Gut mycobiome dysbiosis after sepsis and trauma.

BACKGROUND: Sepsis and trauma are known to disrupt gut bacterial microbiome communities, but the impacts and perturbations in the fungal (mycobiome) community after severe infection or injury, particularly in patients experiencing chronic critical illness (CCI), remain unstudied. METHODS: We assess persistence of the gut mycobiome perturbation (dysbiosis) in patients experiencing CCI following sepsis or trauma for up to two-to-three weeks after intensive care unit hospitalization. RESULTS: We show that the dysbiotic mycobiome arrays shift toward a pathobiome state, which is more susceptible to infection, in CCI patients compared to age-matched healthy subjects. The fungal community in CCI patients is largely dominated by Candida spp; while, the commensal fungal species are depleted. Additionally, these myco-pathobiome arrays correlate with alterations in micro-ecological niche involving specific gut bacteria and gut-blood metabolites. CONCLUSIONS: The findings reveal the persistence of mycobiome dysbiosis in both sepsis and trauma settings, even up to two weeks post-sepsis and trauma, highlighting the need to assess and address the increased risk of fungal infections in CCI patients.

Exosomal microRNA following severe trauma: Role in bone marrow dysfunction.

INTRODUCTION: Severe trauma disrupts bone marrow function and is associated with persistent anemia and altered hematopoiesis. Previously, plasma-derived exosomes isolated after trauma have been shown to suppress in vitro bone marrow function. However, the cargo contained in these vesicles has not been examined. We hypothesized that trauma plasma-derived exosomes exhibit microRNA (miRNA) changes that impact bone marrow function after severe injury. METHODS: Plasma was collected from a prospective cohort study of trauma patients (n = 15; 7 males, 8 females) with hip and/or femur fractures and an Injury Severity Score of ≥15; elective total hip arthroplasty (THA) patients (n = 8; 4 males, 4 females) served as operative controls. Exosomes were isolated from plasma with the Invitrogen Total Exosome Isolation Kit (Thermo Fisher Scientific, Waltham, MA), and RNA was isolated using a miRNeasy Mini Kit (Qiagen, Hilden, Germany). Direct quantification of miRNA was performed by NanoString Technologies on a human miRNA gene panel and analyzed with nSolver with significance defined as p < 0.05. RESULTS: There were no differences in age or sex distribution between trauma and THA groups; the average Injury Severity Score was 23. Trauma plasma-derived exosomes had 60 miRNA identities that were significantly downregulated and 3 miRNAs that were upregulated when compared with THA ( p < 0.05). Twelve of the downregulated miRNAs have a direct role in hematopoiesis regulation. Furthermore, male trauma plasma-derived exosomes demonstrated downregulation of 150 miRNAs compared with male THA ( p < 0.05). Female trauma plasma-derived exosomes demonstrated downregulation of only four miRNAs and upregulation of two miRNAs compared with female THA ( p < 0.05). CONCLUSION: We observed downregulation of 12 miRNAs linked to hematopoiesis along with sexual dimorphism in miRNA expression from plasma-derived exosomes following severe trauma. Understanding sexually dimorphic miRNA expression provides new insight into sex-based changes in postinjury systemic inflammation, immune system dysregulation, and bone marrow dysfunction and will aid us in more precise future potential therapeutic strategies. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level III.

Involvement of extracellular vesicles in the progression, diagnosis, treatment, and prevention of whole-body ionizing radiation-induced immune dysfunction.

Acute radiation syndrome (ARS) develops after exposure to high doses of ionizing radiation and features immune suppression and organ failure. Currently, there are no diagnostics to identify the occurrence or severity of exposure and there are limited treatments and preventative strategies to mitigate ARS. Extracellular vesicles (EVs) are mediators of intercellular communication that contribute to immune dysfunction across many diseases. We investigated if EV cargo can identify whole body irradiation (WBIR) exposure and if EVs promote ARS immune dysfunction. We hypothesized that beneficial EVs derived from mesenchymal stem cells (MSC-EVs) would blunt ARS immune dysfunction and might serve as prophylactic radioprotectants. Mice received WBIR (2 or 9 Gy) with assessment of EVs at 3 and 7 days after exposure. LC-MS/MS proteomic analysis of WBIR-EVs found dose-related changes as well as candidate proteins that were increased with both doses and timepoints (34 total) such as Thromboxane-A Synthase and lymphocyte cytosolic protein 2. Suprabasin and Sarcalumenin were increased only after 9 Gy suggesting these proteins may indicate high dose/lethal exposure. Analysis of EV miRNAs identified miR-376 and miR-136, which were increased up to 200- and 60-fold respectively by both doses of WBIR and select miRNAs such as miR-1839 and miR-664 were increased only with 9 Gy. WBIR-EVs (9 Gy) were biologically active and blunted immune responses to LPS in RAW264.7 macrophages, inhibiting canonical signaling pathways associated with wound healing and phagosome formation. When given 3 days after exposure, MSC-EVs slightly modified immune gene expression changes in the spleens of mice in response to WBIR and in a combined radiation plus burn injury exposure (RCI). MSC-EVs normalized the expression of certain key immune genes such as NFκBia and Cxcr4 (WBIR), Map4k1, Ccr9 and Cxcl12 (RCI) and lowered plasma TNFα cytokine levels after RCI. When given prophylactically (24 and 3 hours before exposure), MSC-EVs prolonged survival to the 9 Gy lethal exposure. Thus, EVs are important participants in ARS. EV cargo might be used to diagnose WBIR exposure, and MSC-EVs might serve as radioprotectants to blunt the impact of toxic radiation exposure.

Chronic early life stress alters the neuroimmune profile and functioning of the developing zebrafish gut.

Chronic early life stress (ELS) potently impacts the developing central nervous and immune systems and is associated with the onset of gastrointestinal disease in humans. Though the gut-brain axis is appreciated to be a major target of the stress response, the underlying mechanisms linking ELS to gut dysfunction later in life is incompletely understood. Zebrafish are a powerful model validated for stress research and have emerged as an important tool in delineating neuroimmune mechanisms in the developing gut. Here, we developed a novel model of ELS and utilized a comparative transcriptomics approach to assess how chronic ELS modulated expression of neuroimmune genes in the developing gut and brain. Zebrafish exposed to ELS throughout larval development exhibited anxiety-like behavior and altered expression of neuroimmune genes in a time- and tissue-dependent manner. Further, the altered gut neuroimmune profile, which included increased expression of genes associated with neuronal modulation, correlated with a reduction in enteric neuronal density and delayed gut transit. Together, these findings provide insights into the mechanisms linking ELS with gastrointestinal dysfunction and highlight the zebrafish model organism as a valuable tool in uncovering how "the body keeps the score."

Suppression of local inflammation via galectin-anchored indoleamine 2,3-dioxygenase.

The treatment of chronic inflammation with systemically administered anti-inflammatory treatments is associated with moderate-to-severe side effects, and the efficacy of locally administered drugs is short-lived. Here we show that inflammation can be locally suppressed by a fusion protein of the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO) and galectin-3 (Gal3). Gal3 anchors IDO to tissue, limiting the diffusion of IDO-Gal3 away from the injection site. In rodent models of endotoxin-induced inflammation, psoriasis, periodontal disease and osteoarthritis, the fusion protein remained in the inflamed tissues and joints for about 1 week after injection, and the amelioration of local inflammation, disease progression and inflammatory pain in the animals were concomitant with homoeostatic preservation of the tissues and with the absence of global immune suppression. IDO-Gal3 may serve as an immunomodulatory enzyme for the control of focal inflammation in other inflammatory conditions.

NUCLEAR FACTOR-ERYTHROID-2-RELATED FACTOR REGULATES SYSTEMIC AND PULMONARY BARRIER FUNCTION AND IMMUNE PROGRAMMING AFTER BURN AND INHALATION INJURY.

ABSTRACT: Major burn injury is associated with systemic hyperinflammatory and oxidative stresses that encompass the wound, vascular, and pulmonary systems that contribute to complications and poor outcomes. These stresses are exacerbated if there is a combined burn and inhalation (B+I) injury, which leads to increases in morbidity and mortality. Nuclear factor-erythroid-2-related factor (NRF2) is a transcription factor that functions to maintain homeostasis during stress, in part by modulating inflammation and oxidative injury. We hypothesized that the NRF2-mediated homeostasis after burn alone and combined B-I injury is insufficient, but that pharmacological activation of the NRF2 pathway has the potential to reduce/reverse acute hyper inflammatory responses. We found that, after burn and B+I injury, Nrf2 -/- mice have higher mortality and exhibit greater pulmonary edema, vascular permeability, and exacerbated pulmonary and systemic proinflammatory responses compared with injured wild-type (WT) controls. Transcriptome analysis of lung tissue revealed specific Nrf2 -dependent dysregulated immune pathways after injury. In WT mice, we observed that B+I injury induces cytosolic, but not nuclear, accumulation of NRF2 protein in the lung microenvironment compared with sham-injured controls. Bardoxolone methyl (CDDO-Me)-containing microparticles (CDDO-MPs) were developed that allow for dilution in saline and stable release of CDDO-Me. When delivered intraperitoneally into mice 1 hour after B+I injury, CDDO-MPs significantly reduced mortality and cytokine dysfunction compared with untreated B-I animals. These data implicate the role of NRF2 regulation of pulmonary and systemic immune dysfunction after burn and B+I injury, and also a deficiency in controlling immune dysregulation. Selectively activating the NRF2 pathway may improve clinical outcomes in burn and B+I patients.

EXTRACELLULAR VESICLES AS REGULATORS OF IMMUNE FUNCTION IN TRAUMATIC INJURIES AND SEPSIS.

ABSTRACT: Despite advancements in critical care and resuscitation, traumatic injuries are one of the leading causes of death around the world and can bring about long-term disabilities in survivors. One of the primary causes of death for trauma patients are secondary phase complications that can develop weeks or months after the initial insult. These secondary complications typically occur because of systemic immune dysfunction that develops in response to injury, which can lead to immunosuppression, coagulopathy, multiple organ failure, unregulated inflammation, and potentially sepsis in patients. Recently, extracellular vesicles (EVs) have been identified as mediators of these processes because their levels are increased in circulation after traumatic injury and they encapsulate cargo that can aggravate these secondary complications. In this review, we will discuss the role of EVs in the posttrauma pathologies that arise after burn injuries, trauma to the central nervous system, and infection. In addition, we will examine the use of EVs as biomarkers for predicting late-stage trauma outcomes and as therapeutics for reversing the pathological processes that develop after trauma. Overall, EVs have emerged as critical mediators of trauma-associated pathology and their use as a therapeutic agent represents an exciting new field of biomedicine.

Performance and efficacy of a recently introduced diamond rotary instrument: Cutting, surface preparation, and cleanability.

STATEMENT OF PROBLEM: Evaluation of the cutting efficiency and effectiveness, surface roughness, and cleanability of a novel rotary instrument is lacking. PURPOSE: The purpose of this in vitro study was to compare the cutting efficiency and effectiveness of a recently introduced diamond rotary instrument containing corundum microspheres with conventional instruments by evaluating the heat generated, surface roughness, and cleanability of each instrument after tooth preparations. MATERIAL AND METHODS: Sound molars (n=225) were used to evaluate cutting efficiency and effectiveness by measuring the heat generated by 3 diamond dental rotary instruments: test instrument (TI), reference instrument (RI), and NTI instrument (NI). Thirty cavity preparations (27 mm3) were prepared, and the thermal change (ΔT) was determined from a thermocouple inserted in the pulp chamber. The surface roughness of the dentin substrate was determined after veneer preparations using scanning white-light interferometry and scanning electron microscope imaging. The cleanability of TI and RI was also determined by comparing the efficacy of 3 conventional disinfection protocols after contaminating the instrument with Gram-positive or Gram-negative oral pathogens. The mean and standard deviation values for thermal change, surface roughness, and colony forming units were calculated at a 95% confidence level, and 1-way ANOVA was used to determine statistical significance (α=.05). RESULTS: The NI instrument had the lowest mean ΔT (1.47 °C). The TI (1.77 °C) and RI (1.85 °C) groups showed statistically similar means (P>.05). The TI presented the lowest surface roughness (1.68 µm), followed by the RI (1.87 µm) (P<.001). The NI resulted in the highest surface roughness (2.17 µm) (P<.001). The disinfection protocols used were more effective on the TI group than on the RI group regardless of organisms and time exposed to the cleaning solution (P<.001). CONCLUSIONS: The novel diamond instrument demonstrated similar cutting efficiency and effectiveness when compared with conventional diamond instruments. However, the novel instrument produced smoother tooth preparations and was easier to clean than the conventional diamond rotary instruments.

Mechanisms of Foreign Body Response Mitigation by Nitric Oxide Release.

Implantable glucose biosensors provide real-time information about blood glucose fluctuations, but their utility and accuracy are time-limited due to the foreign body response (FBR) following their insertion beneath the skin. The slow release of nitric oxide (NO), a gasotransmitter with inflammation regulatory properties, from a sensor surface has been shown to dramatically improve sensors' analytical biocompatibility by reducing the overall FBR response. Indeed, work in a porcine model suggests that as long as the implants (sensors) continue to release NO, even at low levels, the inflammatory cell infiltration and resulting collagen density are lessened. While these studies strongly support the benefits of NO release in mitigating the FBR, the mechanisms through which exogenous NO acts on the surrounding tissue, especially under the condition of hyperglycemia, remain vague. Such knowledge would inform strategies to refine appropriate NO dosage and release kinetics for optimal therapeutic activity. In this study, we evaluated mediator, immune cell, and mRNA expression profiles in the local tissue microenvironment surrounding implanted sensors as a function of NO release, diabetes, and implantation duration. A custom porcine wound healing-centric multiplex gene array was developed for nanoString barcoding analysis. Tissues adjacent to sensors with sustained NO release abrogated the implant-induced acute and chronic FBR through modulation of the tissue-specific immune chemokine and cytokine microenvironment, resulting in decreased cellular recruitment, proliferation, and activation at both the acute (7-d) and chronic (14-d) phases of the FBR. Further, we found that sustained NO release abrogated the implant-induced acute and chronic foreign body response through modulation of mRNA encoding for key immunological signaling molecules and pathways, including STAT1 and multiple STAT1 targets including MAPK14, IRAK4, MMP2, and CXCL10. The condition of diabetes promoted a more robust FBR to the implants, which was also controlled by sustained NO release.

Quantitative Analysis of Biofilm Removal Following Instrumentation with TRUShape and Vortex Blue File Systems: Microbiological Study.

Mesial roots and isthmuses of mandibular molars are difficult areas to obtain adequate disinfection of root canal walls, and consequently microorganisms can survive treatment. The present study compared, through real-time polymerase chain reaction (qPCR), the effectiveness of TRUShape (TS) (Dentsply Tulsa Dental Specialties, Tulsa, OK) and Vortex Blue (VB) (Dentsply Tulsa Dental Specialties, Tulsa, OK) in removing Enterococcus faecalis (E. faecalis) from the mesial canals and isthmuses of mandibular molars. Fifty extracted human lower molars were inoculated with E. faecalis OG1RF for 14 days, and then an initial bacterial sample was collected with paper points from mesiobuccal and mesiolingual canals and isthmuses. The specimens were randomly divided into four groups (n = 10 teeth; 20 canals each), according to instrumentation system: TS 25/0.06, TS 30/0.06, VB 25/0.06 and VB 30/0.06. The remaining 10 teeth were divided between positive control, inoculated teeth without instrumentation or irrigation, and negative controls, teeth without inoculation. After instrumentation, the final sample was taken using paper points and DNA was isolated. Primers specific for E. faecalis were used for qPCR. The bacterial reduction between pre- and post-instrumentation was calculated. One-way analysis of variance (ANOVA) with Bonferroni's multiple-comparisons tests were for statistical analysis with significance of (p < 0.05). All file systems were able to reduce the load of E. faecalis from the prepared root canals, however, TS size 30 removed significantly more bacteria than size 25. Interestingly, regardless of the size, TS files removed significantly more E. faecalis biofilm (p < 0.05) than did VB files (63.7% vs 50.8% for size 25, and 69.5% vs 56% for size 30). In conclusion, when combined with irrigation, TS file system is more effective than VB in reducing E. faecalis biofilms from mesiobuccal and mesiolingual canals and the isthmuses of mandibular molars.

Characterization of the Basal and mTOR-Dependent Acute Pulmonary and Systemic Immune Response in a Murine Model of Combined Burn and Inhalation Injury.

Severe burn injury leads to a cascade of local and systemic immune responses that trigger an extreme state of immune dysfunction, leaving the patient highly susceptible to acute and chronic infection. When combined with inhalation injury, burn patients have higher mortality and a greater chance of developing secondary respiratory complications including infection. No animal model of combined burn and inhalation injury (B+I) exists that accurately mirrors the human clinical picture, nor are there any effective immunotherapies or predictive models of the risk of immune dysfunction. Our earlier work showed that the mechanistic/mammalian target of rapamycin (mTOR) pathway is activated early after burn injury, and its chemical blockade at injury reduced subsequent chronic bacterial susceptibility. It is unclear if mTOR plays a role in the exacerbated immune dysfunction seen after B+I injury. We aimed to: (1) characterize a novel murine model of B+I injury, and (2) investigate the role of mTOR in the immune response after B+I injury. Pulmonary and systemic immune responses to B+I were characterized in the absence or presence of mTOR inhibition at the time of injury. Data describe a murine model of B+I with inhalation-specific immune phenotypes and implicate mTOR in the acute immune dysfunction observed.

Multiplexed Human Gene Expression Analysis Reveals a Central Role of the TLR/mTOR/PPARγ and NFkB Axes in Burn and Inhalation Injury-Induced Changes in Systemic Immunometabolism and Long-Term Patient Outcomes.

Burn patients are subject to significant acute immune and metabolic dysfunction. Concomitant inhalation injury increases mortality by 20%. In order to identify specific immune and metabolic signaling pathways in burn (B), inhalation (I), and combined burn-inhalation (BI) injury, unbiased nanoString multiplex technology was used to investigate gene expression within peripheral blood mononuclear cells (PBMCs) from burn patients, with and without inhalation injury. PBMCs were collected from 36 injured patients and 12 healthy, non-burned controls within 72 h of injury. mRNA was isolated and hybridized with probes for 1342 genes related to general immunology and cellular metabolism. From these specific gene patterns, specific cellular perturbations and signaling pathways were inferred using robust bioinformatic tools. In both B and BI injuries, elements of mTOR, PPARγ, TLR, and NF-kB signaling pathways were significantly altered within PBMC after injury compared to PBMC from the healthy control group. Using linear regression modeling, (1) DEPTOR, LAMTOR5, PPARγ, and RPTOR significantly correlated with patient BMI; (2) RPTOR significantly correlated with patient length of stay, and (3) MRC1 significantly correlated with the eventual risk of patient mortality. Identification of mediators of this immunometabolic response that can act as biomarkers and/or therapeutic targets could ultimately aid the management of burn patients.

Role of Nitric Oxide-Releasing Glycosaminoglycans in Wound Healing.

Two glycosaminoglycan (GAG) biopolymers, hyaluronic acid (HA) and chondroitin sulfate (CS), were chemically modified via carbodiimide chemistry to facilitate the loading and release of nitric oxide (NO) to develop a multi-action wound healing agent. The resulting NO-releasing GAGs released 0.2-0.9 µmol NO mg-1 GAG into simulated wound fluid with NO-release half-lives ranging from 20 to 110 min. GAGs containing alkylamines with terminal primary amines and displaying intermediate NO-release kinetics exhibited potent, broad spectrum bactericidal action against three strains each of Pseudomonas aeruginosa and Staphylococcus aureus ranging in antibiotic resistance profile. NO loading of the GAGs was also found to decrease murine TLR4 activation, suggesting that the therapeutic exhibits anti-inflammatory mechanisms. In vitro adhesion and proliferation assays utilizing human dermal fibroblasts and human epidermal keratinocytes displayed differences as a function of the GAG backbone, alkylamine identity, and NO-release properties. In combination with antibacterial properties, the adhesion and proliferation profiles of the GAG derivatives enabled the selection of the most promising wound healing candidates for subsequent in vivo studies. A P. aeruginosa-infected murine wound model revealed the benefits of CS over HA as a pro-wound healing NO donor scaffold, with benefits of accelerated wound closure and decreased bacterial burden attributable to both active NO release and the biopolymer backbone.

Early expression of IL-10, IL-12, ARG1, and NOS2 genes in peripheral blood mononuclear cells synergistically correlate with patient outcome after burn injury.

BACKGROUND: No methods exist to rapidly and accurately quantify the immune insult created by burn injuries. The development of a rapid, noninvasive clinical biomarker assay that evaluates a burn patient's underlying immune dysfunction and predicts clinical outcomes could transform burn care. We aimed to determine a set of peripheral biomarkers that correlates with clinical outcomes of burn patients. METHODS: This prospective observational study enrolled two patient cohorts within a single burn center into an institutionally approved institutional review board study. Blood draws were performed <48 hours after injury. Initial unbiased immune gene expression analysis compared 23 burn patients and 6 healthy controls using multiplex immune gene expression analysis of RNA from peripheral blood mononuclear cells. We then performed confirmatory outcomes analysis in 109 burn patients and 19 healthy controls using a targeted rapid quantitative polymerase chain reaction. Findings were validated and modeled associations with clinical outcomes using a regression model. RESULTS: A total of 149 genes with a significant difference in expression from burn patients compared with controls were identified. Pathway analysis identified pathways related to interleukin (IL)-10 and inducible nitric oxide synthase signaling to have significant z scores. quantitative polymerase chain reaction analysis of IL-10, IL-12, arginase 1 (ARG1), and inducible nitric oxide synthase demonstrated that burn injury was associated with increased expression of ARG1 and IL-10, and decreased expression of nitric oxide synthase 2 (NOS2) and IL-12. Burn severity, acute lung injury, development of infection, failure of skin autograft, and mortality significantly correlated with expression of one or more of these genes. Ratios of IL-10/IL-12, ARG1/NOS2, and (ARG1-IL-10)/(NOS2-IL-12) transcript levels further improved the correlation with outcomes. Using a multivariate regression model, adjusting for patient confounders demonstrated that (ARG1-IL-10)/(NOS2-IL-12) significantly correlated with burn severity and development of acute lung injury. CONCLUSION: We present a means to predict patient outcomes early after burn injury using peripheral blood, allowing early identification of underlying immune dysfunction. LEVEL OF EVIDENCE: Prognostic/Epidemiological; Level II.

Plasma extracellular vesicles released after severe burn injury modulate macrophage phenotype and function.

Extracellular vesicles (EVs) have emerged as key regulators of immune function across multiple diseases. Severe burn injury is a devastating trauma with significant immune dysfunction that results in an ∼12% mortality rate due to sepsis-induced organ failure, pneumonia, and other infections. Severe burn causes a biphasic immune response: an early (0-72 h) hyper-inflammatory state, with release of damage-associated molecular pattern molecules, such as high-mobility group protein 1 (HMGB1), and proinflammatory cytokines (e.g., IL-1ß), followed by an immunosuppressive state (1-2+ wk post injury), associated with increased susceptibility to life-threatening infections. We have reported that early after severe burn injury HMGB1 and IL-1ß are enriched in plasma EVs. Here we tested the impact of EVs isolated after burn injury on phenotypic and functional consequences in vivo and in vitro using adoptive transfers of EV. EVs isolated early from mice that underwent a 20% total body surface area burn injury (burn EVs) caused similar hallmark cytokine responses in naïve mice to those seen in burned mice. Burn EVs transferred to RAW264.7 macrophages caused similar functional (i.e., cytokine secretion) and immune gene expression changes seen with their associated phase of post-burn immune dysfunction. Burn EVs isolated early (24 h) induced MCP-1, IL-12p70, and IFNγ, whereas EVs isolated later blunted RAW proinflammatory responses to bacterial endotoxin (LPS). We also describe significantly increased HMGB1 cargo in burn EVs purified days 1 to 7 after injury. Thus, burn EVs cause immune outcomes in naïve mice and macrophages similar to findings after severe burn injury, suggesting EVs promote post-burn immune dysfunction.

Characterization of extracellular vesicle miRNA identified in peripheral blood of chronic pancreatitis patients.

Plasma-derived extracellular vesicles (EV) can serve as markers of cell damage/disease but can also have therapeutic utility depending on the nature of their cargo, such as miRNA. Currently, there are challenges and lack of innovations regarding early diagnosis and therapeutic options within different aspects of management of patients suffering from chronic pancreatitis (CP). Use of EV as biomarkers for pancreatic health and/or as adjuvant therapy would make a difference in management of these patients. The aim of this study was to characterize the miRNA cargo of EV purified from the plasma of CP patients and compared to those of healthy participants. EVs were isolated from plasma of 15 CP patients and 10 healthy controls. Nanoparticle tracking analysis was used to determine frequency and size, while NanoString technology was used to characterize the miRNA cargo. Relevant clinical parameters were correlated with EV miRNA cargo. ~ 30 miRNA species were identified to have significantly (p < 0.05) different expression in EV from individuals with CP compared to healthy individuals; ~ 40 miRNA were differentially expressed in EV from pre-diabetic versus non-diabetic CP patients. miR-579-3p, while exhibiting significantly lower (~ 16-fold) expression in CP compared to healthy and lower (~ 24-fold) in CP narcotic users compared to the non-users, is actually enriched (~ 32-fold) within EV in pre-diabetic CP patients compared to non-diabetic CP patients. A unique pattern was identified in female CP patients. These data support the prospect of using a plasma-derived EV cargo to assess pancreatic health and its therapeutic potential in CP patients.

SARS-CoV-2 infection of the oral cavity and saliva.

Despite signs of infection-including taste loss, dry mouth and mucosal lesions such as ulcerations, enanthema and macules-the involvement of the oral cavity in coronavirus disease 2019 (COVID-19) is poorly understood. To address this, we generated and analyzed two single-cell RNA sequencing datasets of the human minor salivary glands and gingiva (9 samples, 13,824 cells), identifying 50 cell clusters. Using integrated cell normalization and annotation, we classified 34 unique cell subpopulations between glands and gingiva. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral entry factors such as ACE2 and TMPRSS members were broadly enriched in epithelial cells of the glands and oral mucosae. Using orthogonal RNA and protein expression assessments, we confirmed SARS-CoV-2 infection in the glands and mucosae. Saliva from SARS-CoV-2-infected individuals harbored epithelial cells exhibiting ACE2 and TMPRSS expression and sustained SARS-CoV-2 infection. Acellular and cellular salivary fractions from asymptomatic individuals were found to transmit SARS-CoV-2 ex vivo. Matched nasopharyngeal and saliva samples displayed distinct viral shedding dynamics, and salivary viral burden correlated with COVID-19 symptoms, including taste loss. Upon recovery, this asymptomatic cohort exhibited sustained salivary IgG antibodies against SARS-CoV-2. Collectively, these data show that the oral cavity is an important site for SARS-CoV-2 infection and implicate saliva as a potential route of SARS-CoV-2 transmission.

Nitric Oxide-Releasing Hyaluronic Acid as an Antibacterial Agent for Wound Therapy.

Taking advantage of their respective wound-healing roles in physiology, the dual activity of hyaluronic acid (HA) and nitric oxide (NO) was combined to create a single-agent wound therapeutic. Carboxylic acid groups of HA (6 and 90 kDa) were chemically modified with a series of alkylamines via carbodiimide chemistry to provide secondary amines for subsequent N-diazeniumdiolate NO donor formation. The resulting NO-releasing HA derivatives stored 0.3-0.6 µmol NO mg-1 and displayed diverse release kinetics (5-75 min NO-release half-lives) under physiological conditions. The 6 kDa HA with terminal primary amines and intermediate release kinetics exhibited broad-spectrum bactericidal activity against common wound pathogens, including planktonic methicillin-resistant Staphylococcus aureus as well as planktonic and biofilm-based multidrug-resistant Pseudomonas aeruginosa. The treatment of infected murine wounds with NO-releasing HA facilitated more rapid wound closure and decreased the quantity of the P. aeruginosa genetic material in the remaining wound tissue. Hyaluronidase readily degraded the HA derivatives, indicating that NO donor modification did not prohibit endogenous biodegradation pathways.

Relevance of Caspase-1 and Nlrp3 Inflammasome on Inflammatory Bone Resorption in A Murine Model of Periodontitis.

This study investigates the role of NLRP3 inflammasome and its main effector Caspase-1 in inflammation and alveolar bone resorption associated with periodontitis. Heat-killed Aggregatibacter actinomycetemcomitans (Aa) was injected 3x/week (4 weeks) into gingival tissues of wild-type (WT), Nlrp3-KO and Caspase1-KO mice. Bone resorption was measured by µCT and osteoclast number was determined by tartrate-resistant acid phosphatase (TRAP) staining. Inflammation was assessed histologically (H/E staining and immunofluorescence of CD45 and Ly6G). In vitro studies determined the influence of Nlrp3 and Caspase-1 in Rankl-induced osteoclast differentiation and activity and on LPS-induced expression of inflammation-associated genes. Bone resorption was significantly reduced in Casp1-KO but not in Nlrp3-KO mice. Casp1-KO mice had increased in osteoclast numbers, whereas the inflammatory infiltrate or on gene expression were similar to those of WT and Nlrp3-KO mice. Strikingly, osteoclasts differentiated from Nlrp3-deficient macrophages had increased resorbing activity in vitro. LPS-induced expression of Il-10, Il-12 and Tnf-α was significantly reduced in Nlrp3- and Casp1-deficient macrophages. As an inceptive study, these results suggest that Nlrp3 inflammasome does not play a significant role in inflammation and bone resorption in vivo and that Caspase-1 has a pro-resorptive role in experimental periodontal disease.

NLRC4 inflammasome has a protective role on inflammatory bone resorption in a murine model of periodontal disease.

There is virtually no information on the role of NLRC4 inflammasome on bone resorption and inflammation associated with periodontitis. Bacterial-associated experimental periodontitis was induced in wild-type (WT) and Nlrc4-KO C57BL/6 mice. 3 µL of a 1 × 109 UFC/mL PBS suspension of heat-killed Gram-negative bacteria were injected (3x/week for 4 weeks) directly into the gingival tissues of WT and Nlrc4-KO mice (n = 6/genotype). Control animals were injected bilaterally (3x/week for 4 weeks) in the same sites with the same volume of the PBS vehicle. Alveolar bone resorption was quantified by µCT. Inflammatory infiltrate in the gingival tissues was assessed qualitatively in H&E-stained slides and by the detection of a pan-leukocyte marker (CD45) and a neutrophil marker (Ly6G) using immunofluorescence. Modulation of Rankl, Mmp-13, Tnf-a, Il-6 and Il-10 expression in the gingival tissues was determined by RT-qPCR. Osteoclastogenesis was assessed in vivo by biochemical staining for TRAP. The relevance of NLRC4 for RANKL-induced osteoclastic differentiation and activity was investigated in vitro using bone marrow-derived macrophages from WT and Nlrc4-KO mice. Bone resorption was significantly greater in Nlrc4-KO mice; however there were no differences between WT and Nlrc4-KO mice on osteoclast numbers and on the inflammatory infiltrate. In vitro, osteoclast activity was significantly enhanced in Nlrc4-deficient macrophages; whereas RANKL-induced differentiation was not affected. Expression of the selected candidate genes was also similarly increased by the induction of experimental periodontal disease, except for the expression of Tnf-alpha and Il-10, which was already significantly higher in the gingival tissues of Nlrc4-KO mice. We conclude that NLRC4 inflammasome has a protective role on inflammatory bone resorption in this experimental model. Furthermore, the bone-sparing effect may be related with the modulation of osteoclast activity.