Keratinocyte Chemoattractant Research Articles

Effects of anesthesia with sevoflurane on outcome parameters in murine experimental studies.

Multiple murine studies modelling the immuno-pathophysiological consequences of trauma, shock, burn or sepsis were performed during the last decades. Almost every animal model requires anesthesia for practical and ethical reasons. Furthermore, often, corresponding control groups involve untreated animals without or with a limited exposure to anesthetics. However, the influences of anesthetic drugs on immuno-pathophysiological reactions remain insufficiently investigated. Therefore, we aimed to closer characterize the anesthetic impact exemplified by sevoflurane on the organ performance in mice and thereby investigate the influence of anesthesia itself on major outcome parameters in animal studies. C57/BL6 mice were subjected either to 270min of sevoflurane narcosis or directly euthanized. Plasma, BAL-fluids, lungs, kidneys, liver and intestine were collected and examined for immunological, functional and morphological changes. Systemic levels of the cytokine keratinocyte chemoattractant (KC) were raised in the narcosis group, while concentrations of high mobility group box protein 1 (HMGB-1) as a major inflammatory marker were reduced. In the lungs, levels of HMGB-1 and interleukin 6 (IL-6) were reduced. In contrast, systemic concentrations of intestinal fatty acid binding-protein (i-FABP) as an intestinal damage marker were elevated. Furthermore, liver-type fatty acid binding-protein (L-FABP) levels were lower in the narcosis animals, and inflammatory markers were reduced in liver tissues. Anesthesia also ameliorated the inflammatory reaction in renal tissues, while plasma levels of urea and creatinine were elevated, reflecting either dehydration and/or impaired renal function. As anesthesia with sevoflurane exhibited distinct effects in different organs, it is difficult to predict its specific impact on targets of interest in in vivo studies. Therefore, further studies are required to clarify the effects of different anesthetic drugs. Overall, the inclusion of a control group subjected to the same anesthesia protocol as the experimental groups of interest seems helpful to precisely define the inherent impact of the anesthetic when investigating immuno-pathophysiologic conditions in vivo.

Neutrophil Depletion Changes the N-Glycosylation Pattern of IgG in Experimental Murine Sepsis.

Sepsis is a life-threatening condition with a rising disease burden worldwide. It is a multifactorial disease and is defined as a dysregulated host response to infection. Neutrophils have been shown to be involved in the pathogenesis of sepsis by exacerbating inflammation. However, the exact effector mechanism of action still remains a mystery. Changes in the glycosylation pattern of the immunoglobulin G (IgG) Fc region are described for several diseases including meningococcal sepsis. In this study, we investigated the possible contribution of neutrophils and neutrophil implication, potentially related to degranulation or neutrophil extracellular trap (NET) formation in changing the IgG Fc N-glycosylation pattern in a murine sepsis model. We have measured the serum level of cytokines/chemokines and immunoglobulins, the serum activity of neutrophil elastase (NE), and analyzed the IgG Fc glycosylation pattern by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) and Lectin enzyme-linked immunosorbent assay (ELISA). We observed an increased activity of NE- and neutrophil-associated cytokines such as keratinocyte chemoattractant (KC) with the development of sepsis. Regarding the IgG Fc N-glycosylation, we observed an increase in fucosylation and α1,3-galactosylation and a decrease for sialyation. Interestingly, these changes were not uniform for all IgG subclasses. After depletion of neutrophils, we saw a change in the exposure of fucose and α2,6-linked sialic acid during the time course of our experimental sepsis model. In conclusion, neutrophils can influence changes in the IgG glycosylation pattern in experimental sepsis.

Cigarette smoke-induced dysbiosis: comparative analysis of lung and intestinal microbiomes in COPD mice and patients

BackgroundThe impact of cigarette smoke (CS) on lung diseases and the role of microbiome dysbiosis in chronic obstructive pulmonary disease (COPD) have been previously reported; however, the relationships remain unclear.MethodsOur research examined the effects of 20-week cigarette smoke (CS) exposure on the lung and intestinal microbiomes in C57BL/6JNarl mice, alongside a comparison with COPD patients’ intestinal microbiome data from a public dataset.ResultsThe study found that CS exposure significantly decreased forced vital capacity (FVC), thickened airway walls, and induced emphysema. Increased lung damage was observed along with higher lung keratinocyte chemoattractant (KC) levels by CS exposure. Lung microbiome analysis revealed a rise in Actinobacteriota, while intestinal microbiome showed significant diversity changes, indicating dysbiosis. Principal coordinate analysis highlighted distinct intestinal microbiome compositions between control and CS-exposed groups. In the intestinal microbiome, notable decreases in Patescibacteria, Campilobacterota, Defferibacterota, Actinobacteriota, and Desulfobacterota were observed. We also identified correlations between lung function and dysbiosis in both lung and intestinal microbiomes. Lung interleukins, interferon-ɣ, KC, and 8-isoprostane levels were linked to lung microbiome dysbiosis. Notably, dysbiosis patterns in CS-exposed mice were similar to those in COPD patients, particularly of Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage 4 patients. This suggests a systemic impact of CS exposure.ConclusionIn summary, CS exposure induces significant dysbiosis in lung and intestinal microbiomes, correlating with lung function decline and injury. These results align with changes in COPD patients, underscoring the important role of microbiome in smoke-related lung diseases.

Neutrophil extracellular trap formation and its implications in nonsteroidal anti-inflammatory drug-induced small intestinal injury.

Nonsteroidal anti-inflammatory drugs (NSAIDs) damage the small intestine via neutrophil infiltration driven by the mucosal invasion of enterobacteria. The antimicrobial function of neutrophils is partially dependent on neutrophil extracellular traps (NETs). Excessive NET formation has been associated with several inflammatory diseases. Here, we aimed to investigate the role of NETs in NSAID-induced small intestinal damage using human samples and an experimental mouse model. Human small intestine specimens were obtained from NSAID users during double-balloon enteroscopy. Wild-type, protein arginine deiminase 4 (PAD4) knockout, and antibiotic-treated mice were administered indomethacin to induce small intestinal injury. The expression of NET-associated proteins, including PAD4, citrullinated histone H3 (CitH3), cell-free DNA, and myeloperoxidase (MPO), was evaluated. The double-positive stained area with CitH3 and MPO, which is specific for neutrophil-derived extracellular traps, was significantly high in the injured small intestinal mucosa of NSAID users. In a mouse model, small intestinal damage developed at 6h after indomethacin administration, accompanied by increased mRNA levels of interleukin-1β and keratinocyte chemoattractant and elevated NET-associated protein levels of PAD4, CitH3, and MPO in small intestine and serum levels of cell-free DNA. Both genetic deletion and pharmacological inhibition of PAD4 attenuated this damage by reducing the mRNA expression of inflammatory cytokines and NET-associated proteins. Furthermore, mice pretreated with antibiotics showed resistance to indomethacin-induced small intestinal damage, with less NET formation. These results suggest that NETs aggravate NSAID-induced small intestinal injury. Therefore, NET inhibition could be a potential treatment for NSAID-induced small intestinal injury.

A comparative study of the digestion behavior and functionality of protein from chia (Salvia hispanica L.) ingredients and protein fractions

Protein derived from chia (Salvia hispanica L.), characterized by a balanced amino acid composition, represents a potentially healthier and environmentally friendly alternative poised for innovation within the plant-based food sector. It was hypothesized that the growing location of chia seeds and processing techniques used might influence protein digestion patterns, which in turn could affect the biological functions of the digestion products. To examine this hypothesis, we assessed the gastrointestinal fate of degummed-defatted flour (DDF), protein concentrate (PC), and isolated albumin (Alb) and globulin (Glo) fractions. Furthermore, we compared the antioxidant and anti-inflammatory activities of the resulting digesta by means of in vitro and cellular assays. Post-gastrointestinal digestion, the PC exhibited elevated levels of soluble protein (7.6 and 6.3 % for Mexican and British PC, respectively) and peptides (24.8 and 27.9 %, respectively) of larger molecular sizes compared to DDF, Alb, and Glo. This can be attributed to differences in the extraction/fractionation processes. Leucine was found to be the most prevalent amino acids in all chia digesta. Such variations in the digestive outcomes of chia protein components significantly influenced the bioactivity of the intestinal digestates. During gastrointestinal transit, British Glo exhibited the best reactive oxygen species (ROS) inhibition activity in oxidative-stressed RAW264.7 macrophages, while Mexican digesta outperformed British samples in terms of ROS inhibition within the oxidative-stressed Caco-2 cells. Additionally, both Mexican and British Alb showed effectively anti-inflammatory potential, with keratinocyte chemoattractant (KC) inhibition rate of 82 and 91 %, respectively. Additionally, Mexican PC and Alb generally demonstrated an enhanced capacity to mitigate oxidative stress and inflammatory conditions in vitro. These findings highlight the substantial potential of chia seeds as functional food ingredients, resonating with the shifting preferences of health-conscious consumers.

Chronic exposure to nanocellulose altered depression-related behaviors in mice on a western diet: The role of immune modulation and the gut microbiome

AimsTo determine if cellulose nanofibrils (CNF) have potential applications as food additives. Materials and methodsMale C57BL/6 mice on a Western diet were exposed to CNF for one month at a dose of 30 mg/kg by gavage. Male NOD mice, a model for type 1 diabetes (T1D), were used in a six-month study. Key findingsSequencing analysis of 16S rRNA genes suggested significant changes in gut microbiome of male C57BL/6 mice exposed to CNF. Analysis of functional metagenomics indicated that many of the functional contents that might be altered following CNF ingestion were associated with lipid and carbohydrate processing. Further studies in NOD mice suggested that there were some decreases in the blood glucose levels during the insulin tolerance test and glucose tolerance test following CNF treatment. However, these small decreases were not considered biologically meaningful as there were no significant changes in either the area under the curve or the first-order rate constant for glucose disappearance. Moreover, serum concentrations of cytokines/chemokines including IL-3, IL-12(p70) and the keratinocyte chemoattractant were increased following chronic exposure to CNF. In addition, behavioral studies suggested that the percentage of immobility time during the tail-suspension test was significantly increased following six months of exposure to CNF in NOD mice, signifying an increase in depression-related behavior. SignificanceCollectively, long-term CNF consumption was associated with changes in the ecology of the gut microbiome, immune homeostasis, and possibly energy metabolism and mental health in male NOD mice on a Western diet.

Exosomes derived from mesenchymal stem cells primed with disease-condition-serum improved therapeutic efficacy in a mouse rheumatoid arthritis model via enhanced TGF-β1 production

BackgroundsRheumatoid arthritis (RA) is a chronic and systemic autoimmune disease characterized by synovial inflammation-mediated progressive destruction of the cartilage and bone, resulting in reduced quality of life. We primed human telomerase reverse transcriptase-overexpressing immortalized human adipose tissue-derived mesenchymal stem cells (iMSCs) with serum derived from a non-human primate RA model and studied the immunomodulatory ability of exosomes obtained from primed iMSCs.MethodsAfter immunophenotyping, nanoparticle tracking analysis, and in vitro functional tests, Dulbecco’s phosphate-buffered saline (dPBS, Group C), exosomes derived from the supernatant of iMSCs (Exo-FBS, Group E), exosomes derived from the supernatant of iMSCs primed with RA serum (Exo-RA, Group F), and methotrexate (Group M) were administered in collagen-induced arthritis (CIA) model mice. dPBS was administered to the normal (N) group for comparison (n = 10/group).ResultsExo-RA had a significantly higher number of exosomes compared to Exo-FBS when measured with nanoparticle tracking analysis or exosome marker CD81, and Transforming growth factor-β1 amounts were significantly higher in Exo-RA than in Exo-FBS. When Exo-FBS or Exo-RA was administered to the collagen-induced arthritis model, serum interleukin (IL)-4 and the proportion of Th2 (CD4+CD25+GATA3+) and M2 (CD11c − CD206+ of CD45+CD64+) cells were significantly increased compared to the control group. Furthermore, Exo-RA could alleviate cartilage damage by significantly lowering the concentrations of proinflammatory cytokines such as tumor necrosis factor-α, keratinocyte chemoattractant, and IL-12p70.ConclusionExosomes derived from disease-condition-serum-primed iMSCs ameliorated cartilage damage in a RA model by enhancing TGF-β1 production, inducing Th2 and M2 polarization and lowering proinflammatory cytokines, TNF-α, KC, and IL-12p70 in the host. Patient-derived serum can be used as an iMSC priming strategy in iMSC-derived exosome treatment of RA.

Isothiocyanate-rich moringa seed extract reduces skin inflammation in mouse ear edema model

BackgroundMoringa (Moringa oleifera Lam.) seed extract (MSE) and its primary bioactive compound, moringa isothiocyanate-1(MIC-1), mitigate inflammation, oxidative stress, diabetes, and cancer in the in vivo rodent models following oral application. PurposeTo investigate the topical anti-inflammatory activity of MSE and purified MIC-1 in a TPA-induced mouse ear edema model. Study DesignThe present study elucidates the topical anti-inflammatory effects and mechanisms of action of MSE, containing 38% of MIC-1 and purified MIC-1 using a mouse ear edema model utilizing 12-O-tetradecanoylphorbol-13-acetate (TPA), as the pro-inflammatory agent. MethodsA time-dependent and dose-dependent response was determined by pretreating CD-1 mice with various doses of MSE and MIC-1, positive control, dexamethasone, or vehicle control, followed by TPA, and the subsequent difference in ear thickness was measured using digital Vernier calipers. The effective doses of MSE and MIC-1were then selected to evaluate the change in weight of the ears using 6 mm biopsy punches and the results were confirmed by microscopy. Inflammatory markers were quantified with Luminex multiplex immunoassay. ResultsMSE and MIC-1 were effective in a dose-dependent manner in a TPA-induced ear edema model, causing a reduction in ear thickness and a 48% and 49% decrease in ear punch weight, respectively. MSE and MIC-1 also caused a reduction in the levels of cytokine and chemokines, interleukin 6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and keratinocyte chemoattractant (KC) in the ear tissue. MSE and MIC-1 reduced IL-6 expression by 84% and 78%, MCP1 by 74% and 73%, and KC by 56% and 43%, respectively. Additionally, the anti-inflammatory effect of MSE and MIC-1 was confirmed by hematoxylin and eosin (H&E) staining, used to assess the thickness of the ear swelling. MSE significantly reduced the thickness of the ears by 20% compared to TPA. ConclusionThese results reveal the topical anti-inflammatory properties of MSE, and MIC-1 likely transmitted via the nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor-kappa B (NF-κB) pathways as mentioned in previous studies. This work also suggests therapeutic uses of MSE and/or MIC-1 for skin inflammation.

Cryptotanshinone alleviates lipopolysaccharide and cigarette smoke-induced chronic obstructive pulmonary disease in mice via the Keap1/Nrf2 axis

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity worldwide. Cigarette smoking, which leads to abnormalities in the airways or alveoli and persistent obstruction of the airway's flow, is a significant risk factor of COPD. Cryptotanshinone (CTS) is the active ingredient in Salvia miltiorrhiza (Danshen) and has many pharmacological properties including anti-inflammatory, antitumor, and antioxidant properties, but its impact on COPD is uncertain. In the present study, the potential effect of CTS on COPD was investigated in a modified COPD mice model induced with cigarette smoke (CS) and lipopolysaccharide (LPS) exposure. CTS significantly reversed the decline in lung function, emphysema, inflammatory cell infiltration, small airway remodeling, pulmonary pathological damage, and airway epithelial cell proliferation in CS- and LPS-exposed mice. Additionally, CTS decreased inflammatory cytokines such as tumor necrosis factor α (TNF α), interleukins IL-6 and IL-1β, and keratinocyte chemoattractant (KC), increased the activities of superoxide dismutase (SOD), Catalase (CAT) and L-Glutathione (GSH), and repressed the expression of protein hydrolases matrix metalloprotein (MMP)− 9 and − 12 in pulmonary tissue and bronchoalveolar lavage fluid (BALF). The protective effects of CTS were also observed in human bronchial epithelial cell line BEAS-2B simulated with cigarette smoke condensate (CSC) and LPS. Mechanistically, CTS can repress the protein level of Keap1, resulting to activation of erythroid 2-related factor (Nrf2), finally alleviating COPD. In summary, the present findings demonstrated that CTS dramatically ameliorates COPD induced by CS and LPS via activating Keap1/Nrf2 pathway.

Angiotensin II type 2 receptor agonist attenuates LPS-induced acute lung injury through modulating THP-1-derived macrophage reprogramming.

Acute respiratory distress syndrome (ARDS) is a devastating respiratory disorder, characterized by overwhelming inflammation in the alveoli without effective pharmacological treatment. We aimed to investigate the effect and mechanism of angiotensin II type 2 receptor (AT2R) agonist, Compound 21 (C21), on the lipopolysaccharide (LPS)-induced acute lung injury (ALI) model. The protective effect of C21 was evaluated via enzyme-linked immunosorbent assay (ELISA), Western blot (WB), real-time PCR, and fluorescence microscopy in LPS-challenged THP1-derived macrophages. Besides, the in vivo efficacy of C21 was assessed using cell counting, ELISA, protein quantification, hematoxylin-eosin (H&E) staining, and WB in an LPS-induced ALI mouse model. The results showed thatC21 significantly inhibited the secretion of pro-inflammatory cytokines (CCL-2, IL-6), overproduction of intracellular ROS, and activation of inflammatory pathways (NF-κB/NLRP3, p38/MAPK) in THP-1 cell-derived macrophages stimulated by LPS. In in vivo study, intraperitoneal injection of C21 could reduce airway leukocytes accumulation and chemokine/cytokine (keratinocyte chemoattractant (KC), IL-6) generation, as well as alleviate diffuse alveolar damage induced by LPS. Conclusively,the AT2R agonist C21 significantly inhibited LPS-stimulated excess inflammatory responses and oxidative stress in macrophages. Meanwhile, C21 could effectively alleviate acute inflammation and tissue damage in the lungs of ALI mice challenged by LPS. The results of this study bring new hope for the early treatment of ALI/ARDS.

Generation and characterization of QLS22001, a humanized monoclonal antibody that neutralizes IL-17A and IL-17F with an extended half-life.

Therapeutic intervention to block IL-17A signaling has proven to be an effective treatment for numerous autoimmune diseases, including psoriasis, psoriatic arthritis, and axial spondylarthritis. Among the IL-17 family members, IL-17F, which shares 55% sequence homology with IL-17A, has been reported to functionally overlap with IL-17A in many inflammatory diseases. In this study, we describe the generation and characterization of QLS22001, a humanized monoclonal IgG1 antibody with an extended half-life and high affinity for both IL-17A and IL-17F. QLS22001 effectively blocks IL-17A and IL-17F mediated signaling pathways both in vitro and in vivo. Briefly, the YTE (M225Y/S254T/T256E) modification was introduced into the Fc fragment of QLS22001 WT Fc to prolong its half-life, and the resulting construct was named QLS22001. Functionally, it significantly inhibits IL-17A- and IL-17F-stimulated signaling in cell-based IL-6 release and reporter assays. The dual neutralization of the endogenous IL-17A and IL-17F produced by Th17 cells, as opposed to the selective blockade of IL-17A alone, results in a greater suppression of inflammatory cytokine secretion, according to in vitro blockade assays. Furthermore, in an in vivo mouse pharmacodynamic study, QLS22001 blocked human IL-17A-induced mouse keratinocyte chemoattractant (KC) release. In cynomolgus monkey pharmacokinetics evaluation, QLS22001 showed linear pharmacokinetic characteristics with a mean half-life of 31.2days, while its parent antibody, QLS22001 WT Fc, had a mean half-life of 17.2days. In addition, QLS22001 does not induce cytokine release in a human whole-blood assay. Collectively, these data provide a comprehensive preclinical characterization of QLS22001 and support its clinical development.

The Effects of Pregnancy on the Pulmonary Immune Response in a Mouse Model of LPS-Induced Acute Lung Injury.

This study evaluated the effect of pregnancy on the pulmonary innate immune response in a mouse model of acute lung injury (ALI) using nebulized lipopolysaccharide (LPS). Pregnant (day 14) C57BL/6NCRL mice and nonpregnant controls received nebulized LPS for 15 minutes. Twenty-four hours later, mice were euthanized for tissue harvest. Analysis included blood and bronchoalveolar lavage fluid (BALF) differential cell counts, whole-lung inflammatory cytokine transcription levels by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), and whole-lung vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and BALF albumin by western blot. Mature bone marrow neutrophils from uninjured pregnant and nonpregnant mice were examined for chemotactic response using a Boyden chamber and for cytokine response to LPS by RT-qPCR. In LPS-induced ALI, pregnant mice had higher BALF total cell (p < 0.001) and neutrophil counts (p < 0.001) as well as higher peripheral blood neutrophils (p < 0.01) than nonpregnant mice, but a similar increase (as compared with unexposed mice) in airspace albumin levels. Whole-lung expression of interleukin 6, tumor necrosis factor-α (TNF-α), and keratinocyte chemoattractant (CXCL1) was also similar. In vitro, marrow-derived neutrophils from pregnant and nonpregnant mice had similar chemotaxis to CXCL1 and N-formylmethionine-leucyl-phenylalanine, but neutrophils from pregnant mice expressed lower levels of TNF (p < 0.001) and CXCL1 (p < 0.01) after LPS stimulation. In uninjured mice, VCAM-1 was higher in lungs from pregnant versus nonpregnant mice (p < 0.05). In this model, pregnancy is associated with an augmented lung neutrophil response to ALI without increased capillary leak or whole-lung cytokine levels relative to the nonpregnant state. This may stem from increased peripheral blood neutrophil response and intrinsically increased expression of pulmonary vascular endothelial adhesion molecules. Differences in lung innate cell homeostasis may affect the response to inflammatory stimuli and explain severe lung disease in respiratory infection during pregnancy. · Inhalation of LPS in midgestation versus virgin mice is associated with increased neutrophilia.. · This occurs without a comparative increase in cytokine expression.. · This may be explained by pregnancy-enhanced pre-exposure expression of VCAM-1 and ICAM-1..

Early effects of bone marrow-derived mononuclear cells on lung and kidney in experimental sepsis

BackgroundIn experimental sepsis, functional and morphological effects of bone marrow-derived mononuclear cell (BMDMC) administration in lung tissue have been evaluated 1 and 7 days after therapy. However, to date no study has evaluated the early effects of BMDMCs in both lung and kidney in experimental polymicrobial sepsis. Material and methodsTwenty-five female C57BL/6 mice were randomly divided into the following groups: 1) cecal ligation and puncture (CLP)-induced sepsis; and 2) Sham (surgical procedure without CLP). After 1 h, CLP animals received saline (NaCl 0.9%) (CLP-Saline) or 106 BMDMCs (CLP-Cell) via the jugular vein. At 6, 12, and 24 h after saline or BMDMC administration, lungs and kidneys were removed for histology and molecular biology analysis. ResultsIn lungs, CLP-Saline, compared to Sham, was associated with increased lung injury score (LIS) and keratinocyte chemoattractant (KC) mRNA expression at 6, 12, and 24 h. BMDMCs were associated with reduced LIS and KC mRNA expression regardless of the time point of analysis. Interleukin (IL)− 10 mRNA content was higher in CLP-Cell than CLP-Saline at 6 and 24 h. In kidney tissue, CLP-Saline, compared to Sham, was associated with tubular cell injury and increased neutrophil gelatinase-associated lipocalin (NGAL) levels, which were reduced after BMDMC therapy at all time points. Surface high-mobility-group-box (HMGB)− 1 levels were higher in CLP-Saline than Sham at 6, 12, and 24 h, whereas nuclear HMGB-1 levels were increased only at 24 h. BMDMCs were associated with decreased surface HMGB-1 and increased nuclear HMGB-1 levels. Kidney injury molecule (KIM)− 1 and IL-18 gene expressions were reduced in CLP-Cell compared to CLP-Saline at 12 and 24 h. ConclusionIn the present experimental polymicrobial sepsis, early intravenous therapy with BMDMCs was able to reduce lung and kidney damage in a time-dependent manner. BMDMCs thus represent a potential therapy in well-known scenarios of sepsis induction. PurposeTo evaluate early bone marrow-derived mononuclear cell (BMDMC) therapy on lung and kidney in experimental polymicrobial sepsis. MethodsTwenty-five female C57BL/6 mice were randomly divided into the following groups: cecal ligation and puncture (CLP)-induced sepsis; and sham (surgical procedure without CLP). After 1 h, CLP animals received saline (CLP-saline) or 106 BMDMCs (CLP-cell) via the jugular vein. Lungs and kidneys were evaluated for histology and molecular biology after 6, 12, and 24 h. ResultsIn lungs, BMDMCs reduced the lung injury score and keratinocyte chemoattractant mRNA expression regardless of the time point of analysis; interleukin-10 mRNA content was higher in CLP-cell than CLP-saline at 6 and 24 h. In kidneys, BMDMCs reduced neutrophil gelatinase-associated lipocalin levels at all time points. BMDMCs decreased surface high mobility group box (HMGB)− 1 but increased nuclear HMGB-1 levels. ConclusionEarly BMDMC therapy reduced lung and kidney damage in a time-dependent manner.

Mucosal Adjuvants Delivered by a Mucoadhesive Patch for Sublingual Administration of Subunit Vaccines.

Among mucosal administration routes for vaccines, the sublingual route has been proven capable of inducing a potent systemic and mucosal immune response. However, the absence of a simple and compliant delivery system and the lack of robust mucosal adjuvants impede the development of sublingual vaccines. Here, we describe a mucoadhesive patch made of a layer-by-layer assembly of polysaccharides, chitosan, and hyaluronic acid. The mucoadhesive patch was covered by adjuvanted nanoparticles carrying viral proteins. We showed that the nanoparticles effectively cross the outer layers of the sublingual mucosa to reach the epithelium. Furthermore, the encapsulated adjuvants, 3M-052 and mifamurtide, targeting toll-like receptor (TLR) 7/8 and nucleotide-binding oligomerization domain-2 (NOD2), respectively, remain fully active after encapsulation into nanoparticles and exhibit a cytokine/chemokine signature similar to the mucosal gold-standard adjuvant, the cholera toxin. However, the particulate adjuvants induced more moderate levels of proinflammatory interleukin (IL)-6 and keratinocyte chemoattractant (KC), suggesting a controlled activation of the innate immune response.

Aerosol physicochemical determinants of carbon black and ozone inhalation co-exposure induced pulmonary toxicity.

Air pollution accounts for more than 7 million premature deaths worldwide. Using ultrafine carbon black (CB) and ozone (O3) as a model for an environmental co-exposure scenario, the dose response relationships in acute pulmonary injury and inflammation were determined by generating, characterizing, and comparing stable concentrations of CB aerosols (2.5, 5.0, 10.0 mg/m3), O3 (0.5, 1.0, 2.0 ppm) with mixture CB + O3 (2.5 + 0.5, 5.0 + 1.0, 10.0 + 2.0). C57BL6 male mice were exposed for 3 h by whole body inhalation and acute toxicity determined after 24 h. CB itself did not cause any alteration, however, a dose response in pulmonary injury/inflammation was observed with O3 and CB + O3. This increase in response with mixtures was not dependent on the uptake but was due to enhanced reactivity of the particles. Benchmark dose modeling showed several-fold increase in potency with CB + O3 compared with CB or O3 alone. Principal component analysis provided insight into response relationships between various doses and treatments. There was a significant correlation in lung responses with charge-based size distribution, total/alveolar deposition, oxidant generation, and antioxidant depletion potential. Lung tissue gene/protein response demonstrated distinct patterns that are better predicted by either particle dose/aerosol responses (interleukin-1β, keratinocyte chemoattractant, transforming growth factor beta) or particle reactivity (thymic stromal lymphopoietin, interleukin-13, interleukin-6). Hierarchical clustering showed a distinct signature with high dose and a similarity in mRNA expression pattern of low and medium doses of CB + O3. In conclusion, we demonstrate that the biological outcomes from CB + O3 co-exposure are significantly greater than individual exposures over a range of aerosol concentrations and aerosol characteristics can predict biological outcome.

Reduction of Emphysema Severity by Human Umbilical Cord-Derived Mesenchymal Stem Cells in Mice.

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality in chronic lung disease patients throughout the world. Mesenchymal stem cells (MSCs) have been shown to regulate immunomodulatory, anti-inflammatory, and regenerative responses. However, the effects of human-umbilical-cord-derived mesenchymal stem cells (hUC-MSCs) on the lung pathophysiology of COPD remain unclear. We aimed to investigate the role of hUC-MSCs in emphysema severity and Yes-associated protein (Yap) phosphorylation (p-Yap) in a porcine-pancreatic-elastase (PPE)-induced emphysema model. We observed that the emphysema percentages (normalized to the total lung volume) measured by chest computed tomography (CT) and exercise oxygen desaturation were significantly reduced by hUC-MSCs at 107 cells/kg body weight (BW) via intravenous administration in emphysematous mice (p < 0.05). Consistently, the emphysema index, as assessed by the mean linear intercept (MLI), significantly decreased with hUC-MSC administration at 3 × 106 and 107 cells/kg BW (p < 0.05). Changes in the lymphocytes, monocytes, and splenic cluster of differentiation 4-positive (CD4+) lymphocytes by PPE were significantly reversed by hUC-MSC administration in emphysematous mice (p < 0.05). An increasing neutrophil/lymphocyte ratio was reduced by hUC-MSCs at 3 × 106 and 107 cells/kg BW (p < 0.05). The higher levels of tumor necrosis factor (TNF)-α, keratinocyte chemoattractant (KC), and lactate dehydrogenase (LDH) in bronchoalveolar lavage fluid (BALF) were significantly decreased by hUC-MSC administration (p < 0.05). A decreasing p-Yap/Yap ratio in type II alveolar epithelial cells (AECII) of mice with PPE-induced emphysema was significantly increased by hUC-MSCs (p < 0.05). In conclusion, the administration of hUC-MSCs improved multiple pathophysiological features of mice with PPE-induced emphysema. The effectiveness of the treatment of pulmonary emphysema with hUC-MSCs provides an essential and significant foundation for future clinical studies of MSCs in COPD patients.

Allergy, inflammation, hepatopathy and coagulation biomarkers in dogs with suspected anaphylaxis due to insect envenomation.

ObjectivesTo compare concentrations of biomarkers of; allergy [mast cell tryptase (MCT) and histamine], inflammation [interleukin (IL)-6,-10, and−18, CXCL8, CCL2, keratinocyte chemoattractant (KC), C-reactive protein (CRP)], endothelial glycocalyx shedding (hyaluronan), coagulation [prothrombin time, activated partial thromboplastin time, fibrinogen concentration, and von Willebrand Factor antigen, protein C (PC) and antithrombin (AT) activity], and hepatopathy [alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and total bilirubin] between dogs with anaphylaxis after suspected insect exposure, dogs with critical illness, and healthy dogs.DesignThis was a single center prospective clinical observational comparative biomarker study that included 25 dogs with anaphylaxis (evidence of insect exposure, acute dermatological signs, and other organ involvement), 30 dogs with other critical illness, and 20 healthy dogs. Differences across groups in biomarker concentrations were tested using one-way ANOVA or Kruskal-Wallis test, with significant P values (<0.05) reported for pairwise differences detected by post-hoc tests. Logistic regression models were used to calculate the area under the receiver operator characteristic curve (AUROC) for discrimination between anaphylaxis and non-anaphylactic illness.ResultsHistamine concentration was significantly higher in the anaphylaxis group than the healthy (P < 0.001) and critically ill groups (P < 0.001), whereas no differences in MCT were detected amongst groups. Biomarker concentrations that were increased relative to healthy dogs in both the anaphylaxis and critically ill groups included IL-10 (P < 0.001 and P = 0.007, respectively), CCL2 (P = 0.007 and P < 0.001, respectively) and AST (both P < 0.001), whereas only the critically ill group had significantly increased CRP (P < 0.001), IL-6 (P < 0.001), KC (P < 0.001), ALP (P < 0.001), and fibrinogen (P = 0.016) concentrations, compared to the healthy group. Only dogs with anaphylaxis had significantly higher hyaluronan (P = 0.021) and ALT (P = 0.021) concentrations, and lower PC (P = 0.030) and AT (P = 0.032) activities, compared to healthy dogs. Both CRP and histamine concentration showed good discrimination between anaphylaxis and other critical illness, with an AUROC of 0.96 (95% CI 0.91–1) and 0.81 (95% CI 0.69–0.93), respectively.ConclusionsThis preliminary study in dogs with anaphylaxis after suspected insect exposure, found evidence of an early innate immune response, glycocalyx shedding and anticoagulant consumption. Both CRP and histamine showed potential clinical utility for differentiation between anaphylaxis and other critical illness.

Enhancing SIRT1 Gene Expression Using Small Activating RNAs: A Novel Approach for Reversing Metabolic Syndrome.

Metabolic syndrome (MetS) is a pathological condition characterized by abdominal obesity, insulin resistance, hypertension, and hyperlipidemia. Sirtuin 1 (SIRT1), a highly conserved histone deacetylase, is characterized as a key metabolic regulator and protector against aging-associated pathologies, including MetS. In this study, we investigate the therapeutic potential of activating SIRT1 using small activating RNAs (saRNA), thereby reducing inflammatory-like responses and re-establishing normal lipid metabolism. SIRT1 saRNA significantly increased SIRT1 messenger RNA (mRNA) and protein levels in both lipopolysaccharide-stimulated and nonstimulated macrophages. SIRT1 saRNA significantly decreased inflammatory-like responses, by reducing mRNA levels of key inflammatory cytokines, such as Tumor Necrosis Factor alpha, Interleukin 1 beta (IL-1β), Interleukin 6 (IL-6), and chemokines Monocyte Chemoattractant Protein-1 and keratinocyte chemoattractant. SIRT1 overexpression also significantly reduced phosphorylation of nuclear factor-κB and c-Jun N-terminal kinase, both key signaling molecules for the inflammatory pathway. To investigate the therapeutic effect of SIRT1 upregulation, we treated a high-fat diet model with SIRT1 saRNA conjugated to a transferrin receptor aptamer for delivery to the liver and cellular internalization. Animals in the SIRT1 saRNA treatment arm demonstrated significantly decreased weight gain with a significant reduction in white adipose tissue, triglycerides, fasting glucose levels, and intracellular lipid accumulation. These suggest treatment-induced changes to lipid and glucose metabolism in the animals. The results of this study demonstrate that targeted activation of SIRT1 by saRNAs is a potential strategy to reverse MetS.

NADPH and xanthine oxidases control induction of inflammatory mediator expression by organic dust in the lung.

Exposure to organic dust in animal and agricultural farms and the ensuing lung inflammation are linked to the development of respiratory diseases. We found previously that elevated production of reactive oxygen species (ROS) by aqueous poultry organic dust extract (hereafter referred to as dust extract) mediates induction of proinflammatory mediators in airway epithelial cells. In the present study, we investigated whether ROS generated by NADPH oxidases (NOX) and xanthine oxidase (XO) controls induction of inflammatory mediators by dust extract and the underlying mechanisms in bronchial epithelial cells. Using chemical inhibitors and siRNA targeted knockdown, we found that NOX1, NOX2, NOX4, and XO-derived ROS regulates induction of proinflammatory mediator levels. Like airway epithelial cells in vitro, NOX inhibitor VAS2870 reduced keratinocyte chemoattractant (KC), IL-6, and TNF-α production and 4-hydroxynonenal (4-HNE) staining induced by dust extract in mouse lungs. VAS2870 inhibition of proinflammatory mediators was associated with reduced NFκB and Stat3 activation indicating that NOX generated ROS activates NFκB and Stat3 to induce proinflammatory gene expression. Dust extract increased the membrane association of p47phox in airway epithelial cells indicating NOX2 activation but had no effect on NOX2 protein levels. In summary, our studies have shown that NOX and XO generated ROS control organic dust induction of proinflammatory mediators in airway epithelial cells via NFκB and Stat3 activation.

Kinetics of the inflammatory response during experimental Babesia rossi infection of beagle dogs

Babesia rossi causes severe morbidity and mortality in dogs in sub-Saharan Africa, and the complications associated with this disease are likely caused by an unfocused, excessive inflammatory response. During this experimental B. rossi study we investigated inflammatory marker and cytokine kinetics during infection and after treatment. We aimed to determine whether infectious dose and treatment would influence the progression of the inflammatory response and clinical disease. Six healthy male beagle dogs formed the study population, one was used to raise the infectious inoculum, three were administered a high B. rossi infectious dose (HD group) and two a low infectious dose (LD group). Clinical examination, complete blood count (CBC) and C-reactive protein (CRP) were determined daily. Cytokines were quantified on stored plasma collected during the study, using a canine specific cytokine magnetic bead panel (Milliplex©). The experiment was terminated and treatment administered when predetermined experimental or humane endpoints were reached. Parasitemia occurred on day 1 and 3 in the HD and LD groups respectively. The rate of increase in parasitemia in the HD group was significantly faster than that seen in the LD group. Significant differences were found in heart rate, blood pressure, interferon gamma (INFγ), keratinocyte chemoattractant (KC), INFγ-induced protein 10 (IP10), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein 1 (MCP1), tumor necrosis factor alpha (TNFα), interleukin 2 (IL-2), IL-6, IL-7, IL-8, IL-10 IL-15, IL-18, CRP, neutrophils and monocytes between groups at multiple time points during the course of the infection. Our findings suggest that the initiation of inflammation occurs before the onset of clinical disease in B. rossi infection and infectious dose influences the onset of the inflammatory response. Treatment enhances the inflammatory response in the immediate post-treatment period which may contribute to disease associated complications. Finally, we found that there is an imbalance in pro/anti-inflammatory cytokine concentrations during infection which may promote parasite replication.