effect of vitamin D supplementation on the development of osteoarthritis (OA) through examining the genetic regulation of TLRs, cytokines, and metalloproteases in chondrocytes as well as the wideness of cartilage in rats with OA. Our results demonstrate that the signaling through TLR-4 is a proinflammatory mechanism in osteoarthritis that drives the upregulation of MMP-3, IL-1ß, and TNF-α gene expression, leading to cartilage degradation and inflammation. Vitamin D supplementation had a protective effect during the onset but not during the chronic stage of OA in the rat model.

Non-typhi Salmonella serovars found in Mexican zoo animals.

The aim of the present study was to determine the bacteriological prevalence of subclinical non-typhi Salmonella infections in zoo animals and to determine the most frequently isolated serovars of the bacteria. A total of 267 samples were analyzed, including fecal samples from zoo animals and rodents, insects (Musca domestica and Periplaneta americana) and samples of the zoo animal's food. Salmonella was detected in 11.6% of the samples analyzed. Characterization of the isolates was performed with serotyping and pulsed-field gel electrophoresis. The following serovars were isolated: S. San Diego, S. Oranienburg, S. Weltevreden, S. Braenderup, S. Derby, S. 6,7, H:en x:- and S. 3,10, H:r:-. The isolates showed seven pulsed-field gel electrophoresis patterns with a Jaccard coefficient≥0.75 indicating a possible common origin. The prevalence of asymptomatic infections caused by Salmonella spp. in zoo animals was high. These findings demonstrate the diversity of Salmonella serovars in several captive wild animal species.

Estudio de la expresión de los receptores de progesterona y estrógeno en el endometrio de ovejas alimentadas con dieta normal y dieta suplementada / Study of the expression of progesterone receptors in the endometrium of ewes with normal and supplementary diet

La influencia de la nutrición sobre la reproducción en ovinos es compleja y los resultados observados son variables. Planes nutricionales bajos pueden ser causa de una reducción de la sensibilidad del endometrio a la progesterona lo que afecta el desarrollo morfofuncional del endometrio en estadios tempranos de la preñez y por lo tanto va en directo detrimento de la sobrevida del embrión. La progesterona y los estrógenos actúan a través de receptores intracelulares específicos que median su efecto fisiológico. Por lo tanto el nivel de expresión de estos receptores es fundamental para el efecto hormonal. El objetivo del presente estudio fue analizar en forma conjunta la expresión de receptores de progesterona (RP) y estrógeno (RE), en el endometrio de ovejas en ciclo alimentadas con dieta suplementada y dieta normal. La expresión de la proteína receptora como del transcrito de ambos receptores se detectó mediante análisis inmunohistoquímico y RT-PCR en Tiempo Real. Los resultados muestran expresión inmunohistoquímica contra ambos receptores en zonas glándulares y carunculares, destacándose una intensa inmunorreacción en núcleo de células estromales y del epitelio glandular. Se detectó mayor expresión del transcrito de ambos receptores en ovejas alimentadas con suplemento respecto a las que no recibieron suplemento alimenticio.

The influence of nutrition on reproduction in sheep is complex and the reported results are variable. Low nutritional programs can cause a reduction in the sensitivity of the endometrium to progesterone which affects morphofunctional development of the endometrium in early stages of pregnancy and therefore is in direct expense of the survival of the embryo. Progesterone and estrogens act through specific intracellular receptors that mediate its physiological effect. Therefore the level of expression of these receptors is essential for the hormonal effect. The aim of this study was to analyze the expression of progesterone receptors (PR) and estrogen (ER) in the endometrium of sheep supplemented diet cycle and fed normal diet. The expression of the receptor protein and the transcript of both receptors was detected by immunohistochemistry and RT-PCR Real Time. The results show immunohistochemical expression against both receptors in caruncular and glandular areas, highlighting an intense immunoreaction in the nucleus of stromal cells and glandular epithelium. Were detected increased expression of the transcript of both receptors in sheep fed on the supplement that did not receive food supplement. We discuss the possible use of this information for applications in breeding programs in sheep breeding.

Humoral immune response in breeding hens and protective immunity provided by administration of purified Salmonella Gallinarum porins.

The current studies were undertaken to assess the ability of humoral immune response in breeding hens to provide protective maternal antibody in the progeny. A highly purified outer membrane protein, 34 kDa, was isolated from a virulent strain of Salmonella Gallinarum. Cross-reactivity was observed between this protein and Salmonella Typhi porins; thus we consider this outer membrane protein as a Salmonella Gallinarum porin. To evaluate passive immunity against Salmonella Gallinarum, 200 broiler breeder hens were immunized with either 10 microg of Salmonella Gallinarum porins, 30 microg of Salmonella Gallinarum porins, or PBS without porins as a control group. Anti-Salmonella Gallinarum porin antibodies were detected in broiler breeder serum and in fertile eggs (P < 0.05). Consequently, chickens from immunized broiler breeder hens were protected between 53 to 70% against challenges of 20 to 500 half-maximal lethal dose of Salmonella Gallinarum (P < 0.001) when compared with control hens that were injected with PBS. These results suggest that Salmonella Gallinarum porins, as those of other Salmonella species, participate in the induction of the passive protective immunity, and the humoral immune response may be one of the mechanisms involved in the establishment of this protection.

Análisis de la expresión del receptor del estrógeno en endometrio de ovejas alimentadas con dieta normal y dieta suplementada / Analysis of the expression of estrogen receptors in the endometrium of ewes fed with normal and supplementary diet

Los efectos de la nutrición en reproducción ovina han sido estudiados por numerosos autores. Principalmente, el interés se ha centrado en los efectos de la calidad de la dieta ya que en ovinos sometidos a planos nutricionales bajos se ha observado anormalidades del embrión, fase lutea inadecuada, deficiencia en el aporte de progesterona al útero y falla en los mecanismos que comprenden el reconocimiento materno a la preñez. Existen trabajos sobre la relación alimentación-hormonas sin embargo la información existente sobre los receptores de hormonas esteroidales endometriales, indispensables para que ellas actúen, es muy escasa. El objetivo del presente estudio fue analizar comparativamente la expresión del receptor de estrógenos (RE) en el endometrio de ovejas en ciclo alimentadas con dieta suplementada y dieta normal. La expresión de la proteína receptora y del transcrito se detectó mediante análisis inmunohistoquímico y RT-PCR en tiempo real respectivamente. Los resultados muestran expresión inmunohistoquímica en zonas glandulares y carunculares, destacándose una intensa inmunorreacción en núcleos de células estromales y del epitelio glandular. Se detecto mayor expresión del transcrito del RE en endometrio de ovejas alimentadas con suplemento respecto a las que no recibieron suplemento alimenticio. Se discute el posible uso de esta información para aplicarla en programas de mejoramiento reproductivo en ovinos.

The effects of nutrition on sheep reproduction have been studied by many authors. The interest has focused on the effects of diet quality in sheepin in which low plans n have low plane nutrition can cause abnormalities of the ovum or the embryo, luteal inadequancy and failure of the supply of progesterone to the uterus or failurein the mechanisms involved maternal of pregnancy. There are papers about the relation food-hormones, however, the existing information on endometrial steroid hormone receptors, which are essential for them to act, is very scarce. The aim of this study was to comparatively analyze the expression of estrogen receptor (ER) in endometrial cycle of sheep fed diet supplemented and normal diet. The expression of the receptor protein and the transcript were detected by immunohistochemical analysis and RT real-time PCR, respectively. The results showed immunohistochemical expression of RE in glandular and carunculares areas, especially in an intense inmunorreacción stromal cell nuclei and glandular epithelium. Detected increased expression of transcripts of RE in sheep fed a supplement with respect to not receiving a dietary supplement. We discuss the possible use of this information for application in breeding sheeps programs.

Escisión de la zona de transformación en lesiones de cérvix: análisis de 1.141 casos / Large loop excision of the cervical transformation zone: analysis of 1.141 cases

Objetivo: Analizar los resultados de la escisión de la zona de transformación del cérvix efectuadas en la Unidad de Patología Cervical del Hospital Guillermo Grant de Concepción. Método: Estudio descriptivo, retrospectivo, de los antecedentes clínicos e histológicos de 1.141 casos de escisión electro quirúrgica efectuados entre 1999 a 2005, con seguimiento mínimo de 18 meses. Resultados: De las 3.004 mujeres derivadas por citología positiva para neoplasia y/o HPV, a 1.141 (37,9%) se les realizó escisión de la zona de transformación; 1.113 con asa electroquirúrgica (97,5%) y 28 (2,5%) cono frío con bisturí. Las lesiones intraepiteliales (LIE) de alto grado fueron las más frecuentes (66,7%), seguidas por las LIE de bajo grado (7,8%); el 6,5% de las pacientes presentan etapas iniciales de carcinoma invasor; 33% de las piezas histológicaspresentaron bordes comprometidos; 18,4% no presentaron lesión histológica, que sumadas a 7,8% de LIE de bajo grado representan un 26,2% de mujeres consideradas como sobretratadas. El porcentaje a 18 meses de seguimiento en mujeres con LIE de alto grado fue de 85,5%. Necesitaron de una segunda intervención 194 pacientes (17%), 110 (9,6%) por persistencia y recidivas de la lesión. Conclusión: Los resultados presentados son satisfactorios en el porcentaje de curación de las LIE de alto grado como en el aporte al diagnóstico precoz de carcinomas del cérvix y similares a estudios publicados tanto nacionales como extranjeros.

Objective: To analyze the results of 1,141 cervical excisions. Method: Descriptive and retrospective study of the clinical charts and biopsies of 1,141 cases with surgical resection of the uterine cervix performed from 1999 to 2005. Only patients with a minimum of 18 months of follow up were included. Results: From 3,004 patients with a positive Papanicolaou smear for neoplastic cells and/or condyloma, 1,113 (97.5%) were treated with loop electrosurgical excisional procedure (LEEP) and 28 (2.5%) with cold-knife conization. The most frequent lesion (66.7%) found was high grade squamous intraepithelial lesions (SIL), follow by low grade SIL (7.8%); 74 patients (6.5%) presented with early stage of squamous cell carcinoma and 377 (33%) of the histological specimens revealed positive margins; 210 (18.4%) of the cases showed no histological lesion. There were a 26.2% of females that were considered to receive over treatment. After 18 months follow up 85.5% of patient were free of high grade SIL. A second surgical treatment was done in 194 patients (17%) including 110 cases (9.6%) with persistent or recurrence of the lesion. Conclusions: The results are satisfactory related to treatment of high grade SIL and it contributes to the early diagnosis of cervical cancer. The results are similar to other national and international series previously published.

A fusogenic peptide expressed on the surface of Salmonella enterica elicits CTL responses to a dengue virus epitope.

Attenuated Salmonella strains are used widely as live carriers of antigens because they elicit both mucosal and systemic immunity against passenger antigens. However, they generally evoke poor cytotoxic T cell (CTL) responses because Salmonella resides within vacuolar compartments and the passenger antigens must travel to the cytosol and be processed through the MHC class I-dependent pathway to simulate CTLs. To address this problem, we designed a fusion protein to destabilize the phagosome membrane and allow a dengue epitope to reach the cytosol. The fusion protein was displayed on the bacterial surface of Salmonella enterica serovar Typhimurium SL3261 through the beta domain of the autotransporter MisL. The passenger alpha domain contained, from the N-terminus, a fusogenic sequence, the NS3 protein 298-306-amino acid CTL epitope from the dengue virus type 2, a molecular tag, and a recognition site for the protease OmpT to release it to the milieu. Display of the fusion protein on the bacterial surface was demonstrated by IFA and flow cytometry using antibodies against the molecular tag. Cleavage of the fusogenic protein-dengue peptide was demonstrated by flow cytometry using OmpT+ Escherichia coli strains. The recombinant Salmonella strains displaying the fusogenic-dengue peptide were able to lyse erythrocytes, induced specific proliferative responses, and elicited CTL responses. These results suggest that the recombinant fusion proteins containing fusogenic sequences provide a promising system to induce CTLs by live vector vaccines.

Resección histeroscópica del septo uterino en mujeres infértiles / Hysteroscopic resection of the uterine septum in infertile women

Análisis retrospectivo de 11 pacientes con útero septado sintomático que fueron tratadas con metroplastía histeroscópica en un período de 10 años. Estas pacientes presentaban un total de 16 embarazos, con 14 abortos de primer trimestre y 2 partos pretérmino inmaduros con neomortinatos, sin niños vivos. El propósito de este estudio fue mostrar la eficacia de la histeroscopia quirúrgica en resolver esta patología y mejorar el pronóstico reproductivo. Se intervinieron 7 pacientes con septo parcial y 4 con septo completo. En 8 casos hubo resección del tabique con resectoscopio y en 3 con tijera histeroscópica flexible. Se obtuvo embarazo de término en 6 pacientes (55 por ciento) con recién nacidos normales. De las 5 pacientes que no embarazaron, 3 tenían infertilidad por factor tuboperitoneal extenso, una sin relación de pareja y otra sin controles. Se demuestra la utilidad de la resección histeroscópica del útero septado en cuanto al menor tiempo de hospitalización, la baja morbilidad y los buenos resultados reproductivos.

Identification of CD4+ T cell epitopes of Taenia solium paramyosin.

T cell mediated response is involved in a protective immune response against experimental cysticercosis conferred by immunization with Taenia solium paramyosin (TPmy) to BALB/c mice. In this study, we analysed the TPmy amino acid sequence for predicted CD4+ T cells epitopes. Five different regions of this protein showed that the residues anchor to bind the I-Ad molecule, synthetic peptides containing these epitopes were evaluated for their ability to induce lymphoproliferative responses of spleen cells from TPmy immunized mice. Among them, Tp176 (amino acids 176-192 sequence DDLQRQMADANSAKSRL) was the immunodominant T cell epitope of TPmy. Delineation of this epitope should facilitate analysis of the role of CD4+ T cell response in experimental cysticercosis.

Identification of two homologous antigenic peptides derived from L1 HPV-16 and 18 proteins specific for the HLA-B*3901 allele.

In this work we present evidence that the homologous peptides IHSMNSTIL and IHSMNSSIL derived from L1 HPV-16 and 18 proteins respectively, and with high specificity for the allele HLA-B*3901, according with an algorithm prediction program, induced T cell stimulation in patients with advanced cervical cancer positive for HPV-16 or 18 infection and for the HLA-B*3901 allele. Interestingly, T lymphocytes derived from a patient with HPV-18 infection and stimulated with the peptide IHSMNSTIL were capable to kill a cervical cancer cell line named Rova, derived from the tumor of the same patient. In addition, the cytotoxic activity was strongly increased when this cell line was previously treated with hrIFN-gamma. These results suggest that the CTL immune response to L1 HPV-16 and 18 protein derived epitopes is maintained in patients with advanced cervical cancer within specific alleles, and opens the possibility that homologous epitopes may be used in the generation of prophylactic vaccines for cervical tumors bearing different HPV-types.

Cellular immune response and Th1/Th2 cytokines in human neurocysticercosis: lack of immune suppression.

Some reports have suggested that human neurocysticercosis (NCC) induces immunosuppression. To test this hypothesis, we performed a study on active cases of NCC who had not received cestocidal or immunosuppressive treatments. We examined blood counts and specific T cell markers (CD3, CD4, and CD8) by flow cytometry and found no differences between patients with NCC and healthy individuals. Both groups responded to concanavalin A (Con A), and patients with NCC responded more to a parasite crude antigen than uninfected individuals. Peripheral blood mononuclear cells were examined for interleukin (IL)-2, interferon-gamma, IL-10, and IL-4 mRNA. Regardless of infection status, more than 60% of individuals synthesized IL-2 mRNA and, less frequently, the other cytokines. These data suggest that immunosuppression does not occur in NCC patients.

Macrophages present exogenous antigens by class I major histocompatibility complex molecules via a secretory pathway as a consequence of interferon-gamma activation.

Macrophages can process and present exogenous antigens on major histocompatibility complex (MHC) class I molecules through an alternative mechanism involving the internalization of antigens and the secretion of peptides loading MHC class I molecules at the cell surface. In this paper, we found that interferon-gamma (IFN-gamma) -activated macrophages infected with Salmonella typhimurum secreted peptides able to load empty MHC Kb molecules on co-cultured TAP-2-deficient RMA-S cells, added as targets for peptide loading. The increase in class I Kb on the RMA-S cells, resulting from the macrophage-derived peptides, exhibited a comparable stability as the direct addition of an exogenous Kb-binding peptide (OVA257-264) to the RMA-S cells. In both cases, the Kb complexes were stable for at least 3 hr after separating the RMA-S cells from the macrophages. The endosomal inhibitors, leupeptin and ammonium chloride, did not inhibit the release of peptides and the increase in Kb staining on the RMA-S cells in the co-culture systems. Brefeldin A also had no effect. P815 cells previously co-cultured with Salmonella-infected macrophages became targets for cytotoxic T lymphocytes isolated from Salmonella-infected BALB/c mice. Taken together, our data suggest that IFN-gamma-activated macrophages process exogenous antigens in an intracellular compartment where serine proteases generate peptides released to the external environment for loading empty MHC class I molecules at the cell surface. This TAP-independent mechanism for the MHC class I presentation may be involved in priming cytotoxic T lymphocytes against intracellular pathogens in vivo.

Simazine removal from water in a continuous bubble column by O3 and O3/H2O2.

Simazine, [2-chloro, 4,6-bis(ethylamino)-1,3,5-s-triazine], a common herbicide found in surface and ground water has been ozonized in continuous flow mode. Typical operating variables in ozonation processes have been investigated. Thus, the ozone dose fed to the system exerted a positive effect, while the gas flow rate did not influence the efficiency of the process provided ozone mass flow rate was kept constant. Increasing the pH led to a higher extension of the free radical degradation of simazine and, therefore, to a higher efficiency of the process. Also, addition of free radical promoters, i.e. hydrogen peroxide, did result in a significant improvement of the simazine removal rate. A first approach to process economy showed the system ozone/hydrogen peroxide as the most advantageous in terms of electrical energy requirements.