Modulation of Toll-like receptor-mediated innate immunity in bovine intestinal epithelial cells by lactic acid bacteria isolated from feedlot cattle.

The ability of lactobacilli isolated from feedlot cattle environment to differentially modulate the innate immune response triggered by Toll-like receptors (TLRs) activation in bovine intestinal epithelial (BIE) cells was evaluated. BIE cells were stimulated with Lactobacillus mucosae CRL2069, Lactobacillus acidophilus CRL2074, Lactobacillus fermentum CRL2085 or Lactobacillus rhamnosus CRL2084 and challenged with heat-stable pathogen associated molecular patterns (PAMPs) from enterotoxigenic Escherichia coli (ETEC) to induce the activation of TLR4 or with polyinosinic:polycytidylic acid (poly(I:C)) to activate TLR3. Type I interferons, cytokines, chemokines and negative regulators of TLR signalling were studied by RT-PCR. L. mucosae CRL2069 significantly reduced the expression of interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1 in BIE cells in the context of TLR3 activation. L. mucosae CRL2069 also reduced the expression of tumour necrosis factor-α, IL-ß, MCP-1, and IL-8 in heat-stable ETEC PAMPs-challenged BIE cells. In addition, reduced expressions of IL-6, MCP-1, and IL-8 were found in BIE cells stimulated with L. rhamnosus CRL2084, although its effect was significantly lower than that observed for the CRL2069 strain. The reduced levels of pro-inflammatory factors in BIE cells induced by the CRL2069 and CRL2085 strains was related to their ability of increasing the expression of TLR negative regulators. L. mucosae CRL2069 significantly improved the expression of A20-binding inhibitor of NFκ-B activation 3 (ABIN-3), interleukin-1 receptor-associated kinase M (IRAK-M) and mitogen-activated protein kinase 1 (MKP-1) while L. rhamnosus CRL2084 augmented ABIN-3 expression in BIE cells. The results of this work suggest that among the studied strains, L. mucosae CRL2069 was able to regulate TLR3-mediated innate immune response and showed a remarkable capacity to modulate TLR4-mediated inflammation in BIE cells. The CRL2069 strain induce the up-regulation of three TLR negative regulators that would influence nuclear factor kB and mitogen-activated protein kinases signalling pathways while reducing the expression of pro-inflammatory cytokines and chemokines. Therefore, L. mucosae CRL2069 is an interesting immunobiotic candidate for the protection of the bovine host against TLR-mediated intestinal inflammatory damage.

Anatomical segmentation of the human medial prefrontal cortex.

The medial prefrontal areas 32, 24, 14, and 25 (mPFC) form part of the limbic memory system, but little is known about their functional specialization in humans. To add anatomical precision to structural and functional magnetic resonance imaging (MRI) data, we aimed to identify these mPFC subareas in histological preparations of human brain tissue, determine sulci most consistently related with mPFC areal boundaries, and use these sulci to delineate mPFC areas in MRIs. To achieve this, we obtained three-dimensional MRI data from 11 ex vivo hemispheres and processed them for cyto- and myelo-architectonic analysis. The architectonic boundaries of mPFC areas were identified in histology and cortical surface length and volumes were measured. Unfolded maps of histologically determined boundaries were generated to identify the association of mPFC areal boundaries with sulci across cases. This analysis showed that cingulate and superior rostral were the sulci most consistently related to mPFC areal boundaries. Based on presence/absence and anastomosis between such sulci, 6 sulci patterns in the 11 hemispheres were found. A further analysis of 102 hemispheres of in vivo MRI scans (N = 51 males, mean ± SD 24.1 ± 3.1 years of age) showed similar sulci patterns, which allowed us to delineate the mFPC areas in them. The volumes of mPFC areas across histological, ex vivo and in vivo MRI delineations were comparable and probabilistic maps generated from the MRIs of the102 hemispheres. Probabilistic maps of mPFC areas were registered to MNI space and are available for regional analysis of functional magnetic resonance imaging data.

Fusion of foreign T-cell epitopes and addition of TLR agonists enhance immunity against Neospora caninum profilin in cattle.

We demonstrated recently that immunization with recombinant Neospora caninum profilin (rNcPRO) induces limited protection and a regulatory T-cell response in mice. The aim of this study was to evaluate the immune response elicited by rNcPRO in cattle and assess a strategy to enhance its immunogenicity, combining the addition of T-cell epitopes and immune modulators. We developed a chimeric recombinant profilin fused to functional T-cell epitopes present in the N-terminal sequence of vesicular stomatitis virus (VSV) glycoprotein G (rNcPRO/G). Groups of three cattle were immunized with two doses (2 weeks apart) of rNcPRO or rNcPRO/G formulated with alum hydroxide or a nanoparticulated soya-based adjuvant enriched with Toll-like receptor (TLR) 2 and TLR9 agonists, aimed to tackle the MyD88 pathway (AVECplus). rNcPRO induced only a primary immune response (IgM mediated), while antibodies in rNcPRO/G-vaccinated animals switched to IgG1 after the booster. The vaccine formulated with rNcPRO/G and AVECplus improved the production of systemic IFN-γ and induced long-term recall B-cell responses. Overall, our study provides data supporting the use of T-cell epitopes from VSV glycoprotein G and TLR agonists to enhance and modulate immunity to peptide antigens in bovines, particularly when using small proteins from parasites for which immune responses are usually feeble.

Dose-dependent immunogenicity of a soluble Neospora caninum tachyzoite-extract vaccine formulated with a soy lecithin/ß-glucan adjuvant in cattle.

Mice immunized with a soluble extract of Neospora caninum tachyzoites (sNcAg) formulated with Providean-AVEC, an aqueous soy-based adjuvant, are fully protected from N. caninum multiplication. Here we evaluated the dose-dependent immunogenicity of this vaccine formulation in cattle. Cattle (N=3 per group) were immunized with two applications (30 days apart) of formulations containing Providean-AVEC and different payloads of sNcAg (100, 50 and 10 µg), that were five to fifty times lower than the only reported study using this same antigen in cattle. Kinetics and magnitude of the vaccine-induced immune responses were dose-dependent. Cattle immunized with 100 µg-sNcAg elicited high-avidity specific antibodies 3 weeks after the primary vaccination while those that received 50 µg of antigen had maximum levels of specific high-avidity antibodies 5 days after the day 30 boost. Vaccination with 10 µg of sNcAg induced comparable antibody responses after 2 weeks post re-vaccination. IgG1 was the predominant isotype in all vaccinated animals. Maximum systemic IFN-γ levels were measured in cattle immunized with 50 and 100 µg-sNcAg (14 ± 2.8 ng/ml). CD4(+)-T cells from vaccinated animals proliferated after sNcAg stimulation in vitro, producing IFN-γ. Recall IFN-γ responses mediated by CD4(+)-T cells were detected up to 140 days post vaccination. Formulations containing Providean-AVEC and 50 µg of sNcAg stimulated broad cellular and humoral immune responses against N. caninum in cattle. The profile and magnitude of the immune response elicited by this vaccine can be modified by the antigen-dose and vaccination schedule. This is the first dose-response study performed in cattle using sNcAg as antigen.

Low ANXA10 expression is associated with disease aggressiveness in bladder cancer.

BACKGROUND: Markers for outcome prediction in bladder cancer are urgently needed. We have previously identified a molecular signature for predicting progression in non-muscle-invasive bladder cancer. ANXA10 was one of the markers included in the signature and we now validated the prognostic relevance of ANXA10 at the protein level. METHODS: We investigated ANXA10 expression by immunohistochemistry using a tissue microarray with 249 Ta and T1 urothelial carcinomas. The expression of ANXA10 was also investigated in an additional set of 97 more advanced tumours. The functional role of ANXA10 in cell lines was investigated by siRNA-mediated ANXA10 knockdown using wound-healing assays, proliferation assays, and ingenuity pathway analysis. RESULTS: Low expression of ANXA10 correlated with shorter progression-free survival in patients with stage Ta and T1 tumours (P<0.00001). Furthermore, patients with more advanced tumours and low ANXA10 expression had an unfavourable prognosis (P<0.00001). We found that ANXA10 siRNA transfected cells grew significantly faster compared with control siRNA transfected cells. Furthermore, a wound-healing assay showed that ANXA10 siRNA transfected cells spread along wound edges faster than control transfected cells. CONCLUSION: We conclude that ANXA10 may be a clinical relevant marker for predicting outcome in both early and advanced stages of bladder cancer.

Repression of KIAA1199 attenuates Wnt-signalling and decreases the proliferation of colon cancer cells.

BACKGROUND: The KIAA1199 transcript is upregulated in colon adenomas and downregulated upon ß-catenin knockdown. METHODS: Transcript profiling was performed on >500 colon biopsies, methylation profiling data were compared with transcript data. Immunohistochemistry assessed KIAA1199 protein expression in 270 stage II/III tumours (>3 years follow-up). The effects of stable KIAA1199 knockdown in SW480 cells (three different constructs) were studied using transcriptional profiling, proliferation and protein analysis. RESULTS: The KIAA1199 transcript was strongly upregulated in 95% of adenocarcinomas. Absent expression in normal mucosa correlated with KIAA1199 promotor methylation. Nuclear and cytoplasmic KIAA1199 protein expression was identified in colon adenocarcinomas and other types of cancers. A subpopulation of patients with tumours strongly expressing KIAA1199 in the nucleus showed a better outcome with regard to recurrence as lung or liver metastases. The KIAA1199 knockdown affected the cell cycle and the Wnt-signalling pathway. Reduced cellular proliferation and decreased KI67, phosphorylated retinoblastoma, ß-catenin and ASCL2 protein expression supported these findings. Eighteen Wnt-signalling genes differentially expressed upon KIAA1199 knockdown correlated with the KIAA1199 expression profile in clinical specimens. CONCLUSION: The KIAA1199 knockdown attenuates the effects of the Wnt/ß-catenin signalling and it may thus be regarded as a regulatory part of this pathway.

Single dilution Avidity-Blocking ELISA as an alternative to the Bovine Viral Diarrhea Virus neutralization test.

This study describes the development and validation of a blocking ELISA that measures avidity of BVDV-specific immunoglobulins (Igs) as an alternative to the classic virus neutralization test. The assay comprises a recombinant soluble E2 glycoprotein as target antigen, a neutralizing serum as detector antibody and a washing-step with a chaotropic agent to determine BVDV-specific Igs avidity. Avidity-Blocking ELISA was validated with 100 negative and 87 positive BVDV-neutralization serum samples from either infected or vaccinated bovines (inactivated commercial vaccines). Specificity and sensitivity of the Avidity-Blocking ELISA were 100% and 98.8%, respectively. The assay was standardized to use a single dilution, so that 90 samples can be tested per plate. Results expressed as Avidity Index (AI) correlated with BVDV neutralizing titers (r=0.94). Unlike the virus neutralization test, the Avidity-Blocking ELISA could discriminate between infected and vaccinated animals (DIVA), suggesting that avidity measurement can be a valuable tool to achieve DIVA compliances. The data show that the avidity of anti BVDV antibodies is related to their capacity to block viral infection in vitro.

The human parahippocampal region: I. Temporal pole cytoarchitectonic and MRI correlation.

The temporal pole (TP) is the rostralmost portion of the human temporal lobe. Characteristically, it is only present in human and nonhuman primates. TP has been implicated in different cognitive functions such as emotion, attention, behavior, and memory, based on functional studies performed in healthy controls and patients with neurodegenerative diseases through its anatomical connections (amygdala, pulvinar, orbitofrontal cortex). TP was originally described as a single uniform area by Brodmann area 38, and von Economo (area TG of von Economo and Koskinas), and little information on its cytoarchitectonics is known in humans. We hypothesize that 1) TP is not a homogenous area and we aim first at fixating the precise extent and limits of temporopolar cortex (TPC) with adjacent fields and 2) its structure can be correlated with structural magnetic resonance images. We describe here the macroscopic characteristics and cytoarchitecture as two subfields, a medial and a lateral area, that constitute TPC also noticeable in 2D and 3D reconstructions. Our findings suggest that the human TP is a heterogeneous region formed exclusively by TPC for about 7 mm of the temporal tip, and that becomes progressively restricted to the medial and ventral sides of the TP. This cortical area presents topographical and structural features in common with nonhuman primates, which suggests an evolutionary development in human species.

SMARCC1 expression is upregulated in prostate cancer and positively correlated with tumour recurrence and dedifferentiation.

BACKGROUND: The identification of new prognostic markers in prostate cancer (PC) is essential to improve patient treatment and management. Data suggest that SMARCC1 protein, a part of the intranuclear SWI/SNF complex which enhances the transactivation of the androgen receptor, is upregulated in PC and therefore a possible candidate marker for PC progression. MATERIALS: Expression of SMARCC1 immunostaining was analysed on a tissue microarray containing specimens from 327 patients with prostate cancer and clinical follow-up information. Furthermore, 30 specimens from patients with benign prostate hyperplasia were included as controls as well as 30 specimens of benign prostate tissue from PC patients. Also, 18 specimens from lymph node metastases were analysed. RESULTS: All benign specimens showed no or minimal staining for SMARCC1. In contrast, 20% of the specimens from patients with non-metastatic and non-recurrent disease showed moderate to marked staining. In 31% of the patients with recurrent disease and in 31% of the patients with metastatic disease we found moderate to strong SMARCC1 immunostaining. In total, 23% of lymph node metastases expressed SMARCC1. SMARCC1 expression was also positively correlated to Gleason score (p<0.05), clinical T stage (p<0.01) and time to recurrence (p<0.001). In a logistic regression analysis, patients with a marked SMARCC1 immunostaining had a significantly elevated odds ratio (OR) of 16 for recurrent cancer and an OR of 4.5 for metastatic disease. Conclusions. Our present results demonstrate an increased expression of SMARCC1 protein in prostate cancer and reveal a positive correlation with tumour dedifferentiation, progression, metastasis and time to recurrence.

Differential expression of DHHC9 in microsatellite stable and instable human colorectal cancer subgroups.

Microarray analysis on pooled samples has previously identified ZDHHC9 (DHHC9) to be upregulated in colon adenocarcinoma compared to normal colon mucosa. Analyses of 168 samples from proximal and distal adenocarcinomas using U133plus2.0 microarrays validated these findings, showing a significant two-fold (log 2) upregulation of DHHC9 transcript (P<10(-6)). The upregulation was more striking in microsatellite stable (MSS), than in microsatellite instable (MSI), tumours. Genes known to interact with DHHC9 as H-Ras or N-Ras did not show expression differences between MSS and MSI. Immunohistochemical analysis was performed on 60 colon adenocarcinomas, previously analysed on microarrays, as well as on tissue microarrays with 40 stage I-IV tumours and 46 tumours from different organ sites. DHHC9 protein was strongly expressed in MSS compared to MSI tumours, readily detectable in premalignant lesions, compared to the rare expression seen in normal mucosa. DHHC9 was specific for tumours of the gastrointestinal tract and localised to the Golgi apparatus, in vitro and in vivo. Overexpression of DHHC9 decreased the proliferation of SW480 and CaCo2 MSS cell lines significantly. In conclusion, DHHC9 is a gastrointestinal-related protein highly expressed in MSS colon tumours. The palmitoyl transferase activity, modifying N-Ras and H-Ras, suggests DHHC9 as a target for anticancer drug design.

[A radiological study of the cervical alterations in Down syndrome. New findings on computerized tomography and three dimensional reconstructions]. / Estudio radiológico de las alteraciones cervicales en el síndrome de Down. Nuevos hallazgos mediante tomografía computarizada y reconstrucciones tridimensionales.

OBJECTIVE: We studied a large proportion of the population in our health district who have Down's syndrome to determine the incidence and variety of changes in the spine and to define the guidelines for preventive diagnosis advisable in relation to atlanto-axial instability, a common disorder in these patients. PATIENTS AND METHODS: First phase: a plain X-ray of the cervical spine in a neutral lateral projection and in flexion in 188 patients, measuring the atlanto-odontoid distance. Second phase: computerized tomography (CT) studies and three dimensional reconstructions in 25 patients (13.3%) chosen at random. The axial cuts from the upper portion of C3 to the occiput were 3 mm in thickness with 3 mm intervals and a standard reconstruction algorithm. RESULTS: The incidences of atlanto-axial instability with an atlodontoid distance (3)5 mm were not comparable with the published series. There was a lower incidence (4.2%), with no difference between measurements in flexion and in the neutral lateral views. There was a greater incidence of malformations than in other reports, including a rare case of os odontoideum and also constant asymmetry of the occipital condyles (100%) in the patients of the CT series and consequently instability of the atlas (96%) and off-centered odontoides (84%). CONCLUSIONS: The study showed that there was deficient asymmetrical development of the occipital bone, which caused different heights of the occipital condyles and led to cervico-cranial mal-position. For study of the degree of error of position and congenital anomalies. We recommend replacing plain X-ray studies by CT with three dimensional reconstructions.

Estudio radiológico de las alteraciones cervicales en el síndrome de down. Nuevos hallazgos mediante tomografía computarizada y reconstrucciones tridimensionales / A radiological study of the cervical alterations in down's syndrome. New findings on computerized tomography and three dimensional reconstructions

Objetivo. Estudiamos gran parte de la población con síndrome de Down de nuestra área sanitaria para establecer la incidencia y variedad de alteraciones de la columna cervical y esclarecer las pautas de diagnóstico preventivo aconsejables en relación con la inestabilidad atloaxoidea, patología frecuente en estos enfermos. Pacientes y métodos. Primera fase: estudio radiológico simple de columna cervical en proyección lateral neutra y flexión a 188 pacientes, medición de las distancias atlodontoideas. Segunda fase: estudio de tomografía computarizada (TC) y reconstrucciones tridimensionales a 25 pacientes (13,3 por ciento) elegidos al azar. Los cortes axiales desde la porción superior de C3 hasta el occipital fueron de 3 mm de grosor con 3 mm de intervalo y algoritmo de reconstrucción estándar. Resultados. Las incidencias de inestabilidad atloaxoidea con distancia atlodontoidea 5 mm no han sido superponibles a las series publicadas, incidencia menor (4,2 por ciento), sin diferencias entre las medidas en flexión y lateral neutra. Hubo una mayor incidencia de malformaciones que otros autores, entre ellas un caso de os odontoideum de rara aparición, así como constante asimetría de los cóndilos occipitales (100 por ciento) en los pacientes de la serie de TC y, consecuentemente, basculación del atlas (96 por ciento) y odontoides descentrada (84 por ciento).Conclusiones. El estudio reveló un deficiente y asimétrico desarrollo del hueso occipital, que condiciona diferente altura en los cóndilos occipitales y provoca una mal posición del cervicocráneo. Para estudiar el grado de malposición y las anomalías congénitas, aconsejamos sustituir el estudio radiológico simple por la TC con reconstrucciones tridimensionales (AU)

Conductas suicidas y alcoholismo en personas sin hogar / Suicide behaviours and alcoholism in homless people

Este trabajo hace referencia a las conductas suicidas de una muestra de pacientes alcohólicos en situación de exclusión social. Es sabido que el consumo abusivo de alcohol ha sido considerado en función de los datos de múltiples estudios como un factor de riesgo de tentativas suicidas y suicidio consumado. Además se ha encontrado que la incidencia de suicidios entre alcohólicos es mucho mayor que en la población general.En nuestro estudio el porcentaje de conductas suicidas es menor que en otros estudios españoles y parece que las conductas suicidas están más relacionadas con la patología concomitante que con la exclusión social (AU)

[Tracheobronchomegaly: an exceptional predisposing factor for pulmonary aspergillomas and massive hemoptysis]. / Traqueobroncomegalia: un factor predisponente excepcional de aspergilomas pulmonares y hemoptisis masiva.

Mounier-Kuhn syndrome--or tracheobronchomegaly--is a rare congenital disorder characterized by significant dilation of the trachea and main bronchi. It is accompanied by ineffective cough and is often complicated by recurrent lung infections and bronchiectasis. Clinical presentation varies widely, ranging from forms with scarce involvement of functional capacity to others that progress to respiratory failure that can prove fatal. We report an exceptional case of massive hemoptysis secondary to the presence of pulmonary aspergillomas and bilateral bronchiectasis in which bronchial arteriography with embolization and surgical resection were insufficient for resolving the complications.

Traqueobroncomegalia: un factor predisponente excepcional de aspergilomas pulmonares y hemoptisis masiva / Tracheobronchomegaly: a rare predisposing factor for pulmonary aspergilloma and massive hemoptysis

El síndrome de Mounier-Kuhn o traqueobroncomegalia es una enfermedad congénita rara, que se caracteriza por una importante dilatación de la tráquea y los bronquios principales. Se acompaña de una tos ineficaz y a menudo complicada con infecciones pulmonares recurrentes y bronquiectasias. Existe un amplio espectro de formas clínicas de presentación, que oscilan entre formas con una escasa afectación de la capacidad funcional y otras progresivas, que evolucionan a una insuficiencia respiratoria que puede ser letal. Presentamos un caso excepcional de hemoptisis masiva secundaria a la presencia de aspergilomas pulmonares y bronquiectasias bilaterales, en la que la arteriografía bronquial con embolización o la resección quirúrgica no fueron suficientes para solucionar estas complicaciones. (AU)

Mutational analysis of Glu272 in elongation factor 1A of E. coli.

In our previous work (Mansilla et al. (1997) Protein Eng. 10, 927-934) we showed that Arg7 of Escherichia coli elongation factor Tu (EF1A) plays an essential role in aminoacyl-tRNA (aa-tRNA) binding. Substitution of Arg7 by Ala or Glu lost this activity. We proposed that Arg7 forms a salt bridge with the charged conserved amino acid Glu272 (Asp284 in Thermus aquaticus) thereby binding the N-terminal region of the protein to domain 2 and thus completing the conformational rearrangement needed for binding aa-tRNA. In this work we have mutated Glu272 to arginine, either alone (Glu272Arg), or in combination with one of the above mentioned mutations (Arg7Glu/Glu272Arg) in order to test this hypothesis. Our results show that, in confirmation of our thesis based on structural knowledge, the substitution of Glu272 (Asp284) decreases the ability of EF1A:GTP to bind aa-tRNA.

Investigation of functional aspects of the N-terminal region of elongation factor Tu from Escherichia coli using a protein engineering approach.

The function of the N-terminal region of elongation factor Tu is still unexplained. Until recently, it has not been visible in electron density maps from x-ray crystallography studies, but the presence of several well conserved basic residues suggest that this part of the molecule is of structural importance for the factor to function properly. In this study, two lysines at positions 4 and 9 were mutated separately to alanine or glutamate. The resulting four point mutants were expressed and purified using the pGEX system. The untagged products were characterized with regard to guanine-nucleotide interaction, intrinsic GTPase activity, and binding of aminoacyl-tRNA (aa-tRNA). The results show that Lys9 is especially strongly involved in the association with guanine nucleotides and the binding of aa-tRNA. Also Lys4 plays a role in the association of GDP and GTP and is also of some importance in aa-tRNA binding. Our results are discussed in structural terms with the conclusion that a complex network of interactions across the interface between domains 1 and 2 with Lys9 being a key residue seems to be important for the fine tuning of the dimensions of the cleft accommodating the acceptor end of aa-tRNA as well as delineating the structure of the effector region.

Mutational analysis of Escherichia coli elongation factor Tu in search of a role for the N-terminal region.

We have mutated lysine 2 and arginine 7 in elongation factor Tu from Escherichia coli separately either to alanine or glutamic acid. The aim of the work was to reveal the possible interactions between the conserved N-terminal part of the molecule, which is rich in basic residues and aminoacyl-tRNA. The enzymatic characterization, comprising GDP and GTP temperature stability assays and measurement of nucleotide dissociation and association rate constants, GTPase activity and aminoacyl-tRNA binding, shows that position 2 is not involved in aminoacyl-tRNA binding, while position 7 is necessary to accomplish this activity. Furthermore, arginine 7 seems to play a role in regulating the binding of GTP. The three-dimensional structure of the ternary complex, EF-Tu:GTP:Phe-tRNAPhe, involving Thermus aquaticus EF-Tu and yeast Phe-tRNA(Phe), shows that Arg7 is in a position which permits salt bridge formation with Asp284, thus binding the N-terminus tightly to domain 2. We propose that this interaction is needed for aminoacyl-tRNA binding, and also for completing the structural rearrangement, which takes place when the factor switches from its GDP to its GTP form.