Intestinal goblet cells maintain the protective epithelial barrier through mucus secretion and yet sample lumenal substances for immune processing through formation of goblet cell associated antigen passages (GAPs). The cellular biology of GAPs and how these divergent processes are balanced and regulated by goblet cells remains unknown. Using high-resolution light and electron microscopy, we found that in mice, GAPs were formed by an acetylcholine (ACh)-dependent endocytic event remarkable for delivery of fluid-phase cargo retrograde into the trans-golgi network and across the cell by transcytosis – in addition to the expected transport of fluid-phase cargo by endosomes to multi-vesicular bodies and lysosomes. While ACh also induced goblet cells to secrete mucins, ACh-induced GAP formation and mucin secretion were functionally independent and mediated by different receptors and signaling pathways, enabling goblet cells to differentially regulate these processes to accommodate the dynamically changing demands of the mucosal environment for barrier maintenance and sampling of lumenal substances. Cells in the gut need to be protected against the many harmful microbes which inhabit this environment. Yet the immune system also needs to ‘keep an eye’ on intestinal contents to maintain tolerance to innocuous substances, such as those from the diet. The ‘goblet cells’ that are part of the gut lining do both: they create a mucus barrier that stops germs from invading the body, but they also can pass on molecules from the intestine to immune cells deep in the tissue to promote tolerance. This is achieved through a ‘GAP’ mechanism. A chemical messenger called acetylcholine can trigger both mucus release and the GAP process in goblet cells.Gustafsson et al. investigated how the cells could take on these two seemingly opposing roles in response to the same signal. A fluorescent molecule was introduced into the intestines of mice, and monitored as it pass through the goblet cells. This revealed how the GAP process took place: the cells were able to capture molecules from the intestines, wrap them in internal sack-like vesicles and then transport them across the entire cell.To explore the role of acetylcholine, Gustafsson et al. blocked the receptors that detect the messenger at the surface of goblet cells. Different receptors and therefore different cascades of molecular events were found to control mucus secretion and GAP formation; this explains how the two processes can be performed in parallel and independently from each other.Understanding how cells relay molecules to the immune system is relevant to other tissues in contact with the environment, such as the eyes, the airways, or the inside of the genital and urinary tracts. Understanding, and then ultimately harnessing this mechanism could help design of new ways to deliver drugs to the immune system and alter immune outcomes. The simple columnar epithelium lining the gastrointestinal tract is an expansive surface exposed to lumenal contents containing innocuous substances from the diet and potentially harmful microbes and their products. Underneath and within this epithelium lies the largest collection of immune cells in the body. It has long been appreciated that the gastrointestinal immune system is not ignorant of the lumenal contents, and in the healthy state gut lumenal contents are sampled to induce adaptive immune responses, characterized by T cell-mediated antigen-specific tolerance. How substances from the lumen are encountered by, or delivered to, the immune compartment is a central concept in mucosal immunity that is incompletely understood, but fundamentally underlies how the immune system can mount tolerogenic responses to substances encountered in this potentially hostile environment.Goblet cells (GCs) are specialized intestinal epithelial cells that have a well-established role in innate immunity through secretion of mucins and maintenance of the mucus layer. The mucus layer provides a first line of defense against physical and chemical injury and protects against pathogen invasion . GCs alsHere, we demonstrate that GAPs form by an endocytic event capturing fluid-phase cargo and often follow secretory granule exocytosis. But in contrast to the fate of fluid-phase endocytosis seen in adjacent enterocytes, GAPs traffic a substantial portion of fluid-phase cargo across the epithelial barrier by transcytosis for capture by underlying phagocytic cells. Further, we find that while ACh induces both mucus secretion and GAP formation in GCs, these processes are not functionally linked and can be performed independently or in parallel to meet the needs of the changing lumenal environment in the small intestine and colon. Together these observations suggest that GAP formation may have evolved as a cell-type specific and specialized endocytic pathway emerging from the basic exocytic machinery of GCs to sample and deliver luminal substances to the mucosal immune system.YFP+ mononuclear phagocytes (MNPs) located beneath the epithelial barrier  using intestinal tissue obtained from mice 1 hr after administration of 10 kDa lysine fixable dextran biotin into the gut lumen. Full thickness tissues were incubated with heavy metal labeled streptavidin and processed for FIB-SEM and imaged at a voxel resolution of 10 nm per slice with >1000 slices encompassing the thickness of a GC. Datasets of four GCs endocytosing lumenal dextran were imaged with this technique. A dataset containing 1199 images encompassing the volume of a GC that had taken up dextran was used to create the 3D model of a GC forming a GAP illustrated in + mucus area per villus or crypt cross section -driven GAP formation in adult mice . In the  section , image EUlex europaeus agglutinin 1 (UEA1+) mucus area per crypt cross section  or UEA1+  mucus area, at all three locations (+ (SI) and UEA1+  GCs  GCs , indicatlon) GCs  consistelon) GCs , suggestFurther correlation of the CCh-induced mucus secretory response with the GAP response between compartments revealed that the SI crypts had the largest reduction of total mucus content per cross section , whereas the SI villus had the largest GAP response to CCh quantified as the percentage of GCs forming GAPs in response to CCh . The SI and distal colon crypts responded to CCh with complete emptying of an average of 3 and 4 GCs per crypt cross section, respectively , and forTo evaluate if the observed regional and spatial differences in ACh-induced mucus secretion and GAP formation were due to the two processes being mediated by different mAChRs, we treated mice with mAChR antagonists and measured the effect on CCh-induced GAP formation and mucus secretion. Our results showed that the mAChR4 antagonist tropicamide had no effect on the CCh-induced mucus secretory response in either the SI villus or crypt compartment , but it We evaluated the expression of mAChRs in the SI and distal colon to correlate the CCh-induced mucus secretory response and the GAP response with expression of the respective receptors. In the SI, the mAChR4 expression increased along the crypt – villus axis, correlating with the higher prevalence of mAChR4-dependent GAP formation in the villi as compared to the crypts . In cont2+ and endocytosis being a Ca2+-dependent process  as well as by chelation of intracellular Ca2+ (BAPTA-AM) in the SI  demonstrating that both baseline and CCh-induced GAP formation are dependent on intracellular Ca2+.Based on previous studies demonstrating that ACh-induced mucus secretion is mediated by elevated levels of intracellular Ca process , we expln the SI  and in tn the SI , indicatn the SI  and distn the SI  was inde2+ that are released in response to mAChR activation include the endoplasmatic reticulum (ER) and acidic organelles such as endosomes/lysosomes, and in the case of GCs, mucin granules that represent a large acidic compartment with high Ca2+ content (2+ release from these intracellular stores occurs via three different pathways mediated by the second messengers inositol trisphosphate (IP3), cyclic ADP ribose (cADPr), and nicotinic acid adenine dinucleotide phosphate (NAADP), each acting on IP3 receptors (IP3R), ryanodine receptors (RyR), and two pore channels (TPC), respectively , cADPr using 8-Br-cADPr, and NAADP using trans-Ned-19 (T-Ned-19), and evaluated the effects on CCh-induced mucus secretion and GAP formation. In the SI villus, despite the CCh secretory response being reversed by chelation of intracellular Ca2+, none of the respective inhibitors reversed the CCh-induced secretory response, suggesting possible redundancy in the signaling pathways inducing this response  and in those not forming GAPs . We observed that CCh induced a significant decrease in the average theca area of GCs forming GAPs at all three locations , indicateca area  and a sheca area  and G, cribution . Given oNotably, in vehicle treated mice, particularly in the SI villus, the theca area of GCs forming GAPs was generally smaller as compared to GCs not forming GAPs compare , consistThe intestinal epithelium is faced with the complex task of acting as a semi-permeable barrier allowing efficient nutrient absorption and controlled exposure of the immune system to lumenal antigens to sustain immune tolerance, while at the same time limiting contact with harmful agents that pass through the gastrointestinal tract. GCs have long been appreciated for their role in barrier function via production and secretion of the mucus layer that covers the intestinal surfaces . RecentlEndocytic retrieval of secretory granule membranes has been shown to occur in various secretory cells including neurons, enterochromaffin cells, pancreatic acinar cells, and endothelial cells  and is l+ triggers neurotransmitter release paralleled by endocytic retrieval of secretory granule membrane and uptake of high molecular weight substances from the extracellular environment. Similar to our observations demonstrating that inhibition of CCh-induced mucus secretion did not affect CCh-induced GAP formation, inhibition of K+ evoked exocytosis did not affect K+-induced endocytic retrieval of secretory granule membrane. The intact endocytic response in response to elevated K+ levels was shown to be mediated by retrieval of previously fused secretory vesicles, thus, although elevated K+ triggered secretory granule exocytosis, retrieval of previously fused granules was enough to sustain the uptake process  is inhibited by activation of the epidermal growth factor receptor (EGFR), which in the proximal colon can occur by GC intrinsic sensing of gut microbial products via Toll-like receptors or by the presence of EGFR ligands in the SI and colon lumen . The benantigens . Thus, fAlthough the focus of the present study was to explore the cellular basis of ACh-induced GAP formation, the ability of GCs to acquire lumenal substances is not restricted to activation of muscarinic receptor signaling and additional ligands and receptors activating cADPr production, such as IL13, have been demonstrated to induce GCs to take up lumenal substances in some settings . This raIn summary, our results define the basis by which GCs sample lumenal antigens for delivery to the immune system and how this process is balanced with mucus secretion within the same cell. Our observations indicate that GCs have evolved a pathway of fluid-phase endocytosis that efficiently serves the transcytotic pathway for non-specific uptake of lumenal substances that are sampled by sub-epithelial phagocytic cells. Compared to neighboring enterocytes, the fluid-phase endocytic GAP pathway of GCs is remarkable for its efficient transport of lumenal substances across the epithelial barrier. Perhaps GAPs evolved from an endocytic system originally used in secretory cell types to recycle secretory granule membranes, but adapted to ensure that during situations when the lumenal environment is toxic for antigen sampling, the GC can suppress GAP formation while retaining its ability to secrete mucus for maintenance of the mucus barrier. In total, our observations reveal the basis by which GCs perform the function of lumenal antigen delivery to the immune system, and provide mechanistic insights into how the critical roles of barrier maintenance and antigen delivery are achieved within the same cell.YFPItgax transgenic mice  were administered i.p. at a dose of 550 µg/kg, 30 min prior to intraluminal injection of 10 kDa lysine fixable TRITC-dextran (12.5 mg/ml) . Tropicamide  was administered at a dose of 100 µg/kg s.c. 20 min prior to intraluminal injection of TRITC-dextran, and every 20 min during the 60 min incubation period. CCh  was administered s.c. at a dose of 125 µg/kg, 5 min following intraluminal injection of TRITC-dextran. FM 1–43FX  was administered intraluminal at a dose of 50 µg/ml, followed by 1 hr incubation. EGTA (2 mM), BAPTA-AM (200 µM), xestospongin C (22 µM), dynasore (150 µM), ciliobrevin D (100 µM) , Dyngo 4a (120 µM) , colchicine (100 µM), cytochalasin D (4 µM), and dimethylenastron (DMEA) (10 µM)  were administered intraluminal 30 min prior to intraluminal injection of TRITC-dextran. In these experiments, the TRITC-dextran solution also contained the respective inhibitors at the concentration described above. 8-Br-cADPr (0.2 mg/kg)  and Trans-Ned19 (5 mg/kg)  were administered i.p. 30 min prior to intraluminal injection of TRITC-dextran and LY294002 (4 mg/kg) was administered i.p. 15 min prior to intraluminal injection of TRITC-dextran.2/5% CO2. Intravital preparation of the SI was performed as previously described (Mice were anesthetized using nebulized isofluorane in 95% Oescribed . Briefly+ (SI)/UEA1+  epithelial cells measuring approximately 20 µm (height) × 5 µm (diameter) traversing the epithelium and containing a nucleus and were enumerated as GAPs per villus/crypt cross section in the SI, and GAPs per crypt cross section in the distal colon.Lysine fixable TRITC or Alexa Fluor 647 conjugated 10 kDa dextran was administered into the SI or distal colon of ketamine/xylasine anesthetized mice. After 1 hr, mice were sacrificed, tissues were dissected, cut open along the mesenteric border, and thoroughly washed with cold PBS before fixation in 10% formalin buffered solution for 2 hr, followed by incubation in 30% sucrose overnight. Tissues were embedded in optimal cutting temperature compound (OCT)  and 6 μm sections were prepared. Sections were stained with fluorescein conjugated WGA  or fluorescein conjugated UEA1  to identify GCs in the SI and distal colon, respectively, and stained with DAPI  to identify the nucleus. Sections were imaged using an Axioskop 2 microscope  or a Nikon n-SIM super-resolution microscope . Obtained images were evaluated using AxioVision, and Imaris, respectively. GAPs were identified as TRITC-dextran filled WGA+ (SI) or UEA1+  GCs theca, and expressed either as GC theca area in µm2 or as the total mucus content per villus/crypt cross section, quantified as the sum of all WGA+/UEA1+ GC theca areas, divided by the area of the villus/crypt cross section, expressed as percentage. Mucus secretion was defined as a significant decrease in the GC theca area, or total mucus content of the villus/crypt cross section comparing vehicle control and the respective treatment groups.Mucus secretion was quantified using Fiji  by measuBriefly, FM 1–43FX was administered into the SI of ketamine/xylasine anesthetized mice. After 1 hr, mice were sacrificed and the SI was dissected, cut open along the mesenteric border, and washed with cold PBS before fixation in 10% formalin buffered solution for 2 hr, followed by incubation in 30% sucrose overnight. Tissues were embedded in OCT, and 6 μm sections were prepared. To evaluate the FM 1–43FX staining pattern in GC, sections were stained with Texas Red conjugated WGA , and DAPI to identify the nucleus, and imaged using an Axioskop 2 microscope. Images were analyzed using AxioVision . To evaluate the FM 1–43FX staining pattern in GCs forming GAPs, FM 1–43FX and dextran-Alexa647 was administered into the SI lumen simultaneously. After 1 hr the mice were sacrified and tissue processes as described above. Tissue sections were stained with DAPI to identify the nucleus and imaged using an LSM700 Axio Examiner Z1 confocal microscope . Acquired images were analyzed using Zen  and Imaris  software.Tissue specimens were washed in cold PBS, fixed in 10% formalin buffered saline for 2 hr, followed by incubation in 30% sucrose overnight. Tissue specimens were embedded in optimal tissue cutting medium and 6-µm-thick section were cut. Sections were rinsed in PBS followed by antigen retrieval in 0.05% citraconic anhydride , permeabilized in 0.05% Triton X-100 or 0.05% saponin (TGN46) , washed in PBS, and blocked in 5% goat serum . Sections were incubated with primary antibodies toward EEA1, Rab7 , Lysozyme, TGN46 , Rab3D , LAMP1, Calnexin , or VAMP-8  at 4°C overnight, rinsed with PBS and incubated with goat anti rabbit or goat anti chicken Alexa Fluor conjugated secondary antibodies  at room temperature for 1 hr. Sections were counterstained with DAPI and imaged using an Axioskop 2 microscope, an LSM700 Axio Examiner Z1 confocal microscope, or an LSM900 with Airyscan 2 microscope . Acquired images were analyzed using Zen  and Imaris  software.SI and distal colon tissue specimens were dissected, fixed for 16 hr in 4% formaldehyde, followed by overnight incubation in 30% sucrose. Tissue specimens were embedded in optimal cutting medium, frozen, and 6 µm thick sections were cut. Cut sections were washed in PBS, followed by single molecule in situ hybridization of Chrm1 (probe: 483441-C2), Chrm3 (probe: 437701), or Chrm4 (probe: 410581-C2) according to manufacturer’s instructions RNAscope fluorescent reagent kit v2  visualized with Opal-570  or CF488A Tyramine . Following the RNAscope protocol, tissue sections were stained with a primary antibody against Epcam  at 4°C overnight followed by incubation using a Alexa Fluor 647 conjugated secondary antibody . Nuclei were counterstained with Hoechst 33528 and a subset of SI sections were stained with Alexa Fluor 555 conjugated WGA , and a subset of distal colon sections were stained with FITC conjugated UEA1  to visualize GCs. Images were obtained by an LSM700 Axio Examiner Z1 confocal microscope .2O2 for 10 min, rinsed with PBS and fixed overnight in 2% glutaraldehyde. Stained tissue specimens were embedded in resin and evaluated using a Zeiss Crossbeam 540 FIB-SEM equipped with a GEMINI II and a Capella Ga-Liquid Metal Ion Source (Ga-LMIS) column . The milling process was set to 10 nm generating a dataset of ~1200 SEM images encompassing the volume of a GC. The acquired dataset was analyzed by Amira . For the 3D visualization, organelles were manually traced and identification of the respective organelles and dextran-containing organelles was performed using Functional Ultrastructure: Atlas of Tissue Biology and Pathology  was administered into the SI of ketamine/xylasine anesthetized mice. After 1 hr, mice were perfused with 4% paraformaldehyde (PFA), followed by fixation in 4% PFA overnight. Tissues were transferred to cold PBS, embedded in 2% agarose, and sectioned using a vibratome; 100 µm thick sections were rinsed in PBS, exposed to glycine, followed by rinsing in PBS and permeabilized in 0.05% saponin. Permeabilized sections were rinsed in PBS, exposed to streptavidin-HRP  and exposed to Ni-DAB  in PBS for 10 min, followed by exposure to Ni-DAB in Hathology . For evaData is presented as mean ± standard error or the mean. Statistical analysis was performed using GraphPad Prism 7 . Analyses between two groups were performed using the Student’s t-test. Comparisons between three groups or more were performed using a one-way ANOVA with Dunnett’s post hoc test for correction of multiple comparisons. A cut-off of p < 0.05 was used for statistical significance. Details regarding the statistical test used in the respective experiments are indicated in the figure legends together with populations size. All experimental groups consist of three or more technical replicates. Results of statistical tests are indicated in the figures and in the text. In this study, the authors present a compelling data regarding how barrier function and immunity are coordinated within the mammalian intestinal system. This paper demonstrates that cells responsible for the secretion of the protective mucous lining of the intestine also sample substances within the intestinal lumen to present to the immune system, and how these two processes are differentially regulated. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Reviewing Editor and Vivek Malhotra as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Intestinal Goblet Cells Sample and Deliver Lumenal Antigens by Regulated Endocytic Uptake and Transcytosis" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) Additional immunolabeling data 2) Enhanced presentation of the FIB SEM data3) Images of the intestinal epithelium following drug treatmentReviewer #1:The authors examined how two processes  are regulated in goblet cells of the small intestine and colon. They find differential regulation of these two processes and that they are not functionally linked. This work involves pharmacological investigation of signaling pathways and calcium regulation, demonstrating distinct responses. The understanding of how these two processes are regulated provides a more detailed understanding of how barrier maintenance and antigen delivery to the immune system are coordinated within the digestive system.1. I would recommend reorganizing the panels in Figure 5 to pair the results of each cell type for GAP formation vs. mucus secretion so that they are more easily compared. For example, panel A would be SI villus secretion and panel B would be SI villus GAP formation.2. Are there any data indicating whether there are differences in the composition of the center vs. lateral regions the apical membrane of goblet cells to provide insight into how these two processes are spatially segregated?Reviewer #2:The authors investigate the formation and cell biology of goblet cell associated antigen passages (GAPs). Using FIB SEM and a fluid phase tracer they provide a 3D model of the structure of a goblet cell including identification of key parts of the trafficking pathway. By TEM they also provide evidence of vesicle uptake by underlying cells. Using pharmacological inhibitors, they provide the first evidence of differential machinery for independent regulation of GAP formation separate from the exocytic machinery necessary for mucin granule exocytosis. They conclude that GAP formation is a transcytosis process based on endocytosis at the edge of the apical side of the goblet cell (in an EEA1 independent manner) followed by trafficking throughout the cytoplasm before exocytosis at the basal side.This is a convincing study providing good evidence of related but separately regulated endocytic and exocytic processes to mediate important physiologically and pathologically relevant functions.StrengthsThe major strength of the manuscript is the in vivo imaging aspects in mice. These provide analysis of the phenomena in situ, adjacent to the appropriate associated gut cells, enterocytes etc. It also allows analysis of the phenomena in discrete sections of the intestinal tract. The quantification of phenomena is robust, careful and thorough and appropriately statistically powered.The study provides good evidence for differences in regulation of GAP formation vs mucin granule exocytosis both in terms of their activation by muscarinic receptors and the downstream signalling from extracellular and intracellular calcium stores. This supports the conclusion that both of these phenomena although related can be differentially controlled. The rationale for the necessity for separate pathways existing is well thought out and logical.The FIB SEM electron microscopy analysis with labelled streptavidin provides a clear and striking delineation of the trafficking route. It convincingly highlights all the structures the authors noted and supports the assertion that a transcytosis route provides a means to safely sample fluid phase material from the gut lumen.WeaknessesThe experiments rely largely on pharmacological interventions to monitor the formation of GAPs and to pick apart the differential regulation of GAP formation vs mucin secretion. These agents will impinge on many intracellular pathways occurring in the cell making effects on GAP formation and mucin secretion potentially both direct and indirect.Images of the effect of some of the drugs and chemicals on goblet cells as they undergo GAP formation and mucin release are likely to be informative as to the direct or indirect nature of the exposure. If an arrest of trafficked dextran is noted at certain points on the surface of the goblet cell, or at discrete points within the cell following dynamin, actin or microtubule inhibition this would indicate more specifically the point at which these pieces of molecular machinery are required. Although exposure of the inner surface of the gut to EGTA is likely to interfere with traffic of Calcium from an extracellular source it is also likely to disrupt intracellular junctions which may have gross and potentially indirect effects.The article is very well written, clear and the subject matter is important.It would be helpful to include more images to support the quantification of the affects you note in Figure 1D. Such images might shed light on the specific role of the inhibited pathways, if for example dextran labelled pools accumulate near the surface, Golgi etc. It will also provide a better idea as to how the drug treatments are more broadly affecting the cells in question.The volume electron microscopy images shown in figure 2 are striking. A movie might help give an idea as to how well the segmentation of the em images fits with the rendered images shown, it might also provide better context to the arrangement of the structures in the goblet cell. Are labelled vesicles present towards the edge of the cell? Are there examples of vesicles elsewhere perhaps in amongst the mucin granules? Also, is it possible to catch endocytic structures forming at the apical edge of the cell as posited.Additional markers of the endocytic pathway associated with GAP formation would also be beneficial. The authors convincingly showed EEA1 is not present, it would be helpful to compare localisation of late endosomes, lysosomes, recycling endosomes, the ER and the TGN. Especially as some of these structures are thought to provide a reservoir for calcium.Associated images of the cells exposed to the calcium related drugs would also be helpful, again to get an idea as to if there are any intermediates of GAPs and to get an idea as to the health of the tissue. The BAPTA-AM appears to actively reduce the numbers of GAPs… Are the effects over the vehicle significant and does this provide further information about their stability and formation?There are typographical errors on line 694 and 760.Generally, this is a well-conceived and interesting study providing good evidence of the cell biology of GAP formation and mucin granule secretion in the goblet cells of mice.Reviewer #3:Combined two-photon in vivo imaging, super-resolution imaging, and FIB-SEM, these authors tried to follow the fate of the fluorescent tracer injected to the gut lumen. They suggested the identity of GAP as results of ACh stimulated endocytic events that are retrograded cross the Golgi network for transcytosis on the other side of the goblet cells. Besides, ACh induced GAP formation and mucin secretion via distinct receptors and signallng pathways.Major strengths: Detailed phamacological experiments clearly established two distinct pathways of ACh induced GAP formation and mucin secretion, which could lead to better understanding of the connection between goblet physiology and intestinal immunity.Weaknesses:1. It is unclear why ACh induces both GAP formation and mucin secretion in the same goblet cell. What would be the benefit of such arrangements?2. The current data are based mostly on phamacological perturbation. I wonder whether the authors could at least show some non-overlapping distributions of mAChR1, mAChR2, mAChR3, and mAChR4.3. Based on imaging data, the authors concluded the transcytosis fate of GAP vesicles. However, partial colocalization of dextran with the trans-Golgi marker and its fuzzy co-localization with Rab3D conferred the evidence, which is weak. We may need more direct evidence to better support this assertion.4. Indeed, the authors have used sophisticated in vivo imaging techniques. However, they did not show the processes of dextran internalization in vivo or any time-dependent changes, which did not seem to fully use the technology's potential. In addition, some images were fuzzy, and no structures can be identified.5. No mention of the blue labels in Figure 1, which I presumably to believe to be nulceus. If that is true, I wonder why red OVA puncta were in the middle of blue nucleus .6. In addition, in Figure 1H, the authors claimed little overlapping between dextran and EEA1. However, structures highlighted by the arrows seem to be dual labeled with both markers.7. The authors have stated that they have obtained four cells underwent FIB-SEM imaging and used one as the example and the left as the replicates to confirm. I would suggest that they shall have some critera/quantifications that are indeed confirmed by the data not shown. Essential revisions:1) Additional immunolabeling data Labeling of mAChR subtypes: Figure 5—figure supplement 1 and 2. Labeling of endocytic markers: Figure 2—figure supplement 1, and Figure 6—figure supplement 1.2) Enhanced presentation of the FIB SEM dataEnhanced presentation of the FIB-SEM data: Video 1 and 2.3) Images of the intestinal epithelium following drug treatmentImages of the epithelium following drug treatment: Figure 1—figure supplement 1, Figure 6E-K, and Figure 7C-I.Reviewer #1:The authors examined how two processes  are regulated in goblet cells of the small intestine and colon. They find differential regulation of these two processes and that they are not functionally linked. This work involves pharmacological investigation of signaling pathways and calcium regulation, demonstrating distinct responses. The understanding of how these two processes are regulated provides a more detailed understanding of how barrier maintenance and antigen delivery to the immune system are coordinated within the digestive system.1. I would recommend reorganizing the panels in Figure 5 to pair the results of each cell type for GAP formation vs. mucus secretion so that they are more easily compared. For example, panel A would be SI villus secretion and panel B would be SI villus GAP formation.We agree with the reviewer and have reorganized Figure 5 according to the suggestions and updated the text to fit the new figure layout. Page 17.2. Are there any data indicating whether there are differences in the composition of the center vs. lateral regions the apical membrane of goblet cells to provide insight into how these two processes are spatially segregated?To our knowledge there are no studies demonstrating differences in the organization of the apical membrane. In goblet cells, the ER is surrounding the theca and one explanation to the spatial segregation of these processes is that they depend on, and access, different pools of calcium.Reviewer #2:[…]The article is very well written, clear and the subject matter is important.It would be helpful to include more images to support the quantification of the affects you note in Figure 1D. Such images might shed light on the specific role of the inhibited pathways, if for example dextran labelled pools accumulate near the surface, Golgi etc. It will also provide a better idea as to how the drug treatments are more broadly affecting the cells in question.We agree with the reviewers and have added images to support the quantification. The added images are found in Figure 1—figure supplement 1, indicated on page 5 lines 115, 118, 120 and 121. We have not found pools of dextran accumulating near the golgi, thus the inhibition appears to be occurring at the initial uptake event. In rare instances we have observed dextran accumulating at the surface of the cell but the events are not common enough for us to quantify.The volume electron microscopy images shown in figure 2 are striking. A movie might help give an idea as to how well the segmentation of the em images fits with the rendered images shown, it might also provide better context to the arrangement of the structures in the goblet cell.We agree with the reviewer and have added 2 Videos to better visualize the 3D mode and the segmentation. Video 1 shows the rendered 3D model, and Video 2 shows the segmentation that the model is based on the EM data.Are labelled vesicles present towards the edge of the cell? Are there examples of vesicles elsewhere perhaps in amongst the mucin granules? Also, is it possible to catch endocytic structures forming at the apical edge of the cell as posited.The labelled vesicles are mainly found at the edge of the cell and a few vesicles are found among the mucin granules. The distribution of vesicles within the cells is now visualized in a clearer way in Video 2, added to the revised manuscript, which demonstrates a Z stack of the FIB-SEM images and the corresponding Z stack of the segmentation that the model is based on. We have observed vesicles containing lumenal tracer at the apical edge of the GAP as shown in Figure 1C, and in the ultrastructural model. We have not captured the initial event of GAP formation in these studies, in part due to the experimental setup evaluating tissue sections one hour after administration of lumenal tracers, and in part because our studies focused on goblet cells definitively forming GAPs, identified by complete filling with lumenal tracer, as opposed to goblet cells potentially endocytosing lumenal substances via other pathways or outcomes.Additional markers of the endocytic pathway associated with GAP formation would also be beneficial. The authors convincingly showed EEA1 is not present, it would be helpful to compare localisation of late endosomes, lysosomes, recycling endosomes, the ER and the TGN. Especially as some of these structures are thought to provide a reservoir for calcium.We agree with the reviewer and have added staining of the late endosome marker Rab7, the lysosome marker LAMP1, the TGN marker TGN46, and the ER marker calnexin to the revised manuscript. These findings are shown in Figure 2—figure supplement 1 on page 32, and Figure 6—figure supplement 1 on page 35 and referenced in the text on page 9 lines 197 -199, 205-209 and on page 20 line 446-451.Associated images of the cells exposed to the calcium related drugs would also be helpful, again to get an idea as to if there are any intermediates of GAPs and to get an idea as to the health of the tissue. The BAPTA-AM appears to actively reduce the numbers of GAPs… Are the effects over the vehicle significant and does this provide further information about their stability and formation?2+. We have added information regarding this in the revised manuscript, page 18, lines 4010 -413. We have not observed intermediates of GAP formation following treatment with the inhibitors. We interpret this to indicate that the inhibitors act on early events in GAP formation, or these events are below the limit of detection of our assays.We agree with the reviewer and have split Figure 6 into Figure 6 on page 19 and Figure 7 on page 21 to include visualization of the effect that the respective inhibitors have on the tissue. The BAPTA-AM treatment does indeed reduce GAP numbers below baseline, demonstrating that both spontaneous and induced GAP formation is dependent on intracellular caThere are typographical errors on line 694 and 760.We apologize and have corrected the typographical error in the revised manuscript.Reviewer #3:Combined two-photon in vivo imaging, super-resolution imaging, and FIB-SEM, these authors tried to follow the fate of the fluorescent tracer injected to the gut lumen. They suggested the identity of GAP as results of ACh stimulated endocytic events that are retrograded cross the Golgi network for transcytosis on the other side of the goblet cells. Besides, ACh induced GAP formation and mucin secretion via distinct receptors and signallng pathways.Major strengths: Detailed phamacological experiments clearly established two distinct pathways of ACh induced GAP formation and mucin secretion, which could lead to better understanding of the connection between goblet physiology and intestinal immunity.Weaknesses:1. It is unclear why ACh induces both GAP formation and mucin secretion in the same goblet cell. What would be the benefit of such arrangements?We can only speculate on the benefit or purpose of such an arrangement. Since the inception of this project four years ago, our thinking on the cellular basis of GAP formation has evolved significantly, as reflected by this work and paralleled by the reviewer’s comment. We too were surprised that GAP formation and secretion were induced by the same stimulus, ACh, and while the processes of secretion and membrane retrieval are inherently linked, GAP formation and ACh induced mucus secretion are not functionally linked or dependent upon each other, and yet can occur in the same cell. We interpret our observations to indicate that GAP formation evolved as an additional, and redundant, pathway of membrane recovery that is specialized to deliver luminal substances to the transcytotic pathway. In other work we have observed that while exogenous ACh  can augment GAP formation, ACh is generally not limiting for GAP formation and GAP formation is largely regulated by inhibiting AChR responsiveness in goblet cells. Incorporating these observations would suggest that goblet cells evolved in an efficient manner to use an available stimulus and the machinery of a highly secretory cell to perform two related but independent tasks allowing them to independently maintain the barrier and/or perform surveillance of the luminal contents. However, we would note that goblet cells may be heterogeneous in their functions, which will require additional studies to dissect.2. The current data are based mostly on phamacological perturbation. I wonder whether the authors could at least show some non-overlapping distributions of mAChR1, mAChR2, mAChR3, and mAChR4.We agree with the reviewers. Immunostaining for the respective receptors using currently available antibodies was unsuccessful. However, we were able to identify mAChR expression using RNAscope for mAChR1, 3 and 4. We did not stain for mAChR2 since this receptor subtype is not discussed in this manuscript. This data is now included in Figure 5—figure supplement 1 on page 33 and Figure 5—figure supplement 2 on page 34 and discussed in the revised manuscript on page 16, lines 372-384.3. Based on imaging data, the authors concluded the transcytosis fate of GAP vesicles. However, partial colocalization of dextran with the trans-Golgi marker and its fuzzy co-localization with Rab3D conferred the evidence, which is weak. We may need more direct evidence to better support this assertion.Nature, 2012) have led us to conclude that the GAP vesicles are transcytosed. However, we acknowledge this concern and are cautious with our interpretation of events at the basolateral surface of GAPs resulting in the transfer of lumenal substances, which will require further studies to fully comprehend.We agree. Rab3D  as well as VAMP8  staining that has been added to the revised manuscript. These markers co-localized with dextran in the lateral and supranuclear portions of goblet cells forming GAPs, but do not extend to vesicles at the basolateral region, suggesting that Rab3D and VAMP8 marks fates of vesicles containing luminal substances proximal to the transcytosis event. The transcytosis fate of the vesicles occurring when GAPs form is supported by the FIB-SEM studies in Figure 2 on page 10, including the TEM images in Figure 2F and G, and the super resolution microscopy in Figure 1C on page 7, which are as granular as these approaches allow. These studies along with prior work demonstrating that LP-APCs acquire both luminal substances and goblet cell proteins from GAPs . However, these studies suffer from the relative low resolution of two photon microscopy that led to the initial incorrect assumptions regarding GAP formation. All studies in this manuscript were performed in vivo and analyzed on fixed tissue sections, with the exception of Figure 1A which is an image from in vivo two photon imaging. As noted previously, the inhibitors used in this study act on GAP formation at early stages, and therefore did not allow us to evaluate intermediate events. In addition, the focus of these studies was on the process of GAP formation, as opposed to events at the apical or basolateral surfaces, so we chose to evaluate tissues after one hour of incubation with luminal dextran, a time point which we have previously observed that most GAPs are fully formed. Further, we acknowledge that the definition of a GAP is delivery of luminal substances to the basolateral surface, and as such delineation of early GAP events vs. other endocytic events recovering membranes, which the literature indicates exists to maintain the secretory potential of highly secretory cells, would be very difficult. In summation, we agree with the reviewer’s comments and acknowledge this limitation, however we believe this work is a solid step forward to delineating GAP formation from other processes in goblet cells.In prior work we have demonstrated GAP formation using in vivo two photon imaging of live anesthetized mice. These time lapse images demonstrate that GAPs form from the apical to basolateral surface through the acquisition of luminal substances .We apologize and have corrected this in the revised version of the manuscript. Figure 1C represents a 3D projection of a z-stack such that the red puncta may be overlaid upon the nucleus. Further it is worth noting that nuclei are not smooth, as can be seen in the FIB-SEM reconstructions and the red puncta may be interspersed between folds of the nucleus. Thus, the red puncta are located near but not in the nucleus. We have added information regarding this in the revised manuscript page 8 line 170.6. In addition, in Figure 1H, the authors claimed little overlapping between dextran and EEA1. However, structures highlighted by the arrows seem to be dual labeled with both markers.We apologize for the lack of clarity. The dual labeling is observed in the enterocytes adjacent to the goblet cells but not in the goblet cells. The text page 6, lines 145-146 have been revised for clarity.7. The authors have stated that they have obtained four cells underwent FIB-SEM imaging and used one as the example and the left as the replicates to confirm. I would suggest that they shall have some critera/quantifications that are indeed confirmed by the data not shown.We agree. We obtained four complete renderings of goblet cells undergoing GAP formation by FIB-SEM. The process of FIB-SEM of a goblet cell requires ~72 hours of continuous imaging  and some attempts to obtain an entire goblet cell were not successful, thus this information does not represent the entirety of the experiments performed. The process of constructing a 3D model of a GAP from a single FIB-SEM experiment required months of computer processing. Therefore, we chose one goblet cell undergoing GAP formation to generate our 3D model, and multiple FIB-SEM and TEM images to confirm this model. We have added a video (Video 2) demonstrating the Z stack of the FIB-SEM images with the corresponding Z stack of the segmented data to better convey the model. Additional information regarding the confirmation of the FIB-SEM data has been added to the Results section, page 8 line 186-187, and the methods section, page 43 lines 914-916 and page 44 lines 917-919.


To identify the prevalence and risk factors for primary Epstein–Barr virus (EBV) infection in human immunodeficiency virus (HIV)-1-positive adult treatment-naïve patients between January 2018 and December 2019 in a state of the Brazilian Amazon region. A total of 268 HIV-1 positive patients and 65 blood donors participated in the study. Epidemiological data were obtained from medical records and through a designed questionnaire. EBV infection was screened by the semiquantitative detection of anti-viral capsid antigen (VCA) EBV IgM and IgG, followed by molecular detection of the EBNA-3C gene. The plasma viral loads of HIV-1 and EBV were quantified using a commercial kit. The prevalence of primary coinfection was 7.12%. The associated risk factors were education level, family income, history of illicit drug use and sexually transmitted infections, homosexual contact and condom nonuse. Approximately 58.5% had late initiation of highly active antiretroviral therapy, which influenced the risk of HIV-EBV 1/2 multiple infection (odds ratio (OR): 4.76; 95% CI 1.51–15.04) and symptom development (p = 0.004). HIV viral load was associated with patient age  and duration of illicit drug use . EBV viral load was associated with younger age . The replication of both viruses was associated with symptom development . The prevalence of HIV/EBV coinfection was lower than that observed in other studies, and social vulnerability and promiscuous sexual behavior were associated risk factors. A long time of HIV-1 infection, without therapy, influenced the risk of coinfection and disease progression. The viral loads of both viruses may be associated with some epidemiological aspects of the population. Herpesviridae, genus Lymphocryptovirus2. Two EBV genotypes are classically known, EBV-1 and EBV-2, whose genetic differences lie in the sequence encoding the viral nuclear antigens 4. Both genotypes have a cosmopolitan distribution, although EBV-1 is more prevalent, especially in Western countries and Asia5, while EBV-2 is more prevalent in Central African countries6.The Epstein–Barr virus (EBV), also known as human gammaherpesvirus 4 (HHV4), belongs to the family 9. In developing countries, primary EBV infection occurs in early childhood and is usually asymptomatic, reaching 97% seroprevalence in adulthood in heterogeneous populations, a value that is similar at the global scale11. In these countries, there is an alert for seroprevalence in children and adolescents with low socioeconomic status12 and for the predominance of infection in individuals with suspected coinfection with human immunodeficiency virus (HIV)-113.In developed countries, EBV infection is common in adolescence, ranging from 0 to 70% in childhood and reaching more than 90% in adulthood; in the latter age group, primary infection is clinically symptomatic16. Coinfection is related to progressive dysfunction events and impaired immune surveillance caused by HIV that favor persistent pathogens17. This factor becomes critical when the infection progresses to AIDS, in which severe immunosuppression results in clinical signs and symptoms related to malignant neoplasms19.Because it is a precursor of lymphatic neoplasms, EBV is identified as one of the factors that contributes to the morbidity of patients infected with HIV-120 and blood tissue22. It is assumed that due to the high incidence of EBV infection during the first 5 years of life, maternal transmission is an important route in maintaining coinfection23. Additionally, microbial translocation and persistent immune activation induced by HIV are factors that may influence EBV replication and the expansion of infected B cells22.Although most studies focus on the pathological basis of viral carcinogenesis, it is still necessary to understand aspects of primary EBV infection in the context of coinfection. In this sense, EBV coinfection has been studied in HIV-1-seropositive children, in whom EBV DNA was isolated from the oral mucosa24. However, there is controversy regarding the detection of EBV in different HIV-1 infection profiles25. A recent study reports higher than 68% primary EBV infection in patients with HIV not on highly active antiretroviral therapy (HAART)26, similar to the rate observed in patients on the HAART regimen27. These findings indicate the complexity of the relationship between viruses that can be attributed to intrinsic factors of coinfection, regardless of the therapeutic status of the patient28.In adults, exposure through unprotected sex can favor HIV/EBV coinfection30. To date, we have only found 1 study, by Jacome-Santos et al., that reported the overall prevalence of coinfection by strictly analyzing the presence of EBV in the oral mucosa of people living with HIV (PLHIV)31. Thus, the aim of the present study was to describe the prevalence of primary EBV infection in HIV-positive adult therapy-naïve and to identify the epidemiological characteristics associated with this profile.There are few scientific studies on the epidemiology of HIV/EBV coinfection in the Brazilian Amazon and northern region of the country. Most research is focused on the carcinogenic effects of viral interactionsThis was an observational, cross-sectional and analytical study conducted in mutual collaboration between the Laboratory of Virology of the Federal University of Pará (LABVIR-UFPA), the Evandro Chagas Institute (IEC) and the Hemotherapy and Hematology Foundation of Pará (HEMOPA), with the selection of participants who regularly resided in municipalities of the state of Pará, Brazil, from January 2018 to December 2019, originating from the Center for Health Care in Acquired Infectious Diseases (CASA DIA), HEMOPA and IEC.32).All participants were clinically evaluated at the respective referral centers and subjected to complementary investigation via serological and molecular biology tests, based on which the participants were categorized as follows: 249 HIV-1 monoinfected patients ; 19 HIV-1-positive patients with primary EBV infection -EBV IgM+; anti-VCA-EBV IgG−; confirmation by molecular biology); and 65 uninfected individuals . All participants were informed about the study objectives and signed an informed consent form. The collected biological samples were stored in a biorepository until use.Participants included in the study were 18 years of age or older, of both sexes, carriers of HIV-1 and/or EBV, antiretroviral therapy-naïve and signed the informed consent form. Participants who did not meet the inclusion criteria or who were on antiviral therapy before sample collection were excluded.Five milliliters of peripheral blood was collected into vacuum tubes containing EDTA as an anticoagulant. DNA was extracted from whole blood following the protocol provided with the QiaAmp DNA Mini Kit .HIV-1 infection was screened by qualitative and simultaneous detection of p24 antigen and anti-HIV-1 and anti-HIV-2 IgG antibodies by enzyme immunoassay ; serological confirmation was performed using an Immunoblot rapid DPP HIV-1/2 kit  following the manufacturer's recommendations. The samples from CASA DIA did not require complementary tests for the diagnosis of HIV-1 because the institution has its own screening panel, to which patients enrolled in the institution are subjected.33 ((1st round) (F: 5′-AGATGGTGAGCCTGACGTG-3′/R: 5′-GCATCCTTCAAAACCTCAGC-3′)) and by Sample et al.3 ((2nd round) (F: 5′-AGAAGGGGAGCGGTGTGTTGT-3′/R: 5′-GGCTGTTTTTGACGTCGGC-3′)) and following the recommended conditions: primers (10 pmol/µL); MgCl2 (50 mM); dNTP (10 mM); and Taq (5U/µL); 1st round cycles—1 cycle of 95° C/3′, 20 cycles of , 1 cycle of 72 °C/7′; and 2nd round cycles—1 cycle of 95° C/3′; 35 cycles of ; 1 cycle of 72 °C/7′. The presence of a 153-bp fragment was considered positive for EBV-1, and the presence of a 246-bp fragment was considered positive for EBV-2. The positive EBV-1 and EBV-2 controls were isolated from the B95 and P3HP1 lymphoid cell lines, respectively.Infection by EBV was screened by semiquantitative detection of anti-EBV IgM and IgG antibodies by enzyme immunoassay  following the manufacturer's recommendations. The identification of EBV genotypes was performed by nested PCR, targeting the EBNA-3C gene, using the primers described by Lorenzetti et al.The HIV-1 plasma viral load was quantified by real-time PCR using an Abbott mSample Preparation System RNA extraction kit and an Abbott Real-Time HIV-1 amplification reagent kit  following the manufacturer’s recommendations.The EBV viral load was quantified by real-time PCR following the protocol provided with the XGEN Master EBV kit . Plasma samples were used to quantify the viral load.The epidemiological data were compared among the study groups using the G test. Fisher’s exact test was applied exclusively in comparisons whose data were arranged in 2 × 2 contingency tables.For significant comparisons, the degree of dependence between the epidemiological data and the study groups was calculated using simple logistic regression for 1 variable and multiple logistic regression for all variables, in which epidemiological data were included as independent variables and the presence or absence of infections as dependent variables. Due to the statistical similarity between the epidemiological profile of HIV-1 monoinfected and HIV-1/EBV coinfected patients, we assumed the presence of monoinfection or coinfection as success (1) and the absence of both as nonsuccess (0). The epidemiological factors that maintained statistical significance in the multiple regression were considered risk factors for monoinfection or coinfection.We also calculated the dependence of the increase in viral load on the epidemiological and behavioral variables of patients. In this context, we calculated the mean viral load values and classified them, for HIV, into low (0), when between undetectable and up to 10,000 RNA copies and high (1), when greater than 10,000 copies, and, for EBV, into low (0), when between undetectable and up to 45 DNA copies, and high (1), when higher than 45 copies. The quantitative epidemiological variables were included in the function as nonbinary data, and exclusively for the length of illicit drug use, we discarded patients with noncontinuous sporadic use and grouped the remaining time of use into less than 1 year, 1 to 5 years, or greater than 5 years.The G test was applied in other analyses of categorical data, such as in the comparison of the profile of sexually transmitted infections (STIs); behavioral changes according to the HIV-1 infection time (since diagnosis) and symptomatology. We used curve fitting to determine the frequency distribution model of cases of monoinfection and coinfection according to the collection time.34.All statistical analyses were performed using the programs GraphPad Prism 3.03  and Bioestat 5.0All methods and experimental protocols were carried out in accordance with regulations 466/2012 and 347/05 of the Brazilian National Health Council and were approved by the Human Research Ethics Committee of Instituto Evandro Chagas . All participants were informed about the study objectives and signed an informed consent form. The collected biological samples were stored in a biorepository until use.2: 0.476), although the increase in HIV-1 monoinfection was slight   : 1.57; 95% CI 1.20–2.05). Only a history of cigarette use  and illicit drug use  were considered risk factors; present use was not relevant because most individuals claimed to have ceased these practices. Most aspects related to sexual practice and sexuality were associated with monoinfection or coinfection: history of STIs  and homosexual contact  had the highest probability ratios, followed by lack of a steady partner  and occasional condom use . An active sex life was associated with the noninfected group , whereas biological sex, history of sexual contact with sex workers and first sexual contact were not associated with infected patients  and showing a greater representation of incomes ranging from 4 to greater than 10 times the minimum wage . Notably, current alcohol consumption was more strongly associated with noninfected patients . A family history of cancer and route of illicit drug administration were not associated with infected patients; most individuals reported no cases of cancer in the family, and for patients with a history of illicit drug use, most ingested them , and high family income was the strongest protective factor   lived for less than 1 month with HIV-1 and sought specific care immediately after diagnosis; however, approximately 51.58% of the patients lived between 1 and more than 12 months with the virus and without the use of HAART. When asked, patients with a longer time since diagnosis reported initial skepticism of the infection. Their point of view changed with the onset of symptoms, including through advice from third parties (data not shown).In an intergroup analysis, we observed that a time of HIV-1 infection greater than 12 months was more frequent in patients coinfected with HIV/EBV-1/2  and that the chance of multiple infections was approximately 5 times higher . Most patients in the HIV and HIV/EBV-2 groups had a recent infection diagnosis (< 1 month) . A time of infection between 1 and 6 months (78%) prevailed among HIV/EBV-1 coinfected patients, but without statistical significance , oligosymptomatic (from 2 to 3 symptoms) and polysymptomatic (from 4 to 5 symptoms). Asymptomatic patients prevailed in our analyses (51.91%); only in the group of patients coinfected with HIV/EBV-1/2 were oligosymptomatic patients predominant (60.0%), but the difference was not statistically significant (p = 0.96) , with other STIs (66.67%), such as herpes 33.35%) and HPV infection (33.33%), prevailing; candidiasis was an alleged condition in 33.5% of the control group  and with the length of illicit drug use in individuals with an illicit drug use history   as markers of EBV infection. The serological panel adopted in the present study was based on the detection of anti-VCA IgM and IgG antibodies, for which we assumed the profile IgM (+) IgG (−) as suspected primary infection, which was confirmed by molecular biology. Although the detection of anti-EBNA IgG antibodies is also suggested as a parameter to distinguish the infectious phases of the disease44. The methodologically closest study did not detect the same clinical profile in patients on HAART in southeastern Brazil45. We emphasize, however, that in the relatively time period included in the present study, the coinfection rate increased approximately sevenfold per month of collection, while the rate of new cases of PLHIV, understood as therapy-naïve based on the continuous care cascade recommended by the Brazilian Ministry of Health46, tended to be stable, with a nonsignificant increase of twofold per month of collection.In Brazil, most studies have focused on the detection of EBV in specific organ sites of PLHIV with particular clinical profiles48, our data warn of an increase in coinfections in PLHIV, a predisposition observed in Brazil49 and internationally50, especially among young men.Although the number of new HIV cases identified is stable nationally, especially in the northern region of the country52. Reports of EBV-2 prevalence in coinfected patients occur in specific clinical conditions after transient reactivation events of cell sites infected by different types of EBV53.We observed a prevalence of EBV-1 among HIV/EBV coinfected patients, confirming the epidemiology of the herpesvirus analyzed in different organ tissues of PLHIV with different clinical profiles56. In the present study, male individuals predominated, as well as a singular representation of homosexual and bisexual orientation, and we did not evaluate ethnic markers, such as self-reported race. Therefore, we assume a multiethnic representation in our sample with a probable predominance of European ancestry, as already described for populations in the Brazilian Amazon58. HIV/EBV-2 coinfection combined with multiple infection by both genotypes accounted for more than half the number of coinfections, a finding that emphasizes the susceptibility to the less frequent EBV genotype or to superinfection in HIV-immunocompromised patients60. It is argued that this is due both to changes in the immunological profile61 and in the exposure behavior of the host62.There is controversy regarding the predominant epidemiological profile among HIV/EBV-1 coinfected patients. Reports indicate that male sex, Caucasian ethnicity and heterosexual preference are factors that influence the distribution of cases66. In the present study, we found a prevalence of factors that are directly and indirectly related to sexual exposure, which makes this the main route of coinfection in the studied context.The epidemiological analysis revealed similarities between the monoinfected and coinfected profiles. Apparently, in the context of HIV coinfection, the presence of primary EBV infection is not associated with specific risk factors. In fact, both infections have points of convergence related mainly to unprotected sexual exposure and possible contact with contaminated blood products68. When functional, education empowers individuals to both reflect on the extent of contact between people and improves their ability to understand and act in favor of preventive health70.The predominant socioeconomic profile was young adult men with a low education level, low family income, and history of illicit drug use. Unsatisfactory education is a risk factor congruent with HIV infection due to the lack of aggregate knowledge about prevention mechanisms of STIs and unprotected sexual exposure71. In the present study, more vulnerable socioeconomic profiles were associated with the risk of infection; in contrast, favorable profiles were prevalent among blood donors, a feature relevant to this altruistic activity72. This finding illustrates the sociodemographic complexity between the factors 'education' and 'income' that result in better conditions of access and social participation. That in Brazil they may be closely related to the way in which the individual is inserted in the community social organization and is motivated to act and support their relationships73. In contrast, low socioeconomic status contributes to less health knowledge and awareness of blood donation needs74.It is argued that the lifestyle associated with a high education level can lead to both an increase and a decrease in the risk of HIV infection, depending on the balance of the different influences on behavior76. In a context of progressive poverty, HIV mortality and morbidity rates were predicted by lower incomes77. Analogously, there is an aggravation of social vulnerability in PLHIV of specific ethnic groups, women, and those with physical health limitations78. In contrast, even in already-infected key populations, a relative increase in income results in improvements in access to basic health care79.It is suggested that most of the factors that predispose an individual to HIV are associated with family or individual income because this creates environmental risks inherent to precarious access to health, the practice of “sex for survival” (sex as a means of earning income), and even the ability to deal with the consequences of the condition81, as observed in the decrease in condom use among NIDU sex workers, even between serodiscordant partners82. A worrying problem is the use of polydrug use combined with unprotected sex with multiple partners83, especially in Brazil, where although HIV prevalence rates among drug users have been declining in recent years48, they can still vary between 10 and 25-fold higher than that estimated in the general population84, and most users do not have basic knowledge about the severity of the infection85.A history of illicit drug use was the greatest risk factor for monoinfection or coinfection in the present study, among which drugs taken orally prevailed. Recent studies have shown that an increase in the number of noninjectable drug users (NIDUs) infected by HIV trigger epidemiological surveillance in the Amazon region because although this group does not expose itself, a priori, to parenteral transmission, they may be at the mercy of other risk variables, especially sexual factors87. However, in developed countries, MSM exposed to high risks report willingness to participate in prophylaxis measures once informed of these prevention efforts88.From the point of view of sexual practice, homosexual or bisexual orientation without the presence of a steady partner and occasional condom use were predominant characteristics among monoinfected and coinfected individuals. In fact, men who have sex with men (MSM) correspond to one of the main groups particularly vulnerable to HIV (key populations) and who historically do not have adequate access to health services. Data suggest that approximately 47% of new infections worldwide were associated with key populations and their sexual partners in 2017; in that evaluation, the MSM group represented 57% of new cases in Western Europe and Central and North America89. In Brazil, recent reports highlight the high prevalence of HIV among MSM, whose main risk factors include environments of vulnerability; stigma and discrimination; behavioral profiles; sexual practices; and lackluster policies and programs91. Notably, there is a low rate of satisfactory knowledge on HIV infection observed among Brazilian MSM92.In developing countries, the high proportion of seropositive MSM who are unaware of their serological status is highly worrying and represents a large gap between basic health care practices and national guidelines93, and a history of STIs is one of the consequences of this practice94, as suggested in the present study. Recent warnings highlight the low rates of disclosure of serological status among sex partners, which is worrisome given the persistence of viral transmissibility, especially when the majority continues to practice unprotected sex95. It is argued that specific social networks may be endorsing this exposure behavior96. In the present study, we showed that unprotected sex with multiple partners, particularly for young men, are relevant attributes in the prevalence of monoinfection or coinfection. Notably, most infected individuals had their first sexual contact in preadolescence (10 to 15 years), a trend that has been observed in previous studies97.As already mentioned, multiple sex partners is one of the characteristics composing the behavioral profiles of MSM98. In Brazil, despite an ongoing policy of free provision of condoms in referral units99, there is still a deficit in adherence to condom use, as suggested in the present study. We do not know whether this scenario is due to poor adherence itself, as a behavioral attitude100, to stigmatization in accessibility101, or to population ignorance regarding the national condom distribution policy. Future studies addressing this issue are essential as a strategic basis for preventive measures in the Brazilian Amazon region.Regarding protected sex, a recent report points to the existence of accessibility barriers for purchasing condoms, even in developed countries, and the need for a multifaceted approach to overcome these barriers62; among these, those diagnosed with syphilis and gonorrhea have the highest rate of HIV coinfection102, similar to our findings. These data are particularly worrying for Brazil because the incidence rates of acquired syphilis have been increasing since 2010103, and there is an estimated global increase in cases of gonorrhea, especially in MSM in developing countries104. The specific detection of these STIs can guide resource-intensive interventions, such as pre-exposure prophylaxis (PrEP), among PLHIV102.A history of STIs was the second factor most strongly associated with monoinfection or coinfection, with a prevalence of reported cases of syphilis and gonorrhea, respectively. Recent studies have shown that the highest risk for STIs is observed in young MSM from the most vulnerable socioeconomic groupsIn summary, we propose a risk/exposure potential matrix for monoinfection or coinfection based on sexual and social risk factors. We showed that the highest risk/exposure occurs among individuals with social vulnerability and promiscuous sexual behavior, among whom condom nonusers were more susceptible to viral infections.105. Alternatively, high education is associated with affordable family income and consequent adoption of safer sexual practices and access to preventive health services106, as suggested for individuals with high purchasing power, whose risk/exposure remained modest regardless of sexual factors.However, condom use had a risk more associated with low education and low family income, similar to that presented among immigrants with similar socioeconomic profiles. In this scenario of preventive failure, we assume that social factors can directly influence the quality of aggregated knowledge about proper condom handling and use and awareness of STI/AIDSWe noted that on a monthly scale, the care and monitoring of HIV infection in a portion of patients was initiated late, with an interval between 1 and 24 months after becoming aware of the infection. However, the majority started the process quickly, approximately 1 month after the primary diagnosis.107. It is argued that in these cases, the immediate initiation of HAART may have favored the gradual immune recovery of patients and impacted mortality108. In Brazil, management guidelines advocate the immediate start of therapeutic regimens regardless of the patient’s clinical or immunological status46.These results are promising given persistent reports of late diagnosis among PLHIV. The greatest concern lies with the increase in the degree of morbidity and mortality inherent to the late initiation of treatment, although a trend toward a decrease in overall lethality is observed in these patients, despite maintaining a high frequency of late or advanced presentation of HIV-infected patients and the evolution of associated factors110. Brazil again stands out for encouraging this practice in official and independent campaigns that aim not only to diagnosis but also to provide information regarding and encourage condom use112.One strategy to encourage the early diagnosis of STIs is to promote testing outside the health setting116; however, little is discussed about the risk behavior profile of this group. In the present study, we observed that the exposure behavior was similar between the different times since diagnosis, indicating that prolonged experience with HIV did not significantly alter the behavioral profile of patients who maintained promiscuous practices related to infection and inherent comorbidities. Although there was a decrease in the percentage of sexual practice in patients for whom treatments was initiated late, most patients had no steady partner and maintained sexual contact with sex workers, some of whom did not use condoms.It is understood that older age and heterosexual orientation are the main factors influencing late start of monitoring in PLHIV118. For EBV, this becomes more specific because the prevalence of EBV-1 and of multiple infection predominate in the most immunosuppressed patients; however, it is not known which factors may be involved in this scenario119.We also show that the risk of HIV/EBV-1/2 multiple infection was higher in patients with longer HIV-1 infection, which emphasizes our warning because coinfections are common in patients who start treatment late, have the highest associated risk rate, and are also correlated with advanced clinical disease stages123. Particularly with EBV, these symptoms are associated with age groups above 18 years old and the host's aggravated immune status64, attributes that were prevalent among coinfected patients in the present study.Regarding the symptomatological aspects, asymptomatic individuals prevailed in all groups. Symptoms, when present, although still noncomplex and clinically nonspecific, were similar between both the initial phase of HIV infection and primary symptomatic EBV infection124 and has secondary benefits126.With the late initiation of HAART, there was a clear progression in coinfection symptoms, evidenced by the increase in cases of oligosymptomatic and polysymptomatic patients with HAART initiation greater than 12 months after the primary diagnosis. Although our results indicate that most patients sought specific medical care at the onset of the disease, we show how neglect of the condition can affect the pathological status of HIV infection, given that antiretroviral therapy initiated as soon as possible delays the progression of the disease and viral transmission127, which favors not only the risk of infection but also intensive therapeutic control128. We suggest that the continued use of these illicit drugs may be conducive to both the acquisition and progression of infection because the time of initiation of HAART was not associated with viral load, which points to a complex interaction between illicit drug use and virological activity, which may be as significant as exposure to therapy129.We observed that some of the epidemiological and behavioral factors were also associated with biological aspects of coinfection. Exclusively for HIV, patients with longer use of illicit drugs were more likely to have a high viral load. Specifically, we noted that most users used cocaine or marijuana, which are associated with effects on the immune system indicative of compromised resistance to HIV132. However, in the present study, all patients were treatment-naïve, unlike those participating in the aforementioned studies, revealing the possible effects of immunosenescence in the face of HIV infection133, even in a predominantly young population.The advancement of age as a risk factor for increased HIV viral load was a finding different from those reported in previous studies135.In contrast, the chances of the EBV viral load increasing were higher in younger patients, in contrast that what was observed in HIV patients. Our findings reflect those observed in other epidemiological studies, in which it is argued that the early acquisition of primary infection results in higher and more sustained levels of EBV viremia. The presence of coinfections may also impair specific immune surveillance for one or both pathogens, leading to worsening of the disease136, an observation more characteristic of the acute phase of infection123. However, studies indicate that viral subtype and chronic immune activation are also aspects that predict the course of progression in populations of different ethnic origins138. For EBV, the plasma viral load is directly proportional to the patient's symptoms, especially within the first 2 weeks of symptom onset; in the acute phase, viral replication is intense in peripheral blood, with the virus contained in infected B cells and a portion being released into the plasma139. In specific clinical cases, the plasma viral load, even when low, is already an effective biomarker141.For both viruses, symptomatology was a factor dependent on viral load; the higher the viral load is, the greater the chances of patients presenting with a polysymptomatic condition, especially in EBV infection. For HIV, in cohorts with a high prevalence of infection, it was shown that viral load is a determining factor of disease progressionBased on the results of the present study, we conclude that the prevalence of primary coinfection with EBV in HIV therapy-naïve patients in a state of the Brazilian Amazon region was lower than that observed in other similar regions. This finding may be associated with the methods used in the different studies, with social vulnerability and promiscuous sexual behavior being the risk factors associated with this population. We warn of the effect of the late initiation of HIV infection monitoring and the consequences of this decision on the risk of coinfections by EBV and disease progression, and we suggest that some epidemiological factors may influence the increase in viral load of both viruses.


In summary, home confinement produced detraining effects in professional football referees associated with hamstring muscle weakness. In this regard, strength-based activities with body loads may be insufficient to avoid muscle weakness and other means  may be necessary to maintain muscle strength. However, the 4-weeks retraining period was sufficient to resolve hamstring muscle weakness induced by the restrictions of home confinement. This information may be helpful in the case of future sport competition suspension or home quarantine due to new waves of COVID-19 pandemic. The COVID-19 pandemic has produced a major disruption for professional football leagues that has affected the physical preparation of both football players and referees. In Spain, health authorities decreed home confinement for eight weeks, supressing the normal training routines of professional referees. After home confinement, referees had four weeks to retrain as the national football league was set to resume matches to complete the 11 games remaining. The aim of the present investigation was to assess changes in eccentric hamstring muscle strength during football competition suspension/resumption due to the COVID-19 pandemic in 21 professional football referees . Eccentric hamstring muscle strength was measured with the Nordic hamstring exercise at four time points. During home confinement, referees presented the lowest value of bilateral eccentric muscle strength (300 ± 14 N). Eccentric muscle strength increased by 13.2 ± 3.7% one week after the end of home confinement (339 ± 16 N;  Several investigations have described the external physical demands of professional football (soccer) refereeing during an official match ,2,3,4. RThe Coronavirus Disease 2019 (COVID-19) pandemic has been a main disruption for professional football leagues . In SpaiHence, professional Spanish football referees were confined at home for 8 complete weeks, and then had 4 weeks of retraining before the resumption of the Spanish football league. Similar to professional football players, professional football referees underwent a congested competitive scenario after the resumption of the competition; it entailed the completion of 11 fixtures in less than six weeks. To the authors’ knowledge, no previous investigation has offered information about the way home confinement produced detraining effects in professional football referees and how physical condition was regained during the retraining and competitive periods. To date, only one investigation has shown that hamstring muscle strength in football players was progressively reduced as the home confinement progressed in duration . That inA total of 23 male professional football referees volunteered to participate in this prospective observational study. Participants were recruited by phone calls and emails and all of them pertain to the refereeing section of the RSFF. Among these, only a group of 21 referees (mean ± standard deviation (SD), age: 33.4 ± 5.1 years; height: 182.4 ± 5.0 cm; body mass: 75.1 ± 4.4 kg; weekly training time: 7.0 ± 0.5 h) completed all testing, consisting of measurements of eccentric hamstring muscle strength at four time-points. As inclusion criteria, participants had to be free of musculoskeletal injuries with no presence of delayed onset muscle soreness in the day of the testing and they had to be active referees during the 2019–2020 season. Two referees were excluded from the study because they did not complete the four measurements. All participants were fully informed of any risks and discomforts associated with the experiments before giving their informed written consent to participate. The study protocol was reviewed and approved by the Institutional Review Board of the European University of Madrid (code: 16/2019/CEICEGC), in accordance with the latest version of the Declaration of Helsinki.In this study, all referees were exposed to the same experimental protocol. This investigation aims to provide data about changes in eccentric hamstring muscle strength during football competition suspension/resumption due to the COVID-19 pandemic in professional football referees. The first measurement of eccentric hamstring muscle strength was performed on 28 March 2020, when referees had undergone home confinement for 13 days (established as the “confinement” measurement). During confinement, the staff of the RSFF provided personalized training programmes and organized individual and group video-based activities to reduce the potential detraining effects. The training program included six training sessions per week and included strength-based activities with body loads, proprioception activities, and endurance-based exercise . Home training protocols were adapted to the conditions of each referee’s residence. Training sessions were intended to produce exercise duration and rating of perceived exertion aligned with referees’ conventional in-season training. The second measurement was performed on 18 May 2020, one week after the measures of home confinement were alleviated (“post-confinement”). The third measurement was performed on 7 June 2020, one-to-four days before the resumption of the national football league (“pre-competition”). This entailed a retraining period of 4 weeks. The fourth measurement occurred on 22 July 2020, three days after the last fixture of the professional football competition was completed (“post-competition”).At all of these four time-points, eccentric hamstring muscle strength was measured using video-recordings of the Nordic hamstring test which were analysed afterwards using a video-based method . For thiMeans ± standard deviations were calculated from the raw database to compare eccentric hamstring muscle strength at four time-points. There were no missing data during the collection of data. A one-way analysis of variance (ANOVA) for repeated measures was used to determine the main effect time and Bonferroni post-hoc tests were applied to detect differences in pairwise comparisons. Cohen’s effect sizes (ES) were also calculated in all pairwise comparisons with the measurement obtained during home confinement to estimate the magnitude of change for strength increase in the remaining measurement time points.p = 0.004) and for the unilateral measurements in the left  and right limb . During confinement, referees presented the lowest value of eccentric muscle strength for the bilateral and unilateral measurements, which were lower than in the remaining measurements (p < 0.050). In comparison to the measurement during confinement, bilateral eccentric muscle strength increased by 13.2 ± 3.7%  in the post-confinement trial and remained stable in the pre-competition  and post-competition measurements . The unilateral measurements followed a similar pattern; in the post-confinement measurement muscle strength increased by 10.6 ± 1.9% in the left limb  and by 14.3 ± 4.9% in the right limb  with respect to the confinement. Hamstring muscle strength was further increased before the onset of the competition in the left limb  although the change did not reach statistical significance in the right limb . After the competition, there was a slight reduction in hamstring muscle strength although without statistical significance with respect to the pre-competition measurement. Still, eccentric muscle strength was higher in the left and right limbs at any time point with respect to the measurement performed during home confinement (ES from 1.6 to 3.2).The aim of the present investigation was to assess changes in eccentric hamstring muscle strength during football competition suspension/resumption due to the COVID-19 pandemic in professional football referees. Although there are no data on eccentric muscle strength prior to the onset of home confinement due to the COVID-19 pandemic, the fluctuation in this variable after home confinement suggests that the quarantine conditions produced detraining effects associated with hamstring muscle weakness. As happened in football players , the exeThis investigation is innovative because it presents, for the first time, that the detraining effects of home confinement were resolved once the referees were allowed to train without restrictions. In this regard, bilateral and unilateral eccentric hamstring muscle strength increased in the post-confinement measurement which occurred only 1 week after home confinement was suspended. In addition, eccentric hamstring muscle strength slightly increased by the pre-competition measurement, suggesting that the retraining period allowed from the end of home confinement to the onset of the football national league was sufficient to resolve hamstring muscle weakness. Perhaps, the high adaptability of the hamstring muscle to different training interventions allowed regaining hamstring muscle strength in a period of 4 weeks ,29,30. FThe current investigation has several limitations. First, football referees were confined at home for eight weeks and they had four weeks to retrain before the resumption of their refereeing activities. The changes in eccentric hamstring muscle strength may be different in situations that involve home confinement or inactivity with longer/shorter durations. Second, although all the referees were confined at home for the same duration, the type of exercise activities that they were able to perform during home confinement was slightly different depending on the equipment referees had available . Last, the sample included only male professional referees and the results should not be translated to other groups of referees.Today, COVID-19 is still a serious respiratory illness with high likelihood of contagion even with the presence of several vaccines and a high proportion of the population vaccinated in most developed countries. Hence, it is likely that home quarantine is needed for some athletes that have been infected with the SARS-CoV-2 or for those who have been in contact with an infected individual. Although the first objective during home confinement is to overcome the infection and the period of transmission, home training should be informed for those with slight symptoms to avoid detraining, particularly for muscle strength. To this regard, strength-based activities with body loads may be insufficient to avoid muscle weakness and the use of weights may be necessary to maintain muscle strength.In summary, the results of this investigation suggest that 8 weeks of home quarantine due to COVID-19 produced hamstring muscle weakness in Spanish professional football referees. The exercise routines performed at home were not effective to prevent a detraining effect in hamstring strength, but football referees rapidly regained hamstring muscle strength once they were allowed to train in sports facilities. A period of ~4 weeks was sufficient to overcome the detraining effects of home confinement on bilateral and unilateral eccentric hamstring muscle strength as determined by the Nordic exercise test.