ST6GAL1 sialyltransferase promotes acinar to ductal metaplasia and pancreatic cancer progression.

The role of aberrant glycosylation in pancreatic ductal adenocarcinoma (PDAC) remains an under-investigated area of research. In this study, we determined that ST6 ß-galactoside α2,6 sialyltransferase 1 (ST6GAL1), which adds α2,6-linked sialic acids to N-glycosylated proteins, was upregulated in patients with early-stage PDAC and was further increased in advanced disease. A tumor-promoting function for ST6GAL1 was elucidated using tumor xenograft experiments with human PDAC cells. Additionally, we developed a genetically engineered mouse (GEM) model with transgenic expression of ST6GAL1 in the pancreas and found that mice with dual expression of ST6GAL1 and oncogenic KRASG12D had greatly accelerated PDAC progression compared with mice expressing KRASG12D alone. As ST6GAL1 imparts progenitor-like characteristics, we interrogated ST6GAL1's role in acinar to ductal metaplasia (ADM), a process that fosters neoplasia by reprogramming acinar cells into ductal, progenitor-like cells. We verified ST6GAL1 promotes ADM using multiple models including the 266-6 cell line, GEM-derived organoids and tissues, and an in vivo model of inflammation-induced ADM. EGFR is a key driver of ADM and is known to be activated by ST6GAL1-mediated sialylation. Importantly, EGFR activation was dramatically increased in acinar cells and organoids from mice with transgenic ST6GAL1 expression. These collective results highlight a glycosylation-dependent mechanism involved in early stages of pancreatic neoplasia.

Interactions between H. pylori and the Gastric Microbiome: Impact on Gastric Homeostasis and Disease.

Like many seemingly inhospitable environments on our planet, the highly acidic human stomach harbors a diverse bacterial microflora. The best-known member of the human gastric flora, Helicobacter pylori, causes a number of gastric diseases, including peptic ulcer disease and gastric adenocarcinoma. In the absence of Helicobacter pylori infection, the gastric microbiota displays some features similar to the oral cavity with Firmicutes the most common phylum, followed by Proteobacteria and Bacteroidetes. When present, H. pylori dominates the gastric microbiome and reduces diversity and composition of other taxa. The composition of the gastric microbiome also is altered in the setting of proton pump inhibitor therapy and gastric neoplasia. This review summarizes foundational and recent studies that have investigated the composition of the human gastric microbiome in a variety of patient groups, with a focus on potential mechanisms involved in regulation of gastric microbial community structure.

Modulation of glycosyltransferase ST6Gal-I in gastric cancer-derived organoids disrupts homeostatic epithelial cell turnover.

Programmed cell death promotes homeostatic cell turnover in the epithelium but is dysregulated in cancer. The glycosyltransferase ST6Gal-I is known to block homeostatic apoptosis through α2,6-linked sialylation of the death receptor TNFR1 in many cell types. However, its role has not been investigated in gastric epithelial cells or gastric tumorigenesis. We determined that human gastric antral epithelium rarely expressed ST6Gal-I, but the number of ST6Gal-I-expressing epithelial cells increased significantly with advancing premalignancy leading to cancer. The mRNA expression levels of ST6GAL-I and SOX9 in human gastric epithelial cells correlated positively with one another through the premalignancy cascade, indicating that increased epithelial cell expression of ST6Gal-I is associated with premalignant progression. To determine the functional impact of increased ST6Gal-I, we generated human gastric antral organoids from epithelial stem cells and differentiated epithelial monolayers from gastric organoids. Gastric epithelial stem cells strongly expressed ST6Gal-I, suggesting a novel biomarker of stemness. In contrast, organoid-derived epithelial monolayers expressed markedly reduced ST6Gal-I and underwent TNF-induced, caspase-mediated apoptosis, consistent with homeostasis. Conversely, epithelial monolayers generated from gastric cancer stem cells retained high levels of ST6Gal-I and resisted TNF-induced apoptosis, supporting prolonged survival. Protection from TNF-induced apoptosis depended on ST6Gal-I overexpression, because forced ST6Gal-I overexpression in normal gastric stem cell-differentiated monolayers inhibited TNF-induced apoptosis, and cleavage of α2,6-linked sialic acids from gastric cancer organoid-derived monolayers restored susceptibility to TNF-induced apoptosis. These findings implicate up-regulated ST6Gal-I expression in blocking homeostatic epithelial cell apoptosis in gastric cancer pathogenesis, suggesting a mechanism for prolonged epithelioid tumor cell survival.

Cytomegalovirus promotes intestinal macrophage-mediated mucosal inflammation through induction of Smad7.

Intestinal macrophages in healthy human mucosa are profoundly down-regulated for inflammatory responses (inflammation anergy) due to stromal TGF-ß inactivation of NF-κB. Paradoxically, in cytomegalovirus (CMV) intestinal inflammatory disease, one of the most common manifestations of opportunistic CMV infection, intestinal macrophages mediate severe mucosal inflammation. Here we investigated the mechanism whereby CMV infection promotes macrophage-mediated mucosal inflammation. CMV infected primary intestinal macrophages but did not replicate in the cells or reverse established inflammation anergy. However, CMV infection of precursor blood monocytes, the source of human intestinal macrophages in adults, prevented stromal TGF-ß-induced differentiation of monocytes into inflammation anergic macrophages. Mechanistically, CMV up-regulated monocyte expression of the TGF-ß antagonist Smad7, blocking the ability of stromal TGF-ß to inactivate NF-κB, thereby enabling MyD88 and NF-κB-dependent cytokine production. Smad7 expression also was markedly elevated in mucosal tissue from subjects with CMV colitis and declined after antiviral ganciclovir therapy. Confirming these findings, transfection of Smad7 antisense oligonucleotide into CMV-infected monocytes restored monocyte susceptibility to stromal TGF-ß-induced inflammation anergy. Thus, CMV-infected monocytes that recruit to the mucosa, not resident macrophages, are the source of inflammatory macrophages in CMV mucosal disease and implicate Smad7 as a key regulator of, and potential therapeutic target for, CMV mucosal disease.

CD103 (αE Integrin) Undergoes Endosomal Trafficking in Human Dendritic Cells, but Does Not Mediate Epithelial Adhesion.

Dendritic cell (DC) expression of CD103, the α subunit of αEß7 integrin, is thought to enable DC interactions with E-cadherin-expressing gastrointestinal epithelia for improved mucosal immunosurveillance. In the stomach, efficient DC surveillance of the epithelial barrier is crucial for the induction of immune responses to H. pylori, the causative agent of peptic ulcers and gastric cancer. However, gastric DCs express only low levels of surface CD103, as we previously showed. We here tested the hypothesis that intracellular pools of CD103 in human gastric DCs can be redistributed to the cell surface for engagement of epithelial cell-expressed E-cadherin to promote DC-epithelial cell adhesion. In support of our hypothesis, immunofluorescence analysis of tissue sections showed that CD103+ gastric DCs were preferentially localized within the gastric epithelial layer. Flow cytometry and imaging cytometry revealed that human gastric DCs expressed intracellular CD103, corroborating our previous findings in monocyte-derived DCs (MoDCs). Using confocal microscopy, we show that CD103 was present in endosomal compartments, where CD103 partially co-localized with clathrin, early endosome antigen-1 and Rab11, suggesting that CD103 undergoes endosomal trafficking similar to ß1 integrins. Dynamic expression of CD103 on human MoDCs was confirmed by internalization assay. To analyze whether DC-expressed CD103 promotes adhesion to E-cadherin, we performed adhesion and spreading assays on E-cadherin-coated glass slides. In MoDCs generated in the presence of retinoic acid, which express increased CD103, intracellular CD103 significantly redistributed toward the E-cadherin-coated glass surface. However, DCs spreading and adhesion did not differ between E-cadherin-coated slides and slides coated with serum alone. In adhesion assays using E-cadherin-positive HT-29 cells, DC binding was significantly improved by addition of Mn2+ and decreased in the presence of EGTA, consistent with the dependence of integrin-based interactions on divalent cations. However, retinoic acid failed to increase DC adhesion, and a CD103 neutralizing antibody was unable to inhibit DC binding to the E-cadherin positive cells. In contrast, a blocking antibody to DC-expressed E-cadherin significantly reduced DC binding to the epithelium. Overall, these data indicate that CD103 engages in DC-epithelial cell interactions upon contact with epithelial E-cadherin, but is not a major driver of DC adhesion to gastrointestinal epithelia.

Characterization of Human Blood Monocytes and Intestinal Macrophages.

Monocytes and macrophages play fundamental roles in defense against microbes, clearance of senescent and dead cells, and immunoregulation. Although blood monocytes are the source of intestinal macrophages in the developed mucosal immune system, blood monocytes and intestinal macrophages from healthy human subjects display distinct phenotypic and functional differences. Blood monocytes can be induced to polarize into M1 and M2 macrophages, whereas intestinal macrophages appear to be terminally differentiated and are unable to undergo such inducible polarization. Nevertheless, in response to local conditions, monocytes differentiated into intestinal macrophages display phenotypic and functional characteristics that enhance their capacity to provide non-inflammatory host defense and participate in local immunoregulation. Using the protocols described here, this unit presents the key phenotypic and functional differences between human blood monocytes and intestinal macrophages, as well as between mouse and human intestinal macrophages. © 2017 by John Wiley & Sons, Inc.

Differential regulation of CD103 (αE integrin) expression in human dendritic cells by retinoic acid and Toll-like receptor ligands.

CD103 (αE integrin) is an important dendritic cell (DC) marker that characterizes functionally distinct DC subsets in mice and humans. However, the mechanism by which CD103 expression is regulated in human DCs and the role of CD103 for DC function are not very well understood. Here, we show that retinoic acid (RA) treatment of human monocyte-derived DCs (MoDCs) increased the ability of the DCs to synthesize RA and induced MoDC expression of CD103 and ß7 at the mRNA and protein level. In contrast, RA was unable to induce the expression of CD103 in primary human DCs isolated from the gastric mucosa. Inhibition of TGF-ß signaling in MoDCs down-regulated RA-induced CD103 expression, indicating that TGF-ß-dependent pathways contribute to the induction of CD103. Conversely, when RA-treated MoDCs were stimulated with live Helicobacter pylori, commensal bacteria, LPS, or a TLR2 agonist, the RA-induced up-regulation of CD103 and ß7 integrin expression was completely abrogated. To determine whether CD103 expression impacts DC priming of CD4+ T cells, we next investigated the ability of CD103+ and CD103─ DCs to induce mucosal homing and T cell proliferation. Surprisingly, RA treatment of DCs enhanced both α4ß7 expression and proliferation in cocultured T cells, but no difference was seen between RA-treated CD103+ and CD103─ DCs. In summary, our data demonstrate that RA, bacterial products, and the tissue environment all contribute to the regulation of CD103 on human DCs and that DC induction of mucosal homing in T cells is RA dependent but not CD103 dependent.

Mother-to-Child HIV-1 Transmission Events Are Differentially Impacted by Breast Milk and Its Components from HIV-1-Infected Women.

Breast milk is a vehicle of infection and source of protection in post-natal mother-to-child HIV-1 transmission (MTCT). Understanding the mechanism by which breast milk limits vertical transmission will provide critical insight into the design of preventive and therapeutic approaches to interrupt HIV-1 mucosal transmission. However, characterization of the inhibitory activity of breast milk in human intestinal mucosa, the portal of entry in postnatal MTCT, has been constrained by the limited availability of primary mucosal target cells and tissues to recapitulate mucosal transmission ex vivo. Here, we characterized the impact of skimmed breast milk, breast milk antibodies (Igs) and non-Ig components from HIV-1-infected Ugandan women on the major events of HIV-1 mucosal transmission using primary human intestinal cells and tissues. HIV-1-specific IgG antibodies and non-Ig components in breast milk inhibited the uptake of Ugandan HIV-1 isolates by primary human intestinal epithelial cells, viral replication in and transport of HIV-1- bearing dendritic cells through the human intestinal mucosa. Breast milk HIV-1-specific IgG and IgA, as well as innate factors, blocked the uptake and transport of HIV-1 through intestinal mucosa. Thus, breast milk components have distinct and complementary effects in reducing HIV-1 uptake, transport through and replication in the intestinal mucosa and, therefore, likely contribute to preventing postnatal HIV-1 transmission. Our data suggests that a successful preventive or therapeutic approach would require multiple immune factors acting at multiple steps in the HIV-1 mucosal transmission process.

Cytomegalovirus enhances macrophage TLR expression and MyD88-mediated signal transduction to potentiate inducible inflammatory responses.

Circulating monocytes carrying human CMV (HCMV) migrate into tissues, where they differentiate into HCMV-infected resident macrophages that upon interaction with bacterial products may potentiate tissue inflammation. In this study, we investigated the mechanism by which HCMV promotes macrophage-orchestrated inflammation using a clinical isolate of HCMV (TR) and macrophages derived from primary human monocytes. HCMV infection of the macrophages, which was associated with viral DNA replication, significantly enhanced TNF-α, IL-6, and IL-8 gene expression and protein production in response to TLR4 ligand (LPS) stimulation compared with mock-infected LPS-stimulated macrophages during a 6-d in vitro infection. HCMV infection also potentiated TLR5 ligand-stimulated cytokine production. To elucidate the mechanism by which HCMV infection potentiated inducible macrophage responses, we show that infection by HCMV promoted the maintenance of surface CD14 and TLR4 and TLR5, which declined over time in mock-infected macrophages, and enhanced both the intracellular expression of adaptor protein MyD88 and the inducible phosphorylation of IκBα and NF-κB. These findings provide additional information toward elucidating the mechanism by which HCMV potentiates bacteria-induced NF-κB-mediated macrophage inflammatory responses, thereby enhancing organ inflammation in HCMV-infected tissues.

HIV-1 envelope glycan moieties modulate HIV-1 transmission.

UNLABELLED: The HIV-1 envelope protein (Env) is heavily glycosylated, with approximately 50% of the Env molecular mass being contributed by N-glycans. HIV-1 Env N-glycans shield the protein backbone and have been shown to play key roles in determining Env structure, surface exposure, and, consequently, antigenicity, infectivity, antibody neutralization, and carbohydrate and receptor binding. Studies of HIV-1 glycosylation have focused mainly on the position of glycosylation, rather than the types of glycans. Also, the role of Env glycan moieties on HIV-1 transmission has not been systematically defined. Using viruses with modified Env glycan content and heterogeneity, we examined the effects of Env glycan moieties on the major events of HIV-1 transmission. Compared to viruses with less oligomannose and more complex Env glycans, viruses with more oligomannose and less complex glycans more efficiently (i) transcytosed across an epithelial cell monolayer, (ii) attached to monocyte-derived macrophages (MDMs), (iii) bound monocyte-derived dendritic cells (MoDCs), and (iv) trans-infected primary lymphocytes via MoDCs. However, viruses with more oligomannose and less complex glycans displayed impaired infectivity in TZMbl cells, MDMs, primary lymphocytes, and fresh human intestinal tissue. Thus, N-linked Env glycans display discordant effects on the major events of HIV-1 transmission, with mature oligosaccharide structures on Env playing a crucial role in HIV-1 infection. Env glycosylation should be taken into consideration in the development of vaccine strategies to interdict HIV-1 transmission. IMPORTANCE: HIV-1 Env N-glycans shield the protein backbone and play key roles in determining Env structure and surface exposure, thereby impacting Env antigenicity, infectivity, antibody neutralization, and carbohydrate and receptor binding. Studies of HIV-1 glycosylation have focused mainly on the position of glycosylation, rather than the types of glycans. In the study described in this report, we investigated systematically the role of Env glycan moieties on HIV-1 transmission. We show that N-linked Env glycans display discordant effects on the major events of HIV-1 transmission. These data indicate that Env glycan moieties impact HIV-1 transmission and that modulation of Env glycan moieties offers a potential strategy for the development of therapeutic or prophylactic vaccines against HIV-1.

Differential glycosylation of envelope gp120 is associated with differential recognition of HIV-1 by virus-specific antibodies and cell infection.

BACKGROUND: HIV-1 entry into host cells is mediated by interactions between the virus envelope glycoprotein (gp120/gp41) and host-cell receptors. N-glycans represent approximately 50% of the molecular mass of gp120 and serve as potential antigenic determinants and/or as a shield against immune recognition. We previously reported that N-glycosylation of recombinant gp120 varied, depending on the producer cells, and the glycosylation variability affected gp120 recognition by serum antibodies from persons infected with HIV-1 subtype B. However, the impact of gp120 differential glycosylation on recognition by broadly neutralizing monoclonal antibodies or by polyclonal antibodies of individuals infected with other HIV-1 subtypes is unknown. METHODS: Recombinant multimerizing gp120 antigens were expressed in different cells, HEK 293T, T-cell, rhabdomyosarcoma, hepatocellular carcinoma, and Chinese hamster ovary cell lines. Binding of broadly neutralizing monoclonal antibodies and polyclonal antibodies from sera of subtype A/C HIV-1-infected subjects with individual gp120 glycoforms was assessed by ELISA. In addition, immunodetection was performed using Western and dot blot assays. Recombinant gp120 glycoforms were tested for inhibition of infection of reporter cells by SF162 and YU.2 Env-pseudotyped R5 viruses. RESULTS: We demonstrated, using ELISA, that gp120 glycans sterically adjacent to the V3 loop only moderately contribute to differential recognition of a short apex motif GPGRA and GPGR by monoclonal antibodies F425 B4e8 and 447-52D, respectively. The binding of antibodies recognizing longer peptide motifs overlapping with GPGR epitope (268 D4, 257 D4, 19b) was significantly altered. Recognition of gp120 glycoforms by monoclonal antibodies specific for other than V3-loop epitopes was significantly affected by cell types used for gp120 expression. These epitopes included CD4-binding site (VRC03, VRC01, b12), discontinuous epitope involving V1/V2 loop with the associated glycans (PG9, PG16), and an epitope including V3-base-, N332 oligomannose-, and surrounding glycans-containing epitope (PGT 121). Moreover, the different gp120 glycoforms variably inhibited HIV-1 infection of reporter cells. CONCLUSION: Our data support the hypothesis that the glycosylation machinery of different cells shapes gp120 glycosylation and, consequently, impacts envelope recognition by specific antibodies as well as the interaction of HIV-1 gp120 with cellular receptors. These findings underscore the importance of selection of appropriately glycosylated HIV-1 envelope as a vaccine antigen.

Ex vivo lung perfusion allows successful transplantation of donor lungs from hanging victims.

BACKGROUND: Donor lungs acquired from victims of asphyxiation by hanging are not routinely used for lung transplantation because of the associated lung injury. Ex vivo lung perfusion (EVLP) is a technique to evaluate marginal donor lungs before transplantation. We report here our experience with the use of EVLP in donor lungs procured from victims of asphyxia by hanging. METHODS: Lungs from 5 donors who became brain dead secondary to hanging were evaluated by EVLP. Donor organs were perfused according to trial protocol. Donor lungs were accepted for transplantation if they maintained a PaO2 greater than or equal to 350 mm Hg, had a clear roentgenogram, and had no significant worsening of physiologic metrics. RESULTS: Perfused organs included single and double lung blocs, and all were perfused without technical incident. Three of the 5 donor organs evaluated met criteria for transplantation after 3 hours of EVLP and were transplanted. Donor organs rejected for transplantation showed either signs of worsening PaO2 or deterioration of physiologic metrics. There were no intraoperative complications in the patients who underwent transplantation, and all were alive at 30 days. CONCLUSIONS: We report here the successful use of EVLP to assess donor lungs acquired from victims of asphyxiation by hanging. The use of EVLP in this particular group of donors has the potential to expand the available donor pool. We demonstrate that EVLP is a viable option for evaluating the function of lung allografts before transplantation and would recommend that all donor lungs obtained from hanging victims undergo EVLP to assess their suitability for transplantation.

Gastric microbiome and gastric cancer.

Cancer of the stomach is the fourth most common cancer worldwide. The single strongest risk factor for gastric cancer is Helicobacter pylori-associated chronic gastric inflammation. Among persons with H. pylori infection, strain-specific components, host immune responses, and environmental factors influence the risk for gastric disease, including adenocarcinoma of the stomach, although only a small proportion of infected persons develop the malignancy. Recent advances in DNA sequencing technology have uncovered a complex community of noncultivatable inhabitants of the human stomach. The interaction between these inhabitants, collectively referred to as the gastric microbiota, and H. pylori likely affects gastric immunobiology and possibly the sequelae of H. pylori infection. Thus, characterization of the gastric microbiota in subjects with and without H. pylori infection could provide new insight into gastric homeostasis and the pathogenesis of H. pylori-associated disease, including gastric cancer.

Vaginal myeloid dendritic cells transmit founder HIV-1.

We report that primary human vaginal dendritic cells (DCs) display a myeloid phenotype and express CD4, CCR5, and CXCR4. Vaginal CD13(+) CD11c(+) DCs rapidly and efficiently bound transmitted/founder (T/F) CCR5-tropic (R5) viruses, transported them through explanted vaginal mucosa, and transmitted them in trans to vaginal and blood lymphocytes. Vaginal myeloid DCs may play a key role in capturing and disseminating T/F R5 HIV-1 in vivo and are candidate "gatekeeper" cells in HIV-1 transmission.

Interactions between HIV-1 and mucosal cells in the female reproductive tract.

Worldwide, the heterosexual route is the prevalent mode of HIV-1 transmission, and the female reproductive tract accounts for approximately 40% of all HIV-1 transmissions. HIV-1 infection in the female reproductive tract involves three major events: entry through the mucosal epithelium, productive infection in subepithelial mononuclear cells, and delivery to lymph nodes to initiate systemic infection. Here, we provide a focused review of the interaction between HIV-1 and mucosal epithelial cells, lymphocytes, macrophages, and dendritic cells in female genital mucosa. Increased understanding of these interactions could illuminate new approaches for interdicting HIV-1 heterosexual transmission.

Reproducable paraplegia by thoracic aortic occlusion in a murine model of spinal cord ischemia-reperfusion.

BACKGROUND: Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury. The current model demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion. METHODS: Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery. Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr. RESULTS: Mice that underwent sham surgery showed no observable hind limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post operative mortality. CONCLUSION: Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.

Brain angiogenesis inhibitor 1 is expressed by gastric phagocytes during infection with Helicobacter pylori and mediates the recognition and engulfment of human apoptotic gastric epithelial cells.

After Helicobacter pylori infection in humans, gastric epithelial cells (GECs) undergo apoptosis due to stimulation by the bacteria or inflammatory cytokines. In this study, we assessed the expression and function of brain angiogenesis inhibitor 1 (BAI1) in the engulfment of apoptotic GECs using human tissue and cells. After induction of apoptosis by H. pylori or camptothecin, there was a 5-fold increase in the binding of apoptotic GECs to THP-1 cells or peripheral blood monocyte-derived macrophages as assayed by confocal microscopy or conventional and imaging flow cytometry. Binding was impaired 95% by pretreating apoptotic cells with annexin V, underscoring the requirement for phosphatidylserine recognition. The phosphatidylserine receptor BAI1 was expressed in human gastric biopsy specimens and gastric phagocytes. To confirm the role of BAI1 in apoptotic cell clearance, the functional domain of BAI1 was used as a competitive inhibitor or BAI1 expression was inhibited by small interfering RNA. Both approaches decreased binding and engulfment >40%. Exposing THP-1 cells to apoptotic cells inhibited IL-6 production from 1340 to <364 pg/ml; however, this decrease was independent of phagocytosis. We conclude that recognition of apoptotic cells by BAI1 contributes to their clearance in the human gastric mucosa and this is associated with anti-inflammatory effects.

Mucosal correlates of protection in HIV-1-exposed sero-negative persons.

Resistance to HIV-1 infection in HIV-1-exposed sero-negative (HESN) persons offers a promising opportunity to identify mechanisms of 'natural' protection. Unique features of the mucosa in particular may contribute to this protection. Here, we highlight several key issues pertaining to the mucosal correlates of protection in HESN persons, including humoral immune responses, mechanisms of mucosal HIV-1 neutralization, immune cell activation, and role of the microbiota in mucosal responses. We also discuss mucosal model systems that can be used to investigate the mechanisms of resistance in HESN subjects. A clear understanding of the mucosal correlates of protection against HIV-1 in HESN persons will provide critical new insights for the development of effective vaccine and microbicide strategies for the prevention of HIV-1 transmission.