Selenomonas sputigena Interactions with Gingival Epithelial Cells That Promote Inflammation.

Increased prevalence and abundance of Selenomonas sputigena have been associated with periodontitis, a chronic inflammatory disease of tooth-supporting tissues, for more than 50 years. Over the past decade, molecular surveys of periodontal disease using 16S and shotgun metagenomic sequencing approaches have confirmed the disease association of classically recognized periodontal pathogens such as Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia while highlighting previously underappreciated organisms such as Filifactor alocis and S. sputigena. Despite abundant clinical association between S. sputigena and periodontal disease, we have little to no understanding of its pathogenic potential, and virulence mechanisms have not been studied. In this study, we sought to characterize the response of gingival epithelial cells to infection with S. sputigena. Here, we show that S. sputigena attaches to gingival keratinocytes and induces expression and secretion of cytokines and chemokines associated with inflammation and leukocyte recruitment. We demonstrate that S. sputigena induces signaling through Toll-like receptor 2 (TLR2) and TLR4 but evades activation of TLR5. Cytokines released from S. sputigena-infected keratinocytes induced monocyte and neutrophil chemotaxis. These results show that S. sputigena-host interactions have the potential to contribute to bacterially driven inflammation and tissue destruction, the hallmark of periodontitis. Characterization of previously unstudied pathogens may provide novel approaches to develop therapeutics to treat or prevent periodontal disease.

Differential Functional Responses of Neutrophil Subsets in Severe COVID-19 Patients.

Neutrophils play a significant role in determining disease severity following SARS-CoV-2 infection. Gene and protein expression defines several neutrophil clusters in COVID-19, including the emergence of low density neutrophils (LDN) that are associated with severe disease. The functional capabilities of these neutrophil clusters and correlation with gene and protein expression are unknown. To define host defense and immunosuppressive functions of normal density neutrophils (NDN) and LDN from COVID-19 patients, we recruited 64 patients with severe COVID-19 and 26 healthy donors (HD). Phagocytosis, respiratory burst activity, degranulation, neutrophil extracellular trap (NET) formation, and T-cell suppression in those neutrophil subsets were measured. NDN from severe/critical COVID-19 patients showed evidence of priming with enhanced phagocytosis, respiratory burst activity, and degranulation of secretory vesicles and gelatinase and specific granules, while NET formation was similar to HD NDN. COVID LDN response was impaired except for enhanced NET formation. A subset of COVID LDN with intermediate CD16 expression (CD16Int LDN) promoted T cell proliferation to a level similar to HD NDN, while COVID NDN and the CD16Hi LDN failed to stimulate T-cell activation. All 3 COVID-19 neutrophil populations suppressed stimulation of IFN-γ production, compared to HD NDN. We conclude that NDN and LDN from COVID-19 patients possess complementary functional capabilities that may act cooperatively to determine disease severity. We predict that global neutrophil responses that induce COVID-19 ARDS will vary depending on the proportion of neutrophil subsets.

The emerging oral pathogen, Filifactor alocis, extends the functional lifespan of human neutrophils.

Periodontitis is a chronic inflammatory infectious disease that affects the integrity of tooth-supporting tissues and has adverse systemic consequences. Advances in sequencing technologies have uncovered organisms that are exclusively found in high numbers in periodontal lesions, such as the gram-positive anaerobic rod, Filifactor alocis. F. alocis can manipulate neutrophil effector functions, which allows the organism to survive within these granulocytes. Several neutrophil functions have been tested in the context of F. alocis challenge, but the effect of the organism on neutrophil apoptosis is still unknown. RNA sequencing of human neutrophils challenged with F. alocis showed that apoptosis pathways were differentially regulated. Compared to media-cultured controls, F. alocis-challenged neutrophils maintain their nuclear morphology, do not stain for Annexin V or 7-AAD, and have decreased DNA fragmentation. Inhibition of apoptosis by F. alocis involved reduced caspase-3, -8, and - 9 activation and upregulation of important anti-apoptotic proteins. Prolonged lifespan was dependent on contact through TLR2/6, and F. alocis-challenged neutrophils retained their functional capacity to induce inflammation for longer timepoints. This is the first in-depth characterization of neutrophil apoptotic programs in response to an oral pathogen and provides key information on how bacteria manipulate immune cell mechanisms to maintain a dysregulated inflammatory response.

Dot/Icm-Dependent Restriction of Legionella pneumophila within Neutrophils.

The Dot/Icm type IV secretion system (T4SS) of Legionella pneumophila is essential for lysosomal evasion and permissiveness of macrophages for intracellular proliferation of the pathogen. In contrast, we show that polymorphonuclear cells (PMNs) respond to a functional Dot/Icm system through rapid restriction of L. pneumophila. Specifically, we show that the L. pneumophila T4SS-injected amylase (LamA) effector catalyzes rapid glycogen degradation in the PMNs cytosol, leading to cytosolic hyperglucose. Neutrophils respond through immunometabolic reprogramming that includes upregulated aerobic glycolysis. The PMNs become activated with spatial generation of intracellular reactive oxygen species within the Legionella-containing phagosome (LCP) and fusion of specific and azurophilic granules to the LCP, leading to rapid restriction of L. pneumophila. We conclude that in contrast to macrophages, PMNs respond to a functional Dot/Icm system, and specifically to the effect of the injected amylase effector, through rapid engagement of major microbicidal processes and rapid restriction of the pathogen. IMPORTANCE Legionella pneumophila is commonly found in aquatic environments and resides within a wide variety of amoebal hosts. Upon aerosol transmission to humans, L. pneumophila invades and replicates with alveolar macrophages, causing pneumonia designated Legionnaires' disease. In addition to alveolar macrophages, neutrophils infiltrate into the lungs of infected patients. Unlike alveolar macrophages, neutrophils restrict and kill L. pneumophila, but the mechanisms were previously unclear. Here, we show that the pathogen secretes an amylase (LamA) enzyme that rapidly breakdowns glycogen stores within neutrophils, and this triggers increased glycolysis. Subsequently, the two major killing mechanisms of neutrophils, granule fusion and production of reactive oxygen species, are activated, resulting in rapid killing of L. pneumophila.

Whole Transcriptome Analysis Reveals That Filifactor alocis Modulates TNFα-Stimulated MAPK Activation in Human Neutrophils.

Periodontitis is an irreversible, bacteria-induced, chronic inflammatory disease that compromises the integrity of tooth-supporting tissues and adversely affects systemic health. As the immune system's first line of defense against bacteria, neutrophils use their microbicidal functions in the oral cavity to protect the host against periodontal disease. However, periodontal pathogens have adapted to resist neutrophil microbicidal mechanisms while still propagating inflammation, which provides essential nutrients for the bacteria to proliferate and cause disease. Advances in sequencing technologies have recognized several newly appreciated bacteria associated with periodontal lesions such as the Gram-positive anaerobic rod, Filifactor alocis. With the discovery of these oral bacterial species, there is also a growing need to assess their pathogenic potential and determine their contribution to disease progression. Currently, few studies have addressed the pathogenic mechanisms used by oral bacteria to manipulate the neutrophil functional responses at the level of the transcriptome. Thus, this study aims to characterize the global changes at the gene expression level in human neutrophils during infection with F. alocis. Our results indicate that the challenge of human neutrophils with F. alocis results in the differential expression of genes involved in multiple neutrophil effector functions such as chemotaxis, cytokine and chemokine signaling pathways, and apoptosis. Moreover, F. alocis challenges affected the expression of components from the TNF and MAPK kinase signaling pathways. This resulted in transient, dampened p38 MAPK activation by secondary stimuli TNFα but not by fMLF. Functionally, the F. alocis-mediated inhibition of p38 activation by TNFα resulted in decreased cytokine production but had no effect on the priming of the respiratory burst response or the delay of apoptosis by TNFα. Since the modulatory effect was characteristic of viable F. alocis only, we propose this as one of F. alocis' mechanisms to control neutrophils and their functional responses.

Redundant and Cooperative Roles for Yersinia pestis Yop Effectors in the Inhibition of Human Neutrophil Exocytic Responses Revealed by Gain-of-Function Approach.

Yersinia pestis causes a rapid, lethal disease referred to as plague. Y. pestis actively inhibits the innate immune system to generate a noninflammatory environment during early stages of infection to promote colonization. The ability of Y. pestis to create this early noninflammatory environment is in part due to the action of seven Yop effector proteins that are directly injected into host cells via a type 3 secretion system (T3SS). While each Yop effector interacts with specific host proteins to inhibit their function, several Yop effectors either target the same host protein or inhibit converging signaling pathways, leading to functional redundancy. Previous work established that Y. pestis uses the T3SS to inhibit neutrophil respiratory burst, phagocytosis, and release of inflammatory cytokines. Here, we show that Y. pestis also inhibits release of granules in a T3SS-dependent manner. Moreover, using a gain-of-function approach, we discovered previously hidden contributions of YpkA and YopJ to inhibition and that cooperative actions by multiple Yop effectors are required to effectively inhibit degranulation. Independent from degranulation, we also show that multiple Yop effectors can inhibit synthesis of leukotriene B4 (LTB4), a potent lipid mediator released by neutrophils early during infection to promote inflammation. Together, inhibition of these two arms of the neutrophil response likely contributes to the noninflammatory environment needed for Y. pestis colonization and proliferation.

Neutrophil Interaction with Emerging Oral Pathogens: A Novel View of the Disease Paradigm.

Periodontitis is a multifactorial chronic inflammatory infectious disease that compromises the integrity of tooth-supporting tissues. The disease progression depends on the disruption of host-microbe homeostasis in the periodontal tissue. This disruption is marked by a shift in the composition of the polymicrobial oral community from a symbiotic to a dysbiotic, more complex community that is capable of evading killing while promoting inflammation. Neutrophils are the main phagocytic cell in the periodontal pocket, and the outcome of the interaction with the oral microbiota is an important determinant of oral health. Novel culture-independent techniques have facilitated the identification of new bacterial species at periodontal lesions and induced a reappraisal of the microbial etiology of periodontitis. In this chapter, we discuss how neutrophils interact with two emerging oral pathogens, Filifactor alocis and Peptoanaerobacter stomatis, and the different strategies deploy by these organisms to modulate neutrophil effector functions, with the goal to outline a new paradigm in our knowledge about neutrophil responses to putative periodontal pathogens and their contribution to disease progression.

Putative Periodontal Pathogens, Filifactor Alocis and Peptoanaerobacter Stomatis, Induce Differential Cytokine and Chemokine Production by Human Neutrophils.

Periodontitis is a highly prevalent infectious disease that affects ~ 50% of the adults in the USA alone. Two Gram-positive anaerobic oral bacteria, Filifactor alocis and Peptoanaerobacter stomatis, have emerged as important periodontal pathogens. Neutrophils are a major component of the innate host response in the gingival tissue, and the contribution of neutrophil-derived cytokines and chemokines plays a central role in disease progression. The pattern of cytokines and chemokines released by human neutrophils upon stimulation with newly appreciated periodontal bacteria compared to the keystone oral pathogen Porphyromonas gingivalis was investigated. Our results showed that both F. alocis and P. stomatis triggered TLR2/6 activation. F. alocis induced significant changes in gene expression of cytokines and chemokines in human neutrophils compared to unstimulated cells. However, except for IL-1ra, neutrophils released lower levels of cytokines and chemokines in response to F. alocis compared to P. stomatis. Furthermore, bacteria-free conditioned supernatant collected from neutrophils challenged with P. stomatis, but not from P. gingivalis or F. alocis, was chemotactic towards both neutrophils and monocytes. Elucidating stimuli-specific modulation of human neutrophil effector functions in the context of dysbiotic microbial community constituents provides valuable information for understanding the pathogenesis of periodontal diseases.

Filifactor alocis manipulates human neutrophils affecting their ability to release neutrophil extracellular traps induced by PMA.

Neutrophils operate at the site of injury or inflammation in the periodontal pocket to ensure periodontal health and clearance of bacterial pathogens. Filifactor alocis is recently identified as a potential periodontal pathogen, and in this study, we assessed the formation of neutrophil extracellular traps (NETs), in response to the presence of the organism . NET formation by human neutrophils was not induced when challenged with F. alocis, independent of opsonization, viability, time, or bacterial dose. F. alocis also failed to induce NETs from TNF-α-primed neutrophils and did not induce the release of extracellular neutrophil elastase. However, significant NET induction was observed when neutrophils were challenged with Streptococcus gordonii or Peptoanaerobacter stomatis, In addition, co-infection studies revealed that the presence of F. alocis with S. gordonii or P. stomatis does not enhance or reduce NETs. Additionally, F. alocis failed to impact pre-formed NETs induced by either S. gordonii or P. stomatis. Pretreatment with F. alocis prior to stimulation with phorbol 12-myristate 13-acetate (PMA), S. gordonii, or P. stomatis revealed that the bacterium is capable of reducing only PMA but not S. gordonii or P. stomatis NET formation. These results indicate that F. alocis manipulates neutrophils, inhibiting the triggering of NET induction.

RNA Polymerase 1 Is Transiently Regulated by Seizures and Plays a Role in a Pharmacological Kindling Model of Epilepsy.

Ribosome biogenesis, including the RNA polymerase 1 (Pol1)-mediated transcription of rRNA, is regulated by the pro-epileptogenic mTOR pathway. Therefore, hippocampal Pol1 activity was examined in mouse models of epilepsy including kainic acid- and pilocarpine-induced status epilepticus (SE) as well as a single seizure in response to pentylenetetrazole (PTZ). Elevated 47S pre-rRNA levels were present acutely after induction of SE suggesting activation of Pol1. Conversely, after a single seizure, 47S pre-rRNA was transiently downregulated with increased levels of unprocessed 18S rRNA precursors in the cornu Ammonis (CA) region. At least in the dentate gyrus (DG), the pilocarpine SE-mediated transient activation of Pol1 did not translate into long-term changes of pre-rRNA levels or total ribosome content. Unaltered hippocampal ribosome content was also found after a 20-day PTZ kindling paradigm with increasing pro-convulsive effects of low dose PTZ that was injected every other day. However, after selectively deleting the essential Pol1 co-activator, transcription initiation factor-1A (Tif1a/Rrn3) from excitatory neurons, PTZ kindling was impaired while DG total ribosome content was moderately reduced and no major neurodegeneration was observed throughout the hippocampus. Therefore, Pol1 activity of excitatory neurons is required for PTZ kindling. As seizures affect ribosome biogenesis without long-term effects on the total ribosome content, such a requirement may be associated with a need to produce specialized ribosomes that promote pro-epileptic plasticity.

Filifactor alocis modulates human neutrophil antimicrobial functional responses.

Filifactor alocis is a newly appreciated pathogen in periodontal diseases. Neutrophils are the predominant innate immune cell in the gingival crevice. In this study, we examined modulation of human neutrophil antimicrobial functions by F. alocis. Both non-opsonised and serum-opsonised F. alocis were engulfed by neutrophils but were not efficiently eliminated. Challenge of neutrophils with either non-opsonised or serum-opsonised F. alocis induced a minimal intracellular as well as extracellular respiratory burst response compared to opsonised Staphylococcus aureus and fMLF, respectively. However, pretreatment or simultaneous challenge of neutrophils with F. alocis did not affect the subsequent oxidative response to a particulate stimulus, suggesting that the inability to trigger the respiratory response was only localised to F. alocis phagosomes. In addition, although neutrophils engulfed live or heat-killed F. alocis with the same efficiency, heat-killed F. alocis elicited a higher intracellular respiratory burst response compared to viable organisms, along with decreased surface expression of CD35, a marker of secretory vesicles. F. alocis phagosomes remained immature by delayed and reduced recruitment of specific and azurophil granules, respectively. These results suggest that F. alocis withstands neutrophil antimicrobial responses by preventing intracellular ROS production, along with specific and azurophil granule recruitment to the bacterial phagosome.

Proapoptotic Requirement of Ribosomal Protein L11 in Ribosomal Stress-Challenged Cortical Neurons.

While impaired ribosomal biogenesis is observed in neurodegenerative diseases, its pathogenic contributions are not clear. For instance, it is well established that in rodent neurons, genetic inhibition of RNA-polymerase 1 that transcribes rRNA results in structural disruption of the nucleolus, neuronal apoptosis, and neurodegeneration. However, in most neurodegenerative diseases, nucleolar morphology is unaffected. It is reported here that in primary cortical neurons from newborn rats, inhibition of ribosomal biogenesis by shRNA-mediated knockdowns of several ribosomal proteins including S6, S14, or L4 resulted in p53-mediated apoptosis despite absence of structural disruption of the nucleolus. Conversely, knockdown of the RP L11, which in nonneuronal systems mediates p53 activation downstream of ribosomal stress, protected neurons against inhibition of ribosomal biogenesis but not staurosporine. Moreover, overexpression of L11 enhanced p53-driven transcription and increased neuronal apoptosis. In addition, inhibition of p53, or L11 knockdown, blocked apoptosis in response to the RNA analog 5-fluorouridine which perturbed nucleolar structure, inhibited ribosomal synthesis, and activated p53. Although the DNA double-strand break (DSB) inducer etoposide activated p53, nucleolar structure appeared intact. However, by activating the DNA damage response kinase ATM, etoposide increased 47S pre-rRNA levels, and enhanced nucleolar accumulation of nascent RNA, suggesting slower rRNA processing and/or increased Pol1 activity. In addition, shL11 reduced etoposide-induced apoptosis. Therefore, seemingly normal morphology of the neuronal nucleolus does not exclude presence of ribosomal stress. Conversely, targeting the ribosomal stress-specific signaling mediators including L11 offers a novel approach to uncover neurodegenerative contributions of deregulated ribosomal synthesis as exemplified in DSB-challenged neurons.

Filifactor alocis Promotes Neutrophil Degranulation and Chemotactic Activity.

Filifactor alocis is a recently recognized periodontal pathogen; however, little is known regarding its interactions with the immune system. As the first-responder phagocytic cells, neutrophils are recruited in large numbers to the periodontal pocket, where they play a crucial role in the innate defense of the periodontium. Thus, in order to colonize, successful periodontal pathogens must devise means to interfere with neutrophil chemotaxis and activation. In this study, we assessed major neutrophil functions, including degranulation and cell migration, associated with the p38 mitogen-activated protein kinase (MAPK) signaling pathway upon challenge with F. alocis. Under conditions lacking a chemotactic gradient, F. alocis-challenged neutrophils had increased migration compared to uninfected cells, indicating that F. alocis increases chemokinesis in human neutrophils. In addition, neutrophil chemotaxis induced by interleukin-8 was significantly enhanced when cells were challenged with F. alocis, compared to noninfected cells. Similar to live bacteria, heat-killed F. alocis induced both random and directed migration of human neutrophils. The interaction of F. alocis with Toll-like receptor 2 induced granule exocytosis along with a transient ERK1/2 and sustained p38 MAPK activation. Moreover, F. alocis-induced secretory vesicle and specific granule exocytosis were p38 MAPK dependent. Blocking neutrophil degranulation with TAT-SNAP23 fusion protein significantly reduced the chemotactic and random migration induced by F. alocis Therefore, we propose that induction of random migration by F. alocis will prolong neutrophil traffic time in the gingival tissue, and subsequent degranulation will contribute to tissue damage.

Requirement of Neuronal Ribosome Synthesis for Growth and Maintenance of the Dendritic Tree.

The nucleolus serves as a principal site of ribosome biogenesis but is also implicated in various non-ribosomal functions, including negative regulation of the pro-apoptotic transcription factor p53. Although disruption of the nucleolus may trigger the p53-dependent neuronal death, neurotoxic consequences of a selective impairment of ribosome production are unclear. Here, we report that in rat forebrain neuronal maturation is associated with a remarkable expansion of ribosomes despite postnatal down-regulation of ribosomal biogenesis. In cultured rat hippocampal neurons, inhibition of the latter process by knockdowns of ribosomal proteins S6, S14, or L4 reduced ribosome content without disrupting nucleolar integrity, cell survival, and signaling responses to the neurotrophin brain-derived neurotrophic factor. Moreover, reduced general protein synthesis and/or formation of RNA stress granules suggested diminished ribosome recruitment to at least some mRNAs. Such a translational insufficiency was accompanied by impairment of brain-derived neurotrophic factor-mediated dendritic growth. Finally, RNA stress granules and smaller dendritic trees were also observed when ribosomal proteins were depleted from neurons with established dendrites. Thus, a robust ribosomal apparatus is required to carry out protein synthesis that supports dendritic growth and maintenance. Consequently, deficits of ribosomal biogenesis may disturb neurodevelopment by reducing neuronal connectivity. Finally, as stress granule formation and dendritic loss occur early in neurodegenerative diseases, disrupted homeostasis of ribosomes may initiate and/or amplify neurodegeneration-associated disconnection of neuronal circuitries.

Oligo-ß-(1 → 3)-glucans: impact of thio-bridges on immunostimulating activities and the development of cancer stem cells.

Recent developments of innovative anticancer therapies are based on compounds likely to stimulate the immune defense of the patients. ß-(1 → 3)-Glucans are natural polysaccharides well-known for their immunostimulating properties. We report here on the synthesis of small oligo-ß-(1 → 3)-glucans characterized by thioglycosidic linkages. The presence of sulfur atom(s) was not only crucial to prolong in vivo immunoactive activities in time, compared to native polysaccharides, but sulfur atoms also had a direct impact on the development of colorectal cancer stem cells. As a result, a short, pure, and structurally well-defined trisaccharidic thioglucan demonstrated similar activities compared to those of natural laminarin.

The relative abundance of oxygen alkyl-related groups in aliphatic domains is involved in the main pharmacological-pleiotropic effects of humic acids.

Despite the rather common presence of humic acid (HA), our full knowledge of its biological effect is still lacking. In this article, we first performed a physicochemical characterization of several HAs, and next, we evaluated their ability to affect interleukin-2 secretion, antibody secretion, wound healing (an in vitro model using HaCaT cells), cancer growth (the Lewis lung carcinoma model), and protection against hepatotoxicity. In all tested reactions, HA showed significant stimulation on immune reactions, including suppression of cancer growth and inhibition of lipopolysaccharide-induced hepatotoxicity. These effects were dependent on its chemical properties. The pleiotropic effects of HA observed in this article suggest the possible role of these compounds in human nutrition.

Procathepsin d involvement in chemoresistance of cancer cells.

BACKGROUND: The role of pCD in cancer has been studied for a long time. We have focused on the hypothesis that increased expression and/or secretion of pCD in cancer cells causes increased chemoresistance to apoptosis inducing molecules. AIM: The aim was to evaluate the effects of pCD expression/release on chemoresistance. MATERIALS AND METHODS: We tested the LC(50) values for various transfectants of breast cancer cell line MDA-MB-231 as well as effects of exogenous additions of pCD, its mutants, pepstatine, antibodies, and Brefeldin on the resistance. RESULTS: We found that pCD levels can be correlated with chemoresistance, the pro-resistant activity seems to be localized outside the cells, proteolytic activity is not involved, and PI3-Akt signaling has an important role in antiapoptotic effects of pCD. CONCLUSION: We can conclude that overexpression of pCD has strong influence on increased resistance of tumor cells. This could, in fact, be an important contribution in the possible use of pCD level determination for prognostic and/or therapeutic purposes.

Comprehensive Evaluation of Anti-hyperglycemic Activity of Fractionated Momordica charantia Seed Extract in Alloxan-Induced Diabetic Rats.

The present study evaluates anti-hyperglycemic activity of fractionated Momordica charantia (bitter gourd) seed extracts. Fasting blood glucose levels were evaluated before and after administration of different fractions of the seed extract. Among the three fractions tested, fraction Mc-3 (15 mg/kg b.wt.) showed the maximum anti-hyperglycemic activity and reduced blood glucose levels in experimental diabetic rats significantly. The activities of the key regulatory enzymes of glucose metabolism (hexokinase, pyruvate kinase, lactate dehydrogenase, and glucose-6-phosphate dehydrogenase) were determined in Mc-3-treated diabetic animals. Once-daily administration of the fraction Mc-3 for prolonged period of 18 days to the experimental diabetic animals did not result in any nephrotoxicity or hepatotoxicity as evident from insignificant changes in biochemical parameters indicative of liver and kidney functions. Further fractionation of the fraction Mc-3 by size exclusion chromatography resulted in a fraction, designated Mc-3.2, possessing anti-hyperglycemic activity. The fraction Mc-3.2 showed the presence of a predominant protein band of ~11 kDa on SDS-PAGE. Loss in anti-hyperglycemic activity of the Mc-3.2 upon protease treatment indicates the proteinaceous nature of the anti-hyperglycemic principles. Overall, the results suggest that Momordica charantia seeds contain an effective anti-hyperglycemic protein(s) which may find application in treatment of diabetes without evident toxic effects.

New 4-deoxy-(1→3)-ß-D-glucan-based oligosaccharides and their immunostimulating potential.

(1→3)-ß-D-Glucans are well-established natural biological immunomodulators. However, problems inherited with the natural origin of these polysaccharides bring about significant setbacks, including batch-to-batch heterogeneity and significant differences based on the source and isolation techniques. In this study, we tried to overcome these problems by preparation of a quantitatively new set of oligo-(1→3)-ß-D-glucan-based synthetic immunomodulators. Some of these non-natural oligosaccharides showed biological activities, such as stimulation of phagocytosis, modulation of gene expression, and anti-cancer activity, which were superior to natural glucans.

Biological properties of (1 → 3)-ß-D-glucan-based synthetic oligosaccharides.

Despite the fact that ß-glucans are well-established immunomodulators, the problems with batch-to-batch heterogeneity remains problematic. The aim of this study was to prepare and evaluate new type of synthetic oligosaccharides. A new family of oligo-(1 → 3)-ß-d-glucans modified on the reducing end was synthesized using a controlled and specific inversion of configuration at C-2 starting from already formed oligo-(1 → 3)-ß-d-glucans. The designed glycosides are characterized by the presence of four or five glucopyranose entities and a mannose residue at the reducing end. To study of the impact of well-defined structural modulations, we used murine and human models to evaluate their immunostimulating potential. These novel oligosaccharides showed strong and long-lasting stimulation of phagocytosis and significant potentiation of synthesis and/or secretion of interleukin (IL-2, IL-4, IL-5, IL-6), tumor necrosis factor-α, and vascular endothelial growth factor. In addition, the oligosaccharides tested showed significant effects on expression of several genes in human fibroblasts and breast cancer cells. From our results it is clear that these synthetic oligosaccharides represent a better alternative to natural ß-glucans.