Calm Under Challenge: Immune-Balancing and Stress-Quenching Effects of Hericium erinaceus Mycelium in Human Immune Cells

Iron is an essential trace element in cell proliferation. Several investigations demonstrate that iron deprivation inhibits cell proliferation. However, the impact of iron on telomerase activity of activated lymphocytes remains unexplained to date. In this study, the effect of iron on the proliferation and telomerase activity of lymphocytes stimulated by phytohemagglutinin (PHA) were investigated. Iron loading was performed by incubating peripheral blood mononuclear cells in 500microM FeSO4.7H2O for 24 h and iron chelation was done by exposing cells to desferrioxamine, a potent iron chelator. The effects of silymarin, a flavonoid with both antioxidant and iron chelating activities, on the proliferation and telomerase activity of PHA-activated lymphocytes were also compared with desferrioxamine. Proliferation and telomerase activity were assessed using BrdU incorporation assay and Telomeric Repeat Amplification Protocol (TRAP), respectively. The proliferations of lymphocytes were significantly inhibited by 10 and 20 microg/ml desferrioxamine in a dose dependent manner, while iron loading recovered suppressed cell proliferation to the normal level. Silymarin at 20 microg/ml significantly increased the proliferation of lymphocytes in both normal and iron-treated conditions. Telomerase activity of lymphocytes was markedly increased by iron treatment and suppressed by desferrioxamine. Conversely, iron treatment had no effect on the telomerase activity of lymphocytes incubated with silymarin. Iron plays a significant role in the proliferation and telomerase activity of lymphocytes. The effects of silymarin on the proliferation and telomerase activity of lymphocytes were completely different from those of desferrioxamine, suggesting that the immunomodulatory effect of silymarin is probably not associated with its iron chelating activity.

Chlorella species are fast-growing microalgae with significant industrial applications. The aim of the present study was to investigate the antitumor, antioxidant, and hemolytic activities of Chlorella sorokiniana UTEX 1230 crude methanol extracts and fractions. Ch. sorokiniana crude methanol extracts and collective fractions (CFs) were obtained from lyophilized biomass by maceration and column chromatography. Antitumor assays against murine lymphoma L5178Y-R and human breast cancer MCF-7 cells were performed by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction technique, using human peripheral blood mononuclear cells (PBMC) as the control group. Antioxidant and hemolytic activities were evaluated using the 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay (DPPH) and erythrocyte hemolysis, respectively. We showed that crude methanol extracts (IC50) increased L5178Y-R and MCF-7 cell growth inhibition, without affecting PBMC. In addition, all evaluated CFs showed significantly higher antioxidant activity than the positive control (ascorbic acid). CF3 and CF4 showed the highest cytotoxicity against L5178Y-R, whereas CF3, CF4, and CF5 caused the highest antitumor activity against MCF-7 cells. CF3, CF4, and CF5 induced significantly higher hemolytic activity compared with all other fractions. CF characterization revealed loliolide, cinnamic acid, methyl dihydrojasmonate, salsalvamide A, 1-monolinolenin, cryptophycin 29, costunolide, riboflavin lumicrome, and germicidin B, which have been related to antitumor and antioxidant activities. In conclusion, we demonstrated that Ch. sorokiniana extracts and fractions possess antitumor and antioxidant potential, without affecting human erythrocytes and PBMC.

Introduction: Hericium erinaceus is known as a medicinal edible mushroom owing to its antimicrobial, antioxidant, anti-tumor and immunomodulatory effects. Helicobacter pylori infection is one of the major health concerns worldwide due to its high rate in global populations, frequent recurrence, and rapid emergence of drug-resistant strains. The present study aims to investigate antioxidant anti-H. pylori and urease inhibitory activities of solvent fractions from H. erinaceus mycelium and culture filtrate. Methods: H. erinaceus mycelium was purely cultured in a liquid medium. A polysaccharide fraction was obtained from the culture filtrate by precipitation with ethanol. The mycelium and culture filtrate were extracted by liquid extraction to obtain solvent-soluble fractions. The antibacterial effects of these fractions were determined using paper disc diffusion and broth microdilution assays. Urease inhibition was determined using the salicylate-hypochlorite method. The antioxidant activity of H. erinaceus was evaluated via 2,2,1-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. Results: The ethyl-acetate (EtOAc) fractions derived from H. erinaceus culture filtrate (fEtOAc Fr.) and mycelium (mEtOAc Fr.) showed the strongest anti-H. pylori activity with MIC (MBC) of 1.25 – 1.5 (5.0 – 7.5) mg/mL and potential urease inhibitory activity with IC50 of 0.34 – 0.35 mg/mL. In addition, fEtOAc Fr. exhibited the greatest antioxidant activity (IC50, 11.83 mg/mL), which was slightly stronger than that of mEtOAc Fr. (IC50, 14.75 mg/mL). Moreover, our study also found that the water fractions from the culture filtrate (fWater Fr.) and the mycelium (mWater Fr.) displayed considerable inhibitory activities against bacterial urease (IC50, 1.26 – 1.40 mg/mL), although they had low or no anti-H. pylori activities and low antioxidant properties. Conclusion: The present study revealed that the EtOAC fractions derived from the H. erinaceus mycelium and culture filtrate potentially have anti-H. pylori, anti-urease and antioxidant activities. These results suggest that H. erinaceus mycelium and culture filtrate could be utilized to develop functional foods and nutraceuticals to prevent H. pylori infection. More research is needed to prove the safety of the H. erinaceus mycelium and culture filtrate fractions and their in vivo efficacy in the treatment of H. pylori infection.

From the paradigm shifting observations of Harvey, Malpighi, and van Leeuwenhoek, blood vessels have become recognized as distinct and dynamic tissue entities that merge with the heart to form a closed circulatory system.1 Vessel structures are comprised predominantly of a luminal layer of endothelial cells that is surrounded by some form of basement membrane, and mural cells (pericytes or vascular smooth muscle cells) that make up the vessel wall. In larger more complex vessel structures the vessel wall is composed of a complex interwoven matrix with nerve components. Understanding the cellular and molecular basis for the formation, remodeling, repair, and regeneration of the vasculature have been and continue to be popular areas for investigation. The endothelium has become a particularly scrutinized cell population with the recognition that these cells may play important roles in maintaining vascular homeostasis and in the pathogenesis of a variety of diseases.2 Although it has been known for several decades that some shed or extruded endothelial cells enter the circulation as apparent contaminants in the human blood stream,3 only more recent technologies have permitted the identification of not only senescent sloughed endothelial cells,4 but also endothelial progenitor cells (EPCs), which have been purported to represent a normal component of the formed elements of circulating blood5 and play roles in disease pathogenesis.6–9 Most citations refer to an article published in 1997 in which Asahara and colleagues isolated, characterized, and examined the in vivo function of putative EPCs from human peripheral blood as a major impetus for generating interest in the field.10 This seminal article presented some evidence to consider emergence of a new paradigm for the process of neovascularization in the form of postnatal vasculogenesis. Since publication of that article, interest in circulating endothelial cells, and particularly EPCs, has soared, …

Rational Design of Immunostimulatory siRNAs

Pseudomonas aeruginosa infections are commonly observed in sepsis, burns, as well as cystic fibrosis (CF). Among the professional phagocytes neutrophils and monocytes are recruited by various chemotactic factors from the cellular environment. Although they provide the first line of host defense excessive neutrophil accumulation seems to be a major cause of pathogenesis during P. aeruginosa infection. Interleukin-8 (IL-8) represents one important chemoattractant for professional phagocytes. To evaluate IL-8 releasability by phagocytes in the context of P. aeruginosa infection and especially of CF, we stimulated human polymorphonuclear neutrophilic granulocytes (PMN) and peripheral blood mononuclear cells (PBMC) as a source for monocytes with clinical P. aeruginosa isolates, with mucoid P. aeruginosa strain (CF3M) and its nonmucoid revertant (CF3), and with purified P. aeruginosa mucoid exopolysaccharide (alginate). A significant increase in IL-8 release as compared to unstimulated cells was observed after an incubation time of 90 min for PMN and after 60 min for PBMC which increased (PMN: up to 60-fold; PBMC: up to 40-fold) over time (up to 4 h). In contrast of PBMC, when PMN were studied, intracellular IL-8 exceeded the IL-8 release in unstimulated as well as in stimulated cells by up to 10-fold. All clinical P. aeruginosa isolates, independent of the clinical source, induced IL-8 release from human PBMC and PMN in a dose- and time-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocompromised mice engrafted with human hematopoietic stem cells (HSC) or peripheral blood mononuclear cells (PBMC) can be promising models of human immune system compartments, useful for studying diseases such as diabetes, lupus, GVHD, and cancer. We have utilized the PBMC-humanized mouse as a platform for xenograft models. To investigate the effect of human antibody (Ab) treatment on immune cells and xenografts using this platform, we examined the infiltrate of human immune cells in A431 xenografts with treatment of an effector function-enhanced anti-tissue factor VIIA (TFVIIA) Ab. Female NSG mice were intravenously engrafted with human PBMC, and subcutaneously implanted two weeks later with A431 human SCC cells. One week post tumor cell implant and thereafter twice weekly, mice received treatment with anti-TFVIIA Ab. Peripheral blood, bone marrow and spleen were analyzed by FACS and terminal tumor samples were analyzed by FACS and immunohistochemistry (IHC). FACS analysis of blood from Ab treated and untreated tumor-bearing mice collected from baseline to termination, showed an overall increase in the frequency of human CD45+, CD3+, CD8+, and CD4+ cells, while the frequency of CD56+ cells remained stable. In comparison, non-tumor bearing mice had increased CD56+ cells and decreased CD3+ cells. FACS analysis of tumors showed a robust increase in the frequency of CD56+ cells and a decrease in the frequency of CD3+ cells with treatment of anti-TF Ab. With treatment, the frequency of CD8+ cells was markedly lower and the frequency of CD4+ cells unchanged. Histological analysis was performed on A431 FFPE xenografts from both the treated and non-treated groups. Hematoxylin and eosin (H&E) staining of the xenografts confirmed the presence of inflammatory cells in the tumors and also showed greater PBMC infiltration of the tumors in the presence of the anti-TFVIIA Ab. Xenografts treated with anti-TFVIIA Ab exhibited greater infiltration of human CD3+, CD8+, and CD57+ cells. There was an increase in cleaved caspase-3 (CC3)+ cells in Ab-treated xenografts, indicating an increase in apoptosis with anti-TFVIIA Ab treatment. In conclusion, anti-TFVIIA Ab treatment causes changes in immune cell populations in xenografts. While the mechanism is unknown, one possibility is that NK migration into the tumor coupled with increased apoptosis with treatment indicates ADCC activity by the NK cells. This would signify some preservation of immune cell function in the PBMC engraftment model, and would enhance studying the effects of experimental antibodies in a more humanized setting. Citation Format: Hillary Millar-Quinn, Brenda Hertzog, Rebecca Hanson, Jeffrey Nemeth, John Alvarez. Human immune cell infiltration of tumors in a PBMC-humanized NSG mouse xenograft model changes with treatment of an anti-tissue factor antibody. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1655. doi:10.1158/1538-7445.AM2014-1655

Shosaiko-to is a Kampo medicine used for the treatment of chronic hepatitis in Japan. Lately, over 200 cases of interstitial pneumonia have been reported resulting from Shosaiko-to therapy, and the number of cases increased when patients were administrated interferon (IFN)-alpha at the same time. However, the mechanisms of this Shosaiko-to implicated interstitial pneumonia are not fully understood. In this study, we examined by flow cytometry analysis the in vitro effects of 7 phenolic compounds (lignans and flavonoids), which were detected from human urine after administration of Shosaiko-to, and IFN-alpha on inducing apoptosis in human lung fibroblasts and peripheral blood mononuclear cells (PBMCs). Among the 7 compounds, baicalein and medicarpin (10 microg/ml) showed significant apoptosis-inducing effects on human PBMCs. In human lung fibroblasts, medicarpin exhibited a significantly higher activity to induce apoptosis compared to the control, and the percentage of cells undergoing apoptosis showed time- and dose-dependent increases. Baicalein (0.1 and 1 microg/ml), liquiritigenin (10 microg/ml) and davidigenin (10 microg/ml) also showed significant effects after 96 h treatment. Whereas, baicalin, oroxylin A and wogonin did not show any effect on inducing apoptosis in PBMCs and fibroblasts. Baicalein and medicarpin significantly inhibited the growth and reduced the viability of lung fibroblasts. IFN-alpha had no apoptosis-inducing effect, and it did not show synergistic interaction with any of the compounds derived from Shosaiko-to on inducing apoptosis in both human lung fibroblasts and PBMCs. These results suggested that phenolic compounds found in human post-administrative urine of Shosaiko-to, especially baicalein and medicarpin, exhibited a direct effect on human lung fibroblasts and immune cells to induce apoptosis.

Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus recently isolated from human prostate cancer and peripheral blood mononuclear cells (PBMCs) of patients with chronic fatigue syndrome (CFS). We and others have shown that host restriction factors APOBEC3G (A3G) and APOBEC3F (A3F), which are expressed in human PBMCs, inhibit XMRV in transient-transfection assays involving a single cycle of viral replication. However, the recovery of infectious XMRV from human PBMCs suggested that XMRV can replicate in these cells despite the expression of APOBEC3 proteins. To determine whether XMRV can replicate and spread in cultured PBMCs even though it can be inhibited by A3G/A3F, we infected phytohemagglutinin-activated human PBMCs and A3G/A3F-positive and -negative cell lines (CEM and CEM-SS, respectively) with different amounts of XMRV and monitored virus production by using quantitative real-time PCR. We found that XMRV efficiently replicated in CEM-SS cells and viral production increased by >4,000-fold, but there was only a modest increase in viral production from CEM cells (<14-fold) and a decrease in activated PBMCs, indicating little or no replication and spread of XMRV. However, infectious XMRV could be recovered from the infected PBMCs by cocultivation with a canine indicator cell line, and we observed hypermutation of XMRV genomes in PBMCs. Thus, PBMCs can potentially act as a source of infectious XMRV for spread to cells that express low levels of host restriction factors. Overall, these results suggest that hypermutation of XMRV in human PBMCs constitutes one of the blocks to replication and spread of XMRV. Furthermore, hypermutation of XMRV proviruses at GG dinucleotides may be a useful and reliable indicator of human PBMC infection.

An understanding of the mechanisms that suppress the human anti-pig cellular response is key for xenotransplantation. We have compared the ability of human regulatory T cells (Tregs) to suppress xenogeneic and allogeneic responses in vitro. Human peripheral blood mononuclear cells (PBMC), CD4+ T cells, or CD4+ CD25- T cells were stimulated with irradiated human or wild type (WT) or alpha1,3-galactosyltransferase gene-knockout (GT-KO) pig PBMC in the presence or absence of human CD4+ CD25 high Tregs. In separate experiments, 5- (and 6)-carboxyfluorescein diacetate succinimidyl ester-labeled human CD4+ T cells were stimulated with human or pig PBMC. The expansion and precursor frequencies of allo- and xenoreactive Tregs were assessed by labeling with FoxP3 mAb and flow cytometric analysis. The responses of human PBMC, CD4+ T cells, and CD4+ CD25- T cells to pig PBMC were stronger than to human PBMC (P<0.05). Human anti-GT-KO responses were weaker than anti-WT responses (P<0.05). Human CD4+ CD25 high Tregs suppressed proliferation of CD4+ CD25- T cells to both human and pig PBMC stimulator cells with the same efficiency. Alloreactive CD4+ CD25+ FoxP3 high responder T cells proliferated more than their xenoreactive counterparts (P<0.05), although xenoreactive CD4+ CD25+ T cells proliferated more than alloreactive cells (P<0.05). There was no difference in precursor frequency between allo- and xeno-reactive CD4+ CD25+ FoxP3 high cells. Human T-cell responses to pig cells are stronger than to allogeneic cells. The human response to GT-KO PBMC is weaker than to WT PBMC. Although human Tregs can suppress both responses, expansion of CD4+ CD25+ FoxP3 high cells against pig PBMC is weaker than against human PBMC. More human Tregs may be required to suppress the stronger xenogeneic response.

Many plant parts have been widely used in the treatment of immune diseases in Vietnam, yet just few of them are known about their mechanism of action. The goal of this study was to investigate the in vitro inhibitory effects of in vitro inhibitory effect of crude ethanol extracts and fractions of thirteen medicinal plants on proliferation of human peripheral blood mononuclear cells (PBMCs), interleukin-2 secretion and antioxidant activity. PBMCs were extracted from the whole blood of healthy volunteers. The effects of thirteen crude extracts in ethanol and twenty four fractionated extracts in chloroform, ethyl acetate and water on in vitro proliferation of PBMCs were evaluated using MTT test. Interleukin - 2 (IL - 2) concentrations secreted by PBMC were determined by ELISA method. The antioxidant capacity of ethanol extracts and fractionated extracts were assessed using the DPPH method. The results showed that out of thirteen crude ethanol extracts, six extracts inhibited PBMC proliferation and two extracts stimulated PBMC proliferation and five extracts had no effect on PBMC proliferation. The inhibitory extracts reduced the amount of IL-2, while the stimulant extracts while the stimulant extracts had no effect on IL-2 secretion compared to the control. The chloroform extract of Wedelia chinesis showed the strongest inhibitory activity with an IC50 concentration 16.1 μg/ml. The chloroform extract of Piper betle showed the strongest DPPH capture capacity with DPPH50 1.94 μg/ml and 2.1 times stronger than vitamin C. In conclusion, the chloroform extract of Wedelia chinensis may be considered for the treatment of autoimmune diseases.

Immunostimulatory and antioxidant activities of a lignin isolated from Conocarpus erectus leaves

Ganoderma lucidum (GL, Lingzhi) has been suggested as a candidate for immunomodulation and cancer treatment. The present study aimed at comparing the different parts of the fruiting body (whole fruiting body, pileus and stipe) of GL as well as Ganoderma spores (sporoderm-broken and -unbroken), with regard to their antitumor and immunomodulatory activities in S-180 sarcoma-bearing mice. The hot water extracts of different parts of GL or the Ganoderma spores were orally administered to the sarcoma-bearing mice. The results showed that GL whole fruiting body, stipe and sporoderm-broken spore possessed stronger inhibitory activities on sarcoma growth when compared with the pileus extract. Higher immunomodulatory activities in terms of enhancing the proliferative responses and the cytokines (IFN-gamma, IL-4 and IL-6) production of spleen lymphocytes were also found in GL stipe and sporoderm-broken spore treatment groups. The sporoderm-broken spores had higher stimulatory effects on mitogen-activated spleen lymphocytes of healthy mice than those of sarcoma-bearing mice. In addition, the immunostimulatory activities of GL hot water extracts and Ganoderma spores were shown to be comparable; hence the latter did not show superiority in efficacy. This is the first comparative study on the immunomodulatory activities of Ganoderma spores and the fruiting body extracts.

Production of Fruiting Body and Antioxidant Activity of Wild Pleurotus

Preclinical studies examining tumor biology and immunity are limited by differences between the mouse and human immune systems. Humanized mouse models offer a unique tool to assess the anti-tumor response by recapitulating aspects of human tumor biology. Humanized mice are created by injecting either human CD34+ hematopoietic stem cells or peripheral blood mononuclear cells (PBMCs) into an immunodeficient mouse strain. Here we utilized a PBMC humanized NCG mouse model to investigate human T-cell mediated anti-tumor response (&amp;gt;95% CD3+ T-cells by day 21). The challenges encountered with this model include the onset of graft versus host disease (GvHD) and donor variability leading to donor specific engraftment rates. Consistency is required with humanized PBMC models to ensure their use as an appropriate research model. Therefore, we examined the relationship between donor selection and engraftment rates as well as the onset of GvHD. Inter-donor variability was assessed by collecting PBMCs from multiple healthy donors. Intra-donor consistency was assessed by collecting PBMCs from the same donors at subsequent collections and comparing to initial human immune cell profiling by flow cytometry. NCG mice were injected with 1 × 107 PBMCs from individual donors. Animals were weighed three times a week to monitor for changes in body weight and general health status. Peripheral blood was collected at day 9, 21 and 42 for flow cytometry screening of human immune cell engraftment (hCD45+, hCD3+, hCD4+ and hCD8+) at the initial collection. Human PBMCs isolated from additional collections from the same donors were also injected into NCG mice and flow cytometry was performed on the new cell lots at day 21. Donors were grouped into categories based on engraftment rates, which were donor dependent. Donor variability is an important aspect to consider when humanizing mice with human PBMCs. PBMC humanized models are important for the study of pre-clinical therapeutics, but donor engraftment rates must be considered when engrafting the mice to create a consistently humanized mouse. Citation Format: Steven Bronson, Jenny Rowe, Christoph Eberle, Stephen Festin. PBMC donor selection affects engraftment rates and onset of graft versus host disease in humanized NCG mice for use in cancer studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 38.