Lysophosphatidic acid down-regulates human RIPK4 mRNA in keratinocyte- derived cell lines.

The tight control of proliferating keratinocytes is vital to the successful function of the skin. Differentiation of dividing cells is necessary to form a skin barrier. The same dividing cells are necessary to heal wounds and when malignant form tumors. RIPK4, a serine-threonine kinase, plays critical roles in these processes. Its loss of function was associated with pathological keratinocyte proliferation and development of squamous cell carcinoma (SCC) in humans and mice. The current study extends previous findings in the importance of RIPK4 in keratinocyte proliferation. A serum-derived phospholipid, lysophosphatidic acid (LPA), was identified as an important biologic inhibitor of RIPK4. LPA functions by inhibiting the transcription of RIPK4 mRNA. LPA treatment led to increased keratinocyte proliferation, and this was compromised in cells with reduced RIPK4 expression. The current study may help to explain the mechanism by which RIPK4 was downregulated during SCC progression and provide insights on RIPK4 functions. It may also allow for targeting of RIPK4 through a natural component of serum.

Window of Susceptibility to Acute Otitis Media Infection.

BACKGROUND: Contemporary, quantitative data are needed to inform recommendations and decision-making regarding referral and surgeon endorsement of tympanostomy tube placement in young children with recurrent acute otitis media (AOM). METHODS: A prospective, observational cohort study of 286 children in a primary care pediatric practice setting, who had at least 1 AOM (range 1-8). Children were followed longitudinally from 6 to 36 months old. AOMs were microbiologically confirmed by tympanocentesis for diagnostic accuracy. A window of susceptibility (WOS) was defined as AOMs closely spaced in time with no gap in occurrence >6 months. For prediction of total number of AOMs, we used a quasi-poisson generalized linear model. RESULTS: Eighty percent of AOMs occurred during child age 6 to 21 months old. Seventy two percent of WOS intervals were <5 months and 97% were <10 months. Clinically applicable models were developed to predict which children would benefit most from tympanostomy tubes. Significant predictors were child age at the first AOM (P < .001) and daycare attendance (P = .03). The age of a child when 2, 3, or 4 AOMs had occurred allowed prediction of the number of additional AOMs that might occur. After insertion of tympanostomy tubes, 16 (52%) of 31 children had no additional AOMs. CONCLUSIONS: Recurrent AOM occurs in a narrow WOS and number of AOMs can be predicted at time of AOM based on child age and daycare attendance. Insertion of tympanostomy tubes likely occurs in many children after the WOS to recurrent AOM has passed or only 1 more AOM may be prevented at most.

Antibiotic Use and Vaccine Antibody Levels.

BACKGROUND: The majority of children are prescribed antibiotics in the first 2 years of life while vaccine-induced immunity develops. Researchers have suggested a negative association of antibiotic use with vaccine-induced immunity in adults, but data are lacking in children. METHODS: From 2006 to 2016, children aged 6 to 24 months were observed in a cohort study. A retrospective, unplanned secondary analysis of the medical record regarding antibiotic prescriptions and vaccine antibody measurements was undertaken concurrently. Antibody measurements relative to diphtheria-tetanus-acellular pertussis (DTaP), inactivated polio (IPV), Haemophilus influenzae type b (Hib), and pneumococcal conjugate (PCV) vaccines were made. RESULTS: In total, 560 children were compared (342 with and 218 without antibiotic prescriptions). Vaccine-induced antibody levels to several DTaP and PCV antigens were lower (P < .05) in children given antibiotics. A higher frequency of vaccine-induced antibodies below protective levels in children given antibiotics occurred at 9 and 12 months of age (P < .05). Antibiotic courses over time was negatively associated with vaccine-induced antibody levels. For each antibiotic course the child received, prebooster antibody levels to DTaP antigens were reduced by 5.8%, Hib by 6.8%, IPV by 11.3%, and PCV by 10.4% (all P ≤ .05), and postbooster antibody levels to DTaP antigens were reduced by 18.1%, Hib by 21.3%, IPV by 18.9%, and PCV by 12.2% (all P < .05). CONCLUSIONS: Antibiotic use in children <2 years of age is associated with lower vaccine-induced antibody levels to several vaccines.

Biomarkers of DNA damage response improve in vitro micronucleus assays by revealing genotoxic mode of action and reducing the occurrence of irrelevant positive results.

We have previously described two flow cytometry-based in vitro genotoxicity tests: micronucleus (MN) scoring (MicroFlow®) and a multiplexed DNA damage response biomarker assay (MultiFlow®). Here, we describe a strategy for combining the assays in order to efficiently supplement MN analyses with a panel of biomarkers that comment on cytotoxicity (i.e. relative nuclei count, relative increased nuclei count, cleaved PARP-positive chromatin and ethidium monoazide-positive chromatin) and genotoxic mode of action (MoA; i.e. γH2AX, phospho-histone H3, p53 activation and polyploidy). For these experiments, human TK6 cells were exposed to each of 32 well-studied reference chemicals in 96-well plates for 24 continuous hours. The test chemicals were evaluated over a range of concentrations in the presence and absence of a rat liver S9-based metabolic activation system. MultiFlow assay data were acquired at 4 and 24 h, and micronuclei were scored at 24 h. Testing 32 chemicals in two metabolic activation arms translated into 64 a priori calls: 42 genotoxicants and 22 non-genotoxicants. The MN assay showed high sensitivity and moderate specificity (90% and 68%, respectively). When a genotoxic call required significant MN and MultiFlow responses, specificity increased to 95% without adversely affecting sensitivity. The dose-response data were analysed with PROAST Benchmark Dose (BMD) software in order to calculate potency metrics for each endpoint, and ToxPi software was used to synthesise the resulting lower and upper bound 90% confidence intervals into visual profiles. The BMD/ToxPi combination was found to represent a powerful strategy for synthesising multiple BMD confidence intervals, as the software output provided MoA information as well as insights into genotoxic potency.

Pneumonia, Sinusitis, Influenza and Other Respiratory Illnesses in Acute Otitis Media-Prone Children.

BACKGROUND: Recurrent acute otitis media in the first years of life can be explained by immune dysfunction. Consequently, it would be expected that otitis-prone (OP) children would be more susceptible to other infectious diseases, especially respiratory infections, since a component of the immune problem involves nasopharyngeal innate immunity. DESIGN: Cohort study with prospective identification of all physician-diagnosed, medically attended respiratory illness visits in children 6 months to 5 years of age to determine the incidence of pneumonia, acute sinusitis, influenza and other bacterial and viral infections among OP compared with non-OP (NOP) children. Tympanocentesis to microbiologically confirm acute otitis media disease. RESULTS: Two hundred eighty-five children were studied. Thirty-nine met a standard definition of stringently defined OP (sOP) determined by tympanocentesis and 246 were NOP. sOP children had increased frequency of presumptive respiratory infections, pneumonia (6-fold higher, P < 0.001), sinusitis (2.1-fold higher, P = 0.026) and influenza (2.9-fold higher, P = 0.002), compared with NOP children. Demographic and risk factor covariate-adjusted fold difference between sOP and NOP children for all respiratory infection illness visits was 2.4-fold (P < 0.00001) at 6-18 months of age, 2.2-fold (P < 0.00001) at 18-30 months of age and at age and 2.4-fold (P = 0.035) higher at 30 to 42 months. For both sOP and NOP children, more frequent medically attended respiratory infection illness visits from 6-18 months of age predicted more frequent visits experienced from 18-60 months of age. CONCLUSIONS: Clinicians should be aware of a significant increased likelihood of bacterial and viral respiratory infection proneness among OP children.

Nasopharyngeal microbiome analyses in otitis-prone and otitis-free children.

OBJECTIVES: About 10-15% children develop frequent acute otitis media (AOM) confirmed by tympanocentesis. These children are designated sOP (stringently defined otitis-prone) because all AOM episodes have been microbiologically confirmed. The cause of otitis-proneness in sOP children is multi-factorial, including frequent otopathogen nasopharyngeal (NP) colonization and deficiency in innate and adaptive immune responses. A largely unexplored contributor to otitis proneness is NP microbiome composition. Since the microbiome modulates otopathogen NP colonization and immune responses, we hypothesized that the NP microbiome composition in sOP children might be dysregulated. METHODS: We performed 16S rRNA sequencing to analyze microbiome composition in 157 NP samples from 28 sOP and 68 AOM-free children when they were 6 months or 12 months old and healthy. Bioinformatic approaches were employed to examine the composition difference between the two populations and its correlation with changes in levels of inflammatory cytokines. RESULTS: A different global microbiome profile and reduced alpha diversity was observed in the NP microbiome of sOP children when 6 months old, compared with that from AOM-free children of the same age. This difference was resolved when groups were compared at 12 months old. We found 4 bacterial genera-Bacillus, Veillonella, Gemella, and Prevotella-correlated with higher levels of pro-inflammatory cytokines in the NP. Those 4 bacterial genera were in lower abundance in sOP compared to AOM-free children. CONCLUSION: Dysbiosis occurs in the NP microbiome of sOP children at an early age even when they were healthy. This dysbiosis correlates with a lower inflammatory state in the NP of these children.

Synchrony in serum antibody response to conserved proteins of Moraxella catarrhalis in young children.

Conserved Moraxella catarrhalis (Mcat) proteins, oligopeptide permease (Opp)A, hemagglutinin (Hag), outer membrane protein (OMP) CD, Pilin A clade 2 (PilA2), and Moraxella surface protein (Msp) 22 have been studied as vaccine candidates. Children who experience frequent acute otitis media (AOM) confirmed with pathogen identification by tympanocentesis are referred to as stringently-defined otitis prone (sOP). Synchrony of serum antibody responses against 5 Mcat proteins, OppA, Hag, OMP CD, PilA2, and Msp22 resulting from nasopharyngeal colonization and AOM was studied for 85 non-otitis prone (NOP) children and 34 sOP children. Changes in serum IgG were quantitated with ELISA. Serum IgG antibody levels against OppA, Hag, OMP CD, and Msp22 rose in synchrony in NOP and sOP children; that is, the proteins appeared equally and highly immunogenic in children at age 6 to 22-25 months old and then leveled off in their rise at 22-25 to 30 months old. In contrast, rises of PilA2 were slow from 6 months old and kept constant and did not level off significantly before 30 months old. OppA, Hag, OMP CD, and Msp22 elicited a synchronous acquisition of naturally-induced serum antibody in young children. A multi-valent Mcat protein vaccine combining OppA, Hag, OMP CD, and Msp22 may exhibit less antigen competition when administered as a combination vaccine in young children.

Examining the Impact of Prior Models in Transmural Electrophysiological Imaging: A Hierarchical Multiple-Model Bayesian Approach.

Noninvasive cardiac electrophysiological (EP) imaging aims to mathematically reconstruct the spatiotemporal dynamics of cardiac sources from body-surface electrocardiographic (ECG) data. This ill-posed problem is often regularized by a fixed constraining model. However, a fixed-model approach enforces the source distribution to follow a pre-assumed structure that does not always match the varying spatiotemporal distribution of actual sources. To understand the model-data relation and examine the impact of prior models, we present a multiple-model approach for volumetric cardiac EP imaging where multiple prior models are included and automatically picked by the available ECG data. Multiple models are incorporated as an Lp-norm prior for sources, where p is an unknown hyperparameter with a prior uniform distribution. To examine how different combinations of models may be favored by different measurement data, the posterior distribution of cardiac sources and hyperparameter p is calculated using a Markov Chain Monte Carlo (MCMC) technique. The importance of multiple-model prior was assessed in two sets of synthetic and real-data experiments, compared to fixed-model priors (using Laplace and Gaussian priors). The results showed that the posterior combination of models (the posterior distribution of p) as determined by the ECG data differed substantially when reconstructing sources with different sizes and structures. While the use of fixed models is best suited in situations where the prior assumption fits the actual source structures, the use of an automatically adaptive set of models may have the ability to better address model-data mismatch and to provide consistent performance in reconstructing sources with different properties.

Effect of pH and oxygen on biofilm formation in acute otitis media associated NTHi clinical isolates.

OBJECTIVES/HYPOTHESIS: Biofilms occur in animal models of acute otitis media (AOM) and in children with recurrent AOM (rAOM) and chronic otitis media with effusion (OME). We therefore studied the ability of nontypeable Haemophilus influenzae (NTHi) strains from children to form biofilms in vitro under conditions we presumed occurred in the middle ear during AOM, rAOM, and OME. STUDY DESIGN: Evaluate NTHi isolates for biofilm formation across a pH range under aerobic, microaerophilic, and anaerobic conditions. METHODS: Using a crystal violet biofilm assay we studied 12 NTHi pediatric clinical isolates to investigate biofilm formation over a pH range of 4.5 to 10 under aerobic, microaerophilic, and anaerobic conditions. RESULTS: Our findings included: 1) not all clinical NTHi strains form biofilms (75% did); 2) the pH of middle ear fluid collected from AOM (n = 170; age range, 4-36 months), rAOM (n = 54; age range, 7-36 months), and OME (n = 30; age range, 9-60 months) subjects tested immediately after withdrawal was similar (mean = 8.0;range 7.0-9.0); 3) biofilms formed optimally at pH 8.0, a finding that is consistent with previous studies by other investigators; 4) biofilms did not form under aerobic conditions as likely occurs in AOM, whereas under microaerophilic and anaerobic conditions biofilm formation was observed as likely occurs during rAOM and OME. CONCLUSIONS: We concluded that biofilm formation by NTHi does not occur in all strains, occurs best where the pH = 8.0 and in anaerobic conditions as likely occurs in children during rAOM and OME. However, biofilm formation is limited or absent under aerobic conditions as likely occurs during AOM. LEVEL OF EVIDENCE: NA.