Identification of salivary autoantibodies as biomarkers of oral cancer with immunoglobulin A enrichment combined with affinity mass spectrometry.

Globally, oral cavity squamous cell carcinoma (OSCC) is one of the most common fatal illnesses. Its high mortality is ascribed to the fact that the disease is often diagnosed at a late stage, which indicates an urgent need for approaches for the early detection of OSCC. The use of salivary autoantibodies (autoAbs) as OSCC biomarkers has numerous advantages such as easy access to saliva samples and efficient detection of autoAbs using well-established secondary reagents. To improve OSCC screening, we identified OSCC-associated autoAbs with the enrichment of salivary autoAbs combined with affinity mass spectrometry (MS). The salivary IgA of healthy individuals and OSCC patients was purified with peptide M-conjugated beads and then applied to immunoprecipitated antigens (Ags) in OSCC cells. Using tandem MS analysis and spectral counting-based quantitation, the level of 10 Ags increased in the OSCC group compared with the control group. Moreover, salivary levels of autoAbs to the 10 Ags were determined by a multiplexed bead-based immunoassay. Among them, seven were significantly higher in early-stage OSCC patients than in healthy individuals. A marker panel consisting of autoAbs to LMAN2, PTGR1, RAB13, and UQCRC2 was further developed to improve the early diagnosis of OSCC.

Transcriptome-wide identification and characterization of the Rab GTPase family in mango.

The Rab GTPase family plays a vital role in several plant physiological processes including fruit ripening. Fruit softening during ripening involves trafficking of cell wall polymers and enzymes between cellular compartments. Mango, an economically important fruit crop, is known for its delicious taste, exotic flavour and nutritional value. So far, there is a paucity of information on the mango Rab GTPase family. In this study, 23 genes encoding Rab proteins were identified in mango by a comprehensive in silico approach. Sequence alignment and similarity tree analysis with the model plant Arabidopsis as a reference enabled the bona fide assignment of the deduced mango proteins to classify into eight subfamilies. Expression analysis by RNA-Sequencing (RNA-Seq) showed that the Rab genes were differentially expressed in ripe and unripe mangoes suggesting the involvement of vesicle trafficking during ripening. Interaction analysis showed that the proteins involved in vesicle trafficking and cell wall softening were interconnected providing further evidence of the involvement of the Rab GTPases in fruit softening. Correlation analyses showed a significant relationship between the expression level of the RabA3 and RabA4 genes and fruit firmness at the unripe stage of the mango varieties suggesting that the differences in gene expression level might be associated with the contrasting firmness of these varieties. This study will not only provide new insights into the complexity of the ripening-regulated molecular mechanism but also facilitate the identification of potential Rab GTPases to address excessive fruit softening.

RAB13 as a novel prognosis marker promotes proliferation and chemotherapeutic resistance in gastric cancer.

Gastric cancer (GC) is still a major lethal gastrointestinal tumor. In this study, we clarified that RAB13, which is a member of Rab GTPase family and responsible for cargos delivery between the Golgi and the plasma membrane, plays critical roles in the proliferation and the chemotherapeutic resistance in GC cells. Analyzing RAB13 expression in GC specimens, we found that its mRNA level was higher in cancerous tissues compared with normal counterparts and this increase was further associated with malignant progression of GC. Moreover, increased RAB13 indicated poor overall survival (OS) and progression free survival (PFS) in GC patients. We then found that deletion of RAB13 inhibited the proliferation and promoted the apoptosis in AGS and NCI-N87 cells, the impairments of viability which was due to reduced amount of RAB13 anchoring the plasma membrane and attenuated cellular response to EGF treatment and the activation of downstream Akt/ERK/mTOR signaling pathways accordingly. Moreover, in vitro experiments showed that RAB13 deletion enhanced the sensitization of AGS and NCI-N87 cells toward cisplatin (CDDP) and 5-fluorouracil (5-FU) treatment respectively. Together, these data demonstrate that RAB13 promotes the proliferation and confers CDDP and 5-FU resistance to GC cells, which provides experimental support to target this protein in future clinical practice.

Proton pumping V-ATPase inhibitor bafilomycin A1 affects Rab7 lysosomal localization and abolishes anterograde trafficking of osteoclast secretory lysosomes.

Osteoclast lysosomes secrete lytic enzymes into bone resorption lacunae, and sort the lysosomal proton pumping vacuolar-type ATPase (V-ATPase) to the plasma membrane to form the acidic environment required for bone digestion. The a3 isoform of V-ATPase is essential for outward trafficking of the secretory lysosomes and interacts physically with Rab7, a small GTPase that regulates trafficking of late endosomes and lysosomes, to recruit it to lysosomes. However, it is unclear whether organelle acidification by V-ATPase is required for the lysosome trafficking. Here, we showed that incubation of osteoclasts with the V-ATPase inhibitor bafilomycin A1 abolished the osteoclast-characteristic peripheral localization of secretory lysosomes, Rab7, and α-tubulin. Although bafilomycin A1 had little or no effect on Rab7 activation and its interaction with a3, treatment with the inhibitor significantly reduced the lysosomal localization of Rab7. Even constitutively active Rab7 did not localize to lysosomes in the presence of the inhibitor. These results suggest that organelle acidification by V-ATPase is required for localization of activated Rab7 to lysosomes.

Autophagy inhibitor Vacuolin-1 interferes with lipid-based small interference RNA delivery.

Autophagy and endocytosis are important pathways regulating macromolecule recycling and regeneration. Small molecule inhibitors are utilized to modulate these pathways and to treat autophagy-related diseases. Vacuolin-1 is a small molecule that can potently and reversibly inhibit autophagy by activating Rab5. In addition, Vacuolin-1 can be applied to inhibit exocytosis in a variety of cell types. Here we report that Vacuolin-1 significantly reduces small interference RNA (siRNA)-mediated gene silencing delivered by liposome transfection reagent or lipid nanoparticles in Hela cells. Vacuolin-1 exhibits the strongest inhibition effect among a few autophagy inhibitors including Chloroquine, Wortmannin, and Bafilomycin A1. We found that siRNAs are over-accumulated intracellularly and colocalized with a late endosome marker Rab7 in Vacuolin-1 treated cells, suggesting Vacuolin-1 inhibits the cytoplasmic release of lipid siRNA complexes from late endosomes. We propose that Vacuolin-1 could potentially be used to control the effects of lipid nanoparticle-based RNAi and gene therapy drugs.

Functional characterization of insect-specific RabX6 of Bombyx mori.

Rab proteins are low-molecular weight (20-25 kDa) monomeric GTPases that are central to the control and regulation of vesicle trafficking. RabX6 is an insect-specific Rab protein that has no close homolog in vertebrates. However, little information about insect-specific Rab proteins is available. In this study, RabX6 was expressed in Escherichia coli and subsequently purified. Antibodies against Bombyx mori RabX6 were produced in rabbits and rats for western immunoblotting and immunohistochemistry. Western blotting of testis tissues revealed two bands, at positions corresponding to a molecular weight of approximately 26 kDa. RabX6-like immunohistochemical reactivity (RabX6-ir) was identified at the face of the testis, not in the spermatogonia, and was specifically detected at a pair of tritocerebral cells of the male brain. Furthermore, RNA interference of RabX6 was shown to decrease testicular growth. These findings suggest that RabX6 is involved in the regulation of testicular growth and male-specific neuropeptide secretion in the brain of B. mori.

Roles of Small GTPases in Acquired Tamoxifen Resistance in MCF-7 Cells Revealed by Targeted, Quantitative Proteomic Analysis.

Development of tamoxifen resistance remains a tremendous challenge for the treatment of estrogen-receptor (ER)-positive breast cancer. Small GTPases of the Ras superfamily play crucial roles in intracellular trafficking and cell signaling, and aberrant small-GTPase signaling is implicated in many types of cancer. In this study, we employed a targeted, quantitative proteomic approach that relies on stable-isotope labeling by amino acids in cell culture (SILAC), gel fractionation, and scheduled multiple-reaction-monitoring (MRM) analysis, to assess the differential expression of small GTPases in MCF-7 and the paired tamoxifen-resistant breast cancer cells. The method displayed superior sensitivity and reproducibility over the shotgun-proteomic approach, and it facilitated the quantification of 96 small GTPases. Among them, 13 and 10 proteins were significantly down- and up-regulated (with >1.5-fold change), respectively, in the tamoxifen-resistant line relative to in the parental line. In particular, we observed a significant down-regulation of RAB31 in tamoxifen-resistant cells, which, in combination with bioinformatic analysis and downstream validation experiments, supported a role for RAB31 in tamoxifen resistance in ER-positive breast-cancer cells. Together, our results demonstrated that the targeted proteomic method constituted a powerful approach for revealing the role of small GTPases in therapeutic resistance.

EhRab21 mobilization during erythrophagocytosis in Entamoeba histolytica.

Rab proteins are present in all eukaryotic lineages and regulate vesicular trafficking. Entamoeba histolytica has approximately 100 genes encoding Rab proteins, among which 16 have homology with human Rab proteins. Human Rab21 participates in integrin recycling, and thus amoebic Rab21 was believed to regulate the mobilization of Ehß1FNR (integrin-like fibronectin receptor related with human integrin ß1). We analyzed the distribution of EhRab21 using a polyclonal antibody produced with a specific peptide against the amoebic Rab protein, using confocal microscopy and specific probes for different organelles. EhRab21 was not associated with Ehß1FNR in fibronectin-stimulated trophozoites. However, EhRab21 was relocalized to lysosomes in erythrophagocytosis assays and was also found in Golgi-positive structures and the nuclear periphery. These results suggest that EhRab21, unlike its human homologue, is not present in the recycling pathway. However, according to the results, EhRab21 may regulate the trafficking between lysosomes and the Golgi apparatus.

[The sequence coverage in different methods of mass spectrometry data analysis obtained on model proteins]. / Stepen' pokrytiia aminokislotnoi posledovatel'nosti pri ispol'zovanii razlichnykh metodov analiza mass-spektrometricheskikh dannykh, poluchennykh na model'nykh belkakh.

The aim of this study was to evaluate sequence coverage of five model proteins (CYB5A, SMAD4, RAB27B, FECH, and CXXC1) by means of shotgun proteomic data analysis employing different methods of data treatment including database-dependent search engines (MASCOT and X!Tandem) and de novo sequencing software ((PEAKS, Novor, and PepNovo+). In order to achieve maximal results, multiprotease hydrolysis including enzymes trypsin, LYS-C, ASPN and GluC was performed in solution and using the FASP method. High resolution mass spectrometry was carried out with a Q EXACTIVE HF hybrid mass spectrometer in the positive ionization mode; parent ions with the highest intensity and a charge range from +2 to +6 were fragmented in the HCD mode. 27 experiments were carried out (hydrolysis with each of 5 enzymes in solution, 4 for the FASP protocol, three technical repeats). Using parameters limiting false identification of peptides, the search engines and de novo sequencing software gave similar results. The degree of sequence coverage was not at least 40%, and in the best cases it reached 80-90%. The use of de novo sequencing software resulted in identification of the Y12H amino acid substitution in one model protein (CYB5A).

Overexpression of Rabl3 and Cullin7 is associated with pathogenesis and poor prognosis in hepatocellular carcinoma.

The expression of Rabl3 and Cullin7 is relevant to the carcinogenesis of certain cancers. However, the relationship of this expression with hepatocellular carcinoma remains unclear. To study the protein expression of Rabl3 and Cullin7 and to evaluate their role in hepatocarcinogenesis, in 162 cases of hepatocellular carcinoma, we used immunohistochemistry to investigate the expression of Rabl3 and Cullin7 in both the cancer tissues and the normal hepatic tissues around the hepatocellular carcinoma. The results demonstrated that the rates of positive Rabl3 and Cullin7 expression were 80.2% and 69.1%, respectively, in hepatocellular carcinoma tissues. However, the rates of positive Rabl3 and Cullin7 expression were 31.5% and 29.0%, respectively, in adjacent normal hepatic tissues. Rabl3 and Cullin7 were expressed at significantly higher rates in hepatocellular carcinoma compared with adjacent normal hepatic tissues (P<.01). The rates of positive Rabl3 and Cullin7 expression were higher in the hepatocellular carcinoma tissues of patients with lymph node metastasis, tumor thrombi in the portal vein and an advanced clinical stage (P<.05). A positive correlation between the expression of Rabl3 and the expression of Cullin7 (r=0.27, P<.001) was also observed in our hepatocellular carcinoma cohort. Moreover, patients with positive expression for both Rabl3 and Cullin7 had a remarkably shorter survival time compared with patients with negative expression for both proteins (P<.05). Therefore, the expression of the Rabl3 and Cullin7 proteins may play an important role in the pathogenesis and progression of hepatocellular carcinoma and could be used as a prognostic indicator in patients with hepatocellular carcinoma.

The Monomeric GTPase Rab35 Regulates Phagocytic Cup Formation and Phagosomal Maturation in Entamoeba histolytica.

One of the hallmarks of amoebic colitis is the detection of Entamoeba histolytica (Eh) trophozoites with ingested erythrocytes. Therefore, erythrophagocytosis is traditionally considered as one of the most important criteria to identify the pathogenic behavior of the amoebic trophozoites. Phagocytosis is an essential process for the proliferation and virulence of this parasite. Phagocytic cargo, upon internalization, follows a defined trafficking route to amoebic lysosomal degradation machinery. Here, we demonstrated the role of EhRab35 in the early and late phases of erythrophagocytosis by the amoeba. EhRab35 showed large vacuolar as well as punctate vesicular localization. The spatiotemporal dynamics of vacuolar EhRab35 and its exchange with soluble cytosolic pool were monitored by fluorescence recovery after photobleaching experiments. Using extensive microscopy and biochemical methods, we demonstrated that upon incubation with RBCs EhRab35 is recruited to the site of phagocytic cups as well as to the nascent phagosomes that harbor Gal/GalNAc lectin and actin. Overexpression of a dominant negative mutant of EhRab35 reduced phagocytic cup formation and thereby reduced RBC internalization, suggesting a potential role of the Rab GTPase in the cup formation. Furthermore, we also performed a phagosomal maturation assay and observed that the activated form of EhRab35 significantly increased the rate of RBC degradation. Interestingly, this mutant also significantly enhanced the number of acidic compartments in the trophozoites. Taken together, our results suggest that EhRab35 is involved in the initial stage of phagocytosis as well as in the phagolysosomal biogenesis in E. histolytica and thus contributes to the pathogenicity of the parasite.

Small GTPase Rab8a-recruited Phosphatidylinositol 3-Kinase γ Regulates Signaling and Cytokine Outputs from Endosomal Toll-like Receptors.

LPS-mediated activation of Toll-like receptor 4 (TLR4) in macrophages results in the coordinated release of proinflammatory cytokines, followed by regulatory mediators, to ensure that this potentially destructive pathway is tightly regulated. We showed previously that Rab8a recruits PI3Kγ for Akt-dependent signaling during TLR4 activation to limit the production of the proinflammatory cytokines IL-6 and IL-12p40 while enhancing the release of the regulatory/anti-inflammatory cytokine IL-10. Here we broaden the array of immune receptors controlled by Rab8a-PI3Kγ and further define the Rab-mediated membrane domains required for signaling. With CRISPR/Cas9-mediated gene editing to stably knock out and recover Rab8a in macrophage cell lines, we match Akt signaling profiles with cytokine outputs, confirming that Rab8a is a novel regulator of the Akt/mammalian target of rapamycin (mTOR) pathway downstream of multiple TLRs. Upon developing a Rab8a activation assay, we show that TLR3 and 9 agonists also activate Rab8a. Live-cell imaging reveals that Rab8a is first recruited to the plasma membrane and dorsal ruffles, but it is retained during collapse of ruffles to form macropinosomes enriched for phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), suggesting that the macropinosome is the location where Rab8a is active. We pinpoint macropinosomes as the sites for Rab8-mediated biasing of inflammatory signaling responses via inducible production of anti-inflammatory cytokines. Thus, Rab8a and PI3Kγ are positioned in multiple TLR pathways, and this signaling axis may serve as a pharmacologically tractable target during infection and inflammation.

TSAP6 is a novel candidate marker of poor survival in metastatic high-grade serous carcinoma.

The objective of this study was to analyze the expression and clinical role of molecules involved in exosome synthesis and secretion in high-grade serous carcinoma, with focus on malignant effusions. The mRNA expression levels of ARF6, nSMase2, TSAP6, Rab27a and Rab27b by quantitative real-time reverse-transcription polymerase chain reaction were analyzed in 103 HGSC effusions and 65 solid specimens (35 ovarian, 30 abdominal metastases). Protein expression of ARF6, nSMase2, TSAP6 and Rab27a by Western blotting was analyzed in 150 specimens (94 effusions, 29 ovarian carcinomas, 27 solid metastases). Secreted ARF6, nSMase2 and Rab27a protein levels in exosomes were analyzed in supernatants from 75 effusions. Expression levels were analyzed for association with anatomic site and clinical parameters, including survival. nSMase2 and TSAP6 mRNA was overexpressed in effusions compared to solid lesions (P<.001 and P=.003, respectively), whereas ARF6, nSMase2, TSAP6 and Rab27a protein was overexpressed in solid specimens (ovarian and peritoneal) compared to effusions (P<.001 for all). Secreted ARF6, nSMase2 and Rab27a levels were found in all effusion supernatants. In univariate survival analysis, higher TSAP6 protein levels in effusions were associated with shorter overall survival (P=.01), a finding which was reproduced in analysis of specimens from patients with pre-chemotherapy effusions tapped at diagnosis (P=.026). Higher levels of exosomal Rab27a protein were significantly related to longer overall survival (P=.025). Molecules which are part of the exosome secretion machinery are differentially expressed in HGSC effusions and solid lesions and are present in effusion supernatant-derived exosomes. TSAP6 and Rab27a may be novel prognostic markers in metastatic HGSC.

Skywalker-TBC1D24 has a lipid-binding pocket mutated in epilepsy and required for synaptic function.

Mutations in TBC1D24 cause severe epilepsy and DOORS syndrome, but the molecular mechanisms underlying these pathologies are unresolved. We solved the crystal structure of the TBC domain of the Drosophila ortholog Skywalker, revealing an unanticipated cationic pocket conserved among TBC1D24 homologs. Cocrystallization and biochemistry showed that this pocket binds phosphoinositides phosphorylated at the 4 and 5 positions. The most prevalent patient mutations affect the phosphoinositide-binding pocket and inhibit lipid binding. Using in vivo photobleaching of Skywalker-GFP mutants, including pathogenic mutants, we showed that membrane binding via this pocket restricts Skywalker diffusion in presynaptic terminals. Additionally, the pathogenic mutations cause severe neurological defects in flies, including impaired synaptic-vesicle trafficking and seizures, and these defects are reversed by genetically increasing synaptic PI(4,5)P2 concentrations through synaptojanin mutations. Hence, we discovered that a TBC domain affected by clinical mutations directly binds phosphoinositides through a cationic pocket and that phosphoinositide binding is critical for presynaptic function.

RAB22A overexpression promotes the tumor growth of melanoma.

Malignant melanoma is the most aggressive type of skin cancer. RAB22A, a member of RAS oncogene family, has been found to be significantly upregulated in multiple human cancers. In the present study, we found that RAB22A mRNA expression was significantly upregulated in melanoma tissues (including 60 primary melanomas and 84 metastatic melanomas) compared to benign nevi (n = 20), which were significantly higher in metastatic melanoma tissues than primary tissues. Immunohistochemistry data further showed that the positive immunoreactivity of RAB22A was detected in 66% (95/144) melanoma tissues, but not in benign nevi. Moreover, high expression of RAB22A was significantly associated with advanced clinical stage in melanoma. Furthermore, patients with high RAB22A expression had shorter overall survival compared those with low expression of RAB22A. In-vitro study showed that RAB22A was also upregulated in melanoma cell lines WM35, A375, WM451, and SK-MEL-1, when compared with the normal melanocyte HM cells. Knockdown of RAB22A significantly reduced the proliferation, migration and invasion of melanoma A375 cells, while overexpression of RAB22A significantly promoted these malignant phenotypes. In addition, RAB22A was found to be a target of miR-203, a tumor suppressive miRNA in melanoma. Besides, miR-203 was downregulated in melanoma tissues and cell lines, when compared with benign nevi and HM cells, respectively. Taken these findings together, our study could validate an oncogenic role of RAB22A in melanoma, suggesting that RAB22A may be a potential therapeutic target for melanoma.

Recombinant Leishmania Rab6 (rLdRab6) is recognized by sera from visceral leishmaniasis patients.

Rab proteins form the largest branch of the Ras superfamily. Rab proteins are key regulators of intracellular vesicular transport and membrane trafficking. Although RabGTPases are well-recognized targets in human diseases but are under-explored therapeutically in the Leishmania parasite. Using a quantitative cytofluorimetric assay, we analyzed the composition and organization of Rab6GTPase protein which was found to be primarily localized on the parasite subpellicular membrane and flagellum due to its association with kinesin motor proteins in the cytoskeletal microtubules. Our aim was to also assess the diagnostic role of recombinant Rab6 protein from Leishmania donovani (rLdRab6) using sera/plasma of Indian visceral leishmaniasis (VL) patients. Receiver-operating characteristic (ROC) curve analysis indicated 100% sensitivity and 100% specificity for rLdRab6-based ELISA which was almost similar in comparison to recombinant K39-based ELISA (95.83% sensitivity and 100% specificity). Sera of patients from another intracellular pathogenic infection, Mycobacterium tuberculosis, did not contain any significant levels of anti-rLdRab6 antibody. Thus rLdRab6 accuracy in visceral leishmaniasis diagnosis makes it a promising antigen for clinical use.

Increased Rab35 expression is a potential biomarker and implicated in the pathogenesis of Parkinson's disease.

Parkinson's disease (PD) is the second common neurodegenerative disease. Identification of biomarkers for early diagnosis and prediction of disease progression is important. The present comparative proteomic study of serum samples using two-dimensional fluorescence differential gel electrophoresis followed by ELISA confirmation demonstrated that protein expression of Rab35 was increased in PD patients compared with matched control subjects and other parkinsonian disorders, progressive supranuclear palsy (PSP) and multiple system atrophy (MSA). The serum level of Rab35 was significantly correlated with the age at onset of PD. The median age of onset in patients with higher Rab35 serum level was 5 years younger than those with lower Rab35 serum level. There was a positive correlation between the Rab35 level and disease duration of PD. Moreover, the protein expression of Rab35 was increased in the substantia nigra but not in the striatum of mouse models of PD, including MPTP-treated mice, rotenone-treated mice, (R1441C) LRRK2 or (G2019S) LRRK2 transgenic mice. Furthermore, overexpression of Rab35 increased the aggregation and secretion of mutant A53T α-synuclein in dopaminergic SH-SY5Y cells. Co-expression of Rab35 with wild-type or A53T α-synuclein in SH-SY5Y cells deteriorated cell death. Our results suggest that Rab35 is potentially useful in the differential diagnosis of parkinsonian disorders and is implicated in the pathogenesis of PD.

The RAB GTPase RABA1e localizes to the cell plate and shows distinct subcellular behavior from RABA2a under Endosidin 7 treatment.

Cytokinesis in plants requires the activity of RAB GTPases to regulate vesicle-mediated contribution of material to the developing cell plate. While some plant RAB GTPases have been shown to be involved in cell plate formation, many still await functional assignment. Here, we report cell plate localization for YFP-RABA1e in Arabidopsis thaliana and use the cytokinesis inhibitor Endosidin 7 to provide a detailed description of its localization compared to YFP-RABA2a. Differences between YFP-RABA2a and YFP-RABA1e were observed in late-stage cell plates under DMSO control treatment, and became more apparent under Endosidin 7 treatment. Taken together, our results suggest that individual RAB GTPases might make different contributions to cell plate formation and further demonstrates the utility of ES7 probe to dissect them.

Exocytosis of Varicella-Zoster Virus Virions Involves a Convergence of Endosomal and Autophagy Pathways.

UNLABELLED: Varicella-zoster virus (VZV) is an extremely cell-associated herpesvirus with limited egress of viral particles. The induction of autophagy in VZV-infected monolayers is easily detectable; inhibition of autophagy leads to decreased VZV glycoprotein biosynthesis and diminished viral titers. To explain how autophagic flux could exert a proviral effect on the VZV infectious cycle, we postulated that the VZV exocytosis pathway following secondary envelopment may converge with the autophagy pathway. This hypothesis depended on known similarities between VZV gE and autophagy-related (Atg) Atg9/Atg16L1 trafficking pathways. Investigations were carried out with highly purified fractions of VZV virions. When the virion fraction was tested for the presence of autophagy and endosomal proteins, microtubule-associated protein 1 light chain (MAP1LC3B) and Ras-like GTPase 11 (Rab11) were detected. By two-dimensional (2D) and 3D imaging after immunolabeling, both proteins also colocalized with VZV gE in a proportion of cytoplasmic vesicles. When purified VZV virions were enumerated after immunoelectron microscopy, gold beads were detected on viruses following incubation with antibodies to VZV gE (∼100%), Rab11 (50%), and LC3B (30%). Examination of numerous electron micrographs demonstrated that enveloped virions were housed in single-membraned vesicles; viral particles were not observed in autophagosomes. Taken together, our data suggested that some viral particles after secondary envelopment accumulated in a heterogeneous population of single-membraned vesicular compartments, which were decorated with components from both the endocytic pathway (Rab11) and the autophagy pathway (LC3B). The latter cytoplasmic viral vesicles resembled an amphisome. IMPORTANCE: VZV infection leads to increased autophagic flux, while inhibition of autophagy leads to a marked reduction in virus spread. In this investigation of the proviral role of autophagy, we found evidence for an intersection of viral exocytosis and autophagy pathways. Specifically, both LC3-II and Rab11 proteins copurified with some infectious VZV particles. The results suggested that a subpopulation of VZV particles were carried to the cell surface in single-walled vesicles with attributes of an amphisome, an organelle formed from the fusion of an endosome and an autophagosome. Our results also addressed the interpretation of autophagy/xenophagy results with mutated herpes simplex virus lacking its ICP34.5 neurovirulence gene (HSVΔ34.5). The VZV genome lacks an ICP34.5 ortholog, yet we found no evidence of VZV particles housed in a double-membraned autophagosome. In other words, xenophagy, a degradative process documented after infection with HSVΔ34.5, was not observed in VZV-infected cells.

Exosome in intestinal mucosal immunity.

Intercellular communication of immune cells is critical to elicit efficient inflammatory responses. In intestinal mucosa, imbalance in pro-inflammatory and anti-inflammatory mediators, especially cytokines and chemokines, characterizes the underlying immune mechanisms of inflammatory bowel disease. Exosomes, small membrane vesicles secreted into the extracellular environment, are emerging as another important intercellular messenger in immune responses. A major recent breakthrough in this field unveils the capacity of exosomes to mediate the functional transfer of genetic materials (mRNAs and miRNAs) between immune cells. RAB27A and RAB27B are two small GTPases involved in exosome secretion. With respect to intestinal mucosal immunity, increased number of RAB27A-positive immune cells and RAB27B-positive immune cells are demonstrated in the colonic mucosa of patients with active ulcerative colitis as compared with that of healthy controls. This indicates the important role of exosome-mediated immune responses in the pathogenesis of inflammatory bowel disease. Here, we will discuss the immune properties of exosomes and recent advances in their function with a special focus on intestinal mucosal immunity.