HIGH FIELD MAGNETIC RESONANCE AND MULTIDETECTOR COMPUTED TOMOGRAPHY OF THE NORMAL CANINE STIFLE UNDER VARYING FLEXION ANGLES

Several new imaging modalities have been described for the evaluation of the canine stifle. However, to our knowledge, imaging of the stifle at varying degree of flexion has not been investigated. Our purpose was to compare high field magnetic resonance (MRI) and computed tomography (CT) of the normal stifle at varying flexion angles to describe and evaluate the visualization of its structures when positioned in four different flexion angles (85\ub0-110\ub0-135\ub0-160\ub0). Six canine hind limbs were imaged at four different flexion angles using a 1.5 Tesla commercial MR unit and a multi-slices CT scanner. For each CT and MRI scan two board certified veterinary radiologists independently evaluated the cranial and the caudal cruciate ligament, the medial and the lateral meniscus, the femoral and tibial cartilage, using a visual assessment score of 0-3 and subjective criteria previously described by Podadera et al. A score of 0 indicated that the structure was not visible. A score of 1 indicated that the structure was visualized partially. A score of 2 indicated that the totality of the structure was identified but poorly demarcated. A score of 3 indicated that the structure was totally visualized and well demarcated. Statistical analysis was performed to evaluate the intermodality difference and the interobserver difference, the effect of each flexion angle and the effect of the plane on the visualization score of the different structures. The visualization scores obtained for MRI were statistically significant different compared to those obtained with CT images. There was statistically significant difference between the two observers. From the pairwise comparison of the different flexion angles the 85\ub0 and 110\ub0 degree of flexion resulted to be the best angles to visualize all the structures. All of the joint structure of the canine stifle can be better identified and evaluated by MRI. Most of the soft tissue structures that were seen on MRI were also identified on CT images using a soft tissue window, but never with the definition that MRI offers. Imaging of the stifle in flexion conditions resulted in better visualization and delineation of the main stile joint structures, with particular focus on the cruciate ligaments and the menisci

The Th1/Tfh-like biased responses elicited by the rASP-1 innate adjuvant are dependent on TRIF and Type I IFN receptor pathways.

Ov-ASP-1 (rASP-1), a parasite-derived protein secreted by the helminth Onchocerca volvulus, is an adjuvant which enhances the potency of the influenza trivalent vaccine (IIV3), even when used with 40-fold less IIV3. This study is aimed to provide a deeper insight into the molecular networks that underline the adjuvanticity of rASP-1. Here we show that rASP-1 stimulates mouse CD11c(+) bone marrow-derived dendritic (BMDCs) to secrete elevated levels of IL-12p40, TNF-α, IP-10 and IFN-β in a TRIF-dependent but MyD88-independent manner. rASP-1-activated BMDCs promoted the differentiation of naïve CD4(+) T cells into Th1 cells (IFN-γ(+)) that was TRIF- and type I interferon receptor (IFNAR)-dependent, and into Tfh-like cells (IL21(+)) and Tfh1 (IFN-γ(+) IL21(+)) that were TRIF-, MyD88- and IFNAR-dependent. rASP-1-activated BMDCs promoted the differentiation of naïve CD4(+) T cells into Th17 (IL-17(+)) cells only when the MyD88 pathway was inhibited. Importantly, rASP-1-activated human blood cDCs expressed upregulated genes that are associated with DC maturation, type I IFN and type II IFN signaling, as well as TLR4-TRIF dependent signaling. These activated cDCs promoted the differentiation of naïve human CD4(+) T cells into Th1, Tfh-like and Th17 cells. Our data thus confirms that the rASP-1 is a potent innate adjuvant that polarizes the adaptive T cell responses to Th1/Tfh1 in both mouse and human DCs. Notably, the rASP-1-adjuvanted IIV3 vaccine elicited protection of mice from a lethal H1N1 infection that is also dependent on the TLR4-TRIF axis and IFNAR signaling pathway, as well as on its ability to induce anti-IIV3 antibody production

Plataforma de compra y venta de entradas para eventos integrada en la tecnología blockchain

Desde la llegada de internet tal y como la conocemos, el sector de las plataformas de ventas de tickets para eventos cómo conciertos, festivales, teatro o deportes, tienen algunos desafíos a nivel de seguridad y fiabilidad con los que a día de hoy siguen teniendo que paliar. La falsificación, la especulación y el fraude han sido tareas sencillas en el sector y en la actualidad seguimos viendo casos con demasiada frecuencia. Tecnologías de almacenamiento de datos y de seguridad en internet novedosas como la blockchain pueden ayudar a crear un mercado de venta de entradas mucho más fiable y eficaz. Hemos aprovechado las características de la blockchain con el objetivo de conseguir que las entradas para eventos no puedan ser falsificadas, duplicadas o revendidas por terceros a precios abusivos, evitando así el fraude que es frecuente en este tipo de servicios. Para demostrarlo, hemos desarrollado un prototipo de plataforma de compra y venta de entradas para eventos integrada en la tecnología blockchain, a la que llamamos Tickbit. El prototipo consta de una plataforma web para clientes y un apartado de gestión para las empresas promotoras de eventos, ambas completamente integradas en la blockchain. También consta de un sistema de validación para que las empresas puedan validar las entradas de los clientes. Hemos podido crear una base sobre la cual empresas de venta de entradas para eventos desarrollarán el futuro de las plataformas de “ticketing”. Los resultados han sido muy positivos, y logramos que el dueño de la entrada tenga la fiabilidad de que su entrada es única, e intransferible. La tecnología blockchain, eso sí, es aún poco accesible para perfiles de clientes no-técnicos y necesita maduración, estabilización y estandarización para poder funcionar óptimamente en un entorno realista.Des de l'arribada d'internet tal com la coneixem, el sector de les plataformes de vendes de tiquets per a esdeveniments com concerts, festivals, teatre o esports, tenen alguns desafiaments a nivell de seguretat i fiabilitat amb els quals avui dia continuen havent de pal·liar. La falsificació, l'especulació i el frau han estat tasques senzilles en el sector i en l'actualitat continuem veient casos amb massa freqüència. Tecnologies d'emmagatzematge de dades i de seguretat en internet noves com la blockchain poden ajudar a crear un mercat de venda d'entrades molt més fiable i eficaç. Hem aprofitat les característiques de la blockchain amb l'objectiu d'aconseguir que les entrades per a esdeveniments no puguin ser falsificades, duplicades o revenudes per tercers a preus abusius, evitant així el frau que és freqüent en aquesta mena de serveis. Per a demostrar-ho, hem desenvolupat un prototip de plataforma de compra i venda d'entrades per a esdeveniments integrada en la tecnologia blockchain, a la qual anomenem Tickbit. El prototip consta d'una plataforma web per a clients i un apartat de gestió per a les empreses promotores d'esdeveniments, ambdues completament integrades en la blockchain. També consta d'un sistema de validació perquè les empreses puguin validar les entrades dels clients. Hem pogut crear una base sobre la qual empreses de venda d'entrades per a esdeveniments desenvoluparan el futur de les plataformes de “ticketing”. Els resultats han estat molt positius, i aconseguim que l'amo de l'entrada tingui la fiabilitat que la seva entrada és única, i intransferible. La tecnologia blockchain, això sí, és encara poc accessible per a perfils de clients no-tècnics i necessita maduració, estabilització i estandardització per a poder funcionar òptimament en un entorn realista.Since the arrival of the internet as we know it, the sector of ticket sales platforms for events such as concerts, festivals, theater or sports, have had some challenges in terms of security and reliability with which they still have to deal today. Falsification, speculation and fraud have been easy tasks in the industry and today we still see cases very frequently. New internet security and data storage technologies such as blockchain can help create a much more reliable and efficient ticketing marketplace. We have taken advantage of the characteristics of the blockchain with the aim of ensuring that tickets for events cannot be counterfeited, duplicated or resold by third parties at abusive prices, preventing the fraud that is frequent in this type of service. To demonstrate this, we have developed a prototype platform for buying and selling tickets for events integrated in blockchain technology, called Tickbit. The prototype consists of a web platform for clients and a management section for events companies, both fully integrated into the blockchain. It also consists of a validation system so that companies can validate customer entries. We have been able to create the basis upon which event ticketing companies will build the future of ticketing platforms. The results have been very positive, and we have achieved that the owner of the ticket has the confidence that his ticket is unique and non-transferable. However, blockchain technology is still not very accessible to non-technical customer profiles and needs to mature, stabilize and standardize in order to function optimally in a realistic environment

Intimin, tir, and Shiga toxin 1 do not influence enteropathogenic responses to Shiga toxin-producing Escherichia coli in bovine ligated intestinal loops

Shiga toxin-producing Escherchia coli (STEC) comprises a group of attaching and effacing (A/E) enteric pathogens of animals and humans. Natural and experimental infection of calves with STEC may result in acute enteritis or subclinical infection, depending on serotype- and host-specific factors. To quantify intestinal secretory and inflammatory responses to STEC in the bovine intestine, serotypes that are associated with human disease (O103:H2 and O157:H7) were introduced into ligated mid-ileal loops in gnotobiotic and conventional calves, and fluid accumulation and recruitment of radiolabeled neutrophils were measured after 12 h. STEC serotype O103:H2, but not serotype O157:H7, elicited strong enteropathogenic responses. To determine if the inflammatory response to STEC O103:H2 in calves requires Shiga toxin 1 or intimate bacterial attachment to the intestinal epithelium, defined mutations were made in the stx1, eae, and tir genes. Our data indicate that some STEC induce intestinal inflammatory responses in calves by a mechanism that is independent of A/E-lesion formation, intimin, or Shiga toxin 1. This may have implications for strategies to reduce STEC carriage in cattle

Multiplex immunofluorescence-guided laser capture microdissection for spatial transcriptomics of metastatic melanoma tissues.

We describe a pipeline for optimized and streamlined multiplexed immunofluorescence-guided laser capture microdissection allowing the harvest of individual cells based on their phenotype and tissue localization for transcriptomic analysis with next-generation RNA sequencing. Here, we analyze transcriptomes of CD3+ T cells, CD14+ monocytes/macrophages, and melanoma cells in non-dissociated metastatic melanoma tissue. While this protocol is described for melanoma tissues, we successfully applied it to human tonsil, skin, and breast cancer tissues as well as mouse lung tissues. For complete details on the use and execution of this protocol, please refer to Martinek et al. (2022)

Concomitant inhibition of PPARγ and mTORC1 induces the differentiation of human monocytes into highly immunogenic dendritic cells.

Monocytes can differentiate into macrophages (Mo-Macs) or dendritic cells (Mo-DCs). The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) induces the differentiation of monocytes into Mo-Macs, while the combination of GM-CSF/interleukin (IL)-4 is widely used to generate Mo-DCs for clinical applications and to study human DC biology. Here, we report that pharmacological inhibition of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) in the presence of GM-CSF and the absence of IL-4 induces monocyte differentiation into Mo-DCs. Remarkably, we find that simultaneous inhibition of PPARγ and the nutrient sensor mammalian target of rapamycin complex 1 (mTORC1) induces the differentiation of Mo-DCs with stronger phenotypic stability, superior immunogenicity, and a transcriptional profile characterized by a strong type I interferon (IFN) signature, a lower expression of a large set of tolerogenic genes, and the differential expression of several transcription factors compared with GM-CSF/IL-4 Mo-DCs. Our findings uncover a pathway that tailors Mo-DC differentiation with potential implications in the fields of DC vaccination and cancer immunotherapy

Transcriptional activation of Jun and Fos members of the AP-1 complex is a conserved signature of immune aging that contributes to inflammaging.

Diverse mouse strains have different health and life spans, mimicking the diversity among humans. To capture conserved aging signatures, we studied long-lived C57BL/6J and short-lived NZO/HILtJ mouse strains by profiling transcriptomes and epigenomes of immune cells from peripheral blood and the spleen from young and old mice. Transcriptional activation of the AP-1 transcription factor complex, particularly Fos, Junb, and Jun genes, was the most significant and conserved aging signature across tissues and strains. ATAC-seq data analyses showed that the chromatin around these genes was more accessible with age and there were significantly more binding sites for these TFs with age across all studied tissues, targeting pro-inflammatory molecules including Il6. Age-related increases in binding sites of JUN and FOS factors were also conserved in human peripheral blood ATAC-seq data. Single-cell RNA-seq data from the mouse aging cell atlas Tabula Muris Senis showed that the expression of these genes increased with age in B, T, NK cells, and macrophages, with macrophages from old mice expressing these molecules more abundantly than other cells. Functional data showed that upon myeloid cell activation via poly(I:C), the levels of JUN protein and its binding activity increased more significantly in spleen cells from old compared to young mice. In addition, upon activation, old cells produced more IL6 compared to young cells. In sum, we showed that the aging-related transcriptional activation of Jun and Fos family members in AP-1 complex is conserved across immune tissues and long- and short-living mouse strains, possibly contributing to increased inflammation with age

Breast cancer instructs dendritic cells to prime interleukin 13–secreting CD4+ T cells that facilitate tumor development

We previously reported (Bell, D., P. Chomarat, D. Broyles, G. Netto, G.M. Harb, S. Lebecque, J. Valladeau, J. Davoust, K.A. Palucka, and J. Banchereau. 1999. J. Exp. Med. 190: 1417–1426) that breast cancer tumors are infiltrated with mature dendritic cells (DCs), which cluster with CD4+ T cells. We now show that CD4+ T cells infiltrating breast cancer tumors secrete type 1 (interferon γ) as well as high levels of type 2 (interleukin [IL] 4 and IL-13) cytokines. Immunofluorescence staining of tissue sections revealed intense IL-13 staining on breast cancer cells. The expression of phosphorylated signal transducer and activator of transcription 6 in breast cancer cells suggests that IL-13 actually delivers signals to cancer cells. To determine the link between breast cancer, DCs, and CD4+ T cells, we implanted human breast cancer cell lines in nonobese diabetic/LtSz-scid/scid β2 microglobulin–deficient mice engrafted with human CD34+ hematopoietic progenitor cells and autologous T cells. There, CD4+ T cells promote early tumor development. This is dependent on DCs and can be partially prevented by administration of IL-13 antagonists. Thus, breast cancer targets DCs to facilitate its development

The bacterial effectors EspG and EspG2 induce a destructive calpain activity that is kept in check by the co-delivered Tir effector

Bacterial pathogens deliver multiple effector proteins into eukaryotic cells to subvert host cellular processes and an emerging theme is the cooperation between different effectors. Here, we reveal that a fine balance exists between effectors that are delivered by enteropathogenic E. coli (EPEC) which, if perturbed can have marked consequences on the outcome of the infection. We show that absence of the EPEC effector Tir confers onto the bacterium a potent ability to destroy polarized intestinal epithelia through extensive host cell detachment. This process was dependent on the EPEC effectors EspG and EspG2 through their activation of the host cysteine protease calpain. EspG and EspG2 are shown to activate calpain during EPEC infection, which increases significantly in the absence of Tir – leading to rapid host cell loss and necrosis. These findings reveal a new function for EspG and EspG2 and show that Tir, independent of its bacterial ligand Intimin, is essential for maintaining the integrity of the epithelium during EPEC infection by keeping the destructive activity of EspG and EspG2 in check