Live Monitoring of Inflammation Reveals Tissue and Sex-specific Responses to Western Diet and Butyrate treatment

Obesity is a current epidemic, affecting millions of individuals worldwide. Chronic obesity is characterized by a low grade systemic inflammation besides not being a classic inflammatory disease. Many studies have tried to identify inflammatory insults dysregulated by a Westernized diet consisted of high fat, high sucrose, and high cholesterol mainly focusing on production and secretion of inflammatory cytokines. The gut microbiome and derived metabolites, including the short-chain fatty acid butyrate, have received increased attention as underlying some of the obesogenic features. In the present work, we utilized a novel biosensor mouse model capable of monitoring in vivo inflammation. We observed tissue- and sex- specific caspase 1 activation patterns in obese mice and treated with butyrate. Our work utilizing a caspase-1 biosensor mouse model, flow cytometry and computational analyses and offers new mechanistic insights underlying the effect of butyrate in obesity and its complications.

Raphanus Sativus Seeds OilArrested In Vivo Inflammation and Angiogenesis Through Down-Regulation of TNF-a

Background: Raphanus sativus is traditionally used as an anti-inflammatory agent. Objectives: The current study was designed to explore the in vivo anti-inflammatory and antiangiogenic properties of Raphanus sativus seeds oil. Methods: Cold press method was used for the extraction of oil (RsSO) and was characterised using GC-MS techniques. Three in vitro antioxidant assays (DPPH, ABTS, and FRAP) were performed to explore antioxidant potential of RsSO. Disc diffusion methods were used to study in vitro antimicrobial properties. In vivo anti-inflammatory properties were studied in both acute and chronic inflammation models. In ovo chicken, a chorioallantoic membrane assay was performed to study antiangiogenic effects. Molecular mechanisms were identified using serum TNF-α ELISA kit and docking tools. Results: GC-MS analysis of RsSO revealed the presence of hexadecanoic and octadecanoic acid. Findings of DPPH, ABTS, and FRAP models indicated relatively moderate radical scavenging properties of RsSO. Oil showed antimicrobial activity against a variety of strains tested. Data of inflammation models showed significant (p < 0.05) anti-inflammatory effects of RsSO in both acute and chronic models. 500 mg/kg RsSO halted inflammation development significantly better (p < 0.05) as compared with lower doses. Histopathological evaluations of paws showed minimal infiltration of inflammatory cells in RsSO-treated animals. Findings of TNF-α ELSIA and docking studies showed that RsSO has the potential to downregulate the expression of TNF-α, iNOS, ROS, and NF-κB, respectively. Moreover, RsSO showed in vivo antiangiogenic effects. Conclusion: Data of the current study highlight that Raphanus sativus seeds oil has anti-inflammatory, and antiangiogenic properties and can be used as an adjunct to standard NSAIDs therapy to reduce its dose and side effects.

A technical note on contamination from PRF tubes containing silica and silicone

Background Platelet-rich fibrin (PRF) has been widely utilized in modern medicine and dentistry owing to its ability to rapidly stimulate neoangiogenesis, leading to faster tissue regeneration. While improvements over traditional platelet rich plasma therapies (which use chemical additives such as bovine thrombin and calcium chloride) have been observed, most clinicians are unaware that many tubes utilized for the production of ‘natural’ and ‘100% autologous’ PRF may in fact contain chemical additives without appropriate or transparent knowledge provided to the treating clinician. The aim of this overview article is therefore to provide a technical note on recent discoveries related to PRF tubes and describe recent trends related to research on the topic from the authors laboratories. Methods Recommendations are provided to clinicians with the aim of further optimizing PRF clots/membranes by appropriate understanding of PRF tubes. The most common additives to PRF tubes reported in the literature are silica and/or silicone. A variety of studies have been performed on their topic described in this narrative review article. Results Typically, PRF production is best achieved with plain, chemical-free glass tubes. Unfortunately, a variety of other centrifugation tubes commonly used for lab testing/diagnostics and not necessarily manufactured for human use have been utilized in clinical practice for the production of PRF with unpredictable clinical outcomes. Many clinicians have noted an increased variability in PRF clot sizes, a decreased rate of clot formation (PRF remains liquid even after an adequate protocol is followed), or even an increased rate in the clinical signs of inflammation following the use of PRF. Conclusion This technical note addresses these issues in detail and provides scientific background of recent research articles on the topic. Furthermore, the need to adequately select appropriate centrifugation tubes for the production of PRF is highlighted with quantitative data provided from in vitro and animal investigations emphasizing the negative impact of the addition of silica/silicone on clot formation, cell behavior and in vivo inflammation.

Induced Experimental Periimplantitis and Periodontitis: What are the Differences in the Inflammatory Response ?

This preliminary study investigates the differences between experimental periodontitis and periimplantitis in a dog model, with a focus on the histopathology, the inflammatory responses and specific immunoregulatory activities, driven by Th1/Th2 positive cells. Twelve dental implants were inserted into the edentulated posterior mandibles of six Beagle dogs and were given twelve weeks time for osseointegration. Experimental periimplantitis and periodontitis (first mandible molar) was then induced using cotton-floss ligatures. Twelve weeks later, alveolar bones were quantitated by cone beam-computer tomography. Histopathological analysis of the inflamed gingiva and of the periodontal tissues was performed by light microscopy, and the Th1/ Th2 cell populations were investigated by flow cytometry. Periimplantitis as well as periodontitis were both found to be associated with pronounced bone resorption effects, both to a similar degree vertically, but with a differential bone resorption pattern mesio-distally, and with a significantly higher and consistent bone resorption result in periimplantitis; however, with a higher variance of bone resorption in periodontitis. The histological appearances of the inflammatory tissues were identical. The percentages of Th1/ Th2 cells in the inflamed gingival tissues of both experimental periimplantitis and periodontitis were also found to be similar. Experimental periodontitis and periimplantitis in the dog model show essentially the same cellular pathology of inflammation. However, bone resorption was found to be significantly higher in periimplantitis; the histopathological changes in the periodontal tissues were similar in both groups, but showed a higher inter-individual variation in periodontitis, and appeared more uniform in periimplantitis. This preliminary study indicates that more focused experimental in-vivo inflammation models need to be developed to better simulate the human pathology in the two different diseases, and in order to have a valuable tool to investigate more specifically how novel treatments/prevention approaches may heal the differential adverse effects on bone tissue and on periodontium in periodontitis and in periimplantitis.

Intense cardiac-targeted small extracellular vesicles-mediated delivery of RAGE siRNA attenuates inflammation in rat myocarditis model

Background Despite advances in the field and new therapeutics being developed, heart disease remains the leading cause of mortality worldwide. Small extracellular vesicles (sEV) are 30–150 nm in diameter and ferry RNA and proteins to cells. sEV are a natural carrier of many signaling molecules and play an important role in heart disease. However, the high probability of off-target effects associated with these carriers is a major barrier to translation into clinical application. Purpose Our aim was to evaluate whether sEVs engineered to express potent cardiac targeting peptides (CTPs) could deliver siRNA to the heart and exert a therapeutic effect. Methods We use vectors encoding LAMP2B (sEV) or CTP-LAMP2B (PsEV) into HEK 293 cells expressing. sEVs were purified from culture media of HEK 293 cells by serial centrifugation followed by tangential flow filtration (TFF) system. sEV and PsEV were loaded with siRNAs by Exo-Fect™ exosome transfection reagent, and were treated into H9C2 rat cardiomyocyte. TNF-alpha were then added to the cells to induce inflammation. And sEVs were intravenously injected into myocarditis rat. Inflammation factors of in vitro and in vivo inflammation model were identified by western blot. Echocardiographic examination was also performed in rat. Results The successful development of PsEVs was analyzed by Western blot and TEM. We observed a 4 fold increase from that of the previously developed sEVs in H9C2 cells and a 200% increase in cardiac-specific delivery without toxicity in rat model. AGE is involved in proinflammatory/pro-apoptotic processes. To block RAGE, we loaded RAGE siRNA (siRAGE) in sEV that had high expression of PsEV. Characteristics of sEVs were maintained despite siRNA load.In inflammatory cell models and rat disease models, PsEV-treated groups significantly reduced molecular levels associated with inflammatory responses such as RAGE, IL-6, TNF-alpha, COX2, HMGB1, and p-p65 / p65. Conclusions Our results suggest that PsEV can potentially serve as a treatment delivery vehicle for heart disease. Scheme Funding Acknowledgement Type of funding source: None

In Vitro and In Vivo Screening of Wild Bitter Melon Leaf for Anti-Inflammatory Activity against Cutibacterium acnes

Cutibacterium acnes (formerly Propionibacterium acnes) is a key pathogen involved in the development and progression of acne inflammation. The numerous bioactive properties of wild bitter melon (WBM) leaf extract and their medicinal applications have been recognized for many years. In this study, we examined the suppressive effect of a methanolic extract (ME) of WBM leaf and fractionated components thereof on live C. acnes-induced in vitro and in vivo inflammation. Following methanol extraction of WBM leaves, we confirmed anti-inflammatory properties of ME in C. acnes-treated human THP-1 monocyte and mouse ear edema models. Using a bioassay-monitored isolation approach and a combination of liquid–liquid extraction and column chromatography, the ME was then separated into n-hexane, ethyl acetate, n-butanol and water-soluble fractions. The hexane fraction exerted the most potent anti-inflammatory effect, suppressing C. acnes-induced interleukin-8 (IL-8) production by 36%. The ethanol-soluble fraction (ESF), which was separated from the n-hexane fraction, significantly inhibited C. acnes-induced activation of mitogen-activated protein kinase (MAPK)-mediated cellular IL-8 production. Similarly, the ESF protected against C. acnes-stimulated mouse ear swelling, as measured by ear thickness (20%) and biopsy weight (23%). Twenty-four compounds in the ESF were identified using gas chromatograph–mass spectrum (GC/MS) analysis. Using co-cultures of C. acnes and THP-1 cells, β-ionone, a compound of the ESF, reduced the production of IL-1β and IL-8 up to 40% and 18%, respectively. β-ionone also reduced epidermal microabscess, neutrophilic infiltration and IL-1β expression in mouse ear. We also found evidence of the presence of anti-inflammatory substances in an unfractionated phenolic extract of WBM leaf, and demonstrated that the ESF is a potential anti-inflammatory agent for modulating in vitro and in vivo C. acnes-induced inflammatory responses.

Modified DNA Aptamers for C-Reactive Protein and Lactate Dehydrogenase-5 with Sub-Nanomolar Affinities

Human C-reactive protein (CRP) and lactate dehydrogenase are important markers in clinical laboratory testing—the former is used to detect in vivo inflammation, and the latter is used to detect cell necrosis and tissue destruction. We developed aptamers that bind to human CRP and human lactate dehydrogenase-5 (LDH-5) with high affinities (dissociation constants of 6.2 pM and 235 pM, respectively), applying the systematic evolution of ligands by exponential enrichment (SELEX) method, and by using a modified DNA library containing the following base-appended base modifications: analog adenine derivative at the fifth position of uracil (Uad), analog guanine derivative at the fifth position of uracil (Ugu), and analog adenine derivative at the seventh position of adenine (Aad). A potential application of these aptamers as sensor elements includes high-sensitivity target detection in point-of-care testing.