RESUMEN
In periparturient dairy cows, immune suppression, resulting in decreased neutrophil numbers and function, leads to increased susceptibility to postpartum conditions such as mastitis, retained placenta, and metritis. Administration of polyethylene glycol-conjugated bovine granulocyte colony stimulating factor (pegbovigrastim, Imrestor; Elanco Animal Health, Greenfield, IN) 7 d before and within 24 h of calving, effectively improves granulocyte production and function in vivo as well as in milk. A recently developed coculture assay was adapted for use with endometrial epithelial cells to assess the effects of pegbovigrastim application on directed granulocyte migration and bactericidal activity in vitro on a per-cell basis in endometrial cell cultures. Granulocytes from treated and untreated periparturient cows (6 and 5 per group, respectively) were evaluated for their ability to migrate to and kill bacteria after treatment, in context of the infected endometrium. We hypothesized that in addition to increasing the absolute concentration of circulating neutrophil granulocytes, pegbovigrastim treatment in vivo alters the ability of granulocytes to migrate to endometrial cells in vitro. The results clearly show a marked increase in the total concentration of granulocytes and monocytes between the 2 treatment groups as early as 2 d after the first injection, and this increased between the samples taken 2 d after calving. No migratory or killing differences were identified between granulocytes of both groups, suggesting that pegbovigrastim-induced granulocytes were as effective as non-induced cells. This may also be due to the absence of negative energy balance in the study animals and leads us to conclude that the positive effects seen in vivo are most likely based on the larger number of granulocytes present rather than a direct effect of pegbovigrastim treatment on the functionality of cells for the parameters tested in this study.
Asunto(s)
Bacterias/inmunología , Bovinos/inmunología , Quimiotaxis de Leucocito/efectos de los fármacos , Endometrio/citología , Factor Estimulante de Colonias de Granulocitos/farmacología , Granulocitos/efectos de los fármacos , Proteínas Recombinantes/farmacología , Animales , Endometrio/inmunología , Metabolismo Energético , Femenino , Granulocitos/inmunología , Recuento de Leucocitos , Leche , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Periodo Posparto/efectos de los fármacos , Embarazo , Distribución AleatoriaRESUMEN
Rare maternally inherited duplications at 15q11-13 are observed in ~1% of individuals with an autism spectrum disorder (ASD), making it among the most common causes of ASD. 15q11-13 comprises a complex region, and as this copy number variation encompasses many genes, it is important to explore individual genotype-phenotype relationships. Cytoplasmic FMR1-interacting protein 1 (CYFIP1) is of particular interest because of its interaction with Fragile X mental retardation protein (FMRP), its upregulation in transformed lymphoblastoid cell lines from patients with duplications at 15q11-13 and ASD and the presence of smaller overlapping deletions of CYFIP1 in patients with schizophrenia and intellectual disability. Here, we confirm that CYFIP1 is upregulated in transformed lymphoblastoid cell lines and demonstrate its upregulation in the post-mortem brain from 15q11-13 duplication patients for the first time. To investigate how increased CYFIP1 dosage might predispose to neurodevelopmental disease, we studied the consequence of its overexpression in multiple systems. We show that overexpression of CYFIP1 results in morphological abnormalities including cellular hypertrophy in SY5Y cells and differentiated mouse neuronal progenitors. We validate these results in vivo by generating a BAC transgenic mouse, which overexpresses Cyfip1 under the endogenous promotor, observing an increase in the proportion of mature dendritic spines and dendritic spine density. Gene expression profiling on embryonic day 15 suggested the dysregulation of mammalian target of rapamycin (mTOR) signaling, which was confirmed at the protein level. Importantly, similar evidence of mTOR-related dysregulation was seen in brains from 15q11-13 duplication patients with ASD. Finally, treatment of differentiated mouse neuronal progenitors with an mTOR inhibitor (rapamycin) rescued the morphological abnormalities resulting from CYFIP1 overexpression. Together, these data show that CYFIP1 overexpression results in specific cellular phenotypes and implicate modulation by mTOR signaling, further emphasizing its role as a potential convergent pathway in some forms of ASD.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Células Dendríticas/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/metabolismo , Trastorno del Espectro Autista/patología , Células Cultivadas , Cromosomas Humanos Par 15 , Variaciones en el Número de Copia de ADN , Células Dendríticas/metabolismo , Células Dendríticas/patología , Espinas Dendríticas/genética , Espinas Dendríticas/patología , Femenino , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Regulación hacia ArribaRESUMEN
Previously, vaccination of cattle with Escherichia coli-expressed bovine lungworm paramyosin (EcPMY) adjuvanted with Quil A resulted in considerable reduction in worm burden and larvae shedding (Strube et al., 2015). To further evaluate the protective potential of PMY, cattle vaccination trials were performed using either E. coli- (EcPMY) or Pichia pastoris-expressed PMY (PpPMY) with different adjuvants (Matrix-Q(™) or Quil A). Combinations EcPMY+Matrix-Q(™) (trial 1), PpPMY+Matrix-Q(™) (trial 2) and PpPMY+Quil A (trial 3) were tested against challenge infections with 2000 Dictyocaulus viviparus larvae. Even though GM worm burden and larvae shedding was lower in almost all vaccinated groups, there were high variations between individuals hampering significant differences. However, in all vaccinated groups, lungworms were significantly shorter compared with those in controls. In vitro stimulation of peripheral blood mononuclear cells (PBMC) with recombinant (r)PMY revealed no significant proliferation following vaccinations or challenge infection. All vaccinated cattle showed a significant rise in specific antibodies, particularly IgG and its subclass IgG1, and detected the native lungworm PMY in immunoblots starting 2 weeks after the first vaccination. The use of a different rPMY-adjuvant combination or combined vaccination with additional recombinant antigens might be a promising future approach towards a new vaccine against lungworms in cattle.
Asunto(s)
Antígenos Helmínticos/inmunología , Enfermedades de los Bovinos/prevención & control , Infecciones por Dictyocaulus/prevención & control , Dictyocaulus/inmunología , Tropomiosina/inmunología , Vacunación/veterinaria , Animales , Anticuerpos Antihelmínticos/inmunología , Antígenos Helmínticos/genética , Bovinos , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Proteínas del Helminto/genética , Proteínas del Helminto/inmunología , Inmunoglobulina G/inmunología , Larva , Leucocitos Mononucleares/inmunología , Masculino , Proteínas Recombinantes , Tropomiosina/genética , Vacunas/inmunología , Levaduras/genética , Levaduras/metabolismoRESUMEN
The composition of the microbiome plays a significant role in the pathogenesis of inflammatory bowel disease (IBD) in humans and chronic enteropathies (CE) in dogs. The administration of probiotic micro-organisms is one way of modulating the microbiome, but experiments elucidating mechanisms of action of probiotics in the intestine of healthy and CE dogs are lacking. The aim of our study was to investigate the effects of different Toll-like receptor (TLR) ligands and Enterococcus faecium (EF) on ex vivo cultured duodenal samples and whole blood (WB) from dogs with food-responsive chronic enteropathy (FRE) when compared to healthy dogs. Biopsy stimulation was performed in 17 FRE and 11 healthy dogs; WB stimulation was performed in 16 FRE and 16 healthy dogs. Expression of TLR2, 4, 5 and 9, IL-17A, IL-22, IFNy, TNFα, IL-4, IL-10, TGFß and PPARy was determined in biopsies by quantitative polymerase chain reaction (PCR). In addition, production of TNFα, IL-10, IFNy and IL-17A protein in WB and biopsy supernatants was assessed by ELISA. Treatment with individual TLR ligands or EF induced a variety of changes in the expression of different TLRs and cytokines, but not necessarily a consistent change with a single stimulating agent. Even though cytokine protein could not be detected in supernatants from ex vivo stimulated biopsies, we found TNFα protein responses in blood to be opposite of the transcriptional responses seen in the biopsies. Stimulation of canine duodenal biopsies with TLR ligands can potentially induce anti-inflammatory gene expression, especially in healthy tissue, whereas the effects of EF were limited.
Asunto(s)
Duodeno/microbiología , Enterococcus faecium , Enfermedades Inflamatorias del Intestino/veterinaria , Probióticos/uso terapéutico , Receptores Toll-Like/agonistas , Animales , Células Cultivadas , Perros , Duodeno/patología , Femenino , Flagelina/farmacología , Enfermedades Inflamatorias del Intestino/microbiología , Enfermedades Inflamatorias del Intestino/terapia , Interleucinas/biosíntesis , Intestinos/microbiología , Intestinos/patología , Lipopéptidos/farmacología , Lipopolisacáridos/farmacología , Masculino , Microbiota , ARN Mensajero/biosíntesis , Células Th17/inmunología , Receptores Toll-Like/biosíntesis , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
OBJECTIVES: Toll-like receptor (TLR)-mediated vascular inflammation, inducible by - amongst other factors - auto-antibodies, is increasingly recognized as a potential mediator of cardiovascular disease. We investigated whether anti-apolipoprotein (Apo)A-1 IgG was associated with a pro-inflammatory cytokine profile in myocardial infarction (MI) patients and whether anti-ApoA-1 IgG elicited a pro-inflammatory response by activating TLRs. METHODS: As surrogate markers of atherosclerotic plaque vulnerability, interleukin (IL)-6, tumour necrosis factor (TNF)-α, matrix metalloproteinase (MMP)-9 and MMP-3 levels were assessed in 221 consecutive MI patients. Using human monocyte-derived macrophages (HMDMs) we investigated (i) the anti-ApoA-1 IgG interaction with TLRs using proximity ligation assay and (ii) anti-ApoA-1 IgG-dependent IL-6/TNF-α production. TLR involvement was further confirmed using HEK293-Blue TLR-2/-4 cells and by computational docking simulations. RESULTS: In MI patients, anti-ApoA-1 IgG positivity was associated with higher levels of IL-6, TNF-α and MMP-9, but lower MMP-3 levels. In in vitro experiments, anti-ApoA-1 antibodies bound to HDMDs in a TLR2-dependent manner, resulting in nuclear translocation of NFκB and a significant increase in TNF-α and IL-6 production. Subsequent functional studies highlighted the importance of CD14 as co-receptor in the anti-ApoA-1 IgG-TLR2-induced cytokine production. Additional bioinformatic studies identified structural homologies between TLR2 and ApoA-1, which may explain the observed cross-reactivity between antibodies against these two molecules. CONCLUSIONS: Anti-ApoA-1 IgG positivity in MI is associated with a high-risk cytokine profile. These auto-antibodies promote inflammation by stimulating the TLR2/CD14 receptor complex, probably because of molecular mimicry, which may contribute to atherosclerosis-related complications in patients.
Asunto(s)
Apolipoproteína A-I/inmunología , Autoanticuerpos/inmunología , Inmunoglobulina G/inmunología , Receptores de Lipopolisacáridos/inmunología , Infarto del Miocardio/inmunología , Receptor Toll-Like 2/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Inflamación/inmunología , Masculino , Persona de Mediana Edad , Infarto del Miocardio/complicacionesRESUMEN
Inflammatory bowel disease (IBD) is thought to be the most common cause of vomiting and diarrhoea in dogs. Although IBD can occur in any canine breed, certain breeds are more susceptible. We have previously shown that polymorphisms in the TLR4 and TLR5 (toll-like receptor) genes are significantly associated with IBD in German Shepherd dogs (GSDs). In order to allow for the development of novel diagnostics and therapeutics suitable for all dogs suffering from IBD, it would be useful to determine if the described polymorphisms are also significantly associated with IBD in other breeds. Therefore, the aim of this study was to investigate whether polymorphisms in the canine TLR4 and TLR5 genes are associated with IBD in other non-GSD canine breeds. The significance of the previously identified non-synonymous single nucleotide polymorphisms (SNPs) in the TLR4 (T23C, G1039A, A1571T and G1807A) and TLR5 genes (G22A, C100T and T1844C) were evaluated in a case-control study using a SNaPSHOT multiplex reaction. Sequencing information from 85 unrelated dogs with IBD consisting of 38 different breeds was compared with a breed-matched control group consisting of 162 unrelated dogs. Indeed, as in the GSD IBD population, the two TLR5 SNPs (C100T and T1844C) were found to be significantly protective for IBD in other breeds (P = 0.023 and P = 0.0195 respectively). Our study suggests that the two TLR5 SNPs, C100T and T1844C could play a role in canine IBD as these were found to be protective factors for this disease in 38 different canine breeds. Thus, targeting TLR5 in the canine system may represent a suitable way to develop new treatment for IBD in dogs.
Asunto(s)
Enfermedades Inflamatorias del Intestino/genética , Polimorfismo Genético , Receptor Toll-Like 5/genética , Animales , Estudios de Casos y Controles , ADN/metabolismo , Perros , Femenino , Predisposición Genética a la Enfermedad , Variación Genética , Masculino , Especificidad de la Especie , Receptor Toll-Like 4/genéticaRESUMEN
Intranasal vaccinations are becoming more important in both human and animal medicine to generate a localized IgA immune response not seen with parenteral vaccinations. This localized IgA response is more effective at reducing pathogen load on the mucosal surface of a potential host. One prerequisite for a successful nasal vaccination is the need to understand the distribution pattern of the nebulized vaccine, which requires an understanding the volume of the nares as well as the mucosal surface area. The exact mucosal surface area of ruminant nares has not yet been investigated. The aim of this concept study is to provide a detailed breakdown of a new method of volumetric rendering that can be used to calculate the volume and mucosal surface area of ruminant nares from computed tomographic images. The program Seg 3D was used to perform semi-automatic segmentation of a CT scan of a 9-month-old lamb head. Threshold segmentation and manual segmentation were used in combination to select the lamb's nasal cavity. The segmentation process yielded a volumetric rendering that was used to calculate the surface area and volume of the lamb's nasal cavity, with the segmentation process was repeated for each individual side of the lamb's nares. The surface area of the mucosal surface of each nostril is approximately 448 cm2, and the volume is approximately 45 cm3. The methodology described in this study successfully calculated the volume and surface area of a lamb's nares using volumetric rendering.
RESUMEN
Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) belong to the same peptide family and exert a variety of biological functions. Both PACAP and VIP have protective effects in several tissues. While PACAP is known to be a stronger retinoprotective peptide, VIP has very potent anti-inflammatory effects. The need for a non-invasive therapeutic approach has emerged and PACAP has been shown to be retinoprotective when administered in the form of eye drops as well. The cell penetrating peptide TAT is composed of 11 amino acids and tagging of TAT at the C-terminus of neuropeptides PACAP/VIP can enhance the traversing ability of the peptides through the biological barriers. We hypothesized that TAT-bound PACAP and VIP could be more effective in exerting retinoprotective effects when given in eye drops, by increasing the traversing efficacy and enhancing the activation of the PAC1 receptor. Rats were subjected to bilateral carotid artery occlusion (BCCAO), and retinas were processed for histological analysis 14 days later. The efficiency of the TAT-bound peptides to reach the retina was assessed as well as their cAMP increasing ability. Our present study provides evidence, for the first time, that topically administered PACAP and VIP derivatives (PACAP-TAT and VIP-TAT) attenuate ischemic retinal degeneration via the PAC1 receptor presumably due to a multifactorial protective mechanism.
Asunto(s)
Antiinflamatorios/farmacología , Fármacos Neuroprotectores/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Retina/efectos de los fármacos , Degeneración Retiniana/tratamiento farmacológico , Péptido Intestinal Vasoactivo/farmacología , Animales , Antiinflamatorios/administración & dosificación , Antiinflamatorios/uso terapéutico , Células CHO , Cricetinae , Cricetulus , Masculino , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/uso terapéutico , Soluciones Oftálmicas , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/química , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Retina/metabolismo , Péptido Intestinal Vasoactivo/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
C-type lectin receptors (CTLR) are cell-surface signalling molecules that recognize a range of highly conserved pathogen molecules and instigate the appropriate immune response. Here, we report the cloning, sequencing, mapping and expression pattern of the bovine C-type lectin domain family 7, member A (CLEC7A; synonyms CLCSF12, Dectin-1). We identified two isoforms, similar to the human system, with a long and short neck. Overall, the organization of the two bovine CLEC7A genes is similar to that of humans and mice. The CLEC7A gene maps on Bos taurus chromosome 5 (BTA5). mRNA transcripts for CLEC7A were detected in bone-marrow cells, monocytes, macrophages and dendritic cells and NK cells, but not in CD4(+) T-cells or CD21(+) B-cells. The increased knowledge of the genomic organization of the bovine CTLR genes may promote our understanding of their evolution and help in the identification of bovine genes underlying disease-resistance traits.
Asunto(s)
Bovinos/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos/genética , Mapeo Cromosómico/veterinaria , Clonación Molecular , Femenino , Lectinas Tipo C , Proteínas de la Membrana/biosíntesis , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Isoformas de Proteínas , ARN/química , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
The uptake of respiratory syncytial virus (RSV) antigen by cattle dendritic cells was investigated. Pathways of antigen uptake were monitored by flow cytometry using specific tracers and by proliferation assays, which were used to measure the presentation of RSV antigen and ovalbumin. Inhibitors that differentially affected pathways were used to distinguish them. Presentation of RSV antigen, but not ovalbumin, was inhibited by phorbol myristate acetate and filipin, which have been reported to inhibit caveolae, but not by cytochalasin D, amiloride, or mannose. These inhibitors have been reported to block macropinocytosis and other actin-dependent uptake mechanisms, endocytic pathways involving clathrin-coated pits, and the mannose receptor. Furthermore, co-localization of RSV antigen and caveolae was observed by confocal microscopy. Thus, the major route for uptake of RSV antigen by cattle dendritic cells is one mediated by caveolae, adding a pathway of antigen uptake by dendritic cells to those established.
Asunto(s)
Antígenos Virales/metabolismo , Células Dendríticas/virología , Endocitosis , Lectinas Tipo C , Lectinas de Unión a Manosa , Virus Sincitiales Respiratorios/metabolismo , Amilorida/farmacología , Animales , Presentación de Antígeno , Brefeldino A/farmacología , Bovinos , Membrana Celular/metabolismo , Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Citocalasina D/farmacología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Filipina/farmacología , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Mananos/farmacología , Receptor de Manosa , Ratones , Microscopía Confocal , Ovalbúmina/metabolismo , Pinocitosis , Receptores de Superficie Celular/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Gammadelta and alphabeta TCR+ T cells share many properties and their interactions are likely to be coordinated and regulated. We provide evidence that cattle gammadelta T cells are able to present antigen to CD4+ T cells. To help elucidate their function gammadelta T cell lines were propagated for extensive characterization. Cells expressed high levels of MHC class II and production of co-stimulatory molecules as evidenced by the binding of a CTLA-4 fusion protein and synthesis of CD80 transcripts. These properties and the presence of a well-developed endosomal compartment indicated the cells might function as antigen-presenting cells. Resting CD4+ T cells from calves immunized with ovalbumin or respiratory syncytial virus-antigen proliferated in response to gammadelta T cells pulsed with antigen that appeared to be endocytosed via clathrin-coated pits or specific receptors.
Asunto(s)
Presentación de Antígeno/fisiología , Células Presentadoras de Antígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Inmunoconjugados , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Linfocitos T/inmunología , Abatacept , Animales , Células Presentadoras de Antígenos/metabolismo , Antígenos CD , Antígenos de Diferenciación/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Antígeno CTLA-4 , Bovinos , Células Cultivadas , Citocinas/biosíntesis , Fluoresceína-5-Isotiocianato/farmacocinética , Antígenos de Histocompatibilidad Clase II/biosíntesis , Memoria Inmunológica/fisiología , Interleucina-2/farmacología , Activación de Linfocitos/fisiología , Ratones , Ovalbúmina/farmacocinética , Fenotipo , ARN Mensajero/metabolismo , Linfocitos T/metabolismoRESUMEN
It has been suggested previously that a deficiency in mucosal immunoglobulin (Ig) A production could be involved in the pathogenesis of chronic enteropathy in German shepherd dogs (GSDs). Recent research has shown that single nucleotide polymorphisms in the gene encoding Toll-like receptor (TLR)-5 are associated with an increased risk of development of chronic idiopathic enteropathy in this breed. IgA is essential for mucosal immunity and studies in mice have linked the interaction of TLR5 with its ligand flagellin to class switching of B cells into IgA-producing plasma cells. We hypothesized that dogs carrying the risk-associated (RA) genotypes for G22A and C100T genes of TLR5 would have a different number of IgA plasma cells in the duodenal and colonic mucosa compared with dogs carrying the risk-protective (RP) genotypes. Thirty-one GSDs were diagnosed with idiopathic chronic enteropathy by clinical exclusion diagnosis and histopathological confirmation. Immunohistochemistry was performed using goat anti-dog IgA primary antibody. Two sections of duodenum, and colon if available, were examined from each animal. Twelve images were captured from each section and IgA-positive cells were counted and expressed per 10,000 µm(2). TLR5 genotypes for the G22A and C100T genes were determined by polymerase chain reaction on blood samples. Numbers of IgA-positive cells in the duodenum and colon were slightly higher than those published previously for GSDs with or without chronic enteropathy (mean in the crypt area of the duodenum 52.6 ± 16.2; mean in the tip of the duodenal villus 51.12 ± 3.83; mean in the base of the duodenal villus 55.02 ± 3.3; mean in the crypt area of the colon 67.4 ± 4.3). There was no correlation between numbers of IgA-positive cells in duodenum or colon between dogs carrying the RA versus the RP alleles of TLR genes. Further studies are needed to assess the production of secretory IgA and its relationship to TLR5 genotypes.
Asunto(s)
Enfermedades de los Perros/genética , Enfermedades Intestinales/veterinaria , Células Plasmáticas/inmunología , Receptor Toll-Like 5/genética , Animales , Enfermedades de los Perros/inmunología , Perros , Predisposición Genética a la Enfermedad , Genotipo , Inmunoglobulina A/sangre , Inmunoglobulina A/inmunología , Inmunohistoquímica , Enfermedades Intestinales/genética , Enfermedades Intestinales/inmunología , Mucosa Intestinal/inmunología , Polimorfismo de Nucleótido SimpleRESUMEN
BACKGROUND: Canine chronic enteropathies (CE) are believed to be caused by an aberrant immune response towards the intestinal microbiome. Administration of probiotics can alleviate colitis in people. In vitro effects of the probiotic Enterococcus faecium NCIMB 10415 E1707 (EF) previously have been evaluated using canine cells (e.g., whole blood, intestinal biopsies), but data on in vivo efficacy are lacking. HYPOTHESIS/OBJECTIVES: Administration of EF to dogs with food-responsive CE will improve clinical outcome and decrease the intestinal inflammatory profile. ANIMALS: Dogs diagnosed with CE were prospectively recruited to receive a hydrolyzed elimination diet plus either a synbiotic product containing EF or placebo for 6 weeks. Both veterinary staff and owners were blinded to the treatment. METHODS: Clinical severity index (CCECAI), clinicopathological data and gene expression using intestinal biopsies (TLR2/4/5/9, IL-17A, IL-22, IL-23p19, RORC, IL-2, IL-12p35, TNFα, IL-4, IFNy, IL-10, TGFß, IL-1ß, IL-18, NLRP3, casp-1, TFF1, TFF3 and PPARy) before and after 6 weeks of treatment were analyzed using linear mixed modeling. RESULTS: Of the 45 cases recruited, 12 finished the clinical trial. Seven received the synbiotic and 5 the placebo product. There was no difference between groups or treatments regarding clinical efficacy, histology scores or expression of any of the investigated genes. CONCLUSIONS AND CLINICAL IMPORTANCE: Standard dietary treatment induced rapid clinical response in all cases. Because the study was underpowered, it was not possible to determine whether or not EF had an additional effect within the time period of 6 weeks.
Asunto(s)
Enfermedades de los Perros/terapia , Enterococcus faecium , Enfermedades Intestinales/veterinaria , Probióticos , Animales , Perros , Femenino , Regulación de la Expresión Génica , Enfermedades Intestinales/microbiología , Enfermedades Intestinales/terapia , Masculino , Proyectos PilotoRESUMEN
The effects of caprine arthritis encephalitis virus (CAEV) infection on cytokine activity of caprine monocytes stimulated with Escherichia coli lipopolysaccharide (LPS) were examined. Compared with supernatants from LPS-stimulated monocytes of CAEV-negative goats, supernatants from CAEV-positive goats stimulated less proliferation of murine thymocytes in the MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) assay, showed about 50% less IL-1 activity on the IL-1-dependent cell line LBRM-33 1 A-5, and showed about 200% more tumor necrosis factor (TNF) activity on the TNF-sensitive murine fibroblast cell line L-929. These results indicate that CAEV infection changes caprine monocyte cytokine responsivity.
Asunto(s)
Virus de la Artritis-Encefalitis Caprina/inmunología , Enfermedades de las Cabras/inmunología , Infecciones por Lentivirus/veterinaria , Monocitos/inmunología , Animales , Línea Celular , Femenino , Fibroblastos , Cabras , Interleucina-1/biosíntesis , Infecciones por Lentivirus/inmunología , Lipopolisacáridos , Ratones , Monocitos/efectos de los fármacos , Sales de Tetrazolio , Tiazoles , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
The effects of bovine leukemia virus (BLV) infection on cytokine activity of bovine monocytes stimulated with Escherichia coli lipopolysaccharide (LPS) were examined. Compared to supernatants of LPS-stimulated monocytes from BLV-negative cows, supernatants from BLV-positive cows contained about four times more interleukin-1 beta (IL-1 beta) (as measured by an enzyme-linked immunosorbent assay (ELISA) for bovine IL-1 beta). Despite their higher IL-1 beta concentration, supernatants from BLV-positive cows stimulated proliferation of murine thymocytes in the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-biphenyl tetrazolium bromide) assay similar to supernatants from BLV-negative cows, but showed about 30% less IL-1 activity than supernatants from BLV-negative cows on the IL-1-dependent cell line LBRM-33 1 A-5, and about five times more tumor necrosis factor (TNF) activity on the TNF-sensitive murine fibroblast cell line L-929. These results demonstrate that BLV infection changes the cytokine response of bovine monocytes to LPS stimulation in vitro. The results are consistent with the assumption that BLV infection leads to the production and secretion of a soluble IL-1 inhibitor by LPS-stimulated peripheral blood monocytes.
Asunto(s)
Leucosis Bovina Enzoótica/inmunología , Virus de la Leucemia Bovina/inmunología , Lipopolisacáridos/farmacología , Monocitos/inmunología , Animales , Bovinos , Sistema Libre de Células/inmunología , Leucosis Bovina Enzoótica/sangre , Femenino , Interleucina-1/sangre , Interleucina-1/farmacología , Activación de Linfocitos/efectos de los fármacos , Monocitos/efectos de los fármacos , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/toxicidadRESUMEN
The effects of IL-12 on the responses of cattle peripheral blood mononuclear cells (PBMC) to bovine respiratory syncytial virus (BRSV) antigen and ovalbumin (OVA) were tested, in vitro. IL-12 did not affect the proliferative responses of PBMC to these antigens but markedly accelerated and augmented the level of IFNgamma secreted. When tested on lymphoblasts rather than resting T-cells IL-12 also enhanced proliferation. In contrast IL-4 and, to greater extent, IL-10 inhibited the response. The effect of IL-12 on IFNgamma synthesis was confirmed at the level of IFNgamma. mRNA expression using Taqman PCR. CD4 and CD8 T-cell populations produced IFNgamma, however, CD4 T-cells comprised the largest contributors to the IFNgamma production. Gamma/delta T-cells did not contribute markedly. A comparison of the species cross-reactivity showed bovine IL-12 was also active in the human system. This study shows that antigen-driven responses in cattle can be significantly influenced by exogenous cytokines and suggests the IL-12/IL-10 balance is crucial for regulation of IFNgamma.
Asunto(s)
Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Interferón gamma/biosíntesis , Interleucina-12/farmacología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Bovinos , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática/veterinaria , Humanos , Interferón gamma/genética , Interleucina-10/farmacología , Interleucina-4/farmacología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Ovalbúmina , Reacción en Cadena de la Polimerasa/veterinaria , ARN Mensajero/metabolismo , Virus Sincitial Respiratorio Bovino , Especificidad de la EspecieRESUMEN
Respiratory syncytial virus (RSV) has been reported to induce the production of chemokines in the airway epithelia. Dendritic cells (DC) are the most potent antigen-presenting cells. They are located throughout the body and release chemokines in response to inflammation and infection. We have investigated the chemokine profile of bovine DC in response to exposure to bovine RSV (BRSV). Transcripts for several chemokines were detected by RT-PCR, subsequently cloned and expressed, and the products analysed by western blotting. To test the effect of the recombinant chemokines on RSV-induced T cell proliferation, DC were pulsed with BRSV, irradiated, and added to purified bovine CD4(+) T cells from RSV-immune cattle in combination with various concentrations of recombinant chemokines, and the proliferative response of the T cells assessed. Eotaxin was the only chemokine, of those investigated, that specifically enhanced the T cell response to BRSV-pulsed DC. Addition of MIP-1alpha to control wells or to wells containing BRSV-pulsed DC had similar effects, suggesting non-specific stimulation of T cells. RANTES and MIP-3alpha did not seem to influence the proliferative response of T cells co-cultured with BRSV-pulsed DC. Thus, although BRSV induced the production of several chemokines by DC, only eotaxin promoted a BRSV specific CD4(+) T cell proliferative response.
Asunto(s)
Quimiocinas/fisiología , Células Dendríticas/fisiología , Activación de Linfocitos , Virus Sincitial Respiratorio Bovino/inmunología , Linfocitos T/inmunología , Animales , Células COS , Bovinos , Movimiento Celular/efectos de los fármacos , Quimiocinas/genética , Humanos , Interferón gamma/biosíntesis , Lipopolisacáridos/farmacología , ARN Mensajero/análisisRESUMEN
The bovine leukaemia virus (BLV) is a retrovirus that infects mainly B lymphocytes of cattle, but proviral DNA can also be isolated from monocytes/macrophages. This study investigated the effect of BLV infection on surface antigens on freshly isolated peripheral blood monocytes and cultured monocyte-derived macrophages, with and without lipopolysaccharide (LPS) stimulation. The effect of BLV infection on phagocytic activity of CD14+ monocytes was also assessed. The percentage of monocytes expressing the surface antigens CD11b, CD32 (FcgammaRII), MHC class II and the surface antigen recognised by mAb DH59B were increased in BLV-positive cattle. In contrast, expression intensity of all markers was low in samples from BLV-positive cattle. CD14+ monocytes from BLV-positive cattle showed less Fcgamma-receptor-mediated phagocytosis compared to monocytes from BLV-negative cattle. After 7 days in culture, there was evidence for shedding/downregulation of surface antigens on monocyte-derived macrophages, in particular on cells from BLV-positive cattle. LPS stimulation decreased the percentage of cells expressing the measured markers in monocyte-derived macrophages taken from BLV-negative cattle, but not in cultures derived from BLV-positive cattle. The results provide further evidence for an altered function of monocytes and macrophages in BLV-infected cattle.
Asunto(s)
Bovinos/inmunología , Leucosis Bovina Enzoótica/inmunología , Virus de la Leucemia Bovina/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Fagocitosis/fisiología , Animales , Anticuerpos Monoclonales , Antígenos CD/inmunología , Células Cultivadas , Citometría de Flujo/veterinaria , Antígenos de Histocompatibilidad Clase II/inmunología , Inmunofenotipificación/veterinaria , Lipopolisacáridos/farmacología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunologíaRESUMEN
Dendritic cells are professional antigen presenting cells derived from the bone marrow and distributed throughout body tissues where they are located in sites that are suitable for antigen uptake. They are central to the induction of immune responses in naive animals and thus have become targets in strategies that are aimed at modulating resistance to infection. Studies in cattle have shown that the dendritic cells are phenotypically heterogeneous and that the different phenotypes have different biological properties. The molecular basis for this variation has begun to be investigated and has led to the identification of a member of the SIRPalpha family of signal regulatory proteins (MyD1) on a subset of dendritic cells in afferent lymph. Uptake of antigen by cattle dendritic cells is by a number of mechanisms that can involve endocytosis via clathrin coated pits or via caveolae as well as macropinocytosis.
Asunto(s)
Bovinos/inmunología , Células Dendríticas/inmunología , Inmunofenotipificación , Señales de Clasificación de Proteína/inmunología , Animales , Clatrina/inmunología , Células Dendríticas/clasificación , Endocitosis/inmunología , Pinocitosis/inmunologíaRESUMEN
The effect of a prolonged low dose infusion of bacterial lipopolysaccharide (LPS) on acute phase-like reactions was examined in heifers. LPS (2 micrograms kg-1 dissolved in 100 ml water), or saline was infused (at 1 ml min-1) intravenously for 100 minutes and blood samples were taken at various times before, during and after the infusion. The serum concentrations of tumour necrosis factor-alpha (TNF alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and serum amyloid A (SAA) and the rectal temperature increased in response to the LPS infusion. Serum TNF alpha increased before the increases in IL-1 beta and IL-6 and remained high from 20 minutes after the onset of the infusion until the end of the sampling period (six hours). The LPS-induced increases in serum IL-1 beta and IL-6 were biphasic. Plasma cortisol and lactate concentrations also increased, and plasma glucose and beta-hydroxybutyrate concentrations decreased in response to the LPS infusion. The similarity of these reactions to changes observed in response to bacterial infections shows that the prolonged infusion of low doses of LPS is a good model for studying the acute phase response to Gram-negative bacterial infection in heifers.