Amphoteric Mannan as an Immune Response Modifier. New Model Immunobiologically Active Candida albicans Mannan-Based Formula.

BACKGROUND: Currently, the incidence and prevalence of serious fungal infections is increasing, especially in immunosuppressed individuals. The co-administration of antibiotic and immunosuppressive therapies has driven the emergence of new multidrug-resistant fungal pathogens. Their significant increase and their ability to form biofilms is associated with rising morbidity and mortality. Research into novel synthetically prepared immunomodulators as potential immune response modifiers and prospective participants in drug delivery systems is of interest. Microbial polysaccharides with zwitterionic charge motifs were shown to be promising candidates. METHODS: Native and ultrasonically treated mannan from the yeast Candida albicans were chemically modified to contain both positive and negative charges in a nearly equimolar ratio mimicking the zwitterionic polysaccharides. RAW 264.7 macrophages and Balb/c mice were subjected as in vitro and in vivo models. Macrophage exposure to the set of amphoteric derivatives of mannan induced a release of Th1, Th2, Th17, and Treg cytokine signature patterns. The functionality of the exposed macrophages was assayed by cell proliferation and phagocytosis. RESULTS: The Th1 and Th17 dominance was over Th2. The phagocytosis and respiratory burst, together with the viability based on cell proliferation supported the bioavailability of formulas. Mouse immunization induced humoral immune responses with high titers of the IgM isotype with the IgM/IgG shift. CONCLUSION: Our study demonstrated the immunobiological activities of amphoteric derivatives of mannan from Candida albicans. Amphoteric derivatives can be considered as bioavailable formulas with an effective immunomodulatory potency, prospectively applied as a subunit formula in the design of a mannan-based platform for drug and vaccine delivery systems.

Metabolite characterization of different palm date varieties and the correlation with their NO inhibitory activity, texture and sweetness.

The aim of this study was to examine the variation in metabolite constituents of five commercial varieties of date fruits; Ajwa, Safawi and Ambar which originated from Madinah, the Iranian Bam and Tunisian Deglet Noor. The differences of metabolome were investigated using proton nuclear magnetic resonance (1H NMR) spectroscopy combined with multivariate data analysis (MVDA). Principal Component Analysis (PCA) revealed clear separation between the date varieties. The Tunisian Deglet Noor demonstrated distinct cluster from the rest of the palm date samples based on the metabolite composition as shown by the pattern observed in Hierarchical Clustering Analysis (HCA) and PCA. Deglet Noor exhibited a significant higher level of sucrose (δ 5.40) and fructose (δ 4.16) in comparison with the other four varieties which can be associated with the distinctive sweet taste of this variety. Dates originated from Madinah and Tunisia exhibited a contrast manner in the amount of xylose and moisture content. These two aspects may contribute towards the soft texture of Tunisian dates. All Madinah dates were found to contain phenolic compounds which were well established as great antioxidant and anti-inflammatory agent. Ajwa dates exerted greater effect in inhibiting the generation of nitric oxide (NO) from the stimulated RAW264.7 cells at 95.37% inhibition. Succinic acid was suggested to have the most significant correlation with the trend of NO inhibitory shown by the selected date palm varieties.

Biological responses of Raw 264.7 macrophage exposed to two strains of Stachybotrys chartarum spores grown on four different wallboard types.

The many benefits of building "green" have motivated the use of sustainable products in the design and execution of the built environment. However, the use of these natural or recycled materials, some of which have been treated with antimicrobials, provides a growth opportunity for microorganisms with the potential to elicit adverse health effects especially in the presence of an antimicrobial. The focus of this research was to determine the effects of Stachybotrys chartarum (strains Houston and 51-11) grown under different conditions on a macrophage cell line (Raw 264.7) using endpoints, including cytotoxicity, and those associated with immunity specifically inflammation and MHC class II expression. The fungi were grown on four different gypsum products, and macrophages were exposed to whole spores of both strains and fragmented spores of strain 51-11. Whole spores of the Houston strain elicited no cytotoxicity with some level of inflammation, while exposure to whole spores of 51-11 caused variable responses depending on the wallboard type supporting the fungal growth. High concentrations of fragmented 51-11 spores primarily resulted in the apoptosis of macrophage with no inflammation. None of the fungal strains caused elevated levels of major histocompatibility complex (MHC) class II expression on the surface of Raw cells. Mycotoxin levels of 51-11 spores from all of the wallboard types measured >250 ng/µL of T2 equivalent toxin based on activity. Collectively, the data demonstrated that all of the wallboard types supported growth of fungi with the ability to elicit harmful biological responses with the potential to negatively impact human health.

Murine RAW Macrophages Are a Suitable Model to Study the CD3 Signaling in Myeloid Cells.

In recent years, a growing body of evidence has shown the presence of a subpopulation of macrophages that express CD3, especially in the context of mycobacterial infections. Despite these findings, the function of these cells has been poorly understood. Furthermore, the low frequency of CD3+ macrophages in humans limits the study of this subpopulation. This work aimed to evaluate the expression of CD3 in a murine macrophage cell line and its potential for the study of CD3 signaling. The murine macrophage cell line RAW was used to evaluate CD3 expression at the transcriptional and protein levels and the effect of in vitro infection with the Mycobacterium bovis Bacillus Calmette-Guérin (BCG) on these. Our data showed that RAW macrophages express CD3, both the ε and ζ chains, and it is further increased at the transcriptional level after BCG infection. Furthermore, our data suggest that CD3 can be found on the cell surface and intracellularly. However, this molecule is internalized constantly, mainly after activation with anti-CD3 stimulus, but interestingly, it is stably maintained at the transcriptional level. Finally, signaling proteins such as NFAT1, c-Jun, and IKK-α are highly expressed in RAW macrophages. They may play a role in the CD3-controlled signaling pathway to deliver inflammatory cytokines such as TNF and IL-6. Our study provides evidence to support that RAW cells are a suitable model to study the function and signaling of the CD3 complex in myeloid cells.

A System Based-Approach to Examine Cytokine Response in Poxvirus-Infected Macrophages.

The poxviruses are large, linear, double-stranded DNA viruses about 130 to 230 kbp, that have an animal origin and evolved to infect a wide host range. Variola virus (VARV), the causative agent of smallpox, is a poxvirus that infects only humans, but other poxviruses such as monkey poxvirus and cowpox virus (CPXV) have crossed over from animals to infect humans. Therefore understanding the biology of poxviruses can devise antiviral strategies to prevent these human infections. In this study we used a system-based approach to examine the host responses to three orthopoxviruses, CPXV, vaccinia virus (VACV), and ectromelia virus (ECTV) in the murine macrophage RAW 264.7 cell line. Overall, we observed a significant down-regulation of gene expressions for pro-inflammatory cytokines, chemokines, and related receptors. There were also common and virus-specific changes in the immune-regulated gene expressions for each poxvirus-infected RAW cells. Collectively our results showed that the murine macrophage RAW 264.7 cell line is a suitable cell-based model system to study poxvirus host response.

Dynamics of the sealing zone in cultured osteoclasts.

Bone resorption by osteoclasts (OCs) depends on the formation and stability of the sealing zone (SZ), a peripheral belt of actin and integrin-based podosomes. Recent studies demonstrated that the SZ is a highly dynamic structure, undergoing cycles of assembly and disassembly. In this study, we explored the mechanisms underlying the regulation of SZ stability and reorganization in OCs cultured on glass slides, and forming an SZ-like podosome belt (SZL). By monitoring this belt in cultured RAW264.7 cells expressing GFP-tagged actin, we show here that SZL stability is usually locally regulated, and its dissociation, occurring mostly in concave segments, is manifested in the loss of both podosome coherence, and actin belt continuity. Double labeling of cells for actin and tubulin indicated that microtubules (MTs) are mostly confined by the inner aspect of the stable SZL-associated actin belt. However, in unstable regions of the SZL, MTs tend to extend radially, across the SZL, toward the cell edge. Disruption of MTs by nocodazole induces SZ disassembly, without affecting individual podosome stability. Inspection of the MT network indicates that it is enriched along stable SZL regions, while bypassing disorganized regions. These results suggest that the SZL is stabilized by MTs flanking its inner aspect, while disruption or misalignment of MTs leads to SZL destabilization. We further demonstrate that the MT-associated protein dynamin2 is involved in the regulation of SZL stability, and dynamin2 knockdown or inactivation cause SZL destabilization.