A Multicenter Training and Interrater Reliability Study of the BASED Score for Infantile Epileptic Spasms Syndrome.

PURPOSE: The best possible outcomes in infantile epileptic spasms syndrome require electroclinical remission; however, determining electrographic remission is not straightforward. Although the determination of hypsarrhythmia has inadequate interrater reliability (IRR), the Burden of AmplitudeS and Epileptiform Discharges (BASED) score has shown promise for the reliable interictal assessment of infantile epileptic spasms syndrome. Our aim was to develop a BASED training program and assess the IRR among learners. We hypothesized moderate or better IRR for the final BASED score and the presence or absence of epileptic encephalopathy (+/-EE). METHODS: Using a web-based application, 31 learners assessed 12 unmarked EEGs (length 1-6 hours) from children with infantile epileptic spasms syndrome. RESULTS: For all readers, the IRR was good for the final BASED score (intraclass correlation coefficient 0.86) and +/-EE (Marginal Multirater Kappa 0.63). For all readers, the IRR was fair to good for all individual BASED score elements. CONCLUSIONS: These findings support the use of our training program to quickly learn the BASED scoring method. The BASED score may be a valuable clinical and research tool. Given that the IRR for the determination of epileptic encephalopathy is not perfect, clinical acumen remains paramount. Additional experience with the BASED scoring technique among learners and advances in collaborative EEG evaluation platforms may improve IRR.

Automated detection of ripple oscillations in long-term scalp EEG from patients with infantile spasms.

Objective.Scalp high-frequency oscillations (HFOs) are a promising biomarker of epileptogenicity in infantile spasms (IS) and many other epilepsy syndromes, but prior studies have relied on visual analysis of short segments of data due to the prevalence of artifacts in EEG. Here we set out to robustly characterize the rate and spatial distribution of HFOs in large datasets from IS subjects using fully automated HFO detection techniques.Approach.We prospectively collected long-term scalp EEG data from 12 subjects with IS and 18 healthy controls. For patients with IS, recording began prior to diagnosis and continued through initiation of treatment with adrenocorticotropic hormone (ACTH). The median analyzable EEG duration was 18.2 h for controls and 84.5 h for IS subjects (∼1300 h total). Ripples (80-250 Hz) were detected in all EEG data using an automated algorithm.Main results.HFO rates were substantially higher in patients with IS compared to controls. In IS patients, HFO rates were higher during sleep compared to wakefulness (median 5.5 min-1and 2.9 min-1, respectively;p = 0.002); controls did not exhibit a difference in HFO rate between sleep and wakefulness (median 0.98 min-1and 0.82 min-1, respectively). Spatially, IS patients exhibited significantly higher rates of HFOs in the posterior parasaggital region and significantly lower HFO rates in frontal channels, and this difference was more pronounced during sleep. In IS subjects, ACTH therapy significantly decreased the rate of HFOs.Significance.Here we provide a detailed characterization of the spatial distribution and rates of HFOs associated with IS, which may have relevance for diagnosis and assessment of treatment response. We also demonstrate that our fully automated algorithm can be used to detect HFOs in long-term scalp EEG with sufficient accuracy to clearly discriminate healthy subjects from those with IS.

Cytokines in Mycoplasma pneumoniae infections.

Mycoplasma pneumoniae (M. pneumoniae) is one of the smallest free-living bacteria known. Along with other unique characteristics of this genus, it lacks the typical peptidoglycan cell wall of most eubacteria. Best known for causing tracheobronchitis and atypical pneumonia in humans, this pathogen also causes a number of extrapulmonary syndromes such as meningitis/encephalitis and arthritis. Recent studies also suggest that infection may be associated with chronic conditions such as asthma. Although the mechanisms of M. pneumoniae pathogenesis remain to be elucidated, one important component of M. pneumoniae infections is the induction of proinflammatory and other cytokines in both acute and chronic conditions. In this review, we survey the induction of cytokines by M. pneumoniae in different model systems, and we discuss the possible role of induced cytokines in M. pneumoniae pathogenesis.

A functional epitope of the pneumococcal surface adhesin A activates nasopharyngeal cells and increases bacterial internalization.

Pneumococcal surface adhesin A (PsaA) is a putative pneumococcal (Pnc) adhesin known to bind to nasopharyngeal (NP) epithelial cells. This study evaluated the effect of peptides within a functional domain of PsaA on NP cells. Detroit 562 NP cells were treated with synthetic peptides derived from PsaA (P4, P6, and P7; 28, 12, and 16 amino acids, respectively). The P4 peptide also binds to NP cells. Analysis of P4-treated NP cells by transmission electron microscopy revealed major cytological changes. Of 9 cytokines analyzed, a 6-fold increase in FGFb secretion at 3 and 6h (11-fold at 12h) was found post-P4 treatment of NP cells. There was a simultaneous reduction in the secreted levels of IL-6, IL-8, and VEGF. We observed enhancement in the adherence of Pnc strains to P4-treated NP cells (2-38-fold increase). Enhancement in adherence (2-fold increase) to P4-treated NP cells was also recorded with other streptococcal species (Streptococcus mitis and Streptococcus pyogenes). Internalization experiments demonstrated that 45% of the adherent bacteria were actually internalized after pretreatment with P4 peptide as compared to controls. Peptide fragments of P4, P6 and P7 did not activate NP cells to the extent of P4 peptide. The P4-mediated enhancement of Pnc adherence was blocked (100%) by anti-P4 antibodies, confirming the specificity of the P4 sequence for NP cell activation. Our data suggests that this functional domain of PsaA contained within the P4 sequence binds and activates NP cells to facilitate Pnc invasion.

Mycoplasma pneumoniae subtype-independent induction of proinflammatory cytokines in THP-1 cells.

Mycoplasma pneumoniae can be divided into two main subtypes depending on the amino acid sequences of the P1 adhesin and the P65 protein, both located in the attachment organelle. Differences between these subtypes in infectivity, virulence and interaction with host cells have not been extensively studied. Using ELISA to measure released protein and real-time PCR to quantify mRNA, we have demonstrated that both M. pneumoniae subtypes significantly increased tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-8 (IL-8) at comparable levels in THP-1 cells over a 72 h period of time. However, subtype 2 induced a statistically significant increase (P<0.001) in the release of interleukin-1beta at 24 h post-infection compared to subtype 1. These data provide evidence that the induction of proinflammatory cytokine gene and protein expression by M. pneumoniae is not dependent on the infecting subtype.

Interference in microsphere flow cytometric multiplexed immunoassays for human cytokine estimation.

The present study describes positive and negative interference of human cytokine measurement in multiplexed bead-based immunoassays. Significant differences in measured IL-6 and TNF-alpha values in 30 normal human plasma samples were apparent depending on whether measurements were with a 2-plex assay or embedded in a multiplex of 8-or more cytokine antibody pairs, as well as among the kits of 3-different vendors. Sample diluents containing proprietary blocking ingredients were shown to greatly affect the outcome of measured cytokine values. Additionally, recovery of IL-6 and TNF-alpha from spiked samples suggests significant negative interference from either endogenous antibodies, soluble receptors or anti-cytokine antibodies in 10% and 26% of samples, respectively. While it is evident that multiplexed immunoassays hold great promise for cytokine profiling, there are still important issues needing further study. Especially needed are universally optimized sample diluents, uniformly calibrated standards with mass values, and internal assay controls, which should greatly facilitate intralaboratory accuracy and precision and interlaboratory comparisons of cytokine measurements. Possible causes of interference and remedies are discussed.

Regulation of proinflammatory cytokines in human lung epithelial cells infected with Mycoplasma pneumoniae.

Mycoplasma pneumoniae is a small bacterium without a cell wall that causes tracheobronchitis and atypical pneumonia in humans. It has also been associated with chronic conditions, such as arthritis, and extrapulmonary complications, such as encephalitis. Although the interaction of mycoplasmas with respiratory epithelial cells is a critical early phase of pathogenesis, little is known about the cascade of events initiated by infection of respiratory epithelial cells by mycoplasmas. Previous studies have shown that M. pneumoniae can induce proinflammatory cytokines in several different study systems including cultured murine and human monocytes. In this study, we demonstrate that M. pneumoniae infection also induces proinflammatory cytokine expression in A549 human lung carcinoma cells. Infection of A549 cells resulted in increased levels of interleukin-8 (IL-8) and tumor necrosis factor alpha mRNA, and both proteins were secreted into culture medium. IL-1 beta mRNA also increased after infection and IL-1 beta protein was synthesized, but it remained intracellular. In contrast, levels of IL-6 and gamma interferon mRNA and protein remained unchanged or undetectable. Using protease digestion and antibody blocking methods, we found that M. pneumoniae cytoadherence is important for the induction of cytokines. On the other hand, while M. pneumoniae protein synthesis and DNA synthesis do not appear to be prerequisites for the induction of cytokine gene expression, A549 cellular de novo protein synthesis is responsible for the increased cytokine protein levels. These results suggest a novel role for lung epithelial cells in the pathogenesis of M. pneumoniae infection and provide a better understanding of M. pneumoniae pathology at the cellular level.

Interleukin-1beta responses to Mycoplasma pneumoniae infection are cell-type specific.

Interleukin-1beta (IL-1beta) is a major proinflammatory cytokine that is involved in many important cellular functions such as proliferation, differentiation, and activation of different cell types. Its mature form is released from the cells in response to various bacterial and viral infections, and it plays a significant role in host defense. Mycoplasma pneumoniae is a small bacterium without a cell wall that causes tracheobronchitis and atypical pneumonia in humans following attachment to respiratory epithelium, as well as extrapulmonary infections. Very little is known about the role of cytokines in pathogenesis or the response of target cells to M.pneumoniae attachment. The purpose of this study was to investigate the ability of M. pneumoniae to induce IL-1beta in human lung epithelial carcinoma A549 and in human monocytic U937 cell lines. Following M. pneumoniae infection, both IL-1beta mRNA and protein were induced in A549 cells vs. no induction in uninfected cells; however, the protein remained inside the A549 cells. Similarly, M. pneumoniae infection strongly increased mRNA and extracellular protein levels in U937 cells, which unlike A549 cells did exhibit baseline constitutive levels. De novo IL-1beta protein expression was verified by cycloheximide studies. M. pneumoniae infection did not affect constitutive caspase-1 mRNA or protein levels in either cell line. Reduced caspase-1 activity in A549 cell lysates suggests the presence of an endogenous caspase-1 inhibitory component in the A549 cells. These collective data confirm previous studies that show that M. pneumoniae is a potent inducer of cytokines following adherence to host target cells, and establish that IL-1beta release in response to M. pneumoniae infection is cell-type specific, thus emphasizing the importance of carefully considering multiple cell types in M. pneumoniae pathogenesis studies involving both immune cells and cytokine release patterns.

Induction of proinflammatory cytokines in human lung epithelial cells during Chlamydia pneumoniae infection.

Chlamydia pneumoniae is an obligate intracellular human pathogen that causes acute respiratory diseases such as pneumonia and bronchitis. Previous studies have established that C. pneumoniae can induce cytokines in mouse and/or human cells, but little information is available on the cytokine response of respiratory epithelial cells, a first line of infection. In this study, heparin treatment of C. pneumoniae significantly reduced its ability to induce interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-alpha) mRNA in human lung carcinoma cells, indicating that cytadherence is an important early stimulus for induction of proinflammatory mediators. Although the IL-8, gamma interferon, and TNF-alpha message was consistently induced by infection of A549 cells not treated with heparin, only an elevation of IL-8 protein was detected in A549 supernatants. A549 IL-beta and IL-6 mRNA and supernatant protein profiles were not significantly changed by infection. Heat or UV inactivation of C. pneumoniae only partially reduced the cytokine response, and inhibition of C. pneumoniae protein or DNA synthesis did not affect its ability to induce cytokine gene expression. To prevent stress-induced cytokine release by the A549 cells, centrifugation was not utilized for infection experiments. These experiments establish the importance of cytadherence in cytokine release by cells of respiratory epithelial origin and suggest that further work in the area of cytokine mediators is warranted to gain valuable pathogenic and therapeutic insights.