Search

Objectives: The pathogenesis of reactive arthritis (ReA) is incompletely understood but may involve aberration(s) in the host's innate immune response towards infecting microbes. We therefore studied the production of interleukin (IL)-1β, a marker of inflammasome activation, and of IL-6, IL-12, IL-23, and tumour necrosis factor (TNF)-a, promoters of T-cell differentiation, by peripheral blood mononuclear cells (PBMNs) and monocyte-derived macrophages from healthy subjects with a history of ReA. Method: The study included 10 human leucocyte antigen (HLA)-B27-positive healthy subjects with previous ReA triggered by Yersinia enterocolitica O:3 infection and 20 healthy reference subjects, of whom 10 were HLA-B27 positive. PBMNs and macrophages were cultured for 18 h with bacterial lipopolysaccharide (LPS), muramyl dipeptide (MDP), Yersinia, or their appropriate combinations. PBMNs were also stimulated with monosodium urate (MSU) crystals. Cytokine levels were measured using an enzyme-linked immunosorbent assay (ELISA) and the Luminex system. Results: IL-1β secretion was similar from cells of the ReA group and from the HLA-B27-positive and -negative reference groups. TNF-α production from macrophages upon co-stimulation of LPS and MDP increased in the order ReA group < HLA-B27-positive reference group < HLA-B27-negative reference group (p for a trend = 0.027). Similarly, Yersiniainduced TNF-α and IL-23 production increased in the same order (p for trend for TNF-α = 0.036; p for trend for IL-23 = 0.026). Conclusions: PBMNs and macrophages from healthy subjects with previous ReA show normal inflammasome activation and low TNF-α and IL-23 production. This low cytokine production may impair bacterial elimination and thereby contribute to the triggering of ReA. [ABSTRACT FROM AUTHOR]

Efficacy and pharmacokinetics of enrofloxacin and flunixin meglumine for treatment of cows with experimentally induced Escherichia coli mastitis. J. vet. Pharmacol. Therap. 25, 251–258. The efficacy of flunixin alone and together with enrofloxacin in treatment of experimental Escherichia coli mastitis was compared using six cows. The cross-over study design was used. Pharmacokinetics of flunixin and enrofloxacin were also studied in these diseased cows. The response of each cow was similar after the first and second challenge and the individual reaction seemed to explain the severity of clinical signs. The most important predictive factor for outcome of E. coli mastitis was a heavy drop in milk yield. Treatment with enrofloxacin and flunixin enhanced elimination of bacteria, but the difference from those receiving flunixin alone was not significant. Two cows, which had received no antimicrobial treatment (Group 1), were killed on day 4 postchallenge. One cow was killed after the first and the other after the second challenge. Cows receiving combination therapy produced 0.9 L more milk per day during the study period than cows which had only received flunixin (P < 0.05). Based on our findings, antimicrobial treatment might be beneficial in the treatment of high-yielding cows in early lactation. The absorption of enrofloxacin was delayed after subcutaneous administration, the mean apparent elimination half-life being about 23 h, whereas after i.v. administration elimination t 1/2 was only 1.5 h. The majority of the antimicrobial activity in milk originated from the active metabolite, ciprofloxacin, which could be measured throughout the 120-h follow-up period after the last subcutaneous administration. No differences were present in the pharmacokinetic parameters of flunixin between treatment groups: mean elimination half-life was 5.7–6.2 h, volume of distribution 0.43–0.49 L/kg and clearance 0.13–0.14 L h/kg. No flunixin or... [ABSTRACT FROM AUTHOR]

Malignant tumors derived from the epithelium lining the nasal cavity region are termed sinonasal cancers, a highly heterogeneous group of rare tumors accounting for 3 - 5 % of all head and neck cancers. Progress with next-generation molecular profiling has improved our understanding of the complexity of sinonasal cancers and resulted in the identification of an increasing number of distinct tumor entities. Despite these significant developments, the treatment of sinonasal cancers has hardly evolved since the 1980s, and an advanced sinonasal cancer presents a poor prognosis as targeted therapies are usually not available. To gain insights into potential targeted therapeutic opportunities, we performed a multiomics profiling of patient-derived functional tumor models to identify molecular characteristics associated with pharmacological responses in the different subtypes of sinonasal cancer., Methods: Patient-derived ex vivo tumor models representing four distinct sinonasal cancer subtypes: sinonasal intestinal-type adenocarcinoma, sinonasal neuroendocrine carcinoma, sinonasal undifferentiated carcinoma and SMARCB1 deficient sinonasal carcinoma were included in the analyses. Results of functional drug screens of 160 anti-cancer therapies were integrated with gene panel sequencing and histological analyses of the tumor tissues and the ex vivo cell cultures to establish associations between drug sensitivity and molecular characteristics including driver mutations., Results: The different sinonasal cancer subtypes display considerable differential drug sensitivity. Underlying the drug sensitivity profiles, each subtype was associated with unique molecular features. The therapeutic vulnerabilities correlating with specific genomic background were extended and validated with in silico analyses of cancer cell lines representing different human cancers and with reported case studies of sinonasal cancers treated with targeted therapies., Conclusion: The results demonstrate the importance of understanding the differential biology and the molecular features associated with the different subtypes of sinonasal cancers. Patient-derived ex vivo tumor models can be a powerful tool for investigating these rare cancers and prioritizing targeted therapeutic strategies for future clinical development and personalized medicine., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Juha K. Rantala is the founder of Misvik Biology Oy, and Noora Lehtinen, Janne Suhonen, Kiesha Rice and Eetu Välimäki are employees of Misvik Biology Oy., (Copyright © 2024. Published by Elsevier Inc.)

The prevalence of tail lesions evaluated at the slaughterhouse varies considerably between herds. These lesions result mainly from tail biting, a harmful behavior with multifactorial origin. This study sought to investigate if a batchwise inspection of tails at slaughterhouse could be a useful method to estimate the animal welfare situation in finishing pig herds, and if so, what type and detail of tail scoring such an inspection should utilize. We investigated the distribution of different types of tail lesions and how well their scoring at slaughterhouse was associated with the situation recorded on-farm by a veterinarian as part of routine herd health visits. We also wanted to determine if animal welfare-related herd-level parameters, recorded by herd veterinarians during herd health visits, are associated with tail scoring at the slaughterhouse. A total of 10,517 pigtails from 84 herds were scored for this study. Herd data were collected from the national health classification register for pig farms in Finland and also included annual herd production quality data collected by the slaughterhouse. The scores of the tails varied considerably between the herds. On average, 48.1% (sd = 19.3) of the tails with an average length of 30.4 cm (sd = 2.7) were fully intact, 37.3% (13.9) had healed (length = 26.4, sd = 5.1 cm), 12.4% (9.0) (length = 28.9, sd = 4.3 cm) had minor acute wounds, and 2.3% (2.1) (length = 24.2, sd = 6.0 cm) had major acute wounds. Proportions of different tail lesions at slaughterhouse were associated with or tended to be associated with the following herd-level parameters in regression models: use of wood as enrichment ( p < 0.1), one health parameter (leg problems other than arthritis, p < 0.05), and long-term animal welfare estimate (annual mortality, p < 0.05). Detailed tail evaluation at the slaughterhouse shows potential in estimating the tail lesions and long-term welfare level on the farm. By recording only one type of tail condition (such as tails with major acute lesions) at the slaughterhouse, it is not possible to estimate the total tail lesion situation in the herds before slaughter. A more detailed scoring similar to the one used in this trial is recommended., Competing Interests: EV and JV are employees of the slaughterhouse company. The University of Helsinki was responsible for data analysis and scientific publication throughout the study. The company involvement did not have any effect on the study design, data collection, interpretation of results, or the writing of the manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Heinonen, Välimäki, Laakkonen, Toppari, Vugts, Fàbrega and Valros.)

Tail biting lesions are a potential measure of on-farm animal welfare, as a large range of stressors increase the risk for tail biting outbreaks. Further, tail biting is a major challenge, as lesions due to tail biting decrease animal welfare and health, as well as production efficiency and carcass quality. The aim of this study was to suggest a tail scoring system for use at slaughterhouses processing undocked pigs, and to link tail lesion scores to meat inspection data. A further aim was to suggest a definition for an intact enough tail. To validate the suggested scoring system we assessed tails before and after scalding and compared results to pathological examinations. In total, 14,433 tails were scored, and 117 tails were collected for pathological examination. After scalding, 49.2% of all tails were scored as fully intact. Of tails with lesions 2.5% were scored as having major acute wounds (>2 cm), while 11.6% had minor acute wounds (<2 cm), and 36.7% healed lesions. Intact tails were on average 31.5 cm (SD 2.5 cm) long. Lesion scored at the slaughter-line agreed well with the pathological assessment. Tail lesions were associated with several meat inspection findings: tails with more severe lesions and of shorter length increased the risk for meat inspection findings to a higher degree. A detailed lesion scoring method helps to identify carcasses at risk for condemnations, as well as being a potential method for on-farm welfare estimation. We suggest that a system for scoring tail lesions in undocked pigs should utilize a combination of scoring of the lesion and measuring the tail length. As bite marks or bruises on an otherwise intact tail were not a concern for meat hygiene, we suggest the definition of an intact enough tail could allow the inclusion of tails with these mild changes. Meat inspection findings in carcasses with tails scored as healed, but with no fresh lesions, and with more than 75% of the average intact length remaining were rather similar to those of fully intact tails. Based on these findings we suggest that a tail of this length, and with no visible fresh lesions could also be considered intact enough., (Copyright © 2020 Valros, Välimäki, Nordgren, Vugts, Fàbrega and Heinonen.)

Extracellular ATP is an endogenous danger signal that is known to activate inflammatory responses in innate immune cells, including macrophages. Activated macrophages start to secrete proteins to induce an immune response, as well as to recruit other immune cells to the site of infection and tissue damage. In this study, we characterized the secretome (i.e., the global pattern of secreted proteins) of ATP-stimulated human macrophages. We show that ATP stimulation activates robust vesicle-mediated unconventional protein secretion, including exosome release and membrane shedding, from human macrophages. Pathway analysis of the identified secreted proteins showed that calpain-related pathways were overrepresented in the secretome of ATP-stimulated cells. In accordance with this, calpains, which are calcium-dependent nonlysosomal cysteine proteases, were activated upon ATP stimulation through a P2X purinoceptor 7 receptor-dependent pathway. Functional studies demonstrated that calpain activity is essential for the P2X purinoceptor 7 receptor-mediated activation of unconventional protein secretion. Unconventional protein secretion was followed by cell necrosis and NLRP3 inflammasome-mediated secretion of the mature form of the proinflammatory cytokine IL-1β. Furthermore, ATP-driven NLRP3 inflammasome activation was also dependent on calpain activity. Interestingly, pro-IL-1β and inflammasome components ASC and caspase-1 were released by ATP-activated macrophages through a vesicle-mediated secretion pathway. In conclusion, to our knowledge, we provide the first global characterization of proteins secreted by ATP-activated human macrophages and show a pivotal role for calpains in the activation of the inflammatory response during ATP exposure., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Viral double-stranded RNA (dsRNA) is the most important viral structure recognized by cytosolic pattern-recognition receptors of the innate immune system, and its recognition results in the activation of signaling cascades that stimulate the production of antiviral cytokines and apoptosis of infected cells. 14-3-3 proteins are ubiquitously expressed regulatory molecules that participate in a variety of cellular processes, and 14-3-3 protein-mediated signaling pathways are activated by cytoplasmic dsRNA in human keratinocytes. However, the functional role of 14-3-3 protein-mediated interactions during viral dsRNA stimulation has remained uncharacterized. Here, we used functional proteomics to identify proteins whose phosphorylation and interaction with 14-3-3 is modulated by dsRNA and to characterize the signaling pathways activated during cytosolic dsRNA-induced innate immune response in human HaCaT keratinocytes. Phosphoproteome analysis showed that several MAPK- and immune-response-related signaling pathways were activated after dsRNA stimulation. Interactome analysis identified RelA-associated inhibitor, high-mobility group proteins, and several proteins associated with host responses to viral infection as novel 14-3-3 target proteins. Functional studies showed that RelA-associated inhibitor regulated dsRNA-induced apoptosis and TNF production. Integrated network analyses of proteomic data revealed that sirtuin1 was a central molecule regulated by 14-3-3s during dsRNA stimulation. Further experiments showed that sirtuin 1 negatively regulated dsRNA-induced NFκB transcriptional activity, suppressed expression of antiviral cytokines, and protected cells from apoptosis in dsRNA-stimulated and encephalomyocarditis-virus-infected keratinocytes. In conclusion, our data highlight the importance of 14-3-3 proteins in antiviral responses and identify RelA-associated inhibitor and sirtuin 1 as novel regulators of antiviral innate immune responses., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Inflammasome is an intracellular molecular platform of the innate immunity that is a key mediator of inflammation. The inflammasome complex detects pathogens and different danger signals, and triggers cysteine protease caspase-1-dependent processing of pro-inflammatory cytokines IL-1β, and IL-18 in dendritic cells and macrophages. Previously, we have shown that water-damaged building associated trichothecene mycotoxins, including roridin A, trigger IL-1β and IL-18 secretion in human macrophages. However, the molecular basis as well as mechanism behind this trichothecene-induced cytokine secretion has remained uncharacterized. Here, we show that the trichothecene-induced IL-1β secretion is dependent on NLRP3 inflammasome in human primary macrophages. Pharmacological inhibition and small interfering RNA approach showed that the trichothecene-induced NLRP3 inflammasome activation is mediated through ATP-gated P2X7 receptor. Moreover, we show that trichothecene-triggered NLRP3 inflammasome activation is dependent on Src tyrosine kinase activity. In addition, gene silencing of c-Cbl, a negative autophagy-related regulator of c-Src, resulted in enhanced secretion of IL-1β and IL-18 in response to trichothecene mycotoxin stimulation in human macrophages. In conclusion, our results suggest that roridin A, a fungal trichothecene mycotoxin, acts as microbial danger signals that trigger activation of NLRP3 inflammasome through P2X7R and Src tyrosine kinase signaling dependent pathway in human primary macrophages., (Copyright © 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Monosodium urate (MSU) is an endogenous danger signal that is crystallized from uric acid released from injured cells. MSU is known to activate inflammatory response in macrophages but the molecular mechanisms involved have remained uncharacterized. Activated macrophages start to secrete proteins to activate immune response and to recruit other immune cells to the site of infection and/or tissue damage. Secretome characterization after activation of innate immune system is essential to unravel the details of early phases of defense responses. Here, we have analyzed the secretome of human primary macrophages stimulated with MSU using quantitative two-dimensional gel electrophoresis based proteomics as well as high-throughput qualitative GeLC-MS/MS approach combining protein separation by SDS-PAGE and protein identification by liquid chromatography-MS/MS. Both methods showed that MSU stimulation induced robust protein secretion from lipopolysaccharide-primed human macrophages. Bioinformatic analysis of the secretome data showed that MSU stimulation strongly activates unconventional, vesicle mediated protein secretion. The unconventionally secreted proteins included pro-inflammatory cytokines like IL-1β and IL-18, interferon-induced proteins, and danger signal proteins. Also active forms of lysosomal proteases cathepsins were secreted on MSU stimulation, and cathepsin activity was essential for MSU-induced unconventional protein secretion. Additionally, proteins associated to phosphorylation events including Src family tyrosine kinases were increased in the secretome of MSU-stimulated cells. Our functional studies demonstrated that Src, Pyk2, and PI3 kinases act upstream of cathepsins to activate the overall protein secretion from macrophages. In conclusion, we provide the first comprehensive characterization of protein secretion pathways activated by MSU in human macrophages, and reveal a novel role for cathepsins and Src, Pyk2, PI3 kinases in the activation of unconventional protein secretion.

Carbon nanomaterials (CNM) are targets of great interest because they have multiple applications in industry but also because of the fear of possible harmful heath effects of certain types of CNM. The high aspect ratio of carbon nanotubes (CNT), a feature they share with asbestos, is likely the key factor for reported toxicity of certain CNT. However, the mechanism to explain this toxicity is unclear. Here we investigated whether different CNM induce a pro-inflammatory response in human primary macrophages. Carbon black, short CNT, long, tangled CNT, long, needle-like CNT, and crocidolite asbestos were used to compare the effect of size and shape on the potency of the materials to induce secretion of interleukin (IL) 1-family cytokines. Our results demonstrated that long, needle-like CNT and asbestos activated secretion of IL-1β from LPS-primed macrophages but only long, needle-like CNT induced IL-1α secretion. SiRNA experiments demonstrated that the NLRP3 inflammasome was essential for long, needle-like CNT and asbestos-induced IL-1β secretion. Moreover, it was noted that CNT-induced NLRP3 inflammasome activation depended on reactive oxygen species (ROS) production, cathepsin B activity, P2X(7) receptor, and Src and Syk tyrosine kinases. These results provide new information about the mechanisms by which long, needle-like materials may cause their harmful health effects. Furthermore, the techniques used here may be of use in future risk assessments of nanomaterials., (© 2011 American Chemical Society)

Background: Chronic inflammation of the arterial wall is a key element in the pathogenesis of atherosclerosis, yet the factors that trigger and sustain the inflammation remain elusive. Inflammasomes are cytoplasmic caspase-1-activating protein complexes that promote maturation and secretion of the proinflammatory cytokines interleukin(IL)-1beta and IL-18. The most intensively studied inflammasome, NLRP3 inflammasome, is activated by diverse substances, including crystalline and particulate materials. As cholesterol crystals are abundant in atherosclerotic lesions, and IL-1beta has been linked to atherogenesis, we explored the possibility that cholesterol crystals promote inflammation by activating the inflammasome pathway., Principal Findings: Here we show that human macrophages avidly phagocytose cholesterol crystals and store the ingested cholesterol as cholesteryl esters. Importantly, cholesterol crystals induced dose-dependent secretion of mature IL-1beta from human monocytes and macrophages. The cholesterol crystal-induced secretion of IL-1beta was caspase-1-dependent, suggesting the involvement of an inflammasome-mediated pathway. Silencing of the NLRP3 receptor, the crucial component in NLRP3 inflammasome, completely abolished crystal-induced IL-1beta secretion, thus identifying NLRP3 inflammasome as the cholesterol crystal-responsive element in macrophages. The crystals were shown to induce leakage of the lysosomal protease cathepsin B into the cytoplasm and inhibition of this enzyme reduced cholesterol crystal-induced IL-1beta secretion, suggesting that NLRP3 inflammasome activation occurred via lysosomal destabilization., Conclusions: The cholesterol crystal-induced inflammasome activation in macrophages may represent an important link between cholesterol metabolism and inflammation in atherosclerotic lesions.

The skin is the primary boundary between the body and the environment. In addition to its properties as a physical barrier, skin keratinocytes actively participate in many defense mechanisms. Viral double-stranded RNA (dsRNA) is the most important viral structure involved in activation of immune response. Intracellular detection of dsRNA by cytoplasmic receptors activates well-characterized antiviral response, as well as pro-inflammatory response and apoptosis of virus-infected cells. Here, we have used quantitative subcellular proteomics to characterize the signaling pathways activated by cytosolic dsRNA recognition pathway in human keratinocytes. Cytoplasmic and mitochondrial proteomes were analyzed using 2-DE in combination with MS, immunoblotting and confocal microscopy. We have identified 239 reproducibly differentially expressed proteins upon dsRNA stimulation. The identified proteins include several key proteins involved in cytoskeletal dynamics, cell signaling, cell death, and stress response. Our analysis provides novel information how the cytokeratin network is disrupted in a caspase-dependent manner upon dsRNA stimulation as well as Encephalomyocarditis virus or Vesicular stomatitis virus infection. We show that this caspase-dependent disruption of cytokeratin is activated by cytoplasmic RNA recognition pathway. In addition, we show that viral infection activates 14-3-3 protein mediated signaling pathways in human keratinocytes which suggest an important role of 14-3-3 proteins in antiviral innate immune response.

Work has been intensified around the integration of textile and measurement technology for physiological measurements in the last years. As a result nowadays it is possible to find available commercial products for cardiovascular personal healthcare monitoring. Most of the efforts have been focused in the acquisition of EKG for cardiovascular monitoring where textile electrodes have shown satisfactory performance. Electrical Bioimpedance is another type of physiological measurement that can be used for personal healthcare monitoring where the integration and the performance of the textile electrodes has not been investigated that thoroughly. In this work, the influence of the textile electrodes on the measurements and on the estimation of the Cole (R(0), R(infinity), f(C) and alpha) and body composition (TBW, ICW, ECW and FFM) parameters has been especially addressed. Complex Spectroscopy 4-electrode wrist-to-ankle electrical bioimpedance measurements taken with conventional Ag/AgCl and textile-electrodes on customized bracelets have been compared and analyzed in the frequency range 3 to 500 kHz. The obtained results suggest that the use of textile electrodes do not influence remarkably on the complex spectral measurements neither in the estimation of Cole nor body composition parameter. In any case any possible effect introduced by the use of textile is smaller than the effect of preparing the skin by the using abrasive conductive paste.