HCMV US2 co-opts TRC8 to degrade the endoplasmic reticulum-resident protein LMAN2L.

The human cytomegalovirus (HCMV) pUS2 glycoprotein exploits the host's endoplasmic reticulum (ER)-associated degradation (ERAD) pathway to degrade major histocompatibility complex class I (MHC-I) and prevent antigen presentation. Beyond MHC-I, pUS2 has been shown to target a range of cellular proteins for degradation, preventing their cell surface expression. Here we have identified a novel pUS2 target, ER-resident protein lectin mannose binding 2 like (LMAN2L). pUS2 expression was both necessary and sufficient for the downregulation of LMAN2L, which was dependent on the cellular E3 ligase TRC8. Given the hypothesized role of LMAN2L in the trafficking of glycoproteins, we employed proteomic plasma membrane profiling to measure LMAN2L-dependent changes at the cell surface. A known pUS2 target, integrin alpha-6 (ITGA6), was downregulated from the surface of LMAN2L-deficient cells, but not other integrins. Overall, these results suggest a novel strategy of pUS2-mediated protein degradation whereby pUS2 targets LMAN2L to impair trafficking of ITGA6. Given that pUS2 can directly target other integrins, we propose that this single viral protein may exhibit both direct and indirect mechanisms to downregulate key cell surface molecules.

The Glycan Ectodomain of SARS-CoV-2 Spike Protein Modulates Cytokine Production and Expression of CD206 Mannose Receptor in PBMC Cultures of Pre-COVID-19 Healthy Subjects.

The ability of recombinant, SARS-CoV-2 Spike (S) protein to modulate the production of two COVID-19 relevant, pro-inflammatory cytokines (IL-6 and IFN-γ) in PBMC cultures of healthy, pre-COVID-19 subjects was investigated. We observed that cytokine production was largely and diversely modulated by the S protein depending on antigen or mitogen stimulation, as well as on the protein source, insect (S-in) or human (S-hu) cells. While both proteins co-stimulated cytokine production by polyclonally CD3-activated T cells, PBMC activation by the mitogenic lectin Concanavalin A (Con A) was up-modulated by S-hu protein and down-modulated by S-in protein. These modulatory effects were likely mediated by the S glycans, as demonstrated by direct Con A-S binding experiments and use of yeast mannan as Con A binder. While being ineffective in modulating memory antigenic T cell responses, the S proteins and mannan were able to induce IL-6 production in unstimulated PBMC cultures and upregulate the expression of the mannose receptor (CD206), a marker of anti-inflammatory M2 macrophage. Our data point to a relevant role of N-glycans, particularly N-mannosidic chains, decorating the S protein in the immunomodulatory effects here reported. These novel biological activities of the S glycan ectodomain may add to the comprehension of COVID-19 pathology and immunity to SARS-CoV-2.

Structure of full-length ERGIC-53 in complex with MCFD2 for cargo transport.

ERGIC-53 transports certain subsets of newly synthesized secretory proteins and membrane proteins from the endoplasmic reticulum to the Golgi apparatus. Despite numerous structural and functional studies since its identification, the overall architecture and mechanism of action of ERGIC-53 remain unclear. Here we present cryo-EM structures of full-length ERGIC-53 in complex with its functional partner MCFD2. These structures reveal that ERGIC-53 exists as a homotetramer, not a homohexamer as previously suggested, and comprises a four-leaf clover-like head and a long stalk composed of three sets of four-helix coiled-coil followed by a transmembrane domain. 3D variability analysis visualizes the flexible motion of the long stalk and local plasticity of the head region. Notably, MCFD2 is shown to possess a Zn2+-binding site in its N-terminal lid, which appears to modulate cargo binding. Altogether, distinct mechanisms of cargo capture and release by ERGIC- 53 via the stalk bending and metal binding are proposed.

Identification and characterization of stromal-like cells with CD207+/low CD1a+/low phenotype derived from histiocytic lesions - a perspective in vitro model for drug testing.

BACKGROUND: Histiocytoses are rare disorders manifested by increased proliferation of pathogenic myeloid cells sharing histological features with macrophages or dendritic cells and accumulating in various organs, i.a., bone and skin. Pre-clinical in vitro models that could be used to determine molecular pathways of the disease are limited, hence research on histiocytoses is challenging. The current study compares cytophysiological features of progenitor, stromal-like cells derived from histiocytic lesions (sl-pHCs) of three pediatric patients with different histiocytoses types and outcomes. The characterized cells may find potential applications in drug testing. METHODS: Molecular phenotype of the cells, i.e. expression of CD1a and CD207 (langerin), was determined using flow cytometry. Cytogenetic analysis included GTG-banded metaphases and microarray (aCGH) evaluation. Furthermore, the morphology and ultrastructure of cells were evaluated using a confocal and scanning electron microscope. The microphotographs from the confocal imaging were used to reconstruct the mitochondrial network and its morphology. Basic cytophysiological parameters, such as viability, mitochondrial activity, and proliferation, were analyzed using multiple cellular assays, including Annexin V/7-AAD staining, mitopotential analysis, BrdU test, clonogenicity analysis, and distribution of cells within the cell cycle. Biomarkers potentially associated with histiocytoses progression were determined using RT-qPCR at mRNA, miRNA and lncRNA levels. Intracellular accumulation of histiocytosis-specific proteins was detected with Western blot. Cytotoxicyty and IC50 of vemurafenib and trametinib were determined with MTS assay. RESULTS: Obtained cellular models, i.e. RAB-1, HAN-1, and CHR-1, are heterogenic in terms of molecular phenotype and morphology. The cells express CD1a/CD207 markers characteristic for dendritic cells, but also show intracellular accumulation of markers characteristic for cells of mesenchymal origin, i.e. vimentin (VIM) and osteopontin (OPN). In subsequent cultures, cells remain viable and metabolically active, and the mitochondrial network is well developed, with some distinctive morphotypes noted in each cell line. Cell-specific transcriptome profile was noted, providing information on potential new biomarkers (non-coding RNAs) with diagnostic and prognostic features. The cells showed different sensitivity to vemurafenib and trametinib. CONCLUSION: Obtained and characterized cellular models of stromal-like cells derived from histiocytic lesions can be used for studies on histiocytosis biology and drug testing.

Detection of metastatic lymph node and sentinel lymph node mapping ​using mannose receptor targeting in in vivo mouse footpad tumor models and rabbit uterine cancer models.

BACKGROUND: This study aimed to evaluate the effectiveness of neo-mannosyl human serum albumin-indocyanine green (MSA-ICG) for detecting metastatic lymph node (LN) and mapping sentinel lymph node (SLN) using mouse footpad uterine tumor models. Additionally, the authors assessed the feasibility of MSA-ICG in SLN mapping in rabbit uterine cancer models. MATERIALS AND METHODS: The authors compared the LN targeting ability of MSA-ICG with ICG. Six mouse footpad tumor models and two normal mice were each assigned to MSA-ICG and ICG, respectively. After the assigned tracers were injected, fluorescence images were taken, and the authors compared the signal-to-background ratio (SBR) of the tracers. A SLN biopsy was performed to confirm LN metastasis status and CD206 expression level. Finally, an intraoperative SLN biopsy was performed in rabbit uterine cancer models using MSA-ICG. RESULTS: The authors detected 14 groin LNs out of 16 in the MSA-ICG and ICG groups. The SBR of the MSA-ICG group was significantly higher than that of the ICG group. The metastatic LN subgroup of MSA-ICG showed a significantly higher SBR than that of ICG. CD206 was expressed at a high level in metastatic LN, and the signal intensity difference increased as the CD206 expression level increased. SLN mapping was successfully performed in two of the three rabbit uterine cancer models. CONCLUSIONS: MSA-ICG was able to distinguish metastatic LN for an extended period due to its specific tumor-associated macrophage-targeting property. Therefore, it may be a more distinguishable tracer for identifying metastatic LNs and SLNs during uterine cancer surgery. Further research is needed to confirm these results.

Mannose Ligands for Mannose Receptor Targeting.

The mannose receptor (MR, CD 206) is an endocytic receptor primarily expressed by macrophages and dendritic cells, which plays a critical role in both endocytosis and antigen processing and presentation. MR carbohydrate recognition domains (CRDs) exhibit a high binding affinity for branched and linear oligosaccharides. Furthermore, multivalent mannose presentation on the various templates like peptides, proteins, polymers, micelles, and dendrimers was proven to be a valuable approach for the selective and efficient delivery of various therapeutically active agents to MR. This review provides a detailed account of the most relevant and recent aspects of the synthesis and application of mannosylated bioactive formulations for MR-mediated delivery in treatments of cancer and other infectious diseases. It further highlights recent findings related to the necessary structural features of the mannose-containing ligands for successful binding to the MR.

Mannose-specific plant and microbial lectins as antiviral agents: A review.

Lectins are non-immunological carbohydrate-binding proteins classified on the basis of their structure, origin, and sugar specificity. The binding specificity of such proteins with the surface glycan moiety determines their activity and clinical applications. Thus, lectins hold great potential as diagnostic and drug discovery agents and as novel biopharmaceutical products. In recent years, significant advancements have been made in understanding plant and microbial lectins as therapeutic agents against various viral diseases. Among them, mannose-specific lectins have being proven as promising antiviral agents against a variety of viruses, such as HIV, Influenza, Herpes, Ebola, Hepatitis, Severe Acute Respiratory Syndrome Coronavirus-1 (SARS-CoV-1), Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV) and most recent Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). The binding of mannose-binding lectins (MBLs) from plants and microbes to high-mannose containing N-glycans (which may be simple or complex) of glycoproteins found on the surface of viruses has been found to be highly specific and mainly responsible for their antiviral activity. MBLs target various steps in the viral life cycle, including viral attachment, entry and replication. The present review discusses the brief classification and structure of lectins along with antiviral activity of various mannose-specific lectins from plants and microbial sources and their diagnostic and therapeutic applications against viral diseases.

High-Mannose Oligosaccharide Hemimimetics that Recapitulate the Conformation and Binding Mode to Concanavalin A, DC-SIGN and Langerin.

The "carbohydrate chemical mimicry" exhibited by sp2 -iminosugars has been utilized to develop practical syntheses for analogs of the branched high-mannose-type oligosaccharides (HMOs) Man3 and Man5 . In these compounds, the terminal nonreducing Man residues have been substituted with 5,6-oxomethylidenemannonojirimycin (OMJ) motifs. The resulting oligomannoside hemimimetic accurately reproduce the structure, configuration, and conformational behavior of the original mannooligosaccharides, as confirmed by NMR and computational techniques. Binding studies with mannose binding lectins, including concanavalin A, DC-SIGN, and langerin, by enzyme-linked lectin assay and surface plasmon resonance revealed significant variations in their ability to accommodate the OMJ unit in the mannose binding site. Intriguingly, OMJMan segments demonstrated "in line" heteromultivalent effects during binding to the three lectins. Similar to the mannobiose (Man2 ) branches in HMOs, the binding modes involving the external or internal monosaccharide unit at the carbohydrate binding-domain exist in equilibrium, facilitating sliding and recapture processes. This equilibrium, which influences the multivalent binding of HMOs, can be finely modulated upon incorporation of the OMJ sp2 -iminosugar caps. As a proof of concept, the affinity and selectivity towards DC-SIGN and langerin were adjustable by presenting the OMJMan epitope in platforms with diverse architectures and valencies.

Mannose oligosaccharide recognition of CGL1, a mannose-specific lectin containing DM9 motifs from Crassostrea gigas, revealed by X-ray crystallographic analysis.

CGL1 is a mannose-specific lectin isolated from the Pacific oyster Crassostrea gigas, and it belongs to the DM9 domain protein family. Each subunit of the CGL1 dimer consists of a tandem repeat of DM9 motifs, which were originally found in the Drosophila melanogaster genome. The CGL1 protomer contains two carbohydrate-binding sites: a high-affinity site A and a low-affinity site B. An assay using dendrimers containing oligomannose from yeast (Saccharomyces cerevisiae) revealed that CGL1 exhibited significantly higher affinity for mannotetraose (Man4) compared to mannobiose (Man2) and mannotriose (Man3). To investigate its oligomannose-recognition mechanism, X-ray crystallographic analyses of CGL1/oligomannose complexes were performed. In the CGL1/Man2 and CGL1/Man3 complexes, Manα1-2Man and Manα1-2Manα1-2Man, respectively, were primarily bound to site A, interacting with the non-reducing mannose residue. On the other hand, in the CGL1/Man4 crystal, Man4 (Manα1-2Manα1-2Manα1-6Man) was bound at both site A and site B at the non-reducing and reducing ends, thus linking adjacent CGL1 molecules with crystallographic symmetry. These findings suggest that CGL1 can recognize both the non-reducing and reducing mannose residues of mannose oligosaccharides at its two distinct carbohydrate-binding sites. This enables efficient complex formation, making CGL1 a pattern-recognition molecule capable of recognizing diverse structures of mannose-containing carbohydrate chains.

Mannose-Binding Lectins as Potent Antivirals against SARS-CoV-2.

The SARS-CoV-2 entry into host cells is mainly mediated by the interactions between the viral spike protein (S) and the ACE-2 cell receptor, which are highly glycosylated. Therefore, carbohydrate binding agents may represent potential candidates to abrogate virus infection. Here, we evaluated the in vitro anti-SARS-CoV-2 activity of two mannose-binding lectins isolated from the Brazilian plants Canavalia brasiliensis and Dioclea violacea (ConBR and DVL). These lectins inhibited SARS-CoV-2 Wuhan-Hu-1 strain and variants Gamma and Omicron infections, with selectivity indexes (SI) of 7, 1.7, and 6.5, respectively for ConBR; and 25, 16.8, and 22.3, for DVL. ConBR and DVL inhibited over 95% of the early stages of the viral infection, with strong virucidal effect, and also protected cells from infection and presented post-entry inhibition. The presence of mannose resulted in the complete lack of anti-SARS-CoV-2 activity by ConBR and DVL, recovering virus titers. ATR-FTIR, molecular docking, and dynamic simulation between SARS-CoV-2 S and either lectins indicated molecular interactions with predicted binding energies of -85.4 and -72.0 Kcal/Mol, respectively. Our findings show that ConBR and DVL lectins possess strong activities against SARS-CoV-2, potentially by interacting with glycans and blocking virus entry into cells, representing potential candidates for the development of novel antiviral drugs.

ROMP-based Glycopolymers with High Affinity for Mannose-Binding Lectins.

Well-defined, highly reactive poly(norbornenyl azlactone)s of controlled length (number-average degree of polymerization DPn¯ = 10 to 1,000) were made by ring-opening metathesis polymerization (ROMP) of pure exo-norbornenyl azlactone. These were converted into glycopolymers using a facile postpolymerization modification (PPM) strategy based on click aminolysis of azlactone side groups by amino-functionalized glycosides. Pegylated mannoside, heptyl-mannoside, and pegylated glucoside were used in the PPM. Binding inhibition of the resulting glycopolymers was evaluated against a lectin panel (Bc2L-A, FimH, langerin, DC-SIGN, ConA). Inhibition profiles depended on the sugars and the degrees of polymerization. Glycopolymers from pegylated-mannoside-functionalized polynorbornene, with DPn¯ = 100, showed strong binding inhibition, with subnanomolar range inhibitory concentrations (IC50s). Polymers surpassed the inhibitory potential of their monovalent analogues by four to five orders of magnitude thanks to a multivalent (synergistic) effect. Sugar-functionalized poly(norbornenyl azlactone)s are therefore promising tools to study multivalent carbohydrate-lectin interactions and for applications against lectin-promoted bacterial/viral binding to host cells.

Mannose binding lectin-associated serine protease-1 is a novel contributor to myocardial ischemia/reperfusion injury.

BACKGROUND: The lectin pathway has been demonstrated to play a critical role in the pathological process of myocardial ischemia/reperfusion injury (IRI). Mannose-binding lectin (MBL)-associated serine protease-1 (MASP-1), especially different from other components of the lectin pathway, mediates proinflammatory and procoagulant reactions independent of complement cascades. However, the role of MASP-1 in myocardial IRI remains unknown so far. METHODS: Myocardial IRI was established with 45 min ischemia and 24 h reperfusion in mice. C1 inhibitor, as the natural inhibitor of MASP-1, was administrated at 20 IU/Kg via tail vein 5 min before surgical operation. Cardiac function and myocardial infarct size were assessed. Myocardial histology and fibrosis were evaluated by H&E and Masson staining, respectively. Deposition of MASP-1, expression of PAR-1/4 and neutrophil extracellular traps (NET) were investigated on myocardium tissue by IHC staining. Cell apoptosis was detected by TUNEL assay. Levels of myocardial enzymes and proinflammatory cytokines were determined by ELISA. RESULTS: Inhibition of MASP-1 with C1 INH improved cardiac function and alleviated myocardium tissue injury (infarct size, enzymes, histology and fibrosis) after myocardial IRI. Deposition of MASP-1 and expression PAR-1, as well as NET formation in myocardial tissue were suppressed by MASP-1 inhibitor, while PAR-4 was elevated. Levels of apoptosis, HMGB-1 and IL-6 were lower after blocking MASP-1. Yet, IL-8 and TNF-α remained unchanged. CONCLUSIONS: MASP-1, as a new contributor, played a critical role in myocardial IRI. Inhibition of MASP-1 protected myocardial tissue from IRI probably via regulation of PARs/NET pathway. This may provide a novel target strategy against myocardial IRI.

Mannose-binding lectin suppresses macrophage proliferation through TGF-ß1 signaling pathway in Nile tilapia.

Mannose-binding lectin (MBL) is a multifunctional pattern recognition molecule, which not only mediates the recognition of pathogenic microorganisms and their products, playing an important role in innate immune defense, but also participates in adaptive immune responses of mammalian. However, it's related immune mechanism remains limited, especially the regulation of cell proliferation in early vertebrates. In this study, OnMBL was found to bind to kidney macrophages (MФ) from Nile tilapia (Oreochromis niloticus). Interestingly, OnMBL was able to reduce the proliferation of activated-MФ by regulating the cell cycle, arresting a large number of cells in the G0/G1 phase, and increasing the probability of apoptosis. More importantly, we found that the inhibition of cell proliferation by OnMBL was closely related to the evolutionarily conserved canonical transforming growth factor-beta 1 (TGF-ß1) signaling pathway. Mechanistically, OnMBL could significantly increase the expression of TGF-ß1, activate and regulate the downstream Smad-dependent pathway to reduce the MФ proliferation, thereby maintaining cellular homeostasis in the body's internal environment. This study represents the first description regarding the regulatory mechanisms of the MBL on cell proliferation in teleost fish, which provides a novel perspective on the understanding of the multiple function and evolutionary origins of C-type lectins in the immune system.

A novel peptide derived from the mannose binding lectin inhibits LPS-activated TLR4/NF-κB signaling and suppresses ocular inflammation.

Uveitis is a major cause of vision impairment worldwide. Current treatments have limited effectiveness but severe complications. Mannose binding lectin (MBL) is an important protein of the innate immune system that binds to TLR4 and suppresses LPS-induced inflammatory cytokine secretion. MBL-mediated inhibition of inflammation via the TLR4 pathway and MBL-derived peptides might be a potential therapeutics. In this study, we designed a novel MBL-derived peptide, WP-17, targeting TLR4. Bioinformatics analysis was conducted for the sequence, structure and biological properties of WP-17. The binding of WP-17 to THP-1 cells was analyzed using flow cytometry. Signaling molecules were analyzed by western blotting, and activation of NF-κB was measured by immunofluorescence-histochemical analysis. Effects of WP-17 were studied in vitro using LPS-stimulated THP-1 cells and in vivo in endotoxin-induced uveitis (EIU). Our results showed that WP-17 could bind to TLR4 expressed on macrophages, thus downregulating the expression levels of MyD88, IRAK-4, and TRAF-6, and inhibiting the downstream NF-kB signaling pathway and LPS-induced expression of TNF-α and IL-6 in THP-1 cells. Moreover, in EIU rats, intravitreal pretreatment with WP-17 demonstrated significant inhibitory effects on ocular inflammation, attenuating the clinical and histopathological manifestations of uveitis, reducing protein leakage and cell infiltration into the aqueous humor, and suppressing TNF-α and IL-6 production in ocular tissues. In summary, our study provides the first evidence of a novel MBL-derived peptide that suppressed activation of the NF-кB pathway by targeting TLR4. The peptide effectively inhibited rat uveitis and may be a promising candidate for the management of ocular inflammatory diseases.

Capture and neutralization of SARS-CoV-2 and influenza virus by algae-derived lectins with high-mannose and core fucose specificities.

We first investigated the interactions between several algae-derived lectins and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We created lectin columns using high-mannose (HM)-type glycan-specific lectins OAA and KAA-1 or core fucose-specific lectin hypninA-2 and conducted binding experiments with SARS-CoV-2. The results showed that these lectins were capable of binding to the virus. Furthermore, when examining the neutralization ability of nine different lectins, it was found that KAA-1, ESA-2, and hypninA-2 were effective in neutralizing SARS-CoV-2. In competitive inhibition experiments with glycoproteins, neutralization was confirmed to occur through HM-type or core fucose-type glycans. However, neutralization was not observed with other lectins, such as OAA. This trend of KAA-1 and ESA-2 having the neutralizing ability and OAA not having it was also similar to influenza viruses. Electron microscopy observations revealed that KAA-1 and hypninA-2 strongly aggregated SARS-CoV-2 particles, while OAA showed a low degree of aggregation. It is believed that the neutralization of SARS-CoV-2 involves multiple factors, such as glycan attachment sites on the S protein, the size of lectins, and their propensity to aggregate, which cause inhibition of receptor binding or aggregation of virus particles. This study demonstrated that several algae-derived lectins could neutralize SARS-CoV-2 and that lectin columns can effectively recover and concentrate the virus.

A combination of the HLA-DRB1*03 phenotype and low plasma mannose-binding lectin predisposes to autoantibody formation in women with recurrent pregnancy loss.

Introduction: It is documented that a series of autoantibodies can be detected with increased frequency in women with recurrent pregnancy loss (RPL) and they may impact the pregnancy prognosis negatively. It is unknown whether the autoantibodies per se or the basic immune disturbances underlying autoantibody production, are the reason for this association. Our group has previously found that some genetically determined immunological biomarkers are associated with RPL and the same biomarkers are also in various degrees known to predispose to autoantibody production. The aim of this study was to clarify whether the RPL-associated immunogenetic biomarkers are associated with positivity for three major classes of autoantibodies associated with RPL. Methods: In 663 patients with RPL in whom we had results for HLA-DRB1 typing and plasma mannose-binding lectin (p-MBL) measurement, it was investigated whether there is a correlation between positivity for the autoantibodies: anticardiolipin antibodies, ß2 glycoprotein I antibodies, and lupus anticoagulant (jointly called antiphospholipid antibodies), thyroid-peroxidase antibodies, and antinuclear antibodies and each of the HLA-DRB1 alleles HLA-DRB1*03 or HLA-DRB1*07 either alone or in combination with low p-MBL defined as ≤500 µg/l. Results: Although slightly higher frequencies of positivity of two or more autoantibodies were seen in patients with either p-MBL ≤500 µg/l or being positive for HLA-DRB1*03, none were significantly associated. However, in patients with the combination of low p-MBL and HLA-DRB1*03, presence of at least one autoantibody was significantly more frequent than in patients with no such combination (OR= 2.4; 95% CI 1.2-5.0, p = 0.01). In an analysis of which autoantibodies were most strongly associated with the low p-MBL/HLA-DRB1*03 combination, antinuclear antibodies were significantly more frequent in these patients (OR 2.0; 95% CI 1.0-3.9, p=0.05) whereas the other autoantibodies were also positively but more weakly associated with this combination. Discussion: In conclusion, to clarify the pathogenetic background, underlying immunogenetic factors should be examined in autoantibody positive RPL patients (as well as other patients with autoimmune diseases) but the genetic background may be complex.

Size-advantage of monovalent nanobodies against the macrophage mannose receptor for deep tumor penetration and tumor-associated macrophage targeting.

Rationale: Nanobodies (Nbs) have emerged as an elegant alternative to the use of conventional monoclonal antibodies in cancer therapy, but a detailed microscopic insight into the in vivo pharmacokinetics of different Nb formats in tumor-bearers is lacking. This is especially relevant for the recognition and targeting of pro-tumoral tumor-associated macrophages (TAMs), which may be located in less penetrable tumor regions. Methods: We employed anti-Macrophage Mannose Receptor (MMR) Nbs, in a monovalent (m) or bivalent (biv) format, to assess in vivo TAM targeting. Intravital and confocal microscopy were used to analyse the blood clearance rate and targeting kinetics of anti-MMR Nbs in tumor tissue, healthy muscle tissue and liver. Fluorescence Molecular Tomography was applied to confirm anti-MMR Nb accumulation in the primary tumor and in metastatic lesions. Results: Intravital microscopy demonstrated significant differences in the blood clearance rate and macrophage targeting kinetics of (m) and (biv)anti-MMR Nbs, both in tumoral and extra-tumoral tissue. Importantly, (m)anti-MMR Nbs are superior in reaching tissue macrophages, an advantage that is especially prominent in tumor tissue. The administration of a molar excess of unlabelled (biv)anti-MMR Nbs increased the (m)anti-MMR Nb bioavailability and impacted on its macrophage targeting kinetics, preventing their accumulation in extra-tumoral tissue (especially in the liver) but only partially influencing their interaction with TAMs. Finally, anti-MMR Nb administration not only allowed the visualization of TAMs in primary tumors, but also at a distant metastatic site. Conclusions: These data describe, for the first time, a microscopic analysis of (m) and (biv)anti-MMR Nb pharmacokinetics in tumor and healthy tissues. The concepts proposed in this study provide important knowledge for the future use of Nbs as diagnostic and therapeutic agents, especially for the targeting of tumor-infiltrating immune cells.

TRPV4 Activation Increases the Expression of CD207 (Langerin) of Monocyte-Derived Langerhans Cells without Affecting their Maturation.

Langerhans cells (LCs) are the sole professional antigen-presenting cell normally found in the human epidermal compartment. Research into their physiological role is hindered by the fact that they are invariably activated during isolation from the skin. To overcome this challenge, we turned to a monocyte-derived LC (moLC) model, which we characterized with RNA sequencing, and compared the transcriptome of moLCs with that of donor-matched immature dendritic cells. We found that moLCs express markers characteristic of LC2 cells as well as TRPV4. TRPV4 is especially important in the skin because it has been linked to the conservation of the skin barrier, immunological responses, as well as acute and chronic itch, but we know little about its function on LCs. Our results show that TRPV4 activation increased the expression of Langerin and led to increased intracellular calcium concentration in moLCs. Regarding the functionality of moLCs, we found that TRPV4 agonism had a mitigating effect on their inflammatory responses because it decreased their cytokine production and T-cell activating capability. Because TRPV4 has emerged as a potential therapeutic target in dermatological conditions, it is important to highlight LCs as, to our knowledge, a previously unreported target of these therapies.

Anticancer Activity of Mannose-Specific Lectin, BPL2, from Marine Green Alga Bryopsis plumosa.

Lectin is a carbohydrate-binding protein that recognizes specific cells by binding to cell-surface polysaccharides. Tumor cells generally show various glycosylation patterns, making them distinguishable from non-cancerous cells. Consequently, lectin has been suggested as a good anticancer agent. Herein, the anticancer activity of Bryopsis plumosa lectins (BPL1, BPL2, and BPL3) was screened and tested against lung cancer cell lines (A549, H460, and H1299). BPL2 showed high anticancer activity compared to BPL1 and BPL3. Cell viability was dependent on BPL2 concentration and incubation time. The IC50 value for lung cancer cells was 50 µg/mL after 24 h of incubation in BPL2 containing medium; however, BPL2 (50 µg/mL) showed weak toxicity in non-cancerous cells (MRC5). BPL2 affected cancer cell growth while non-cancerous cells were less affected. Further, BPL2 (20 µg/mL) inhibited cancer cell invasion and migration (rates were ˂20%). BPL2 induced the downregulation of epithelial-to-mesenchymal transition-related genes (Zeb1, vimentin, and Twist). Co-treatment with BPL2 and gefitinib (10 µg/mL and 10 µM, respectively) showed a synergistic effect compared with monotherapy. BPL2 or gefitinib monotherapy resulted in approximately 90% and 70% cell viability, respectively, with concomitant treatment showing 40% cell viability. Overall, BPL2 can be considered a good candidate for development into an anticancer agent.