Water extract of Cayratia albifolia C.L.Li root relieves zymosan A-induced inflammation by restraining M1 macrophage polarization.

BACKGROUND: Cayratia albifolia C.L.Li (CAC) is a traditional Chinese herbal medicine used to treat inflammatory diseases. Our laboratory has firstly reported that the water extract from CAC relieved lipopolysaccharide (LPS)-induced inflammation, however stronger evidence is still needed to prove its anti-inflammatory effects and the mechanisms involved are also ambiguous. PURPOSE: This study sought to provide more evidence for the application of CAC in alleviating infectious inflammation and disclose novel pharmacological mechanisms. METHODS: Mice were injected with zymA into their paws or peritoneal cavities, and then treated with CAC. ELISA, immunofluorescence and flow cytometry were performed to detect the cytokines (IL-1ß, IL-6, TNF-α and IL-10) generation, the cell infiltration, and the CD86 or CD206 expression of macrophages. Then in vitro assays were performed on bone marrow-derived macrophages (BMDMs) and peritoneal macrophages (PMs) to detect their expression of iNOS, arg-1 and the cytokines above. On mechanisms, western blotting (WB), electrophoretic mobility shift assay (EMSA) and flow cytometry were carried out to measure NF-κB transcriptional activity, mitochondrial bioactivity and the mTORC1 activation when BMDMs were stimulated by zymA and treated with CAC. Finally, the chemical components consisted in the extract were analyzed by LC-MS. RESULTS: 200 mg/kg CAC clearly inhibited zymA induced mouse paw edema and reduced the contents of IL-1ß, IL-6 and TNF-α rather than IL-10 in local tissues. CAC also reduced CD86 but not CD206 in macrophages in situ. Through in vitro experiments, it was discovered that CAC reduced the protein and mRNA levels of IL-1ß, IL-6 and TNF-α, and also inhibited iNOS expression, but showed no influence on IL-10 and arg-1 in macrophages. We found CAC reduced NF-κB transcriptional activity, down-regulated mitochondrial membrane potential and ROS levels, and inhibited mTORC1 activity. Finally, we identified 15 major compounds in the extract, among which 4-guanidinobutyric acid and kynurenic acid were the most abundant. CONCLUSION: This study provides further evidence that CAC significantly reduces zymA induced infectious inflammation. In addition, this novel data revealed that CAC restrained M1 rather than promoting M2 macrophages polarization via multi-target inhibitory effects, based on its potentially active components.

Zymosan enhances in vitro phagocyte function and the immune response of mice infected with Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is the major etiologic agent of Paracoccidioidomycosis (PCM), the most frequent human deep mycosis in Latin America. It is proposed that masking of ß-glucan in P. brasiliensis cell wall is a critical virulence factor that contributes to the development of a chronic disease characterized by a long period of treatment, which is usually toxic. In this context, the search for immunomodulatory agents for therapeutic purposes is highly desirable. One strategy is to use pattern recognition receptors (PRRs) ligands to stimulate the immune response mediated by phagocytes. Here, we sought to evaluate if Zymosan, a ß-glucan-containing ligand of the PRRs Dectin-1/TLR-2, would enhance phagocyte function and the immune response of mice challenged with P. brasiliensis. Dendritic cells (DCs) infected with P. brasiliensis and treated with Zymosan showed improved secretion of several proinflammatory cytokines and expression of maturation markers. In addition, when cocultured with splenic lymphocytes, these cells induced the production of a potential protective type 1 and 17 cytokine patterns. In macrophages, Zymosan ensued a significant fungicidal activity associated with nitric oxide production and phagolysosome acidification. Importantly, we observed a protective effect of Zymosan-primed DCs delivered intranasally in experimental pulmonary PCM. Overall, our findings support the potential use of ß-glucan-containing compounds such as Zymosan as an alternative or complementary antifungal therapy. LAY SUMMARY: We report for the first time that Paracoccidioides brasiliensis-infected phagocytes treated with Zymosan (cell wall extract from bakers' yeast) show enhanced cytokine production, maturation, and fungal killing. Also, Zymosan-primed phagocytes induce a protective immune response in infected mice.

Doxorubicin Hydrochloride Loaded Zymosan-Polyethylenimine Biopolymeric Nanoparticles for Dual 'Chemoimmunotherapeutic' Intervention in Breast Cancer.

OBJECTIVE: To utilize nanoparticles produced by condensation of zymosan (an immunotherapeutic polysaccharide) with pegylated polyethylenimine (PEG-PEI) for dual intervention in breast cancer by modulating tumor microenvironment and direct chemotherapy. METHOD: Positively charged PEG-PEI and negatively charged sulphated zymosan were utilized for electrostatic complexation of chemoimmunotherapeutic nanoparticles (ChiNPs). ChiNPs were loaded with doxorubicin hydrochloride (DOX) for improved delivery at tumor site and were tested for in-vivo tolerability. Biodistribution studies were conducted to showcase their effective accumulation in tumor hypoxic regions where tumor associated macrophages (TAMs) are preferentially recruited. RESULTS: ChiNPs modulated TAMs differentiation resulting in decrement of CD206 positive population. This immunotherapeutic action was furnished by enhanced expression of Th1 specific cytokines. ChiNPs also facilitated an anti-angiogenetic effect which further reduces the possibility of tumor progression and metastasis.

The use of Zymosan A and bacteria anchored to tumor cells for effective cancer immunotherapy: B16-F10 murine melanoma model.

The idea of using killed microorganisms or their parts for a stimulation of immunity in the cancer immunotherapy is very old, but the question of interactions and binding of these preparations to tumor cells has not been addressed so far. The attachment of Zymosan A and both Gram-positive and Gram-negative bacteria to tumor cells was tested in in vivo experiments. This binding was accomplished by charge interactions, anchoring based on hydrophobic chains and covalent bonds and proved to be crucial for a strong immunotherapeutic effect. The establishment of conditions for simultaneous stimulation of both Toll-like and phagocytic receptors led to very strong synergy. It resulted in tumor shrinkage and its temporary or permanent elimination. The role of neutrophils in cancer immunotherapy was demonstrated and the mechanism of their action (frustrated phagocytosis) was proposed. Finally, therapeutic approaches applicable for safe human cancer immunotherapy are discussed. Heat killed Mycobacterium tuberculosis covalently attached to tumor cells seems to be promising tool for this therapy.

Low dose zymosan ameliorates both chronic and relapsing experimental autoimmune encephalomyelitis.

Zymosan has previously been reported to have both pro-inflammatory and anti-inflammatory effects. Here we demonstrate that low dose zymosan prevented or reversed chronic and relapsing paralysis in EAE. In suppressing CNS autoimmune inflammation, zymosan not only regulated APC costimulator and MHC class II expression, but also promoted differentiation of regulatory T cells. Following adoptive transfer of zymosan-primed CD4(+) T cells, recipient mice were protected from EAE. In contrast, a MAPK inhibitor and a blocker of ß-glucan, reversed the effects of zymosan. These results demonstrate that zymosan may be a promising beneficial agent for Multiple Sclerosis (MS).

Effect of zymosan on antioxidant and immune function of S180 tumor-bearing mice.

The objective is to study the effect of zymosan on antioxidant and immune function of S(180) tumor-bearing mice. Seventy Kunming mice were randomly divided into seven groups: a normal control group (NC), a tumor control group (TC), three dose groups of zymosan (low, medium, high), a cyclophosphamide (Cy) group, and a combination of zymosan and Cy group. The S(180) tumor-bearing mice model was established by the inoculation of cancer cell suspension subcutaneously in the mouse's right anterior limb. At the 19th day, malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) activity in liver homogenate were analyzed. The reverse transcriptase-polymerase chain reaction was used to determine the mRNA expression levels of IL-2, TNF-α, and TGF-ß(1). The activity of GSH-Px and SOD in the liver increased with the dose of zymosan, whereas the activity of MDA significantly decreased in the higher-dose groups of zymosan, compared to the TC group (P < 0.01). In the zymosan groups, mRNA expression levels in tissues of S(180) tumor-bearing mice were significantly higher for TNF-α and IL-2, but lower for TGFß(1) than in the Cy or TC group (P < 0.01). The high-dose of zymosan markedly showed a depressant effect on S(180) tumor, enhanced by the action of Cy that increased mRNA expression levels of TNF-α and IL-2. The mechanism of zymosan on the inhibition of tumor growth may be due to its ability to enhance the antioxidant and immune function in a dose-dependent manner.

Roles for TGF-beta and programmed cell death 1 ligand 1 in regulatory T cell expansion and diabetes suppression by zymosan in nonobese diabetic mice.

Zymosan is a complex fungal component shown to be capable of both promoting and suppressing the development of autoimmune disorders in mice. In this study, we show that a single injection of zymosan just prior to diabetes onset can significantly delay the progression of disease in NOD mice. Zymosan treatment of NOD mice induced the production of biologically active TGF-beta from cells infiltrating the pancreas and was associated with expansion of programmed cell death 1 ligand 1(+)TGF-beta(+) macrophages and Foxp3(+) regulatory T cells in vivo. Neutralization of either TGF-beta or programmed cell death 1 ligand 1 abrogated the protective effects of zymosan. Zymosan acted through TLR2 as well as ERK and p38 MAPK to induce macrophage secretion of TGF-beta and promotion of Foxp3(+) regulatory T cells in vitro and in vivo.

Controlling pathological pain by adenovirally driven spinal production of the anti-inflammatory cytokine, interleukin-10.

Gene therapy for the control of pain has, to date, targeted neurons. However, recent evidence supports that spinal cord glia are critical to the creation and maintenance of pain facilitation through the release of proinflammatory cytokines. Because of the ability of interleukin-10 (IL-10) to suppress proinflammatory cytokines, we tested whether an adenoviral vector encoding human IL-10 (AD-h-IL10) would block and reverse pain facilitation. Three pain models were examined, all of which are mediated by spinal pro-inflammatory cytokines. Acute intrathecal administration of rat IL-10 protein itself briefly reversed chronic constriction injury-induced mechanical allodynia and thermal hyperalgesia. The transient reversal caused by IL-10 protein paralleled the half-life of human IL-10 protein in the intrathecal space (t(1/2) approximately 2 h). IL-10 gene therapy both prevented and reversed thermal hyperalgesia and mechanical allodynia, without affecting basal responses to thermal or mechanical stimuli. Extra-territorial, as well as territorial, pain changes were reversed by this treatment. Intrathecal AD-h-IL10 injected over lumbosacral spinal cord led to elevated lumbosacral cerebrospinal fluid (CSF) levels of human IL-10, with far less human IL-10 observed in cervical CSF. In keeping with IL-10's known anti-inflammatory actions, AD-h-IL10 lowered CSF levels of IL-1, relative to control AD. These studies support that this gene therapy approach provides an alternative to neuronally focused drug and gene therapies for clinical pain control.

Adjuvant treatment for complement activation increases the effectiveness of photodynamic therapy of solid tumors.

Phototoxic lesions generated in tumor tissue by photodynamic therapy (PDT) are recognized by the host as a threat to the integrity and homeostasis at the affected site. Among the canonical pathways invoked by the host for dealing with this type of challenge is the activation of the complement system, integrating proteins that serve as molecular sensors of danger signals produced by PDT and those initiating signalling cascades coupled into the network of inflammatory and immune responses. Since the activated complement system is a salient participant of the antitumor response produced by PDT, it is worth exploring whether its manipulation can be exploited for the therapeutic benefit. Using mouse tumor models, the present study examined the potential of representative complement-activating agents to act as effective adjuvants to PDT. Tumor-localized treatment with zymosan, an alternative complement pathway activator, reduced the recurrence-rate of PDT-treated tumors, markedly increasing the percentage of permanent cures. In contrast, a similar treatment with heat aggregated gamma globulin (complement activator via the classical pathway) was of no significant benefit as a PDT adjuvant. Systemic complement activation with streptokinase treatment had no detectable effect on complement deposition at the tumor site without PDT, but it augmented the extent of complement activity in PDT-treated tumors. This finding based on immunohistochemistry analysis explains the results of tumor therapy experiments, which showed that systemic treatment with streptokinase or a similar agent, urokinase, enhances the PDT-mediated tumor response. Zymosan and streptokinase administrations produced no beneficial results with PDT of tumors growing in complement-deficient mice. This study, therefore, establishes the potential of complement-activating agents to serve as effective adjuvants to PDT for cancer treatment.

Continuous dialysis with bicarbonate/lactate-buffered peritoneal dialysis fluids results in a long-term improvement in ex vivo peritoneal macrophage function.

To circumvent the potentially negative consequences of long-term exposure to unphysiologic acidic lactate-buffered peritoneal dialysis fluids (PDF), neutral pH solutions buffered with bicarbonate/lactate have recently been introduced in phase 2 and 3 clinical trials. This study examines the longitudinal changes in peritoneal macrophage (PMØ) function in patients dialyzed continuously with either lactate (LPD; 40 mM lactate, pH 5.2)-buffered or bicarbonate/lactate (TBL; 25 mM/15 mM bicarbonate/lactate, pH 7.3)-buffered PDF. Before the study, during the run in period of a phase 3 clinical trial, all patients had been taking LPD for at least the previous 18 wk. At the beginning of the study (day 0), both constitutive and serum-treated zymosan (STZ) stimulated tumor necrosis factor alpha (TNF-alpha) synthesis were assessed in PMØ isolated from 12-h dwell effluent (with 1.36% glucose) in all patients. The patients were subsequently randomized to either continuous TBL or LPD therapy and PMØ function was assessed after further 3- and 6-mo periods in all patients. At all time points measured STZ induced a dose-dependent increase in PMØ TNF-alpha secretion (P = 0.043 versus control for doses greater than 100 microg/ml). In patients continuously dialyzed with LPD, constitutive PMØ TNF-alpha synthesis levels (mean +/- SEM, pg/10(6) PMØ per18 h, n = 5 patients) were 154 +/- 65, 261 +/- 60, and 101 +/- 99 at 0, 3, and 6 mo, respectively. Stimulated STZ (1000 microg/ml) levels were 1340 +/- 519, 1046 +/- 586, and 758 +/- 250 at 0, 3, and 6 mo, respectively. In patients dialyzed with TBL, constitutive PMØ TNF-alpha synthesis levels (pg/10(6) PMØ per 18 h, n = 5 patients) were 300 +/- 136, 106 +/- 35, and 213 +/- 62 at 0, 3, and 6 mo, respectively. Stimulated STZ (1000 microg/ml) levels were 1969 +/- 751, 1541 +/- 330, and 2670 +/- 671 at 0, 3, and 6 mo, respectively. At 6 mo, STZ-stimulated PMØ TNF-alpha synthesis was significantly higher in patients treated with TBL compared with those treated with LPD (P = 0.0035). These data suggest that in patients continuously dialyzed with a neutral pH solution, there is a long-term improvement in PMØ function compared with patients on conventional therapy. Better PMØ function suggests improved host defense status and may affect the peritoneum's susceptibility to infection and potentially reduce the negative consequences of repeated intraperitoneal inflammation on long-term membrane function.

Beta-glucan, a "specific" biologic response modifier that uses antibodies to target tumors for cytotoxic recognition by leukocyte complement receptor type 3 (CD11b/CD18).

beta-Glucans were identified 36 years ago as a biologic response modifier that stimulated tumor rejection. In vitro studies have shown that beta-glucans bind to a lectin domain within complement receptor type 3 (CR3; known also as Mac-1, CD11b/CD18, or alphaMbeta2-integrin, that functions as an adhesion molecule and a receptor for factor I-cleaved C3b, i.e., iC3b) resulting in the priming of this iC3b receptor for cytotoxicity of iC3b-opsonized target cells. This investigation explored mechanisms of tumor therapy with soluble beta-glucan in mice. Normal mouse sera were shown to contain low levels of Abs reactive with syngeneic or allogeneic tumor lines that activated complement, depositing C3 onto tumors. Implanted tumors became coated with IgM, IgG, and C3, and the absent C3 deposition on tumors in SCID mice was reconstituted with IgM or IgG isolated from normal sera. Therapy of mice with glucan- or mannan-rich soluble polysaccharides exhibiting high affinity for CR3 caused a 57-90% reduction in tumor weight. In young mice with lower levels of tumor-reactive Abs, the effectiveness of beta-glucan was enhanced by administration of a tumor-specific mAb, and in SCID mice, an absent response to beta-glucan was reconstituted with normal IgM or IgG. The requirement for C3 on tumors and CR3 on leukocytes was highlighted by therapy failures in C3- or CR3-deficient mice. Thus, the tumoricidal function of CR3-binding polysaccharides such as beta-glucan in vivo is defined by natural and elicited Abs that direct iC3b deposition onto neoplastic cells, making them targets for circulating leukocytes bearing polysaccharide-primed CR3. Therapy fails when tumors lack iC3b, but can be restored by tumor-specific Abs that deposit iC3b onto the tumors.

Effect of interferon-alpha (IFN alpha) on various human tumor xenografts.

The growth of some human tumor xenografts (3 out of 8, melanoma, non-Hodgkin's lymphoma, squamous cell carcinoma) was successfully--but moderately and temporarily--inhibited, when interferon-alpha (EGIS, Hungary) was given for 10 days. The route of administration (intratumoral or intraperitoneal) was usually not a decisive factor. An attempt to potentiate IFNa action with Zymozan or Cyclophosphamide did not succeed.

Protection of mice against Babesia microti with cord factor, COAM, zymosan, glucan, Salmonella and Listeria.

Cord factor (trehalose 6-6' dimycolate). COAM (chlorite-oxidized oxyamylose), zymosan, glucan, Salmonella enteritidis 11RX and Listeria monocytogenes were found to protect mice against subsequent infection with Babesia microti, an intra-erythrocytic protozoan parasite. This protection was not observed after injection of Staphylococcus epidermidis, a viridans group Streptococcus, thioglycollate, or colloidal carbon. All the agents which protect against B. microti have also been reported to induce non-specific protection against experimental tumours. The parasites appear to die inside circulating red cells. This implies that these can exert non-specific protection against this parasite through the mediation of a soluble factor.