These EEGs have a high degree of artifact contamination and often short recording lengths. As both sample sizes and EEG channel densities increase, traditional processing approaches like manual data rejection are becoming unsustainable. Moreover, such subjective approaches preclude standardized metrics of data quality, despite the heightened importance of such measures for EEGs with high rates of initial artifact contamination. There is presently a paucity of automated resources for processing these EEG data and no consistent reporting of data quality measures. To address these challenges, we propose the Harvard Automated Processing Pipeline for EEG (HAPPE) as a standardized, automated pipeline compatible with EEG recordings of variable lengths and artifact contamination levels, including high-artifact and short EEG recordings from young children or those with neurodevelopmental disorders. HAPPE processes event-related and resting-state EEG data from raw files through a series of filtering, artifact rejection, and re-referencing steps to processed EEG suitable for time-frequency-domain analyses. HAPPE also includes a post-processing report of data quality metrics to facilitate the evaluation and reporting of data quality in a standardized manner. Here, we describe each processing step in HAPPE, perform an example analysis with EEG files we have made freely available, and show that HAPPE outperforms seven alternative, widely-used processing approaches. HAPPE removes more artifact than all alternative approaches while simultaneously preserving greater or equivalent amounts of EEG signal in almost all instances. We also provide distributions of HAPPE's data quality metrics in an 867 file dataset as a reference distribution and in support of HAPPE's performance across EEG data with variable artifact contamination and recording lengths. HAPPE software is freely available under the terms of the GNU General Public License at  Electroencephalography (EEG) is a sensitive means to noninvasively capture neurophysiological activity with clinical and basic science utility across a number of fields. However, during acquisition, the EEG signal is contaminated by both experimental artifacts like electrical interference and electrode displacement, and participant-induced artifacts like eye and muscle movements. These artifact signals are in many cases far more prominent than the neurophysiological signal, significantly distorting EEG as a measure of brain function if left uncorrected , and protocols typically have far shorter EEG collection times than those with healthy adults to accommodate reduced tolerance for testing  is to provide an automated, standardized approach for processing these classes of EEG data. Specifically, HAPPE is designed for data with high levels of artifact or very short recording length , although the pipeline may appropriately be used with longer or less-contaminated data. HAPPE integrates Matlab-based  code with freely available academic software, including EEGLAB functions  Matlab files for resting-state EEG, and the data may have differing identifying variable names across files. HAPPE reads EGI-exported simple binary files for event-related EEG presently. However, users can easily modify the importing code to read any file format for any resting-state or event-related EEG that EEGLAB accepts. An individual HAPPE run should include only resting-state data or only event-related data, and users must specify one or the other as the input file type. HAPPE is currently compatible with EEG layouts of 64 and 128 channels. Each run of HAPPE must include files collected with the same channel layout (company and electrode number) and users must specify the appropriate channel layout in a given HAPPE run. Users who wish to include different EEG channel layouts within a single pipeline run can easily do so by accessing HAPPE through the Batch EEG Automated Processing Platform (BEAPP) software, available at HAPPE consists of the following processing steps for EEG data:FilteringEEG channel subset selectionElectrical (line) noise removalBad channel rejectionWavelet-enhanced thresholding (W-ICA)ICA with automated component rejectionAutomated segment rejection Segmentation Interpolation of bad channels for each epoch Interpolation of bad channelsRe-referencingBelow, the implementation of each processing step in HAPPE is described in detail.All files are subject to a 1 Hz high-pass filter. The filter removes non-stationary signal drift across the recording and serves as a pre-processing step for the electrical noise removal and ICA steps that follow 2 data samples to undergo ICA decomposition , that is, 983.04 s of recording  to be reliably decomposed with ICA.Users must specify a subset of 19 channels corresponding to the International 10–20 system electrodes (without the Cz reference electrode) for their channel layout (for use later with automated ICA artifact rejection) and any additional channels they wish to be processed in HAPPE. Channels that are not provided in the subset list are removed from subsequent processing and cannot be recovered later. For example, for data from a 128-channel net where the user selects 50 channels, the post-HAPPE processed data will contain only data for those 50 selected channels. Channel subset selection facilitates the use of ICA within the context of short EEG recordings where robust ICA decomposition is unlikely if all of the high-density channels are inputs. Typically, ICA decomposes signal into the same number of signal sources as net channels (assuming no channel interpolation has occurred yet) . A 5-min recording with 75,000 samples would easily provide adequate data samples. The 19 channels from the 10–20 electrodes must be included in the total number of electrodes users select . It should be noted that if the length and sampling rate of the EEG files in a study permit ICA decomposition on the entire number of channels, then the full set of channels may be entered in the user input as the channel “subset.”However, short EEG recordings, like those usually captured with developmental populations, and especially short EEG recordings made with high-density channel layouts , do not provide enough data samples for reliable ICA decomposition without data dimension reduction. Therefore, HAPPE implements channel subset selection to improve the robustness of the ICA decomposition for these types of data. The number of channels that can be processed in a single HAPPE run will therefore depend on how long a user's EEG recordings are and the sampling rate during acquisition. For example, selecting 40 out of 128 channels to process abaseline EEG recording with a 250 Hz sampling rate would require 48,000 samples for adequate ICA  steps that follow.HAPPE removes electrical noise  from the EEG through the multi-taper regression approach implemented by the CleanLine program Mullen, . Multi-tHAPPE identifies and removes channels, including channels with high impedances or displacement during recording. HAPPE labels such channels as “bad channels,” and their data are not included in further processing or analyses. HAPPE determines bad channels by evaluating the normed joint probability of the average log power from 1 to 125 Hz across the user-specified subset of included channels. Channels whose probability falls more than 3 standard deviations from the mean are removed as bad channels. The bad channel evaluation is performed twice per data file, as channels that would otherwise be manually identified as bad channels  were found to remain in the data after the initial joint probability evaluation, but were successfully identified during the second evaluation during HAPPE development. Channels removed as bad channels have their data interpolated from nearby channels in a later processing step (after ICA decompositions) to preserve the complete user-selected channel set for post-processing analyses.For studies with lower levels of EEG artifact, data segments with obvious artifact contamination, especially non-stereotyped artifact , are commonly rejected before performing ICA as an artifact rejection approach  wavelet. The Coiflets wavelet family was selected because it has been found to provide optimal extraction of neural from artifact signal across both typical and epileptic EEG recordings , a machine-learning algorithm that evaluates the ICA-derived components . The local skewness feature identifies components with time series outliers, where higher skewness values indicate likely artifact .The next three features all rely on information from the frequency domain.2. Log alpha power: The second feature is the average log power in the alpha band (defined as 8–13 Hz). Brain-derived components typically manifest robust levels of alpha-band power, whereas artifact-driven components do not . The application of this feature to developmental data is discussed in detail below.3. Lambda: The third feature, lambda, captures the degree to which a component's power spectrum deviates from the prototypical 1/f distribution observed in cerebral-derived components. The power for six frequencies across the power spectrum are sampled for each component to generate its spectrum power curve and calculate lambda. This feature is particularly sensitive to muscle artifact, which typically manifests as a power spectrum with very poor fit to the 1/f distribution .4. Fit error: Similarly, the fourth feature, fit error, represents the mean squared error of the approximation of the f distribution to each component's distribution specifically in the 8–15 Hz range that captures alpha band power and the transition to beta band power. The fit error feature is nonspecific to artifact types but instead serves as a generalized marker of artifact probability.The last two features make use of spatial information to detect artifactual components.5. Range within pattern: Specifically, the fifth feature, range within pattern, takes the (log) difference between the largest and smallest activation magnitudes across the scalp for a component, where artifact components typically exhibit larger range within patterns .6. Current density norm: The final feature, current density norm, makes use of the 10–20 channel locations input to MARA and reflects the solution to source-modeling the component using a model that was designed to fit cerebral-based activity . Since external artifacts were not meant to be modeled with this approach, artifact-driven components return very high current density norms (reflecting overly complex source models). The current density norm feature is similarly non-specific to certain types of artifact components, but instead captures general artifact probability.Together, these 6 features address a comprehensive range of artifacts observed in independent components.MARA was not trained specifically on developmental or patient data, but several findings support its application in these contexts. First, the anatomical correlations of the 10–20 electrodes that MARA uses to calculate its spatial features are highly consistent across infant and adult brains , data can be segmented around events as specified by user inputs. For data without event markers , regularly marked segments of any duration specified by the user are generated from the start of the EEG file for the duration of the recording.After segmentation, several additional artifact-reduction options are available, although users may also segment their data without applying the following options. Post-segmentation artifact reduction parameters that the user chooses may depend on the number of available segments, as well as the extent of artifact contamination remaining in individual segments after prior preprocessing steps.Users with relatively short data files, for whom segment rejection would lead to an unacceptably low remaining number of segments for analysis, may choose an optional post-segmentation step involving the interpolation of data for channels determined to be artifact-contaminated within each individual segment, as implemented by FASTER software , or for users who wish to avoid interpolating data within individual segments, the second optional step is segment rejection based on both amplitude and joint probability criteria. Amplitude-based rejection is useful for high-amplitude artifacts like eye blinks, while joint probability-based rejection catches other classes of artifacts, especially high-frequency artifacts like muscle artifact. Together, these two criteria are an effective and time-efficient combination for determining artifact-contaminated segments. This combination has previously been used with developmental EEG data . The ROI-channel subset option allows users to tailor segment rejection for a specific ROI analysis if multiple ROIs were included in the channels selected for HAPPE processing.For all HAPPE runs (regardless of segmentation options), any channels removed during the bad channel rejection processing step are now subject to spherical interpolation  of their signal. Channel interpolation repopulates data for the complete channel subset specified by the user and reduces bias in re-referencing if the average re-reference option is selected. The identity of all interpolated channels, if any, for a file are recorded in HAPPE's processing report for users who wish to monitor the percentage or identity of interpolated channels in their datasets before further analysis.HAPPE's final processing step is to re-reference the EEG data. The user may specify either re-referencing using an average across all channels  or using a channel subset of one or multiple channels. For both re-referencing options, only channels within the user-specified channel set selected for HAPPE processing can be used for re-referencing. Rereferencing also reduces artifact signals that exist consistently across electrodes, including residual line-noise.HAPPE generates several folders containing EEG files that are located within the user-specified folder of files for processing. EEG files are saved out after several intermediate processing steps so that users can explore in-depth and visualize how those steps affected the EEG signal in their own datasets. The intermediate files include minimally-processed EEG data, post-wavelet-thresholded data, data post-ICA with the component information intact, post-component rejection EEG data, and if segmentation parameters are selected, files with post-segmentation EEG data. HAPPE outputs fully-processed files that are suitable inputs for further analyses  in one of several formats to increase compatibility with other software for data visualizations or statistical analyses. HAPPE also outputs the HAPPE processing report (described below) for the file batch, and, if users ran HAPPE in the semi-automated setting, an image for each EEG file containing the fully-processed EEG's power spectrum.For each run, HAPPE generates a report table of descriptive statistics and data metrics for each EEG file in the batch in a single spreadsheet to aid in quickly and effectively evaluating pipeline performance and data quality across participants within or across studies  . The user may also easily tabulate the descriptive statistics for remaining segments to report in their manuscript's Methods section .Next, the percentage of channels contributing uninterpolated data (“good channels”) and the identity of interpolated channels are provided. Users wishing to limit the amount of interpolated data in further analyses can easily identify files for removal using these two metrics. The distributions of the percent of good channels for the developmental sample included here are visualized in Figure The number and percent of the independent components rejected as artifact components by MARA after the post-wavelet-thresholded ICA decomposition are also given. These measures may be useful for evaluating how ICA with MARA performs on the user's data across files, since poor artifact segregation from neural signal would result in MARA rejecting most components consistently across files. The distributions of the percent of components rejected in the developmental sample included here are visualized in Figure A complementary metric to the component rejection variables is the measure of variance in the data retained after MARA rejection relative to before MARA rejection of components. Here, the retained variance across all electrodes is calculated using the compvar function . It should be noted that these values are derived before any segment rejection or segment-level channel interpolation occurs, so these metrics will overestimate the artifact levels in the fully-processed data if either segment rejection or interpolation options have been selected. Still, the artifact probability metrics, especially the median and mean artifact probabilities, may inform which files remain too artifact-contaminated to contribute to further analyses. Distributions for the median and mean artifact probabilities for the developmental sample included here are provided . The 39-channel subset selected for preprocessing contains bilateral frontal and temporal channels (in addition to the 10–20 channels), as these locations typically have the most extreme artifact levels, and therefore provide the greatest challenge to HAPPE's performance. To illustrate both the degree of artifact that occurs in these example files, and HAPPE performance for the same EEGs, data during the first 30 s of several EEGs is provided before and after HAPPE processing Figure . TherefoData quality and HAPPE's effectiveness for this dataset are first discussed by way of the HAPPE processing report metrics. The first evaluation point for assessing data quality is the number of channels contributing uninterpolated data to further analysis . In the present dataset, an average of 88% of the channels from the user-selected channel subset for each file were retained as good channels for further processing. Only one file contained fewer than 80% good channels , with most of the bad channels covering the right frontal region of interest (ROI) for that file. This file may merit removal from further analysis depending on the ROI(s) to be examined . For this processing example exclusive of specific statistical analyses, baselineEEG11 is retained.Next, the effectiveness of W-ICA and ICA in segregating and rejecting artifact from the neurophysiological signal are evaluated using the post-MARA data metrics. For this sample of 10 files, on average, 42% of the components were rejected per file during HAPPE, comparable to both the rate of MARA component rejection in adult EEG , a sufficient number of segment samples for calculating power  is a comprehensive processing pipeline that, like HAPPE, takes raw EEG files as inputs and produces fully-processed EEG data suitable for analyses. The remaining six alternative approaches consist of artifact-rejection steps. Therefore, for these alternative artifact-rejection approaches, data are processed in HAPPE for all steps besides the alternative rejection steps. Each approach's performance is evaluated using the data quality metrics provided in the HAPPE processing report for consistency across methods Figure . SpecifiThe ICA alternative approach remains fully-automatable, and simply removes the wavelet-thresholding step from the processing sequence of HAPPE. That is, data are fed directly into a single ICA decomposition for the purpose of rejecting artifact. Like HAPPE, this alternative approach does not sacrifice any data segments during artifact rejection, an appealing feature for processing short data recordings. Data were processed in HAPPE for the initial steps of filtering, channel subset selection, line noise removal through CCleanLine, and bad channel detection, as these steps may all improve ICA decomposition performance  = 5.31, p = 0.00058, n = 10]. For half of the files (5), only a single component was preserved as neurophysiological signal, an unacceptably low retention rate. The ICA approach also removed a significantly greater amount of the variance from the data than HAPPE , leaving only 24.8% of the data's variance on average, barely above the rejection threshold for evaluating HAPPE. Next, to assess the quality of the retained data post-MARA rejection across approaches, the median and mean artifact probability metrics were compared. Despite the higher rates of component rejection, the remaining components after the ICA alternative approach had median and mean artifact probabilities about twice as large as the components after HAPPE processing . Applying even the more liberal retained-artifact probability threshold of 0.3 used to evaluate HAPPE performance, the ICA approach resulted in 3 files meeting criteria for removal from further analyses, compared to 0 files in HAPPE. Overall, the alternative ICA approach without an artifact rejection step preceding it (like W-ICA in HAPPE) performed much more poorly on the developmental files relative to HAPPE, rejecting far more components but also retaining higher artifact levels in the data.First, independent component rejection rates and retained variance were compared across approaches as an indicator of how well the ICA segregated artifact from signal. The ICA alternative approach rejected significantly more components (86.3%) compared to HAPPE (42%) , preserving 2.9 times the number of segments retained through manual rejection . The manual approach left 3 files with fewer than 20 segments, which may be insufficient for further post-processing analyses. The manual approach also resulted in MARA rejecting significantly more ICA components than HAPPE , and retained significantly less data variance after component rejection , keeping only about half of the variance retained through HAPPE on average. Indeed, 3 files processed with the manual approach did not retain the minimum of 20% of the data variance to be included in post-processing analyses . Despite the higher rates of data rejection, the manual approach also had median and mean artifact probabilities more than twice as large as those obtained in HAPPE, . Accordingly, half of the files had artifact probabilities above the 0.3 threshold for rejection from further analyses. Therefore, in addition to the disadvantage of requiring manual input for each file, the manual approach did not result in either greater retained data or better retained data quality compared with HAPPE.HAPPE retained more EEG data than the manual rejection approach across all measures. First, as the manual approach involves segment rejection, the number of retained data segments was compared to the retained segment count after the optional, automated segment rejection step in HAPPE. HAPPE retained a significantly greater number of data segments relative to the manual segment rejection approach  and retained far less EEG data variance  on the ASR-treated data than in HAPPE. The ASR approach also retained higher mean  and median  artifact levels in the data post-component rejection compared to HAPPE. Consequently, the ASR approach resulted in half of the example files meeting either the retained variance or retained artifact criteria for removal from further analyses, Thus, by rejecting more EEG data but also retaining higher levels of artifact, the ASR approach performed less successfully than HAPPE across all measures in the context of developmental resting-state EEG files.HAPPE retained more EEG data than the ASR approach across all measures. Although ASR was designed for brief “bursts” of artifact in otherwise clean data, due to the high degree of artifact contamination in the developmental data, the ASR approach interpolated an average of 35.7% of the EEG data per file, which may constitute a prohibitively high interpolation rate. Even with the high rate of artifact interpolation, MARA rejected far higher percentages of independent components  and retained significantly more EEG data variance . However, FASTER also retained far higher mean  and median  artifact levels in the data, with mean artifact levels almost 6 times higher and median artifact levels more than 8 times higher than those found in HAPPE -processed data. Every one of the example files met artifact retention criteria for removal from further analyses post-FASTER processing. This pattern of high levels of retained data together with high levels of retained artifact through FASTER processing is consistent with incomplete artifact segregation and rejection. Thus, in the context of developmental EEG files, HAPPE outperforms FASTER in rejecting EEG artifact.The FASTER approach retained more data than HAPPE, but also retained much higher rates of artifact. Specifically, FASTER rejected a lower percentage of independent components  and retained far less EEG data variance than HAPPE . Moreover, the FASTER-MARA approach resulted in higher mean  and median  artifact levels in the preserved components compared to HAPPE. Indeed, 6 of the 10 example files met either the retained variance or retained artifact criteria for removal from further analyses using the FASTER-MARA approach. Therefore, HAPPE retained EEG data variance while rejecting EEG artifact more successfully than the FASTER-MARA approach for these developmental EEG files.Next, we tested whether combining the FASTER approach with MARA independent component rejection would lead to improved artifact rejection for developmental EEG files compared to the combination of W-ICA with MARA rejection in HAPPE. Data were processed as above in FASTER [see section Fully Automated Statistical Thresholding for EEG Artifact (FASTER)] and all retained independent components were then subjected to MARA classification and rejection. The combined FASTER-MARA approach rejected significantly more components , although it did not reject significantly fewer components than HAPPE . However, ADJUST retained higher mean  and median  artifact levels compared to HAPPE, and half of the example files met the artifact retention criteria for removal from further analysis with the ADJUST approach. Taken together, this pattern of increased data variance retention along with increased artifact levels suggests more incomplete artifact segregation and rejection during ADJUST compared with HAPPE, most likely due to the EMG artifacts that ADJUST was not designed to robustly eliminate.The ADJUST approach retained somewhat more EEG data variance than HAPPE  or the amount of EEG variance retained after rejection . However, SASICA retained significantly higher mean  artifact levels after component rejection compared with HAPPE. Indeed, half of the data files met the artifact retention criteria for removal from further analysis with the SASICA approach. There was also a trend-level difference with higher median artifact levels in retained data through SASICA compared with HAPPE . Thus, while SASICA retains comparable amounts of EEG variance, the higher level of retained artifact makes it less successful than HAPPE processing developmental data.SASICA did not significantly differ from HAPPE in the amount of EEG signal retained post-component rejection by any metric. That is, SASICA did not statistically differ from HAPPE in either the percent of components rejected [EEG recordings like those collected with developmental populations present particular challenges from a data processing perspective, as they typically contain a high degree of artifact contamination, can by necessity be shorter than recordings collected in adults, and are often recorded in the absence of polygraphic signals for localizing physiological artifact. Multiple toolboxes and pipelines exist for various steps of EEG processing , but these softwares are often optimized for conditions that are not met for these EEG classes due to data's constraints. To date, there is a paucity of resources tested with or targeting EEG data with high-artifact levels, short recording lengths, or absent physiological signal co-recordings. As demonstrated above, direct application of contemporary processing strategies used with typically-functioning adults, like ICA, without additional pre-processing considerations are not effective under these short-length, high-noise conditions. However, as fields move toward larger sample sizes with higher-density EEG channel layouts, the traditional manual data rejection approach used in many labs is becoming unsustainable. Moreover, despite the heightened importance of EEG data quality monitoring and reporting in fields with the highest rates of artifact contamination, no standardized metrics are currently systematically referenced in the literature  on the entire channel set and then pass a subset of the resulting PCA components to ICA (instead of channel-level data). It should be noted, though, that the PCA approach introduces nonlinearities into the data , and through selecting a PCA component subset, some amount of brain-origin signal is discarded with the rejected components  (Free Software Foundation, LG-D conceived of HAPPE and drafted the manuscript. LG-D, AM contributed and tested code. LG-D, AM, CW, and AL contributed to the design of HAPPE, analysis, and interpretation of data, critically revised the manuscript for intellectual content, and approved the final version for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


During the twenty-first century, drug discovery is expanding rapidly and a large number of chemical moieties are recognized. Many of them are poorly soluble and hence related biopharmaceutical constraints are to be addressed systematically. Among novel approaches to resolving biopharmaceutical issues, micro- and nano-emulsified systems serve as the best strategy for delivering both hydrophobic and hydrophilic drugs owing to their greater solubilization and transportation capabilities. Of late, the unique physical and biopharmaceutical properties of these liquid isotropic homogenous systems have gained substantive research importance. In addition nano/micro lipid systems share structural and functional similarity with that of the physiological lipids which offer better tolerance ability in the body. In this context, this article provides information on the historical emergence of particulate emulsified systems, importance and rationale of selection of carriers. It also encompasses the physicochemical principles that are responsible for the elevation of therapeutic outcomes of delivery systems. Detailed and schematic absorption of these drug delivery systems is explained here. Gastro-intestinal biochemistry necessary in the understanding of digestion process, lipolytic products formed, micellar structures, enzymes, transporters, mechanism of cell uptake involved after subsequent oral absorption are also emphasized. In addition, this article also explains disposition and pharmacokinetic properties of emulsified systems with real-time therapeutic research outcomes. The influence of biochemical compositions and biopharmaceutical principles on absorption and disposition patterns of ME/NEs was described in the article for the interest of readers and young researchers. With the advent of several emerging novel chemical entities, delivery of high molecular weight and poorly soluble bioactive molecules remains a challenging task. Therapeutic efficacy of low soluble molecules (BCS Class II and IV) is generally high and hence the design of oral delivery systems with improved dissolution and permeability characteristics cannot be ignored  However,In times of yore, many attempts were made to deliver lipophilic/hydrophobic moieties for better therapeutic outcomes. Of late, Oral Emulsified Lipid Delivery Systems (OELDS) was instigated with sulphonamide lipid emulsions . In suchBased on the potential considerations of ME/NEs as an appealing substitute, the review was undertaken to emphasize typical biopharmaceutical aspects of oral absorption, including enzymes, transporters, and carriers involved therein. Distribution and elimination attributes of ME/NEs are also discussed here. Although both the emulsified particulate systems differ in droplet size, composition, and physicochemical properties, there is a firm connection in their in-vivo performance. Therefore, both the emulsion systems and their subtypes, self-micro/nano emulsifying drug delivery systems are explicated together.Findings of Hoar and Schulman in 1940’s have led to the concept of the microemulsion, which formed a clear homogenous fluid of milky emulsion with hexanol. Later, the term microemulsion was coined by Schulman in 1959 based on the droplet size (100–600 nm). Since then the term has modified and defined, according to, on many occasions .Micro/nanoemulsions (ME/NEs) share the similarities in definition, as they are isotropic, homogenous liquid systems composed of oil, amphiphile and water contents, very often included with a modifier like cosurfactant or cosolvent  to constME/NEs including SMEDDS and SNEDDS are strategically significant due to the protection of lipophilic drugs against degradation in GIT, translocation across epithelial barriers by extending transit time and specified absorptive pathways. Performance of ME/NEs is typically depended on the class/type of system and composition .Oils as carriers firstly meant for hydrophobic drugs to sink, long, medium and short chain triglycerides  were predominantly used in the preparation of ME/NEs with the rank order of MCTs>LCTs>SCTs . Type ofDue to typical structural significance, amphiphiles reduce the interfacial tension and impart thermodynamic stability in dispersions. Droplet size of hetero-dispersions is affected by HLB value, type, and concentration of surfactant . SurfactBeing nonionic, polysorbate is less toxic and unaffected by changes in pH, ionic strength and promotes the formation of ultrafine droplets  whereas,Short-to-medium-chain (C3-C8) alcohols such as methanol, ethanol, isopropyl alcohol, carbitol, PEG’s, glycerin, propylene glycol, etc. have the potential to increase the emulsification efficiency by reducing the bending stress at an interface and allow film flexibility to different curvatures for the formation of ME/NE’s. They primarily increase the mobility of hydrocarbon tail and allow greater penetration of oil at the interface , 26. TraMucins, long (200–1000 nm) heterogenous polydispersed filaments of glycosylated proteins are protecting GI mucosa from pH fluctuations, pathogens, mechanical irritation and toxins , 29. TheThe solubility of lipophilic drugs at absorption site depends on selected lipid vehicles as they enhance solvation capacity of the GI fluids by forming swollen colloidal micelles. These solubilized forms of drugs (intact micelles) undergo certain mesomorphic transformations which are due to during dispersion of GIT contents, dilution of contents by various endogenous secretions and effect of drug-chemical stability by various digestive processes. Absorption of ME/NEs takes place from the free drug concentration that is in equilibrium with the solubilized colloidal reservoir rather than from intact micelles .P < 5 passively diffused from the lumen into the systemic circulation by enterocytes [P > 5 are re-esterified to triglycerides in enterocytes as represented in Fig. Mostly, absorption of ME/NEs is high in small intestinal lumen rather than in other parts of GIT since it contains biliary lipids . MCTs with amphiphilic nature present in particulate emulsified systems undergo lipolysis at lumen of the intestine into free fatty acids and monoglycerides. These products have the ability to coalesce readily with bile lipids and form micelles. Further, fatty acids of 10–12 carbon atoms and drugs with lop erocytes –41. FattA pH-stat automatic titration unit model demonstrated the lipolysis of ME/NEs and subsequent phases which alters the release profile and fate of a drug in GIT. The further extent of digestion affects release profiles of the drug located in the core of ME/NEs rather than shell , 48. CarATP-binding cassette (ABC) transporters and solute carriers are notable intestinal transporters which regulate the transport of molecules into and out of enterocytes. Cytochrome P-450 3A4 (CYP3A4), multidrug efflux pump and P-glycoprotein (P-gp) are present high levels at villus tip enterocytes of the small intestine. P-gp is ABC transporter and highly expressed on the brush-border membrane of columnar epithelial cells. The efflux transport of particulate emulsified systems into enterocytes is affected by these local transporters. However, efflux inhibition is anticipated with membrane fluidization, induced conformational changes and cholesterol depletion . PEG 400Cellular surfaces are dominated by negatively charged sulfated proteoglycans that play an important role in cellular proliferation, migration, and motility. Particulate emulsified systems with higher surface charge, bind strongly to the cell membrane with electrostatic interactions between the anionic membrane and cationic nanodroplets, thus increasing the cellular uptake. After adsorption onto the cellular membrane, the uptake occurs possibly via pinocytosis, nonspecific or receptor-mediated endocytosis or phagocytosis –63. AbsoM cells of the lymphoid follicle-associated epithelium represent a potential portal for intestinal drug delivery because of their high transcytotic capacity and ability to transport particulates . ParticlAs demonstrated in Fig. Being submicron sized systems, ME/NEs exhibit prolonged circulation . A majorEmulsions poor in cholesteryl ester but rich in free cholesterol show remnant-like behavior, whereas emulsions with rich cholesteryl ester but poor in free cholesterol were metabolized like nascent chylomicron particles. Emulsions of defined composition and with known metabolic behavior should be of value not only to probe lipoprotein metabolism but perhaps also as vehicles for the delivery of hydrophobic drugs to targeted organs. Subsequent dosing of emulsion, droplets acquire apoproteins  from high-density lipoproteins and very low-density lipoproteins of blood. Apo-CII acts as lipoprotein lipase (LPL) activator, which leads to hydrolysis of triglycerides, whereas apo-E helps in hepatic removal of remnants .ME/NEs, being prolonged circulation entities display regular elimination patterns with increased half-life generally. Droplet sizes of 50–100 nm were removed by the liver and spleen, polymeric micelles smaller than 5 and 5–10 nm were easily eliminated through the renal glomeruli . Such kiParticulate emulsified delivery systems are being gradually more used to protect bioactives against the intense environment, to improve molecular targets, stability and to increase bioavailability. Molecular action of selected lipophilic drugs when associated with oil core or amphiphiles of the ME/NE systems is ascribed to transfer the drug molecules into the targeted cells by fusion of oil droplet with the cell membranes through lipid exchange or by endocytosis of the oil droplets . If the Essential PUFA transporters of an abluminal membrane of the endothelial cells of BBB allowed the CNS acting essential PUFA (omega-3 and omega-6), a lipophilic moiety into the brain . NonioniCurcumin encapsulated by a combination of cationic surfactants such as cetyl and dodecyl trimethylammonium bromide was found to be intact even against alkaline hydrolysis  and bioamax and AUC [Oral administration of ME of cyclosporine A resulted in increased bioavailability with not many side effects in hepatic impaired patients  and stab and AUC . Thus thME/NEs are the most dynamic and novel appealing platforms for poorly soluble moieties with expected therapeutic outcomes. In view of the fact, development of ME/NEs is not an easy task for the formulators wherein, they require utmost attention on selection and usage of carriers/integrants of the delivery system. Thus, this article prolifically presents the information on carriers, composition and their in-vitro and in-vivo functions. It was successful to illustrate on the micellization of particulates with luminal contents and subsequent intestinal/lymph absorption. Impact of biochemical and biopharmaceutical principles on absorption and disposition patterns of ME/NEs was presented well herein for the interest of readers and young researchers.


A 66-year-old woman presented with progressive lancinating pain and sensory deficits attributable to a myelopathy of unclear etiology. Spinal cord magnetic resonance imaging showed a longitudinally extensive T2-hyperintense lesion of the dorsal columns. Comprehensive serum, urine, and cerebrospinal fluid analyses failed to identify an etiology. Empiric intravenous methylprednisolone and intravenous immunoglobulin were of no benefit and serial screens for an occult malignancy were negative. She developed dysesthesias and allodynia affecting her entire body and lost the use of her arms and legs due to severe sensory ataxia that was steadily progressive from onset. She opted against additional aggressive medical management of her condition and passed away on hospice eleven months after symptom onset. Autopsy revealed findings most consistent with polyphasic spinal cord ischemia affecting the dorsal and lateral white matter tracts and, to a lesser extent, adjacent gray matter. The underlying etiology for the progressive vasculopathy remains unknown. Spinal cord ischemia affecting the posterior spinal cord is rare and to our knowledge this case represents the only instance of a progressive spinal cord tractopathy attributable to chronic spinal cord ischemia. Autopsy findings suggest that chronic, progressive microvasculopathy should be considered in the differential diagnosis of a dorsal column myelopathy when alternative, more common, etiologies have been ruled out.A 66-year-old woman presented with lancinating pain, allodynia, and numbness that steadily ascended from her feet to the level of her umbilicus over a three-month period. There was no clinical history to suggest an episodic or relapsing-remitting course. Her past medical history was significant for tobacco use, hypertension, poorly controlled type II diabetes mellitus, chronic kidney disease, chronic obstructive pulmonary disease, breast cancer , and a ruptured hepatic abscess . She had no known history of nitrous oxide exposure. Her neurologic examination showed normal mental status and cranial nerves and a normal motor exam when utilizing visual feedback to guide movement of the muscle groups being tested. She had no visual field deficits on bedside testing and there were no other clinical signs or symptoms of optic neuropathy. Joint position sense was severely impaired, with a corresponding severe sensory ataxia and impaired upper and lower extremity vibration sense . Reflexes were symmetrically brisk (3+). Bilateral Babinski and Hoffman's signs were elicited.MRI of the cervical and thoracic spine was obtained at the time of presentation and demonstrated mild, diffuse atrophy of the cord and a diffuse hyperintense signal on T2-weighted images within the dorsal columns, extending from the craniocervical junction to T12 . A low c “Differential Diagnosis of Chronic Myelopathy”). A lesion involving the dorsal columns symmetrically and extending over so many spinal levels with commensurate cord atrophy might suggest a metabolic process such as subacute combined degeneration from vitamin B12 deficiency or copper-deficiency myeloneuropathy. However, chronic sequelae from syphilis  or HIV (vacuolar myelopathy) could have the same appearance. In contrast, homogenous extension over such a long spinal segment, without contrast enhancement, would be unusual for a demyelinating disease, such as multiple sclerosis or neuromyelitis optica (NMO), or for a neoplasm. Although intravascular B cell lymphoma (IVBCL) can present with longitudinally extensive spinal cord lesions, this neoplasm would be expected to cause expansion of the cord and is rarely predominantly tract specific [The differential diagnosis for a dorsal column predominant “tractopathy” is broad and includes infectious, inflammatory/autoimmune/demyelinating, metabolic, toxic, and vascular  etiologies , infectious , viral ), and autoimmune etiologies  was nondiagnostic . NeurosyPostmortem examination confirmed the cause of death as bilateral pneumonia with multiple flora and foreign material (consistent with aspiration) and found severe diffuse atherosclerosis within the aorta from the arch to the bifurcation, with evidence of ulceration. No neoplasm was detected. Gross examination of the spinal cord revealed atrophy and mild discoloration. Microscopic examination of the spinal cord showed a complex polyphasic myelopathy affecting dorsal and lateral white matter tracts and, to a lesser extent, adjacent gray matter. More specifically, the dorsal columns showed degeneration in a medial-to-lateral gradient, with evidence of acute, subacute, and chronic changes. The more acute/subacute changes included a neutrophilic infiltrate limited to the medial portions of the dorsal columns, appearing in association with acute/subacute necrosis, reactive/reparative vasculature, activated microglia, and numerous foamy macrophages . The chrOutside the areas of injury, blood vessels in the spinal cord showed only moderate arteriolosclerosis, without inflammation or thrombosis. Similar arteriolosclerosis was evident in the brain and meninges. The brainstem showed only very focal coarse vacuolation of the right spinothalamic tract and ventral spinocerebellar tract in the lateral medulla; other ascending and descending tracts were unremarkable. Sections of sampled dorsal root ganglia showed no evidence of nodules of Nageotte or of ganglion cell damage or loss. The feeding vessels of the vertebral and spinal arteries, which originate from the aorta, were not collected during prosection, so subsequent histopathologic examination of these vessels was not possible.This pattern of spinal cord injury, involving the dorsal columns in a gradient of severity from medial (worst) to lateral (less severe) and also involving the dorsolateral white matter, correlated with the clinical history of ascending sensory symptoms. Coupled with damage to gray matter, this pattern also raised suspicion for chronic ischemia/infarction as an etiology for the patient's myelopathy.This case is unique and instructive for both the spatial  and temporal (subacute-to-chronic and steadily progressive) pattern of myelopathy, presumed secondary to a polyphasic ischemic process. Typically, spinal cord infarction presents with an acute onset of deficits that vary based on the territories involved. Overall, nontraumatic spinal cord ischemia or infarction accounts for only ~1% of total strokes . UsuallySigns and symptoms of spinal cord ischemia may vary in intensity and evolve over a few days before deficits become fixed. This patient presented with a subacute-to-chronic history of pain and severe sensory ataxia, and MRI findings supported localization to the posterior cord; accordingly, our initial differential diagnosis focused on common causes of this form of myelopathy. This patient did not fit the typical case of spinal cord infarction as she had no acute inciting event and it is uncommon for spinal cord infarction to preferentially involve the dorsal columns , 22, 23.The underlying etiology of this patient's apparent polyphasic spinal cord infarction remains unclear. The lack of an inflammatory CSF profile, nodular contrast enhancing lesions in the spine , nondiagnostic paratracheal lymph node biopsy guided by results of a full body PET scan, and lack of pathologic features of noncaseating granulomas on complete autopsy, all argued against neurosarcoidosis. The patient's serum anti-aquaporin-4 IgG was negative and other than radiologic evidence of a longitudinally extensive lesion of the dorsal columns there were no clinical features suggestive of NMO or NMOSD. Radiological and postmortem examinations identified severe atherosclerosis in the aorta and visceral blood vessels, but there was no definitive evidence of thrombosis or vasculitis to explain the more acute changes. Given the symmetric, tract specific changes on imaging, symmetric clinical deficits, and the progressive course, a paraneoplastic myelopathy remains possible, despite the lack of an identified occult malignancy at autopsy or on PET/CT scan. Although VGCC autoantibodies have been reported in cases of paraneoplastic myelopathy they are not specific to this disorder; and, given the low titers, this finding has unclear clinical significance , 16. NonChronic, progressive microvasculopathy should be considered in the differential diagnosis of a progressive myelopathy with imaging evidence of a dorsal column tractopathy when alternative, more common, etiologies have been ruled out. Given the strong suspicion for an occult malignancy in our case and the strong clinical resemblance to previously reported cases of paraneoplastic dorsal column myelopathy, we propose that an immune mediated microvasculopathy be considered in the list of potential pathophysiologic mechanisms for paraneoplastic myelopathies. To our knowledge, no comparable cases exist in the literature and additional cases are necessary to further test this hypothesis, emphasizing the importance of postmortem examinations in this unfortunate patient population.


During the 2014–2016 West Africa Ebola Virus Disease (EVD) epidemic, the public health community had concerns that sexual transmission of the Ebola virus (EBOV) from EVD survivors was a risk, due to EBOV persistence in body fluids of EVD survivors, particularly semen. The Sierra Leone Ebola Virus Persistence Study was initiated to investigate this risk by assessing EBOV persistence in numerous body fluids of EVD survivors and providing risk reduction counseling based on test results for semen, vaginal fluid, menstrual blood, urine, rectal fluid, sweat, tears, saliva, and breast milk. This publication describes implementation of the counseling protocol and the key lessons learned.The Ebola Virus Persistence Risk Reduction Behavioral Counseling Protocol was developed from a framework used to prevent transmission of HIV and other sexually transmitted infections. The framework helped to identify barriers to risk reduction and facilitated the development of a personalized risk-reduction plan, particularly around condom use and abstinence. Pre-test and post-test counseling sessions included risk reduction guidance, and post-test counseling was based on the participants’ individual test results. The behavioral counseling protocol enabled study staff to translate the study’s body fluid test results into individualized information for study participants.The Ebola Virus Persistence Risk Reduction Behavioral Counseling Protocol provided guidance to mitigate the risk of EBOV transmission from EVD survivors. It has since been shared with and adapted by other EVD survivor body fluid testing programs and studies in Ebola-affected countries. The 2014–2016 West Africa Ebola Virus Disease (EVD) epidemic was large and widespread, affecting thousands of people across Guinea, Liberia, and Sierra Leone. Prior to this epidemic, there were limited data on persistence of Ebola virus in body fluids of EVD survivors and the potential risk that viral persistence may pose for Ebola virus transmission, including possible sexual transmission. This paper documents the development and implementation of a behavioral counseling protocol to facilitate adoption of risk reduction behaviors among male and female EVD survivors enrolled in the Sierra Leone Ebola Virus Persistence Study. This behavioral counseling protocol, composed of pre-test, delivery of results, and post-test counseling, enabled study staff to translate the study’s body fluid test results into individualized information and preventive action for study participants. Risk reduction behavioral counseling became an important EVD epidemic control measure. The 2014–2016 West Africa Ebola Virus Disease (EVD) epidemic was large and widespread, with at least 28,616 probable and suspected cases of EVD and 11,310 deaths across Guinea, Liberia, and Sierra Leone , 6.Given limited pre-existing data, the potential for EBOV to persist in survivors was identified as an important research topic early in the epidemic. There was also a programmatic need to develop methods to test body fluids of EVD survivors and counsel them on risk reduction practices. This need became even more clear when a woman from Liberia tested positive for EBOV infection, and epidemiologic investigation found that her only exposure was unprotected vaginal intercourse with a male EVD survivor whose semen tested positive for EBOV RNA by real time reverse transcriptase polymerase chain reaction (qRT-PCR) 199 days after he first became symptomatic with EVD , 8. SequPrior to the EVD epidemic, the Demographic Health Survey conducted in 2013 in Sierra Leone showed that condom use was low, with 5% of female and 13% of male respondents reporting having used a condom in the past 12 months. Twenty-five percent of men and 6% of women reported having two or more sex partners in the last 12 months, and 23% of men and 5% of women reported concurrent sexual partnerships. Multiple and concurrent sexual partnerships were highest among older married men with low education . Human IIn May 2015, the Sierra Leone Ebola Virus Persistence Study was launched to investigate EBOV persistence in the body fluids of EVD survivors in Sierra Leone .The standard operating procedure for the behavioral counseling protocol included EBOV persistence pre-and post-test counseling as well as HIV pre-and post-test counseling. EBOV persistence pre-test counseling is an introduction to EBOV testing and risk reduction advice prior to testing, while post-test counseling is a delivery of tailored guidance based on qRT-PCR test results. During the first visit, participants were first seen by a nurse who described the study, obtained informed consent, and facilitated the demographic, behavioral, and clinical questionnaire. After specimen collection, each participant visited a counselor for pre-test counseling. In this session, counselors educated participants about qRT-PCR and virus isolation testing methods; asked participants about their sexual behavior, intimate relationships, and life stressors including experiences with stigma; gave a male and female condom demonstration; and referred to survivor care services if needed. Rapid HIV testing and counseling was offered to each participant at their first visit after specimen collection, and, if they did not initially consent, at every visit thereafter.During visit 2, held two weeks after initial specimen collection at the first visit, counselors and participants met for post-test counseling. In the first part of post-test counseling, counselors delivered the prior visit’s qRT-PCR test results for each specimen: positive, negative, indeterminate, or no interpretation  . InsteadFollowing the delivery of test results for specimens collected during the previous visit, participants provided a second set of specimens for qRT-PCR testing and then visited the counselor for further post-test counseling. This order was used to prevent delays in study flow. During the second part of post-test counseling, counselors reviewed test results, delivered behavioral guidance for any positive qRT-PCR fluid, developed a personalized risk reduction plan and negotiated risk reduction strategies, gave a condom demonstration, and distributed condoms.Participants continued to provide specimens and receive post-test counseling for all test results. Study visits continued until participants received two consecutive negative qRT-PCR test results on all body fluids, at which time participants were discharged from bi-weekly study visits, and asked to come back for a follow-up visit with specimen collection after three and six months. The counseling provided at these latter follow-up visits followed the same messaging as described above. Positive virus isolation results were delivered when they became available, which occurred several months after the initial specimen was collected, as these specimens were sent to the United States for testing. If virus isolation results were positive, participants were told that “the virus found in your specimen when it was tested was alive and possibly infectious”.At all sessions, counselors asked participants to report any medical, psychosocial, or other issues they may have been experiencing for referral to community-based services where available. Additionally, if a participant could not provide a specimen at any visit, the counselor discussed specimen collection strategies and scheduled another visit for the participant .As prior evidence showed that EBOV could persist in semen , male EVSexual risk reduction behavioral guidance was tailored for each type of body fluid. Participants were given different guidance for fluids that posed a greater risk for sexual transmission of EBOV due to contact during sexual activity  as compared to fluids for which there was likely to be less contact during sexual activity  . Sexual http://www.cdc.gov/meningitis/viral.html). This guidance was modified for a low resource setting, taking into consideration limited access to clean water and sustained power sources, and lack of flushing toilets, bleach, or other materials and practices used in standard IPC protocols.For non-sexual risk reduction behavioral guidance, the study team reviewed general infection prevention and control (IPC) guidelines for other pathogens that are spread through contact with infectious body fluids such as viral meningitis and hepatitis B  post-symptom onset . ResultsEVD survivors in West Africa have reported experiencing a myriad of difficulties including stigma from family, friends, and community members, loss of employment, housing, and social networks, and numerous mental or physical health sequelae , 23, 24.We observed great receptivity to condom demonstrations, with many participants informing research staff that they had not participated in a condom demonstration prior to joining the study. Given the low national rates of condom use among men and women in Sierra Leone , exposurCommunicating the risk of sexual transmission of EBOV to study participants was challenging as there was limited scientific evidence regarding the length of persistence and the infectiousness of positive qRT-PCR body fluids. In this study, we observed that communicating uncertainty in a transparent way appeared to facilitate trust between participants and the research study. For example, following the report of an EVD survivor in the United Kingdom who relapsed with EVD meningitis , the behIn the course of the study, we identified a need to clearly and simply explain the detection method of the qRT-PCR assay to participants. We developed a “mango tree” analogy comparing detection of EBOV-specific RNA primers (short target sequences) in the body fluids of Ebola survivors via qRT-PCR testing to trying to detect a whole mango tree  by being able to detect only the mango fruits or the leaves (the target RNA primers for EBOV). Detecting only fruits or leaves, one cannot determine whether the tree is indeed intact and alive just as qRT-PCR testing can only determine whether the target RNA primers are detected or undetected in a body fluid specimen, and not whether the virus is infectious. Detecting only one of the two target RNA sequences (an indeterminate test result), could also be explained using this analogy, and also to explain possible variation in test results using different qRT-PCR assays with differing target sequences. This addition to the counseling script was very helpful for study participants to conceptualize the test, the different results they encountered, and the associated counseling messages.A flexible protocol was also instrumental in optimizing participant flow during the study. For example, some participants preferred to visit the counselor prior to specimen collection for more detailed instructions on specimen collection processes or for extra encouragement to continue with the testing process. Talking points were developed as reference materials for study counselors as the original scripts were lengthy. Because we believed that participants should receive their previous test results before deciding whether to donate specimens for testing at the current visit, there was a delay between delivery of test results and post-test counseling and explanation of guidance. Future iterations of similar counseling protocols may be able to concurrently deliver testing results and post-test counseling if a rapid diagnostic test for detection of Ebola RNA in the body fluids of Ebola survivors becomes available.The EVD epidemic had a devastating impact on the economic and social ties of communities across Sierra Leone , 21, 24.The mental health infrastructure of Sierra Leone is underdeveloped. There is limited capacity to identify and treat mental health disorders in the general public or EBOV-affected populations . When thAfter observing initial high rates of HIV testing uptake in the first phase of the study, acceptance of HIV testing declined among study participants during the first visit. We hypothesize that participants may have felt anxious about their EBOV qRT-PCR tests and did not want to learn their HIV test results at the same time. We re-trained study counselors on HIV testing and counseling procedures and encouraged them to offer HIV tests at follow-up study visits and observed a rise in uptake of HIV testing. More formative research is needed to determine how best to incorporate both HIV and EBOV testing and counseling in future EVD epidemics.One limitation of our behavioral counseling protocol is that, due to time constraints during study visits, we did not employ couples’ counseling. Future development of similar EBOV persistence behavioral counseling protocols may consider the inclusion of couples’ counseling to facilitate correct and consistent condom use, particularly for those participants who repeatedly test positive . ConsideAnother limitation of the protocol is that we developed and implemented risk reduction guidance when relatively little was known about the persistence of Ebola RNA in the body fluids of survivors, or how qRT-PCR results related to the risk of transmitting Ebola to others. Since the implementation of this behavioral counseling protocol, additional data on the persistence of Ebola RNA in semen has become available from multiple Ebola-affected countries –31. ThesA behavioral counseling protocol that pairs test results with risk reduction behavioral guidance might help mitigate transmission risks associated with body fluids of EVD survivors in which EBOV has been detected. Risk reduction behavioral counseling is rapidly becoming an integral part of addressing the sexual transmission risk in this EVD epidemic and in filovirus outbreaks moving forward; its utility will likely also be swiftly demonstrated for other pathogens where virus persistence in body fluids may pose a risk for continued transmission from survivors . A qualiAs of July 2016, the Ebola Virus Persistence Risk Reduction Behavioral Counseling Protocol has been used for more than 220 male and 120 female participants. The protocol has been adapted for use by other body fluid testing programs for EVD survivors, including the Government of Sierra Leone’s CPES  and Liberia’s Men’s Health Screening Program . LessonsS1 Checklist(DOC)Click here for additional data file.S1 Protocol(DOCX)Click here for additional data file.


The onset of cancer is unavoidably accompanied by suppression of antitumor immunity. This occurs through mechanisms ranging from the progressive accumulation of regulatory immune cells associated with chronic immune stimulation and inflammation, to the expression of immunosuppressive molecules. Some of them are being successfully exploited as therapeutic targets, with impressive clinical results achieved in patients, as in the case of immune checkpoint inhibitors. To limit immune attack, tumor cells exploit specific pathways to render the tumor microenvironment hostile for antitumor effector cells. Local acidification might, in fact, anergize activated T cells and facilitate the accumulation of immune suppressive cells. Moreover, the release of extracellular vesicles by tumor cells can condition distant immune sites contributing to the onset of systemic immune suppression. Understanding which mechanisms may be prevalent in specific cancers or disease stages, and identifying possible strategies to counterbalance would majorly contribute to improving clinical efficacy of cancer immunotherapy. Here, we intend to highlight these mechanisms, how they could be targeted and the tools that might be available in the near future to achieve this goal. Immune suppression is an area of great interest in cancer immunotherapy. Although it has been acknowledged that tumor cells spontaneously induce T cell activation through the expression of specific antigens, and that these responses are likely blunted in vivo by immune escape mechanisms, an impressive tumor control that can be achieved by neutralizing immunosuppressive mechanisms  could not be foreseen even by the most optimistic scientists . As a maEach of the steps composing this “immunosuppressive cycle” , if appr+CD14+HLA-DRlow/−CD15−, and a polymorphonuclear population (PMN-MDSCs), CD14−CD11b+CD15+(or CD66b+), formerly called granulocytic-MDSCs [MDSCs, a heterogeneous mixture of myeloid cells, play a crucial role in both local and systemic immune suppression. At tumor sites, MDSCs provide a microenvironment favoring tumor cell survival and metastasis. In fact, in addition to exerting immune suppression, they possess strong angiogenic functions and directly promote tissue remodeling. Human MDSCs include a monocytic population (M-MDSCs), CD11bic-MDSCs . Several independent studies demonstrated that the different subsets of MDSCs accumulate in the blood and at the tumor sites of cancer patients . While tGiven the strong and multifaceted pro-tumor activity of MDSCs, their association with clinical features and their crucial ability to hamper the patients’ response to therapy, the targeting of these cells represents a promising approach to improve tumor treatment. In this regard, strategies are mainly focused on reduction of MDSC levels in the blood by blocking their expansion from hematopoietic precursors, promotion of MDSC differentiation, and inhibition of their suppressive functions. It is worth noting that blocking MDSC recruitment through chemokine receptor blockade has been recently identified as a promising new strategy in preclinical and clinical studies ,23. In tIn addition to drugs targeting STAT3, other compounds have been shown to block the effector functions of patients’ derived MDSCs in vitro and in preclinical models. Celecoxib (Cyclooxygenase-2 inhibitor) suppresses the activation of melanoma derived M-MDSCs and increases the frequency of cytotoxic T lymphocytes (CTLs) . In anim+ cells, while increasing HLA-DR+ cells and IL-12 and IFN-γ plasma levels. This testifies to an increased activation of DCs and T cells in head and neck cancer patients [Other strategies of MDSC targeting include attempts in inducing their differentiation. For this purpose, All-trans retinoic acid (ATRA) and 25-hydroxyvitamin D(3) have been administered to renal cell cancer and head and neck squamous cell carcinoma patients in pilot studies. Results showed an effective induction of the differentiation of myeloid cells. Of note, 25-hydroxyvitamin D(3) reduced the number of immune suppressive CD34patients ,42. In rpatients . Finallypatients . Thus, rIn conclusion, MDSCs represent a diversified population of cells for both phenotype and functions. They play a crucial role in immunosuppressive circuits, and, to date, several strategies are available for limiting their frequency, survival and functions . Many ofTregs are fundamental for the maintenance of peripheral tolerance and to prevent autoimmunity. However, they can infiltrate human tumors and limit antitumor immunity and immunotherapy effects . SeveralSome traditional anti-cancer drugs have been shown to interfere with Treg activity. The most common chemotherapeutic agent studied for this purpose is cyclophosphamide (CTX). The rationale for investigating the effects of CTX on Tregs is that Tregs residing in the tumor microenvironment are highly proliferating. Actually, only the metronomic administration of CTX seems to possess immunomodulatory effects on Tregs in patients with advanced cancers ,51. Howe++CD14+), thus with an indirect mechanism [Enthusiasm is increasing for the effect of the FDA approved ICI, anti-CTLA-4 and -PD-1 mAbs, which are improving the overall survival (OS) of patients with several types of advanced malignancies, even if the majority of patients (about 70%) still fail to respond to these treatments. Immune checkpoints (IC) can be expressed not only by effector T cells but also by Tregs. While the role of ICI on effector T cells is largely documented , their aechanism . Evidencechanism . Positivechanism . In cont+ Treg subset characterized by the expression of LAG-3 [+ T cells expressing both LAG-3 and PD-1 are endowed with impaired effector function. Dual blockade of LAG-3 and PD-1 during the T cell priming phase can restore the antitumor function of NY-ESO-1 specific CD8+ T cells [A key goal would be to unravel mechanisms or pathways selectively used by Tregs confined to the tumor microenvironment but not in their activity of “normal” immune control. While investigating Treg subpopulations specifically enriched in cancer patients, we identified a discrete CD4of LAG-3 . These h T cells , but the T cells . These eIn summary, to counteract immune suppression mediated by Tregs, the main focus should be placed on contrasting their suppressive activity, rather than on elimination of these cells. This could be achieved through the administration of ICI targeting molecules not only expressed by effector T cells but also by Tregs like PD-1, or in combination with novel drugs targeting Treg molecules such as LAG-3 . Production and release of extracellular vesicles (EVs), devoted to intercellular communication, is a feature shared by normal as well as transformed cells. Since their discovery more than three decades ago, EV studies have dissected their multifaceted roles, mainly depending on their cellular origin. The accumulating evidence prompted possibilities of intervention for the employment of these vesicles as therapeutic targets, agents or vehicles and biomarkers in diseases not limited to cancer. EVs appear to be suitable natural carriers for drugs or other therapeutic agents. At the same time, these vesicles can be “shaped” by insertion of molecules for different purposes, such as targeting. The large family of EVs includes various members, including exosomes, microvesicles, ectosomes, apoptotic bodies and large oncosomes . Each veThe exploitation of EVs, especially exosomes, as acellular vehicles for the stimulation of antitumor immune responses in vivo has gained major interest starting from more than a decade ago. The attraction of exosomes as an immunotherapeutic tool is based on studies showing that exosomes produced by immune cells (including DCs and B lymphocytes) display functional major histocompatibility complex (MHC) class I and II as well as co-stimulatory and cell adhesion molecules. Zitvogel et al. demonstrated for the first time in 1998 that exosomes secreted by bone marrow-derived DCs, which were challenged with tumor-derived peptides, activated CTLs and caused the eradication of established tumors in mice . AdminisThe possibility of loading specific molecules, drugs and genetic material into and onto EVs has attracted major interest for their exploitation as therapeutic shuttles to tumor sites . Our uniAmong the multitude of approaches currently under evaluation, the capture of tumor exosomes aimed at dampening their detrimental effects on the immune system appears as an interesting strategy. Selective elimination of exosomes from the body implicates their recognition, this being a particularly difficult task. In this view, Gobbo and coworkers developed an interesting strategy based on the expression of hsp70 primarily by cancer exosomes, as detected in the plasma of cancer patients. With the help of an hsp70 binding aptamer, these authors were able to demonstrate the inhibition of MDSC activation mediated by hsp70 carrying exosomes binding to TLR2 expressed by MDSCs .The discovery of genetic material inside of the extracellular vesicles led to enormous development in EV research. In fact, this opened up new functional aspects of these vesicles, offering new possibilities for therapeutic targeting approaches. The loading of EVs with selected miRNAs appears to be a major strategy with potential translation into the clinics. Currently, the miRNA selection is mainly aimed at inhibiting tumor growth. To date, few approaches actually target cells involved in immune suppression. In fact, the targeting of therapeutic vesicles to immune cells might represent a new challenge. However, to overcome immune suppression in cancer, we might learn from the induction of immune suppression in autoimmunity diseases, such as contact allergies. Here, the targeting of immune cells is the principal objective, as demonstrated by a recent study showing the delivery of a chosen inhibitory miRNA to target effector T cells in an antigen-specific manner by a surface coating of antibody light chains . AdditioIt becomes evident that EVs, depending on their cells of origin and their content and composition, could be utilized to overcome immune suppression in cancer patients mainly by contrasting their immune suppressive effects through elimination, if originating from tumor and immune suppressive cells, or by exploiting their potential as shuttles to distant sites to target immune suppressive cells like Tregs and MDSCs to revert immune suppressive status. In this regard, due to their bioavailability, the exploitation of EVs, especially exosomes, appears as an attractive alternative for drug or molecule delivery purposes. Moreover, EVs can be targeted to reach not only the tumor site, but also immune suppressive cells present in the tumor microenvironment. It could be envisaged that, in the near future, exosome therapeutics might be composed of different “ingredients” aimed at docking specific targets and modulating the suppressive milieu.+-ATPases, Na+-H+ antiporters, and H+-linked monocarboxylate transporters), leading to decreased pH in the tumor microenvironment [The mechanisms linking chronic inflammation and cancer are not fully clarified, but certainly involve multiple interactions between cancer and stroma immune cells. These complex networks are hypothesized to be driven by a variety of soluble factors including proinflammatory cytokines/chemokines, directly inhibiting tumor immunity and/or polarizing resident, or recruited myeloid cells toward immunosuppressive or protumorigenic phenotypes . Howeverironment . + T cell infiltrate in tumor lesions and significantly potentiate the therapeutic efficacy of a cancer vaccine or adoptive immunotherapy [Acidity can profoundly mold immune responses, impairing T cell proliferation and function, but also favoring the differentiation of proinflammatory stromal cells . Recent otherapy , supportotherapy . In addiotherapy ,100. Furotherapy . On the otherapy ,103,104.In addition to vATPases, several pH regulators such as carbonic anhydrases (CA) IX and XII expressed in cancers have been mostly investigated for their role in tumor cell proliferation and survival . NeverthAn easy albeit non-specific approach to counteract tumor acidity is represented by bicarbonate monotherapy, which has been shown to impair growth of some cancers in preclinical models, and to contrast tumor development in prostate cancer-prone mice . More reEnormous progress has been achieved over the last few years in the field of cancer immunotherapy, thanks to the identification of the right tools to unleash adaptive and innate immunity and its potency in controlling tumor growth and progression. Nevertheless, the pathways of immune suppression addressed so far, mostly related to the upregulation of immune checkpoints, involve only a subset of the complex cross-talk networking progressively established by cancer cells with host environments during disease development. Although many of these mechanisms might be common features of tumor cells, as clearly demonstrated by broad efficacy of PD-1/PD-L1 blockade across different cancer types, some of them could instead be linked to the histological nature of the transformed tissue, the surrounding organ, and the biophysical/biochemical properties of the microenvironment. Furthermore, the reaction that the immune system establishes in response to diverse patterns of immune suppression might be finely tuned in different settings, resulting in multiple molecular pathways that could display histotype- and even patient-specificity. Understanding these intricate and dynamic scenarios more in depth, and, most importantly, developing proper means for clinical intervention will surely pave the way to an effective immune modulation, for a comprehensive way to cure cancer by counteracting immune suppression.


The etiology has been controversial, with theories including vascular degenerative changes or a reaction to vegetable material; ultrastructural studies and experimentally induced lesions in animals favor the latter. This lesion is typically seen in the oral cavity, with only rare reports in the gastrointestinal tract and gallbladder. We herein describe five cases of pulse granulomas identified in these sites. All cases contained foreign-body giant cells and vegetable debris within or near hyaline rings. Pulse granulomas may form mass lesions but are usually an incidental finding on microscopic examination. In incidentally detected cases, recognition of pulse granulomas can suggest a mural abscess, fistula, or perforation of the gut, findings which may not be grossly apparent. The presence of vegetable material in all five cases further supports an exogenous pathogenesis.Hyaline rings with admixed multinucleated giant cells characterize pulse granulomas; the term The etiology has been controversial, with theories including vascular degenerative changes or a reaction to vegetable material; ultrastructural studies and experimentally induced lesions in animals favor the latter. This lesion is typically seen in the oral cavity, with only rare reports in the gastrointestinal tract and gallbladder. We herein describe five cases of pulse granulomas identified in these sites. Case  1 was a 72-year-old female with a friable colonic mass suspicious for malignancy. Cases  2 and 3 were a 62-year-old female and 59-year-old male with peridiverticular pulse granulomas in the sigmoid colon. Case  4 was a 52-year-old male with pulse granulomas in appendiceal serosa and a coloenteric fistula tract secondary to perforated sigmoid diverticulitis. Case  5 was a 66-year-old male with pulse granulomas of the gallbladder wall in the setting of cholecystocolonic fistula secondary to chronic calculous cholecystitis. All of the cases exhibited convoluted hyaline rings varying in appearance from loose rings infiltrated by neutrophils to densely hyalinized, thick-walled rings  or a reaction to exogenous vegetable material, particularly cellulose . The majWhile usually an incidental finding, pulse granulomas of the gastrointestinal tract and gallbladder are useful to suggest or confirm a defect in the tubular gut. In these five cases, identification of pulse granulomas confirmed the presence of mural abscesses, fistulous tracts, or frank perforation of the intestinal wall; in some of the cases, these findings were not apparent on gross pathologic examination. Additionally, Gonzalez  recentlyGiven the distinctive appearance of the hyaline rings, the differential diagnostic considerations for pulse granuloma are fairly limited. The presumably early lesions have abundant acute inflammation, the rings have a compartmentalized structure suggestive of vegetable origin, and there is abundant free vegetable material. Cases with densely hyalinized, paucicellular rings with scant vegetable material may potentially resemble vascular amyloid deposits. In most cases, the corrugated shape and clustered distribution of the rings, presence of foreign-body giant cells, and associated vegetable material suggest the correct diagnosis. In problematic cases, special stains for amyloid would allow these entities to be distinguished. Liesegang rings may also enter the differential diagnosis, as these consist of eosinophilic material, overlap in size with pulse granulomas, and are often seen in a background of inflammatory cells and multinucleated giant cells. However, Liesegang rings are characterized by a laminated appearance with radially oriented striations. Perhaps most importantly, the granulomatous inflammatory response inherent to pulse granulomas may cause concern for Crohn's disease; this is particularly true in the gastrointestinal tract, where pulse granulomas are also associated with fistulizing disease. In peridiverticular pulse granulomas, distinction from Crohn's disease may be further confounded by architectural distortion, crypt injury with cryptolytic granulomas, and transmural inflammation related to diverticular colitis. In such cases, the recognition of diverticular disease in the segment and absence of well-formed epithelioid granulomas would prevent misclassification as Crohn's disease.In summary, herein we have described five cases of pulse granulomas involving the gastrointestinal tract and the gallbladder. The importance of recognizing this entity, particularly in the gastrointestinal tract, is that these lesions commonly mimic malignancy and other mass-forming lesions such as sclerosing mesenteritis . It is i


Hepatocellular carcinoma is one of the fatal malignancies and is considered as the third leading cause of death. Mutations, genetic modifications, dietary aflatoxins, or impairments in the regulation of oncogenic pathways may bring about liver cancer. An effective barrier against hepatotoxins is offered by gut-liver axis as a change in gut permeability and expanded translocation of lipopolysaccharides triggers the activation of Toll-like receptors which stimulate the process of hepatocarcinogenesis. Prebiotics, nondigestible oligosaccharides, have a pivotal role to play when it comes to inducing an antitumor effect. A healthy gut flora balance is imperative to downregulation of inflammatory cytokines and reducing lipopolysaccharides induced endotoxemia, thus inducing the antitumor effect. Hepatocellular carcinoma (HCC) ranks sixth amongst the most widely recognized malignancies and it makes up the third leading cause of cancer related deaths on a global level . WHO desThe health of gut and liver is paramount to nutrient absorption and controlling certain chief metabolic activities. Liver is provided with a double blood supply, by the courtesy of hepatic artery and portal vein . ProductThe LPS (lipopolysaccharide) level is sensitive to overgrowth in small intestinal bacteria, changed composition of microbiota, and increased intestinal permeability . EndotoxThe gut microbial composition in the liver is often seen as a stimulator to an increased activity of liver Toll-like receptors (TLRs), a class of proteins sensitive to some structurally conserved molecules derived from the microbiota . Variousα, which is triggered by TLR4 activation brought about by LPS and other pathogen-associated molecular patterns (PAMPs). Furthermore, the neoplasia of lymphotoxin-induced HCC is also enhanced by the activated NF-kB in mouse [Epiregulin, along with other mediators, creates a protumorigenic medium in an already established inflammatory microenvironment, thus paving the way for HCC. TLR4 are known to promote HCC at advanced stages of liver disorders . Myriadsin mouse . By utilin mouse .α production [The activation of downstream signals is effectively prevented by a lipid A analogue, eritoran tetrasodium (E5564), which works by binding itself to internal pocket of MD-2 (coreceptor of TLR4). Inflammation induced by LPS is also significantly brought under control by E5564 that also has importance for endurance in a sepsis model . Some otoduction . TAK-242oduction . In a nuoduction .HCC is advanced to a next stage as gut microbiota are an important contributive factor by virtue of gut-liver axis . HoweverPrebiotics have a central importance in maintaining a healthy intestinal microflora balance. They are categorized under the umbrella of nonabsorbent and indigestible food ingredients like lactulose which is known to promote growth and activity of various gut friendly microbiota , 27. The Prevotella and Xylanibacter, increases the population of Bifidobacterium, the clostridial cluster XIVa, and Faecalibacterium prausnitzii, and makes conditions harsh for harmful bacteroides like Firmicutes and Enterobacteriaceae [ Bifidobacteria and Lactobacilli. Based on some experiments, certain fungal products have also been identified to be used as prebiotic agents in future. Some traditional Chinese therapies include using Hirsutella sinensis (the anamorph of Cordyceps sinensis), Antrodia cinnamomea, and Ganoderma lucidum as an energy booster; water mycelium extracts of this fungus have been known to reduce LPS-induced endotoxemia when used on high-fat diet mice [On the basis of chemical structure, the inulin-type fructans (ITF) and the galactooligosaccharides (GOS) are the two major groups of prebiotics . Dietaryeriaceae . One of iet mice   can be brought about by triggering an increased fabrication of SCFAs in gut which can be easily achieved by incorporating diet supplemented with 10% (w/v) XOS-supplemented in the regular meals to increase Bifidobacterium colonies throughout the intestine to a remarkably great number. The same increase in the number of Bifidobacteria and Lactobacilli colonies can also be achieved in vitro by administering acidic oligosaccharides obtained from apple pectin which also creates an increased concentration of acetic, propionic, and lactic acid [β-glucosidase and β-glucuronidase [Nondigestible oligosaccharides make up the majority of prebiotics identified and tested in labs so far , 58. Lactic acid . This deronidase .κ-Carrageenan oligosaccharides also showed immunomodulation effects on S180-bearing mice along with antitumor activities, extracted from Kappaphycus striatum [Immune system receives a multitude of benefits, when exposed to oligosaccharides, along with successfully inhibiting cancer metastasis and carrying on certain other activities like complement activation and immunological activities , thus mastriatum   are known to exhibit antitumor activities by regulating the function of immune system and conducting direct actions against tumor cells [ Trametes versicolor, Phellinus linteus, Poria cocos, Lycium barbarum, and Atractylodes macrocephala [Uplifting the efficacy of chemotherapy has always been a challenge and botanical polysaccharides hold a special significance as they make a rich source for adjuvants, antitumor, and immunomodulating agents , 68. Mancharides , 69, Astor cells –72. The ocephala . Pleurotus tuber-regium) and sporoderm-broken germinating spores (SBGS) of Reishi exert a significant antitumor effect, specifically in prevention of revival or metastasis of cancerous cells. It mitigates the toxic and side effects of chemotherapy and radiotherapy in some patients [ Pleurotus ostreatus also have antitumor activity against Hela tumor cells [Polysaccharides immunomodulating properties also include the enhanced proliferation of lymphocytes and antibody production,  as well patients  Moreoveror cells   linkage with additional b-(1/6) branch points . The ant Bifidobacteria [Fructans are the most widely used prebiotics among others . Oligofrbacteria . Inulin-bacteria , 82.The gut microbiota influence progression of BaF3 cells by altering its metabolome. Due to increased gut microbiota derived metabolites which specifically target liver tissue, propionate is higher in the portal vein of rats fed with ITF and mediates a protective effect  and enteButyrate and other short chain fatty acids upset the cell cycle in human cancer cells by inhibiting proliferation, inducing differentiation, and cell death –88. IntrAdministration of lactulose accelerates posthepatectomized liver regeneration in rats by inducing hydrogen, which may result from offsetting oxidative stress and inflammatory response . LactuloCellular immunity plays an important role in tumor immunity. There is mainly the secretion of Th1 cytokines in a body having strong antitumor immunity, while the body's antitumor immunity is inhibited when there is mainly the secretion of Th2 cytokines. IFN-g and IL-4 are representative cytokines, respectively, produced by Th1 and Th2 cells. Significantly greater level of IFN-g and no difference in the level of IL-4 suggested that Th1/Th2 cell polarization evidently shifted to Th1 cells, and patient's antitumor immunity was significantly enhanced after the administration of lactulose . Many otSeveral evidences have suggested the role of prebiotics in alteration of gut microbiota and reduction of procarcinogenic factors in liver. So, in the light of antitumor properties of prebiotics, it may be suggested that modulation of gut flora by prebiotics may represent novel strategies to prevent progression of chronic liver disorders to HCC. However, further studies are still needed to confirm and clarify the possible mechanisms involved and we may hope for the development of new therapeutic strategies to prevent HCC in near future. In conclusion, this data suggests that prebiotics may prove economical and safer antitumor agents against hepatocarcinogenesis.


Human-associated microbial communities include prokaryotic and eukaryotic organisms across high-level clades of the tree of life. While advances in high-throughput sequencing technology allow for the study of diverse lineages, the vast majority of studies are limited to bacteria, and very little is known on how eukaryote microbes fit in the overall microbial ecology of the human gut. Human-associated microbial communities include prokaryotic and eukaryotic organisms across high-level clades of the tree of life. While advances in high-throughput sequencing technology allow for the study of diverse lineages, the vast majority of studies are limited to bacteria, and very little is known on how eukaryote microbes fit in the overall microbial ecology of the human gut. As recent studies consider eukaryotes in their surveys, it is becoming increasingly clear that eukaryotes play important ecological roles in the microbiome as well as in host health. In this perspective, we discuss new evidence on eukaryotes as fundamental species of the human gut and emphasize that future microbiome studies should characterize the multitrophic interactions between microeukaryotes, other microorganisms, and the host. Humans coevolved alongside a multikingdom gut microbial ecosystem that includes microbial eukaryotes (microeukaryotes) among ancestral inhabitants of the human gut . The gutCandida albicans, are known to elicit strong immune responses  . These nesponses . HoweverMicroeukaryotes have been traditionally studied for their parasitic (protozoans) or pathogenic (fungi) relationships with the host, although they are increasingly being recognized to include commensal and potentially beneficial members of the gut microbiome . Here, wIn light of the ecological and disease-related evidence discussed here, we emphasize the imperative need to include microeukaryotes in all future human gut microbiome studies and to prioritize the characterization of specific bacterial-eukaryotic interactions in the gut.Saccharomyces, Candida, and Malassezia as the most common genera in healthy North Americans. Our own studies in 250 healthy rural Mexicans, using 18S as a marker gene, found Saccharomyces, Pichia, and Aspergillus as the most abundant genera (unpublished observations). This finding suggests that, just like the fecal bacterial community, the fungal composition differs between sociogeographical settings. Compositional differences can also be detected during early infancy, as revealed by comparing an early-life microbiome study in North American babies by Fujimura et al.  of ocieties , 21–23. eties 21–, 24–26 aes 21–24––29 indivs 21–24–– and withs 21–24––. We mades 21–24––. Althougn et al.  reportedn et al. . Future iversity .Blastocystis sp. likely exerts a significant impact on smaller prey organisms such as bacteria.Although much remains to be elucidated on how microeukaryotes can support bacterial diversity, multitrophic network stability has been shown to be highly dependent on the size ratio between trophic levels . As poteGut microbes are implicated in the pathogenesis of immune-mediated diseases, such as inflammatory bowel disease (IBD) and asthma, in which a causal association has been confirmed in relevant models of disease , 36. UntCandida species was significantly higher in CD individuals than in healthy first-degree relatives and positively correlated with levels of anti-Saccharomyces antibodies (ASCA) -induced and T-cell transfer-mediated colitis (Microbiome studies in pediatric  and aduls (ASCA) , an antis (ASCA) . Studies colitis .Microbiome studies in prospective infant cohorts have also revealed gut fungal alterations preceding asthma development. We recently discovered marked fungal dysbiosis in 3-month-old Ecuadorian babies associated with subsequent asthma risk. In this study , as wellWhile previously overlooked, the mycobiome is altered after antibiotic treatment , a risk Blastocystis sp. as a potential ecosystem engineer of the gut microbiome, likely capable of influencing the overall microbiome structure, as well as interacting and modulating the host immune system. Future ecological experiments should test this for Blastocystis sp., as well as other protozoan species more commonly found in nonwesternized settings.As modern sequencing technology continues to increase the pace and depth of taxonomic and functional characterizations of the human gut microbiome, emergent studies have provided exciting new findings that involve microbial eukaryotes. A misconception of the overall importance of microeukaryotes is their reduced abundance in comparison to bacteria. However, the impact of a species on community structure and function is not necessarily proportional to its relative abundance. Macro- and microecology studies provide plenty of examples of the strong structuring consequences of cascading effects of low-abundance species, crossing multiple trophic levels and modulating the relative abundance of many species , 44. CurGiven the evidence discussed here, we propose that, just like current microbiome studies must account for the effects of diet, age, antibiotics, inflammation, and other variables known to influence the microbial community, new human microbiome studies should integrate and account for the effect of microbial eukaryotes. Finally, given the crucial interrelationship between the infant gut microbiome and many host physiological developmental processes, it is of utmost importance to understand how early-life patterns of diversity occur, how they are influenced by interkingdom interactions, and how these community patterns influence infant development. This will provide a more integrated view on gut microbiomes, the factors that shape them, and the mechanisms by which they relate to health and disease.