MicroRNA-27a-3p enhances the inflammatory phenotype of Juvenile Idiopathic Arthritis fibroblast-like synoviocytes.

BACKGROUND: Juvenile Idiopathic Arthritis (JIA) is the most prevalent chronic pediatric rheumatic disorder. In joints of JIA patients, aggressive phenotypic changes in fibroblast-like synoviocytes (FLS) of the synovial lining play a key role in inflammation. MicroRNAs are dysregulated in rheumatoid arthritis and JIA, including miR-27a-3p. However, it is not understood if miR-27a-3p, enriched in JIA synovial fluid (SF) and leukocytes, alters FLS function. METHODS: Primary JIA FLS cells were transfected with a miR-27a-3p mimic or a negative control microRNA (miR-NC) and stimulated with pooled JIA SF or inflammatory cytokines. Viability and apoptosis were analyzed by flow cytometry. Proliferation was evaluated using a 3H-thymidine incorporation assay. Cytokine production was assessed by qPCR and ELISA. Expression of TGF-ß pathway genes was determined using a qPCR array. RESULTS: MiR-27a-3p was constitutively expressed in FLS. Overexpression of miR-27a-3p caused increased interleukin-8 secretion in resting FLS, and interleukin-6 was elevated in SF-activated FLS compared to miR-NC. Furthermore, stimulation with pro-inflammatory cytokines augmented FLS proliferation in miR-27a-3p-transfected FLS relative to miR-NC. Expression of multiple TGF-ß pathway genes was modulated by overexpression of miR-27a-3p. CONCLUSIONS: MiR-27a-3p significantly contributes to FLS proliferation and cytokine production, making it a potential candidate for epigenetic therapy that targets FLS in arthritis.

Differentially Expressed Inflammation-Regulating MicroRNAs in Oligoarticular Juvenile Idiopathic Arthritis.

OBJECTIVE: To evaluate microRNA expression in synovial fluid (SF), plasma, and leukocytes from patients with juvenile idiopathic arthritis (JIA). METHODS: MicroRNA expression in pooled JIA plasma and SF was assessed by absolute quantitative droplet digital PCR array. The results were validated in individual patient samples. MicroRNA content in leukocytes and extracellular vesicles was evaluated by real-time PCR in JIA blood and SF. Blood microRNA expression was compared with healthy controls (HCs). Principal component analysis was used to profile JIA plasma and SF microRNAs, and the potential biological consequences of microRNA dysregulation were investigated by pathway analysis. RESULTS: MiR-15a-5p and miR-409-3p levels were higher in JIA plasma than in HC plasma. JIA SF contained elevated levels of miR-21-5p, miR-27a-3p, miR-146b-5p, miR-155-5p, and miR-423-5p, and decreased miR-192-5p and miR-451a, compared to JIA plasma. Extracellular vesicle analysis demonstrated variable encapsulation among selected microRNAs, with only miR-155-5p being represented substantially in extracellular vesicles. SF leukocytes also had higher expression of miR-21-5p, miR-27a-3p, miR-146b-5p, and miR-155-5p, and lower expression of miR-409-3p and miR-451a, relative to blood. No differences were observed between JIA and HC blood leukocytes. Clusters of microRNAs were commonly altered in JIA joint fluid and leukocytes compared to JIA blood samples. In silico analysis predicted that differentially expressed microRNAs in JIA target the transforming growth factor (TGF)-ß pathway. CONCLUSION: The expression of multiple microRNAs is dysregulated in JIA both locally and systemically, which may inhibit the TGF-ß pathway. These findings advance our knowledge of JIA immunopathogenesis and may lead to the development of targeted therapies.

High Dose Intravenous IgG Therapy Modulates Multiple NK Cell and T Cell Functions in Patients With Immune Dysregulation.

Intravenous immunoglobulin (IVIG) is an effective immunomodulatory treatment for immune dysregulation diseases. However, the mechanisms by which it reduces systemic inflammation are not well understood. NK cell cytotoxicity is decreased by IVIG in women with reduced fertility, but IVIG effects on NK cells in immune dysregulation are less clear. We hypothesized that IVIG modulation of lymphocyte function, especially in NK cells, is important for resolution of inflammation. Our aim was to identify IVIG-induced changes in a cohort of patients with Kawasaki disease (KD) and those that occur broadly in pediatric patients with various immune dysregulatory diseases. Peripheral blood mononuclear cells (PBMCs) of patients with KD or autoimmune/inflammatory diseases were phenotyped pre and post high dose IVIG treatment by flow cytometry. In KD patients, after IVIG infusion Treg cell frequency and the proportion of activated CD25+ immunoregulatory CD56bright NK cells was increased, and multiple lymphocyte subsets showed increased expression of the lymphoid tissue homing receptor CD62L. Importantly, IVIG treatment decreased the frequency of cells expressing the degranulation marker CD107a among cytotoxic CD56dim NK cells, which was reflected in a significant reduction in target cell killing and in decreased production of multiple pro-inflammatory mediators. Interestingly, the activating receptor CD336 was expressed on a higher proportion of CD56bright NK cells after IVIG in both KD and autoimmune/inflammatory patients while other NK receptors were increased differentially in each cohort. In autoimmune/inflammatory patients IVIG induced the proliferation marker CD71 on a higher percentage of CD56dim NK cells, and in contrast to KD patients, CD107a+ cells were increased in this subset. Furthermore, when PBMCs were stimulated ex vivo with IL-2 or Candida antigen in autologous plasma, more of the CD4+ T cells of KD patients expressed CD25 after IVIG therapy but fewer cytotoxic T cells were degranulated based on CD107a expression. In summary, IVIG treatment in patients with immune dysregulation has multiple effects, especially on NK cell subsets and CD4+ T cells, which are compatible with promoting resolution of inflammation. These novel findings provide insight into the immunomodulatory actions of IVIG in autoimmune and inflammatory conditions.

Universal Healthcare Coverage Does Not Ensure Adherence to Initial Colorectal Cancer Screening Guidelines.

INTRODUCTION: Colorectal cancer is the second leading cause of cancer deaths in the USA, and screening tests are underutilized. The aim of this study was to determine the proportion of individuals at average risk who utilized a recommended initial screening test in a universal healthcare coverage system. MATERIALS AND METHODS: This is a retrospective cohort study of active duty and retired military members as well as civilian beneficiaries of the Military Health System. Individuals born from 1960 to 1962 and eligible for full benefits on their 50th birthday were evaluated. Military rank or rank of benefits sponsor was used to determine socioeconomic status. Adherence to the U.S. Preventive Services Task Force guidelines for initial colorectal cancer screening was determined using "Current Procedural Terminology" and "Healthcare Common Procedure Coding System" codes for colonoscopy, sigmoidoscopy, fecal occult blood test, and fecal immunohistochemistry test. Average risk individuals who obtained early screening ages 47 to 49 were also identified. RESULTS: This study identified 275,665 individuals at average risk. Of these, 105,957 (38.4%) adhered to screening guidelines. An additional 19,806 (7.2%) individuals were screened early. Colonoscopy (82.7%) was the most common screening procedure. Highest odds of screening were associated with being active duty military (odds ratio [OR] 3.63, 95% confidence interval [CI] 3.43 to 3.85), having highest socioeconomic status (OR 2.37, 95% CI 2.31 to 2.44), and having managed care insurance (OR 4.36, 95% CI 4.28 to 4.44). CONCLUSIONS: Universal healthcare coverage does not ensure initial colorectal cancer screening utilization consistent with guidelines no does it eliminate disparities.

Highly efficient serum-free manipulation of miRNA in human NK cells without loss of viability or phenotypic alterations is accomplished with TransIT-TKO.

Natural killer (NK) cells are innate lymphocytes with functions that include target cell killing, inflammation and regulation. NK cells integrate incoming activating and inhibitory signals through an array of germline-encoded receptors to gauge the health of neighbouring cells. The reactive potential of NK cells is influenced by microRNA (miRNA), small non-coding sequences that interfere with mRNA expression. miRNAs are highly conserved between species, and a single miRNA can have hundreds to thousands of targets and influence entire cellular programs. Two miRNA species, miR-155-5p and miR-146a-5p are known to be important in controlling NK cell function, but research to best understand the impacts of miRNA species within NK cells has been bottlenecked by a lack of techniques for altering miRNA concentrations efficiently and without off-target effects. Here, we describe a non-viral and straightforward approach for increasing or decreasing expression of miRNA in primary human NK cells. We achieve >90% transfection efficiency without off-target impacts on NK cell viability, education, phenotype or function. This opens the opportunity to study and manipulate NK cell miRNA profiles and their impacts on NK cellular programs which may influence outcomes of cancer, inflammation and autoimmunity.

Distribution of Legionella and bacterial community composition among regionally diverse US cooling towers.

Cooling towers (CTs) are a leading source of outbreaks of Legionnaires' disease (LD), a severe form of pneumonia caused by inhalation of aerosols containing Legionella bacteria. Accordingly, proper maintenance of CTs is vital for the prevention of LD. The aim of this study was to determine the distribution of Legionella in a subset of regionally diverse US CTs and characterize the associated microbial communities. Between July and September of 2016, we obtained aliquots from water samples collected for routine Legionella testing from 196 CTs located in eight of the nine continental US climate regions. After screening for Legionella by PCR, positive samples were cultured and the resulting Legionella isolates were further characterized. Overall, 84% (164) were PCR-positive, including samples from every region studied. Of the PCR-positive samples, Legionella spp were isolated from 47% (78), L. pneumophila was isolated from 32% (53), and L. pneumophila serogroup 1 (Lp1) was isolated from 24% (40). Overall, 144 unique Legionella isolates were identified; 53% (76) of these were Legionella pneumophila. Of the 76 L. pneumophila isolates, 51% (39) were Lp1. Legionella were isolated from CTs in seven of the eight US regions examined. 16S rRNA amplicon sequencing was used to compare the bacterial communities of CT waters with and without detectable Legionella as well as the microbiomes of waters from different climate regions. Interestingly, the microbial communities were homogenous across climate regions. When a subset of seven CTs sampled in April and July were compared, there was no association with changes in corresponding CT microbiomes over time in the samples that became culture-positive for Legionella. Legionella species and Lp1 were detected frequently among the samples examined in this first large-scale study of Legionella in US CTs. Our findings highlight that, under the right conditions, there is the potential for CT-related LD outbreaks to occur throughout the US.

On-DNA Pd and Cu promoted C-N cross-coupling reactions.

Encoded library technology (ELT) is a novel hit identification platform synergistic with HTS, fragment hit ID and focused screening. It provides both an ultra high-throughput and a cost-efficient tool for the discovery of small molecules that bind to protein targets of pharmaceutical interest. The success of ELT relies heavily on the chemical diversity accessed through DNA-encoded library (DEL) synthesis. We developed unprecedented Pd and Cu(i) promoted C-N cross-coupling reactions between a DNA-conjugated aryl iodide and primary amines. These reported reactions have strong potential for application in DNA-encoded library (DEL) synthesis.

Frequency and long-term follow-up of trapped fourth ventricle following neonatal posthemorrhagic hydrocephalus.

OBJECTIVE Intraventricular hemorrhage (IVH) is a common complication of premature neonates with small birth weight, which often leads to hydrocephalus and treatment with ventriculoperitoneal (VP) shunting procedures. Trapped fourth ventricle (TFV) can be a devastating consequence of the subsequent occlusion of the cerebral aqueduct and foramina of Luschka and Magendie. METHODS The authors retrospectively reviewed 8 consecutive cases involving pediatric patients with TFV following VP shunting for IVH due to prematurity between 2003 and 2012. The patients ranged in gestational age from 23.0 to 32.0 weeks, with an average age at first shunting procedure of 6.1 weeks (range 3.1-12.7 weeks). Three patients were managed with surgery. Patients received long-term radiographic (mean 7.1 years; range 3.4-12.2 years) and clinical (mean 7.8 years; range 4.6-12.2 years) follow-up. RESULTS The frequency of TFV following VP shunting for neonatal posthemorrhagic hydrocephalus was found to be 15.4%. Three (37.5%) patients presented with symptoms of posterior fossa compression and were treated surgically. All of these patients showed signs of radiographic improvement with stable or improved clinical examinations during postoperative follow-up. Of the 5 patients treated conservatively, 80% experienced stable ventricular size and 1 patient experienced a slight increase (3 mm) on imaging. All of the nonsurgical patients showed stable to improved clinical examinations over the follow-up period. CONCLUSIONS The frequency of TFV among premature IVH patients is relatively high. Most patients with TFV are asymptomatic at presentation and can be managed without surgery. Symptomatic patients may be treated surgically for decompression of the fourth ventricle.

Impact of supplemental vitamin K1 administration on postoperative blood component requirements after craniosynostosis repair: a prospective, placebo-controlled, randomized, blinded study.

Total cranial vault craniosynostosis repairs often require additional blood transfusions in the intensive care unit. Vitamin K1 participates in hepatic production of procoagulant proteins, and body stores of vitamin K1 are limited and dietary dependent. Surgical stress and diet interference may place infants at risk for vitamin K deficiency. Through design of a surgically stratified, randomized, placebo-controlled, blinded pilot study, we evaluated impact of vitamin K1 supplementation on coagulation parameters in infants after craniosynostosis repair. Patients received intramuscular vitamin K1 or placebo coincident with surgical incision. Serum vitamin K1 levels, protein induced in vitamin K absence-prothrombin, and factor VII were obtained at predetermined intervals after surgery. Patients received blood products in the intensive care unit in accordance with transfusion thresholds. Fifteen patients (vitamin K1 = 6, placebo = 9) completed the study procedures. Despite group assignment, patients received an average of 3 postoperative transfusions. Variations were observed with respect to intraoperative resuscitation of patients between comparably trained pediatric anesthesiologists. Thirty-three percent of patients were vitamin K1 deficient on 1 or more laboratory specimens. All breast-fed patients became deficient. Compared with placebo, elevated serum vitamin K1 levels at 6, 12, and 24 hours in the active drug group (P < 0.0001) were not associated with increased factor VII levels or reduced need for postoperative blood products. However, lack of a standardized intraoperative resuscitation plan may contribute to postoperative coagulopathy and is a major study limitation.