Lectin Receptors Research Articles

Paired C-type lectin receptors mediate specific recognition of divergent oomycete pathogens in C. elegans

Innate immune responses can be triggered upon detection of pathogen- or damage-associated molecular patterns by host receptors that are often present on the surface of immune cells. While invertebrates like Caenorhabditis elegans lack professional immune cells, they still mount pathogen-specific responses. However, the identity of host receptors in the nematode remains poorly understood. Here, we show that C-type lectin receptors mediate species-specific recognition of divergent oomycetes in C.elegans. A CLEC-27/CLEC-35 pair is essential for recognition of the oomycete Myzocytiopsis humicola, while a CLEC-26/CLEC-36 pair is required for detection of Haptoglossa zoospora. Both clec pairs are transcriptionally regulated through a shared promoter by the conserved PRD-like homeodomain transcription factor CEH-37/OTX2 and act in sensory neurons and the anterior intestine to trigger a protective immune response in the epidermis. This system enables redundant tissue sensing of oomycete threats through canonical CLEC receptors and host defense via cross-tissue communication.

The Dectin-1 and Dectin-2 clusters: C-type lectin receptors with fundamental roles in immunity.

The ability of myeloid cells to recognize and differentiate endogenous or exogenous ligands rely on the presence of different transmembrane protein receptors. C-type lectin receptors (CLRs), defined by the presence of a conserved structural motif called C-type lectin-like domain (CTLD), are a crucial family of receptors involved in this process, being able to recognize a diverse range of ligands from glycans to proteins or lipids and capable of initiating an immune response. The Dectin-1 and Dectin-2 clusters involve two groups of CLRs, with genes genomically linked within the natural killer cluster of genes in both humans and mice, and all characterized by the presence of a single extracellular CTLD. Fundamental immune cell functions such as antimicrobial effector mechanisms as well as internalization and presentation of antigens are induced and/or regulated through activatory, or inhibitory signalling pathways triggered by these receptors after ligand binding. In this review, we will discuss the most recent concepts regarding expression, ligands, signaling pathways and functions of each member of the Dectin clusters of CLRs, highlighting the importance and diversity of their functions.

The protein segregase VCP/p97 promotes host antifungal defense via regulation of SYK activation.

C-type lectin receptors (CLRs) are essential to execute host defense against fungal infection. Nevertheless, a comprehensive understanding of the molecular underpinnings of CLR signaling remains a work in progress. Here, we searched for yet-to-be-identified tyrosine-phosphorylated proteins in Dectin-1 signaling and linked the stress-response protein valosin containing protein (VCP)/p97 to Dectin-1 signaling. Knockdown of VCP expression or chemical inhibition of VCP's segregase activity dampened Dectin-1-elicited SYK activation in BMDMs and BMDCs, leading to attenuated expression of proinflammatory cytokines/chemokines such as TNF-α, IL-6 and CXCL1. Biochemical analyses demonstrated that VCP and its cofactor UFD1 form a complex with SYK and its phosphatase SHP-1 following Dectin-1 ligation, and knockdown of VCP led to a more prominent SYK and SHP-1 association. Further, SHP-1 became polyubiquitinated upon Dectin-1 activation, and VCP or UFD1 overexpression accelerated SHP-1 degradation. Conceivably, VCP may promote Dectin-1 signaling by pulling the ubiquitinated SHP-1 out of the SYK complex for degradation. Finally, genetic ablation of VCP in the neutrophil and macrophage compartment rendered the mice highly susceptible to infection by Candida albicans, an observation also phenocopied by administering the VCP inhibitor. These results collectively demonstrate that VCP is a previously unappreciated signal transducer of the Dectin-1 pathway and a crucial component of antifungal defense, and suggest a new mechanism regulating SYK activation.

Single-Cell Transcriptomics Unveils Skin Cell Specific Antifungal Immune Responses and IL-1Ra- IL-1R Immune Evasion Strategies of Emerging Fungal Pathogen Candida auris.

Candida auris is an emerging multidrug-resistant fungal pathogen that preferentially colonizes and persists in skin tissue, yet the host immune factors that regulate the skin colonization of C. auris in vivo are unknown. In this study, we employed unbiased single-cell transcriptomics of murine skin infected with C. auris to understand the cell type-specific immune response to C. auris. C. auris skin infection results in the accumulation of immune cells such as neutrophils, inflammatory monocytes, macrophages, dendritic cells, T cells, and NK cells at the site of infection. We identified fibroblasts as a major non-immune cell accumulated in the C. auris infected skin tissue. The comprehensive single-cell profiling revealed the transcriptomic signatures in cytokines, chemokines, host receptors (TLRs, C-type lectin receptors, NOD receptors), antimicrobial peptides, and immune signaling pathways in individual immune and non-immune cells during C. auris skin infection. Our analysis revealed that C. auris infection upregulates the expression of the IL-1RN gene (encoding IL-1R antagonist protein) in different cell types. We found IL-1Ra produced by macrophages during C. auris skin infection decreases the killing activity of neutrophils. Furthermore, C. auris uses a unique cell wall mannan outer layer to evade IL-1R-signaling mediated host defense. Collectively, our single-cell RNA seq profiling identified the transcriptomic signatures in immune and non-immune cells during C. auris skin infection. Our results demonstrate the IL-1Ra and IL-1R-mediated immune evasion mechanisms employed by C. auris to persist in the skin. These results enhance our understanding of host defense and immune evasion mechanisms during C. auris skin infection and identify potential targets for novel antifungal therapeutics.

Analysis of Differential Alternative Splicing in Largemouth Bass After High Temperature Exposure.

Temperature is one of the critical factors affecting the physiological functions of fish. With ongoing global warming, changes in water temperature have a profound impact on fish species. Alternative splicing, being a significant mechanism for gene expression regulation, facilitates fish to adapt and thrive in dynamic and varied aquatic environments. Our study used transcriptome sequencing to analyze alternative splicing in largemouth bass gills at 34 °C for 24 h. The findings indicated an increase in both alternative splicing events and alternative splicing genes after high temperature treatment. Specifically, the comparative analysis revealed a total of 674 differential alternative splicing events and 517 differential alternative splicing genes. Enrichment analysis of differential alternative splicing genes revealed significant associations with various gene ontology (GO) terms and KEGG pathways, particularly in immune-related pathways like necroptosis, apoptosis, and the C-type lectin receptor signaling pathway. These results emphasize that some RNA splicing-related genes are involved in the response of largemouth bass to high temperatures.

BLNK negatively regulates innate antifungal immunity through inhibiting c-Cbl-mediated macrophage migration

B cell linker protein (BLNK) is crucial for orchestrating B cell receptor-associated spleen tyrosine kinase (Syk) signaling. However, the role of BLNK in Syk-coupled C-type lectin receptor (CLR) signaling in macrophages remains unclear. Here, we delineate that CLRs govern the Syk-mediated activation of BLNK, thereby impeding macrophage migration by disrupting podosome ring formation upon stimulation with fungal β-glucans or α-mannans. Mechanistically, BLNK instigates its association with casitas B-lineage lymphoma (c-Cbl), competitively impeding the interaction between c-Cbl and Src-family kinase Fyn. This interference disrupts Fyn-mediated phosphorylation of c-Cbl and subsequent c-Cbl-associated F-actin assembly. Consequently, BLNK deficiency intensifies CLR-mediated recruitment of the c-Cbl/phosphatidylinositol 3-kinase complex to the F-actin cytoskeleton, thereby enhancing macrophage migration. Notably, mice with monocyte-specific BLNK deficiency exhibit heightened resistance to infection with Candida albicans, a prominent human fungal pathogen. This resistance is attributed to the increased infiltration of Ly6C+ macrophages into renal tissue. These findings unveil a previously unrecognized role of BLNK for the negative regulation of macrophage migration through inhibiting CLR-mediated podosome ring formation during fungal infections.

Mannose coated selenium nanoparticles normalize intestinal homeostasis in mice and mitigate colitis by inhibiting NF-κB activation and enhancing glutathione peroxidase expression

Impaired intestinal homeostasis is a major pathological feature of inflammatory bowel diseases (IBD). Mannose and selenium (Se) both demonstrate potential anti-inflammatory and anti-oxidative properties. However, most lectin receptors bind free monosaccharide ligands with relatively low affinity and most Se species induce side effects beyond a very narrow range of dosage. This has contributed to a poorly explored therapies for IBD that combine mannose and Se to target intestinal epithelial cells (IECs) for normalization gut homeostasis. Herein, a facile and safe strategy for ulcerative colitis (UC) treatment was developed using optimized, mannose-functionalized Se nanoparticles (M-SeNPs) encapsulated within a colon-targeted hydrogel delivery system containing alginate (SA) and chitosan (CS). This biocompatible nanosystem was efficiently taken up by IECs and led to increased expression of Se-dependent glutathione peroxidases (GPXs), thereby modulating IECs’ immune response. Using a mouse model of DSS-induced colitis, (CS/SA)-embedding M-SeNPs (C/S-MSe) were found to mitigate oxidative stress and inflammation through the inhibition of the NF-kB pathway in the colon. This stabilized mucosal homeostasis of IECs and ameliorated colitis-related symptoms, thereby providing a potential new approach for treatment of IBD.

Nanopore-based full-length transcriptome sequencing of the skin in Pseudopleuronectes yokohamae identifies novel antimicrobial peptide genes

The marbled flounder (Pseudopleuronectes yokohamae) is highly esteemed for its exceptional nutritional value and delicious taste. However, this species has extremely limited transcriptome data, which can offer priceless information for disease protection. In the study, the skin transcriptomic sequencing of P. yokohamae revealed 7.72 GB of clean data using the Nanopore sequencing platform. The results revealed 30,498 transcripts of functional annotations in the P. yokohamae transcriptome. All transcripts were searched in eight functional databases. A total of 10,337 ORFs were obtained, of which 6081 complete ORFs accounted for 58.83% of all predicted CDS. Moreover, 10,195 SSRs were detected. Meanwhile, the non-pecific immunity pathways were investigated for better understanding of the immunological reaction in P. yokohamae, and seven innate immune pathways were identified. The innate-immune related genes were highly expressed in the NOD-like receptor signaling pathway, followed by the C-type lectin receptor signaling pathway, Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway and Cytosolic DNA-sensing pathway. In this study, four families of antimicrobial peptides (AMPs) in P. yokohamae were analysed for the first time, including piscidins, hepcidins, liver-expressed antimicrobial peptide and defensins. Seven AMPs, including Pypleurocidin-like WF3, Pypleurocidin-like WFX, Pyhepcidin 1, Pyhepcidin-like 1, PyLEAP-2, Pybeta-defensin and Pybeta-defensin-like 1, were further identified. The seven AMPs showed a highly identity in their cDNA and genomic structures and an inducible expression pattern preferable to skin in response to pathogens. The transcriptomic data and investigation of AMPs from P. yokohamae promote a deeper awareness of fish mucosal immunity and provide information in the prevention of fish diseases.

Mannose and Lactobionic Acid in Nasal Vaccination: Enhancing Antigen Delivery via C-Type Lectin Receptors.

Nasal vaccines are a promising strategy for enhancing mucosal immune responses and preventing diseases at mucosal sites by stimulating the secretion of secretory IgA, which is crucial for early pathogen neutralization. However, designing effective nasal vaccines is challenging due to the complex immunological mechanisms in the nasal mucosa, which must balance protection and tolerance against constant exposure to inhaled pathogens. The nasal route also presents unique formulation and delivery hurdles, such as the mucous layer hindering antigen penetration and immune cell access. This review focuses on cutting-edge approaches to enhance nasal vaccine delivery, particularly those targeting C-type lectin receptors (CLRs) like the mannose receptor and macrophage galactose-type lectin (MGL) receptor. It elucidates the roles of these receptors in antigen recognition and uptake by antigen-presenting cells (APCs), providing insights into optimizing vaccine delivery. While a comprehensive examination of targeted glycoconjugate vaccine development is outside the scope of this study, we provide key examples of glycan-based ligands, such as lactobionic acid and mannose, which can selectively target CLRs in the nasal mucosa. With the rise of new viral infections, this review aims to facilitate the design of innovative vaccines and equip researchers, clinicians, and vaccine developers with the knowledge to enhance immune defenses against respiratory pathogens, ultimately protecting public health.

Coarse-Graining the Recognition of a Glycolipid by the C-Type Lectin Mincle Receptor.

Macrophage inducible Ca2+-dependent lectin (Mincle) receptor recognizes Mycobacterium tuberculosis glycolipids to trigger an immune response. This host membrane receptor is thus a key player in the modulation of the immune response to infection by M. tuberculosis and has emerged as a promising target for the development of new vaccines against tuberculosis. The recent development of the Martini 3 force field for coarse-grained (CG) molecular modeling allows the study of interactions of soluble proteins with small ligands which was not typically modeled well with the previous Martini 2 model. Here, we present a refined approach detailing a protocol for modeling interactions between a glycolipid and its receptor at a CG level using the Martini 3 force field. Using this approach, we studied Mincle and identified critical parameters governing ligand recognition, such as loop flexibility and the regulation of hydrophobic groove formation by calcium ions. In addition, we assessed ligand affinity using free energy perturbation calculations. Our results offer mechanistic insight into the interactions between Mincle and glycolipids, providing a basis for the rational design of molecules targeting this type of membrane receptors.

The Immune Defense Response and Immune-Related Genes Expression in Macrobrachium nipponense Infected with Decapod Iridescent Virus 1 (DIV1).

Macrobrachium nipponense is a significant cultivated species in China. However, decapod iridescent virus 1 (DIV1), as a newly discovered crustacean-lethal virus, has resulted in significant financial losses for the M. nipponense industry. In order to examine the immunological response of M. nipponense to DIV1, we conducted transcriptome analysis of the hepatopancreas from M. nipponense infected with DIV1 using RNA-seq. RNA sequencing analysis identified a combined total of 41,712 assembled unigenes, and 7014 genes that showed differential expression were identified in the group infected with DIV1, compared to the control group. Among these DEGs, 3952 were found to be up-regulated, while 3062 were down-regulated; many well-characterized DEGs were involved in innate immune defense, particularly involving the C-type lectin receptor signaling pathway, complement and coagulation cascades, phagosome, lysosome and PPAR signaling pathway. Moreover, the expression levels of well-known immune-related genes (dorsal, wnt6, lectin, caspase, integrin, hsp70) in the hepatopancreas and hemolymph were investigated by Quantitative real-time PCR (qRT-PCR), and the findings demonstrated a significant increase in gene expression in the hepatopancreas and hemolymph at various time points after infection. The results acquired in this study offered further comprehensive understanding of the immunological response of M. nipponense to DIV1 infection.

Exploration of the Relationship between Polycystic Ovary Syndrome and Recurrent Pregnancy Loss Based on Bioinformatics.

Recurrent Pregnancy Loss (RPL) and Polycystic Ovary Syndrome (PCOS) are both common diseases involving women of childbearing age, and their pathogenesis is still not sufficiently known. This study aimed to explore the relationship between RPL and PCOS in bioinformatics. Two expression chips, GSE86241 (obtained from 8 PCOS patients and 9 healthy controls) and GSE73025 (obtained from 5 RPL patients and 5 healthy controls), were downloaded from the Gene Expression Omnibus (GEO) database. We used the GEO database to analyze the gene expression profiles of PCOS and RPL to identify the intersection of abnormal miRNA expression, predicted the target genes of the intersecting miRNAs from miRDB, miRTarBase, and TargetScan databases, and then incorporated the miRNA-mRNA modulation network. By using the string database, the PPI network was built, which could screen the Hub genes and enrich them for analysis. Ultimately, the critical miRNA-mRNA regulatory network was set on the basis of the relationship between hub genes and miRNA. A total of 39 significantly altered miRNAs of PCOS and 137 significantly altered miRNAs of RPL were obtained, three miRNAs (miR-767-5p, miR-3196, and miR-187-3p), five signaling pathways (PI3K-Akt, p53, Toll-like receptor, C-type lectin receptor, and TNF signaling pathways), and six Hub genes (CASP8, PIK3R1, ADAMTS2, ADAMTS3, COL3A1, and MDM2) were found to be related to the development and progression of two diseases. More importantly, all Hub genes were regulated by miR-767-5p. This research clarifies the possible relationship between miRNA and mRNA with PCOS and RPL for the first time. It provides a basis for illustrating the pathogenic mechanism and a target of therapies for these two diseases.

Natural variation in maize ZmLecRK1 fine-tunes co-receptor interactions to boost immunity

Pattern recognition receptors (PRRs) are integral to plant immunity, functioning as the first line of defense against pathogens. Among these, receptor kinases (RKs) play a critical role in recognizing external signals and initiating immune responses. Recent studies by Li et al. (2024) have identified the G-type lectin receptor kinase ZmLecRK1 in maize as essential for resistance to Pythium aphanidermatum and other fungal pathogens. A key finding is that a resistant variant of ZmLecRK1, which carries an alanine at position 404, has emerged in maize, while the ancestral form, with a serine at this position, is conserved across most grasses. This amino acid substitution significantly influences the interaction with the co-receptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1 (BAK1), enhancing immune complex formation and subsequent defense signaling. This work underscores the importance of genetic variation in enhancing disease resistance, offering potential strategies for crop improvement through targeted genetic modification.

Yiqi Yangxue formula inhibits cartilage degeneration in knee osteoarthritis by regulating LncRNA-UFC1/miR-34a/MMP-13 axis

Ethnopharmacological relevanceKnee osteoarthritis (KOA) is a prevalent and disabling clinical condition affecting joint structures worldwide. The Yiqi Yangxue formula (YQYXF) is frequently prescribed in clinical settings for the treatment of KOA. Existing research has primarily focused on alterations in drug metabolism, with limited investigation into the epigenetic effects of YQYXF, particularly in relation to non-coding RNA. Aim of the studyExploring the effects of YQYXF on critical factors of long chain non-coding RNA UFC1/miR-34a/matrix metalloproteinase-13 (MMP-13) axis and their interrelationships. MethodsUHPLC-QE-MS technology was used to identify the YQYXF ingredients in rat serum. KEGG and GO analysis were performed on the targets of blood components acting on KOA using a database. Simultaneously, a protein interaction network was constructed using target proteins and metabolites to identify the core components and key pathways of YQYXF. The KOA rat model was established using an improved Hulth method. SPF SD rats were randomly divided into normal group, sham surgery group, model group, celecoxib capsules group (18 mg/kg), YQYXF low, medium and high dose groups (4.6g/kg, 9.2g/kg, 18.4g/kg). Observe the synovial and cartilage tissues of rats using pathological methods. RT-PCR was used to detect the levels of UFC1, miR-34a, and MMP-13 in cartilage. Immunohistochemistry was used to detect the levels of MMP-13 and ADAMTS-5 in cartilage. ELISA method was used to detect the levels of MMP-13 and ADAMTS-5 in serum. In addition, we further validated the regulation of crucial factor expression levels of UFC1/miR-34a/MMP-13 axis in rat chondrocytes and degenerative chondrocytes of KOA patients by YQYXF, providing a basis for its treatment of KOA. ResultsThe compounds that YQYXF enters the bloodstream mainly contain flavonoids and phenylpropanoid compounds. The core components that act on OA include quercetin, fisetin, and demethylweldelolactone. The main target pathways are the IL-17 signaling pathway, lipid and atherosclerosis, cellular sensitivity, inflammatory mediator regulation of TRP channels, TNF signaling pathway, relaxin signaling pathway and C-type lectin receptor signaling pathway. YQYXF inhibited the expression of miR-34a and MMP-13 mRNA, and reduced the protein levels of MMP-13 and ADAMTS-5. In vitro studies have confirmed that 20% YQYXF serum promoted UFC1 and reduce miR-34a levels. In addition, miR-34a in sh-UFC1+10% YQYXF serum and sh-UFC1+20% YQYXF serum groups significantly decreased compared to the sh-UFC1 group. ConclusionThe anti-KOA cartilage degeneration effect of YQYXF might be related to inhibiting cell apoptosis and promoting cell proliferation, which regulated the lncRNA-UFC1/miR-34a/MMP-13 axis.

Galectin-9 activates host immune response and improve immunoprotection of Onychostoma macrolepis against Aeromonas hydrophila infection

Galectin-9 (Gal-9) belongs to a family of the glycan-binding proteins (GBPs) and is known to restrict bacterial activity via interacting with pathogen associated molecular pattern (PAMPs). However, the underlying immune mechanism of endogenous Gal-9 on fish against bacterial infection is still unclear. In this study, effect of Gal-9 from Onychostoma macrolepis (OmGal-9) on expression of immune related-genes were measured by HEK293T. The immune response of O. macrolepis with OmGal-9 overexpression to Aeromonas hydrophila (A. hydrophila) infection (1.65×108 CFU/mL) were evaluated by tissue bacterial load, fish survival rate and transcriptome analysis. The results showed that OmGal-9 displayed a punctate distribution in the nucleus and cytoplasm of HEK293T cells. Compared to cells transfected with the empty vector (EV) group, recombinant plasmid pEGFP-Gal9 treatment (Gal9) significantly down-regulated the expression of immune-related genes TNFα, STAT3, MyD88, LCK, and p52 of HEK293T cells stimulated with LPS at 24 h, while up-regulated IκBα and caspase1 (P < 0.05). The activities of catalase (CAT), superoxide dismutase (SOD), the total antioxidant capacity (T-AOC), alkaline phosphatase (AKP), acid phosphatase (ACP), and lysozyme (LZM) of O.macrolepis were significantly increased on 7 days in Gal9 group compared to EV group (P < 0.05). The bacterial load of liver, spleen, and kidney of O.macrolepis infected with A. hydrophila in Gal9 group at 24 h significantly lower than that in EV group (P < 0.05), and the survival rate has increased from 15% to 35%. A comparative transcriptome analysis between the Gal9 and EV group identified 305 differentially expressed genes (DEGs). The analysis showed that OmGal-9 might play important regulatory in glycolysis / gluconeogenesis, fatty acid degradation, and ascorbate and aldarate metabolism. Moreover, the immune-related DEGs were predominantly enriched in eleven pathways, with the most important three of them being linked to innate immunity: NOD-like, C-type lectin and Toll-like receptor signaling pathway. Taking together, OmGal-9 can enhance the resistance of fish to bacterial diseases by improving immune system function and activating immune-related pathways.

Characteristics of lectin receptors on erythrocyte membranes of patients with glioblastoma

Aim. This work is devoted to the search the abnormalities of the receptor-lectin reactions associated with glioblastoma for diagnostic purposes. Methods. Red blood cell (RBC) specimens were taken from 22 persons distributed into the two cohorts: donors without an oncological diagnosis; patients with glioblastoma (GB). Panel of 9 commercial lectins (LECTINOTEST, Lviv, Ukraine) was used: ConA, WGA, PHA-P; PHA-E; PHA-L, SNA, VAA, STA, and LABA. Also the original lectin from persimmon sepals (named PSL) was used. It was obtained by isoelectric focusing and ammonium sulfate fractionation. Intensity of lectin-receptor interactions was determined in reaction of hemagglutination. Results. Тwo lectins showed some directed divergence in patients from donors. For wheat germ agglutinin (WGA) сhanges in the intensity of lectin-receptor binding were downward. In the case of persimmon L (PSL) the alternative result was observed: the intensity of hemagglutination showed a trend to exceed the level of control. Conclusions. Some individual features in the reaction of erythrocytes with a set of lectins are observed in all examined persons. For two studied lectins, namely WGA and PSL, unidirectional deviations were observed in the reaction of erythrocytes of patients with GB compared to individuals without an oncological diagnosis.

Preparation, Characterization and Evaluation of Monosaccharide Tagged Nanostructured Lipid Carriers for Targeting of Etravirine to Macrophagic AIDS Viral Reservoirs.

Purpose of the study: The present study aimed to develop and examine monosaccharide-decorated nanoparticles specifically designed for macrophage targeting of etravirine. Methods: Nanostructured lipid carriers (NLC) were prepared with stearylamine as such or after tagging with mannose and galactose as one of the lipid components of NLC. Prior to synthesis, a docking study was used to analyze the potential for interaction of the stearylamine- sugar conjugates with respective lectin receptors. Mannose and galactose-conjugated stearylamine were separately synthesized. Fourier-transform infrared spectroscopy (FTIR), mass spectrometry (MS) and nuclear magnetic resonance (NMR) studies were used to assess successful reactions. Etravirine-loaded non-targeted nanostructured lipid carriers (NLCs) were synthesized with the emulsification solvent evaporation method and characterized for parameters such as zeta potential, in-vitro release, entrapment efficiency and particle size. To create targeted nanoparticles, stearylamine was replaced with either of the conjugated lipids in the formulation. For stability, all NLC dispersions were lyophilized. In-vitro investigations included cellular uptake and cytotoxicity assessments in the RAW 264.7 macrophage cell line. Following intravenous administration of the NLCs in Wistar rats, in-vivo biodistribution was evaluated. Results: The docking studies showed good binding potential of both conjugated lipids for respective receptors. Evaluation of synthesized products showed successful conjugation of sugars to stearylamine. The synthesized nanoparticles demonstrated impressive etravirine loading capabilities and exhibited a circular shape with a mean size of approximately 260 nm. Drug release from all NLC formulations persisted for over 24 hours and remained stable after lyophilization. In-vitro evaluations in the RAW 264.7 macrophage cell line revealed that mannosylated nanoparticles had enhanced uptake when compared to non-targeted and galactosylated counterparts. In-vivo plasma and tissue distribution studies corroborated the in-vitro findings, as the mannosylated NLCs displayed a higher distribution into organs with a rich presence of macrophages. Conclusion: The studies indicate that while sugar tagging of nanoparticles can prove to be an effective strategy for targeting drugs to macrophagic tissue for tackling latent AIDS virus, the choice of sugar is critical.

Formulation Attributes Impact Immune Profile of an Oral and Thermostable COVID-19 Subunit Vaccine.

While approved vaccines for COVID-19 provide protection against severe disease and death, they have limited efficacy in the prevention of infection and virus transmission. Mucosal immunity is preferred over systemic immunity to provide protection at the point of entry against pathogens such as SARS-CoV-2. VaxForm has developed an oral vaccine delivery platform that elicits mucosal and systemic immune responses by targeting immune cells in the gut through C-type lectin receptors. The technology consists of microencapsulating the vaccine with an enteric polymer, which also results in enhanced thermostability. This article describes the formulation development and in vivo testing of a novel protein-based oral COVID-19 vaccine using this technology. Results demonstrate successful induction of immune response in mice and showed that the particle size of the vaccines following administration can impact the ratio of mucosal to systemic response. Immunogenicity and thermostability of liquid suspension and dry powder versions of the vaccine were compared in mice. The liquid suspension vaccine showed excellent heat resistance by maintaining immunogenicity after 14 days of storage at 60 °C. While further investigation is needed to determine correlates of protection and duration of response for mucosal immunity, this study demonstrates the vaccine's potential as a COVID-19 booster to enhance mucosal protection in humans and improve global access by lowering the cost of production, removing cold-chain requirements, and allowing self-administration.

Systematic Evaluation of Regiochemistry and Lipidation of Aryl Trehalose Mincle Agonists.

The Macrophage-Inducible C-type Lectin receptor (Mincle) plays a critical role in innate immune recognition and pathology, and therefore represents a promising target for vaccine adjuvants. Innovative trehalose-based Mincle agonists with improved pharmacology and potency may prove useful in the development of Th17-mediated adaptive immune responses. Herein, we report on in vitro and in silico investigations of specific Mincle ligand-receptor interactions required for the effective receptor engagement and activation of Th17-polarizing cytokines. Specifically, we employed a library of trehalose benzoate scaffolds, varying the degree of aryl lipidation and regiochemistry that produce inflammatory cytokines in a Mincle-dependent fashion. In vitro interleukin-6 (IL-6) cytokine production by human peripheral blood mononuclear cells (hPBMCs) indicated that the lipid regiochemistry is key to potency and maximum cytokine output, with the tri-substituted compounds inducing higher levels of IL-6 in hPBMCs than the di-substituted derivatives. Additionally, IL-6 production trended higher after stimulation with compounds that contained lipids ranging from five to eight carbons long, compared to shorter (below five) or longer (above eight) carbon chains, across all the substitution patterns. An analysis of the additional cytokines produced by hPBMCs revealed that compound 4d, tri-substituted and five carbons long, induced significantly greater levels of interleukin-1β (IL-1β), tumor necrosis factor- α (TNF-α), interleukin-23 (IL-23), and interferon- γ (IFN-γ) than the other compounds tested in this study. An in silico assessment of 4d highlighted the capability of this analogue to bind to the human Mincle carbohydrate recognition domain (CRD) efficiently. Together, these data highlight important structure-activity findings regarding Mincle-specific cytokine induction, generating a lead adjuvant candidate for future formulations and immunological evaluations.

Intranasal Administration of Bedaquiline-Loaded Fucosylated Liposomes Provides Anti-Tubercular Activity while Reducing the Potential for Systemic Side Effects.

Liposomal formulations of antibiotics for inhalation offer the potential for the delivery of high drug doses, controlled drug release kinetics in the lung, and an excellent safety profile. In this study, we evaluated the in vivo performance of a liposomal formulation for the poorly soluble, antituberculosis agent, bedaquiline. Bedaquiline was encapsulated within monodisperse liposomes of ∼70 nm at a relatively high drug concentration (∼3.6 mg/mL). Formulations with or without fucose residues, which bind to C-type lectin receptors and mediate a preferential binding to macrophage mannose receptor, were prepared, and efficacy was assessed in an in vivo C3HeB/FeJ mouse model of tuberculosis infection (H37Rv strain). Seven intranasal instillations of 5 mg/kg bedaquiline formulations administered every second day resulted in a significant reduction in lung burden (∼0.4-0.6 Δlog10 CFU), although no differences between fucosylated and nonfucosylated formulations were observed. A pharmacokinetic study in healthy, noninfected Balb/c mice demonstrated that intranasal administration of a single dose of 2.5 mg/kg bedaquiline liposomal formulation (fucosylated) improved the lung bioavailability 6-fold compared to intravenous administration of the same formulation at the same dose. Importantly, intranasal administration reduced systemic concentrations of the primary metabolite, N-desmethyl-bedaquiline (M2), compared with both intravenous and oral administration. This is a clinically relevant finding as the M2 metabolite is associated with a higher risk of QT-prolongation in predisposed patients. The results clearly demonstrate that a bedaquiline liposomal inhalation suspension may show enhanced antitubercular activity in the lung while reducing systemic side effects, thus meriting further nonclinical investigation.