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Modulation of cytokines production, granzyme B and perforin in murine CIK cells by Ganoderma lucidum polysaccharides
Abstract
Ganoderma lucidum polysaccharides (Gl-PS) have shown a variety of immune modulating effects. Culture of immunologic effector cells termed cytokine induced killer (CIK) cells is dependent on exogenous cytokines. It is not known about effects of Gl-PS on cytokine production, perforin and granzyme B expression in CIK cells. We made use of CIK cells as a means to investigate interaction between Gl-PS and cytokines, and explore mechanism of Gl-PS acting on proliferation and anti-tumor activity of CIK cells. The results suggested that Gl-PS (400 or 100 μg/ml) promoting CIK cells proliferation and cytotoxicity were relevant to enhancing IL-2, TNF production, protein and mRNA expression of granzyme B and perforin in CIK cells through synergizing cytokines in decreasing doses of IL-2 and anti-CD3 by 75 and 50%, and maybe were irrelevant to nitric oxide (NO). These results confirmed that Gl-PS was a promising biological response modifier (BRM) and immune potentiator.
... G. lucidum is traditionally used for the prevention and treatment of a large number of diseases, including many forms of cancer. G. lucidum extracts and polysaccharides were also proven to have, along with antitumor, also immune potentiating activities (14,15). G. lucidum polysaccharides were shown to induce apoptosis, inhibit cell proliferation and suppress cell migration of highly invasive human prostate cancer cells. ...
... G. lucidum polysaccharides were shown to induce apoptosis, inhibit cell proliferation and suppress cell migration of highly invasive human prostate cancer cells. The mechanism of antitumor activity of G. lucidum polysaccharides proceeded through stimulation of host defence responses (14,15). Our earlier ...
The use of plant derived essential oils as a major therapeutic agent to treat various diseases has gained great
momentum recently. According to the available literature data essential oils have proven to be a potential
alternative to synthetic drugs especially because of their antimicrobial properties against various pathogenic
microorganisms. These volatile oils are extracted from leaves, bark, stem, fruits, flowers of the plants by different
distillation methods. Annona genus represents a large number of essential oil-bearing plants and literature
analysis reveals that these oils have promising pharmacological activities too. Chemical fingerprinting of these
essential oils indicates the presence of monoterpenes, sesquitepenes and oxygenated sesquitepenes as the
prominent classes of compounds. Major essential oil constituents identified are bicyclogermacrene, germacrene
D, α-pinene, β-pinene, (E)-caryophyllene, caryophyllene oxide, spathulenol, linalool etc. Geographical variations
may alter other components in essential oils that seems to be more specific to a particular Annona plant. This
article will give an overview of potential plants from Annona genus, their essential oil composition and pharmacological activities.
... Labrafil V R M 1944CS was received as a gift from Gattefoss e Co., Ltd (Nanterre Cedex, France). As described previously, Ganoderma lucidum polysaccharide was prepared by water extraction and purified by alcohol precipitation by our group (Zhu et al., 2006, Zhu et al., 2012Wang et al., 2018). Chitosan (MW 5 kDa) was purchased from Jinan Haidebei Marine Bioengineering Co., Ltd (Jinan, China). ...
... To validate the feasibility of GLP as a surfactant, we replaced RH40 with equivalent GLP in the preparation of MEs(PS-GLP). GLP was prepared by classical water extraction and alcohol precipitation method, and dialyzed by a 14,000 kD dialysis membrane in double distilled water for 24 h to eliminate small molecules interfering (Zhu et al., 2006, Zhu et al., 2012Wang et al., 2018). Furthermore, the experiment found that GLP has no obvious absorption peak at 260 in ultraviolet spectrum, suggesting no detectable nucleic acid existed in GLP. ...
The aim of this study is to explore the influence of Ganoderma lucidum-derived polysaccharides (GLP) to coix oil-based microemulsion on pharmaceutical performance and anti-lung cancer treatment. GLP-integrated coix oil-based microemulsion (MEs(PS-GLP)) exhibited a clear spherical shape, small particle size, and good hydrodynamics similar to the coix oil-based microemulsion, but showed a lower zeta potential and a better stability. Fluorescence resonance energy transfer analysis presented that GLP was integrated with microemulsion as a single system. Notably, the average molecular distance between polysaccharide and microemulsion was approximately 1.7 nm. The half-maximal inhibitory concentration of MEs(PS-GLP) against A549 cells was about 119 μg/mL. In vivo imaging studies showed that introduction of GLP promoted the tumor-specific accumulation of microemulsion in comparison with controls. In vivo, antitumor results showed that MEs(PS-GLP) markedly inhibited the tumor growth of A549-bearing xenograft nude mice and obviously improve the serum immune index. Collectively, this study demonstrates the potential mechanism of spatial relation between polysaccharides and microemulsion and validates the significances of GLP on tumoral accumulation and antitumor efficacy.
... There is considerable evidence to support the immunostimulating activities of G. lucidum via induction of cytokines and enhancement on immunological effector (Wang et al. 1997;Zhu and Lin 2006). Different components from G. lucidum were proved to enhance the proliferation and maturation of T and B lymphocytes, splenic mononuclear cells, NK cells, and dendritic cells in vitro and in animal studies in vivo (Bao et al. 2001;Cao and Lin 2002;Zhu, Chen, and Lin 2007;Ma et al. 2008). ...
... In addition to polysaccharides, a lanostane triterpenoid, ganoderic acid Me, inhibited tumor growth and metastasis of Lewis lung carcinoma in "T helper 1 responder" C57BL/6 mice by enhancing immune function in terms of IL-2 and IFN-γ expression and NK cell activity ). Zhu and Lin (2006) used cytokine-induced killer (CIK) cells to investigate the interaction between GL-PSs and cytokines, which mediated cell proliferation and antitumor activity. The cytotoxicity of CIK cells was correlated well with the expression of perforin and granzyme B induced by IL-2 and anti-CD3. ...
... Lakshmi et al. [44] studied the antimutagenic activity of methanolic extract of G. lucidum and its effect on hepatic damage caused by benzo [a] pyrene [44]. [47] showed that G. lucidum polysaccharides (Gl-PS) have a variety of immune modulating effects. These polysaccharides can modulate of cytokine production, granzyme B and perforin in murine CIK cells [47]. ...
... [47] showed that G. lucidum polysaccharides (Gl-PS) have a variety of immune modulating effects. These polysaccharides can modulate of cytokine production, granzyme B and perforin in murine CIK cells [47]. Liu et al. [48] found out that the triterpenoids fraction of G. lucidum might be a useful ingredient in the treatment of benign prostatic hyperplasia [48]. ...
Herbal medicines which formed the basis of health care throughout the world since the earliest days of mankind are still attracting more and more attention within the context of health care provision and health sector reform. Recording of their clinical, pharmaceutical and economic value is encouraging for international trading, through it varies widely between countries. A number of plant based traditional system of medicines have been in use in India and also in many parts of the world for ages. Before the advent of modern medicine, the traditional systems of medicine were playing a central role in healthcare. According to an estimate, majority of the world population, especially in the developing countries.
Numerous legends surrounding reishi mushroom provide an historical record which spans 2000 years. Traditionally, it was used in China by Taoist monks to promote a centered calmness, improve meditative practices, and attain a long and healthy life.
... Zhu et al reported the effect of polysaccharides with a molecular weight of >500 kDa, isolated from GL polysaccharides, in the promotion of granzyme B expression in cytokine-induced killer (CIK) cells (55). In that study, the promotion of granzyme B expression by GL polysaccharides, at the protein and mRNA level in vitro was demonstrated, but the in vivo effect of the polysaccharide was not fully elucidated. ...
Ganoderma, a medicinal mushroom with various physiological activities, has been extensively investigated regarding its effectiveness. The aim of the present study was to examine the effects of a subcritical water extract of Ganoderma (SWEG) on the immune system. The use of subcritical water with a higher temperature and pressure than hot water allows efficient elution of components from natural products. As an evaluation of the effectiveness of SWEG, a cell proliferation and a cell differentiation test were carried out using A-6 cells, a model of hematopoietic stem cells. Furthermore, an oral administration test in mice was conducted to examine the effects of SWEG on the number and function of immune cells. As a result, SWEG was revealed to promote both self-renewal and differentiation into immune cells such as T cells and natural killer (NK) cells in experiments with A-6 cells. These results were not obtained in experiments using hot water extract of Ganoderma lucidum and Ganoderma sinense. The oral administration test in mice demonstrated that SWEG increased hematopoietic precursor cells, immature B cells, and NK cells in the bone marrow, and T cells in the thymus. In addition, SWEG enhanced the immune functions in the spleen by promoting granzyme B expression and NK cell activity. SWEG was demonstrated to be a food material that acts on HSCs and regulates immunity in vivo.
... There is ample evidence of Ganoderma ability to boost immune monitoring, balance the immune system, and improve the diagnosis and destruction of cancer cells in the body [18]; [10]; [19]; [20]. Immuno system amplification is a major function of Ganoderma lucidum used in cancer patients. ...
Cancer has always been one of the leading causes of death worldwide, characterized by the proliferation and abnormal growth of cancer cells. While it is not yet clear how normal cells turn into cancer cells, several suggestions have been made that they may be due to viral sources or perhaps a change in the body's natural defenses (immune system). Recently, it has been suggested that certain individuals are genetically predisposed to cancer. It is therefore not surprising that at one age a person's immune system becomes vulnerable to certain types of cancer. Ganoderma lucidum is a medicinal fungus with woody tissue and a branch of basidiomycetes that has been used since ancient times to prevent and treat various diseases and increase health. The purpose of this study is to review some of the past scientific research on the anti-cancer properties of Ganoderma lucidum.
... Важливим напрямом досліджень у наш час є визначення можливості застосування препаратів грибів як лікувальних засобів при онкологічних захворюваннях. Одним із механізмів інгібування росту пухлин екстрактами грибів є підсилення функцій неспецифічного та адаптивного імунітету [7,11]. Найбільш вивченими видами грибів, які містять біологічні речовини, що здатні стимулювати імунну систему та пригнічувати розвиток новоутворень, є Ganoderma lucidum (рейші), Cordyceps sinensis, Lentinus edodes (шиітаке), Grifola frondosa (мейтаке), Hericium erinaceus та Agaricus blazei (химемацутаке) [8]. ...
The effect of the cultural medium and an extract of mycelium of the Leucoagaricus macrorhizus mushroom on the oxygen dependent metabolism of peritoneal macrophages of mice has been investigated. It has been shown that the cultural medium influenced on the indices of the oxygen dependent metabolism of the phagocytic cells under study. The greatest effect was demonstrated by the cultural medium in a concentration of 100 and 200 μg/ml, raising the oxygendependent metabolism by 168 % and 143 % respectively. An extract of mycelium in a concentration of 50 μg/ml lowered the oxygen-dependent metabolism by 11 % compared to the control value, an addition of other concentrations did not cause reliable changes.
... f allergenic proteins are heavily dependent upon the kinds of saccharides (Rao et al., 2018). Glycation can not only weaken the allergenicity of allergenic proteins through destroying and masking epitopes but also enhances the allergenicity of allergenic proteins via the generation of glycation products as new potent epitopes (Mueller et al., 2013;X. Zhu & Lin, 2006). Fu et al. (2019) indicated that after the reaction with GOS, the allergenicity of Penaeus chinensis tropomyosin was reduced by up to 60%. ...
Ovomucoid (OVM), known as the major allergen in egg white, has gained increasing concerns in industrialized countries. Here, we found the deglycosylation and Maillard reaction with galactooligosaccharide (GOS) and fructooligosaccharide (FOS) can induce conformational transformation of OVM from other structures (β‐turn, strang, and random coils) to α‐helix. We also introduced an approach to reduce the allergenicity of Gallus domesticus OVM by Maillard reaction with GOS and FOS. However, the OVM glycated by mannosan (MOS) and deglycosylated OVM exhibited higher allergenicity than native OVM. Therefore, GOS and FOS, especially GOS, could be applied in the reduction of the potential allergenicity of OVM through glycation. Furthermore, these findings may provide new insights into the development of hypoallergenic egg products.
Practical Application
In this study, the allergenicity and conformation of OVM treated with deglycosylation and glycation (GOS, FOS, and MOS) were investigated. The results would provide a better understanding of the effects of deglycosylation and Maillard reaction with different reducing sugars on the molecular characteristics of OVM and further provide new insights into the development of hypoallergenic egg products.
... Важливим напрямом досліджень у наш час є визначення можливості застосування препаратів грибів як лікувальних засобів при онкологічних захворюваннях. Одним із механізмів інгібування росту пухлин екстрактами грибів є підсилення функцій неспецифічного та адаптивного імунітету [7,11]. Найбільш вивченими видами грибів, які містять біологічні речовини, що здатні стимулювати імунну систему та пригнічувати розвиток новоутворень, є Ganoderma lucidum (рейші), Cordyceps sinensis, Lentinus edodes (шиітаке), Grifola frondosa (мейтаке), Hericium erinaceus та Agaricus blazei (химемацутаке) [8]. ...
Abstract. The effect of the cultural medium and an extract of mycelium of the Leucoagaricus macrorhizus mushroom
on the oxygen dependent metabolism of peritoneal macrophages of mice has been investigated. It has been shown that the
cultural medium influenced on the indices of the oxygen dependent metabolism of the phagocytic cells under study. The
greatest effect was demonstrated by the cultural medium in a concentration of 100 and 200 μg/ml, raising the oxygen�dependent metabolism by 168 % and 143 % respectively. An extract of mycelium in a concentration of 50 μg/ml lowered
the oxygen-dependent metabolism by 11 % compared to the control value, an addition of other concentrations did not cause
reliable changes.
Key words: Leucoagaricus macrorhizus, culture medium, extract of mycelium, peritoneal macrophages, oxygen�dependent metabolism.
... The fruit body of Ganoderma lucidum (Leyss ex Fr) Karst, also known as Reishi mushroom (Lingzhi, Ganoderma), is one of the most popular herbal dietary supplements used by cancer patients around the world. 15 Preclinical findings reveal that Reishi mushroom (Lingzhi) has chemopreventive, tumoricidal, and immunostimulating abilities, [16][17][18][19][20][21][22][23][24] increases the overall survival of tumor bearing mice, 25 alleviates chemotherapy-induced side effects, [26][27][28] and has a possible synergistic effect with cisplatin. 21 A number of clinical studies have suggested that G lucidum is well tolerated among cancer patients, has potential effects on immune modulation, and improves QoL and survival outcomes. ...
Background
Cancer patients often experience decreased quality of life during chemotherapy. This study aimed to determine the preliminary efficacy and safety of Reishi & Privet Formula (RPF) for maintaining quality of life among patients with non–small cell lung cancer (NSCLC) undergoing chemotherapy.
Methods
We conducted a phase II randomized, double-blind, placebo-controlled clinical trial in China. Adults with NSCLC scheduled to receive chemotherapy were randomly assigned (3:1 ratio) to receive oral RPF (3.36 g/day) or placebo daily for 6 weeks. The main outcome was the Functional Assessment of Cancer Therapy–Lung (FACT-L). We evaluated RPF’s safety profile using the Common Terminology Criteria for Adverse Events and assessed changes in outcome measures from baseline to weeks 3 and 6 using a linear mixed effects model.
Results
We enrolled 82 participants across 8 cancer centers in China. The median age was 59 years, 56 (68%) had advanced cancer. Compared with the placebo group, the RPF group had nonstatistically significant higher quality of life as measured by the FACT-L total score ( P = .086) over 2 cycles of chemotherapy. The RPF group was associated with a nonsignificant better general health ( P = .050) and emotional well-being ( P = .090) than the placebo group. Adverse events rates did not differ between groups.
Conclusions
This study demonstrated preliminary safety and suggests a promising trend in RPF’s effect on maintaining quality of life and emotional well-being among NSCLC patients undergoing chemotherapy. Future adequately powered randomized-controlled trials are needed to verify the efficacy and safety of RPF in cancer patients undergoing chemotherapy.
... The watersoluble extract of this mushroom is known to inhibit 15 types of bacteria. In 2008, Keypour [14,15] . The polysaccharides present in G. lucidum can function as an immunomodulator both in laboratory as well as in open condition. ...
Mushrooms are used as a nutritional rich food and used as traditional medicine to cure many life-threatening diseases since long ago. It is regarded as "Food of gods" by the Romans and a "wonder herb for the warriors" by the Greek and an "elixir of life by the Chinese". Among all major mushrooms, Ganoderma lucidum is the utmost important one with various therapeutic and pharmaceutical properties. The mycelium, spores and the fruiting bodies carry the medicinal properties. It is more popular in Asian countries but the amazing attributes have widespread throughout the world. It is the new focus of interest in modern pharmacological and biochemical research in recent years. The antimicrobial, anti-diabetic, anti-aging, antioxidant, anti-allergic, anti-inflammatory, immunomodulating functions of mushroom made it more popular among the drug industries. The existence of potential bioactive compounds such as polysaccharides, triterpenoids, proteins, peptides etc have amazing health benefits against hepatitis, hypertensions, asthma, gastric, insomnia etc. Ganoderma lucidum is proved as one of the best anti-cancer agents from decades due to its inhibitory effect on cancerous and tumour cells, protects from cell proliferation etc. This review describes about all the amazing health benefits of G. lucidum. But more research is required in near future to discover novel bioactive compounds and mass production of this medicine which will help the mankind.
... (2007) Polysaccharides of G. lucidum stimulate apoptosis, hamper cell propagation and restrain cell relocation of extremely persistent cells of human prostate cancer. The antitumor activity of G. lucidum polysaccharides mechanism is advanced all the way through stimulus of host-resistance response (Lu et al., 2004;Zhu & Lin, 2006). Having least cytotoxicity of ganodermas, their utilization may raise the prospect of immune confrontation and diminished toxicity. ...
From the time immemorial, Ganoderma lucidum generally called as Reishi, is utilized as healthcare constituent, well-being, and longevity in developing countries. It is a basidiomycete rot fungus thus, most important resource for the manufacture of innate fungal medicines. Because of its medicinal value Ganoderma has been utilized for the cure and healing of different diseases for several years. With the use of murine and human cell lines on anticancer characteristics of G. lucidum have been verified by numerous studies, both in vitro and in vivo examinations. Rigorous investigations proved that G. lucidum presents a range of pharmacological active constituents, namely, anticancer, cardioprotective, hepatoprotective, antioxidant, immune modulatory, antihypertensive, and antidiabetic, etc. Furthermore, these experimented anticancer constituents extracted from Ganoderma have encouraged its convention to cancer patients together with chemotherapy. Discovery of innate resource proposed for new and fresh bioactive composites proved to be a promising source of drugs over the ancient times, presenting medicines or lead compounds of substantial remedial prospective. Ganoderma is found to have wide-ranging relevance for curing cancer through the immune system regulation. Ganoderma exercises multiple mechanisms for the destruction of cancer cells and perhaps has probable curative exercise for the anticipation as well as management of the deadly disease. This book chapter is designed to assist the communities improved understanding of variety and prospective of macro fungi paving way to additional systematic examination and therefore, extra-efficient management and employment.
... Важливим напрямом досліджень у наш час є визначення можливості застосування препаратів грибів як лікувальних засобів при онкологічних захворюваннях. Одним із механізмів інгібування росту пухлин екстрактами грибів є підсилення функцій неспецифічного та адаптивного імунітету [7,11]. Найбільш вивченими видами грибів, які містять біологічні речовини, що здатні стимулювати імунну систему та пригнічувати розвиток новоутворень, є Ganoderma lucidum (рейші), Cordyceps sinensis, Lentinus edodes (шиітаке), Grifola frondosa (мейтаке), Hericium erinaceus та Agaricus blazei (химемацутаке) [8]. ...
Abstract. The effect of the cultural medium and an extract of mycelium of the Leucoagaricus macrorhizus mushroom on the oxygen dependent metabolism of peritoneal macrophages of mice has been investigated. It has been shown that the cultural medium influenced on the indices of the oxygen dependent metabolism of the phagocytic cells under study. The greatest effect was demonstrated by the cultural medium in a concentration of 100 and 200 μg/ml, raising the oxygen- dependent metabolism by 168 % and 143 % respectively. An extract of mycelium in a concentration of 50 μg/ml lowered the oxygen-dependent metabolism by 11 % compared to the control value, an addition of other concentrations did not cause reliable changes.
Key words: Leucoagaricus macrorhizus, culture medium, extract of mycelium, peritoneal macrophages, oxygen- dependent metabolism.
... CML), it is difficult to predict how glycation of allergen with different saccharides affects the allergenicity. immunosuppressive effects on lymphocyte proliferation and immune activity (Zhu & Lin, 2006), while there are very few reports about their effects on allergenicity (Fu, Wang, Wang, Ni, & Wang, 2019). This study investigated how glycation of shrimp TM by functional oligosaccharides affected its allergenicity. ...
... These lymphocytes were pulsed with P815 tumor antigens during the stage of antigen presentation and the reported mechanisms of cytotoxicity involved the IFNγ and granzyme B pathways. In addition, the found that GLPS (400 or 100 mg/mL), which promoted CIK cell proliferation and cytotoxicity, enhanced IL-2 and TNF production, and the protein and mRNA expression of granzyme B and perforin in CIK cells through a synergistic interaction with cytokines, decreasing doses of IL-2 and anti-CD3 by 75 and 50%, respectively, which might be irrelevant to nitric oxide (NO) (Zhu and Lin, 2006 ...
Ganoderma is a significant source of natural fungal medicines and has been used for the treatment of various diseases for many years. However, the use of Ganoderma in cancer immunotherapy is poorly elucidated. In this study, we have analyzed 2,398 English-language papers and 6,968 Chinese-language papers published between 1987 and 2017 by using bibliometrics. A steady growth in the number of publications was observed before 2004, followed by an exponential increase between 2004 and 2017. The most common category for publications about Ganoderma was “Pharmacology & Pharmacy,” in which immunomodulation (25.60%) and cancer treatment (21.40%) were the most popular subcategories. Moreover, we have provided an overview of the bioactive components and combinatorial immunomodulatory effects for the use of Ganoderma in the treatment of cancer, including the major pathways of immune cells. Immunomodulatory protein and polysaccharides are the key bioactive factors responsible for cancer immunotherapy, and the NF-κB and MAPK pathways are the most comprehensively investigated major pathways. Our results indicate that Ganoderma has a broad-spectrum application for the treatment of cancer through the regulation of the immune system. This review provides guidance for future research into the role of Ganoderma in cancer immunotherapy.
... [25] Polysaccharides isolated from botanical sources represent an important class of compounds because a diversity of biological activities have been associated with them, including antitumor, [26] antioxidant, [27] anti-inflammatory, [28] and immunomodulating agent. [29,30] They can modulate the immune responses via their stimulatory activity on macrophages which enhance phagocytosis, antigen processing ability, production of reactive oxygen species and nitric oxide (NO), and secretion of cytokines and chemokines, such as tumor necrosis factor-α (TNF-α), interferon (IFN)-γ, interleukin (IL)-1β, IL-6, IL-8, IL-12, and IFN-β2. [31,32] Flavonoids are the most common group of secondary plant metabolites that are widely distributed throughout the plant kingdom. ...
Objective: The present study aims to optimize the extraction conditions of polysaccharides and flavonoids from a polyherbal preparation consisting of three kinds of Chinese medicinal herbs, Codonopsis pilosula, Crataegus pinnatifida, and Lycium barbarum, and evaluation of its immunomodulatory activity in immunosuppressed mice. Materials and Methods: An orthogonal design (L9 [3]4) was constructed to achieve the optimal extraction conditions. The immunomodulatory action of the polyherbal preparation was studied at three doses of 10, 20, and 40 mL/kg/day orally by measuring splenocyte proliferation in mice model of cyclophosphamide-induced immunosuppression. Results: The chosen parameters, including the ratio of solvent-to-raw material, duration of extraction, and extraction times, were the fundamental variables that influenced the extract yields. The highest yield of total polysaccharides content was 54.3 mg/mL when the ratio of solvent-to-raw material, duration of extraction, and number of extractions were 12:1, 1.5 h, and 3, respectively. The maximum extraction yield of the flavonoids was 3.5 mg/mL when the ratio of solvent-to-raw material was 12:1, the extraction time was 2 h, and the number of extractions was 3. The prescription screening showed that the impact of the polyherbal preparation on the splenocyte proliferation capacity was more pronounced than its disassembled components. Oral administration of the polyherbal preparation could significantly increase the concanavalin A-stimulated mouse spleen cells proliferation in a dose-dependent manner. Conclusion: These findings suggested that the polyherbal preparation possesses potential for augmenting the immune activity due to the polysaccharide and flavonoid content in these herbal medicines.
... Polysaccharides from G. lucidum was a promising biological response modifier and immune potentiator [1]. It was found that the polysaccharide extracts from the mycelium of G. lucidum had anti-tumor effects against fibro sarcoma in male and female mice and inhibited the metastasis of a tumor to the lung [2]. The fruit bodies of G. lucidum grow slowly in nature, their growth is restricted to a specific area. ...
The carcinostatic substance in Ganoderma lucidum (Fr.) Karst (Polyporaceae) is a water soluble polysaccharides (WSP) which might be useful in immunotherapy. Attempt to produce effective substances from cultured mycelia is important to carry out since solid cultivation is a time consuming and quality fluctuating. The effects of cultivating conditions on the water soluble polysaccharides content of G. Lucidum mycelium were investigated in submerged flask cultures. Culture from fruiting bodies was maintained on potato dextrose-agar slope. Slopes were inoculated and incubated at 30°C for 7 days, and stored at 4°C. The flask experiments were performed in 100 ml erlenmeyer flasks containing 20 ml of the sterilized media. Actively growing mycelia (1 piece, 5 mm X 5 mm) from a newly prepared slant culture (about 7 days incubation at 30°C) were inoculated into the flask. The pH was measured and adjusted to the desired value by addition of either 4 M HCl or 2.5 M NaOH. Incubation temperature were 20, 25, and 30°C. At the end of inoculation period (14 days) mycelium consisting of individual pellets was harvested and wash for the analysis. WSP content was analysed using phenol-sulfuric acid method. The optimal initial pH for metabolite production would depend on the culture medium. Generally, high values of pH, such as 9, negatively affect both cell growth and WSP production. The optimum temperature range for the high G. lucidum mycelium and WSP production were found to be 25 – 30 °C at pH values 5 – 7 in both of media.
... Spleen was aseptically removed from sacrificed rat (6 groups) in PBS and total RNA was extracted using TRIzol Reagent (Invitrogen, USA) [40]. The concentration of RNA was quantified by UV spectrophotometer (Lark UV 2500, Chennai, India). ...
Indigofera tinctoria and Scoparia dulcis are being widely used in Indian folk medicine for the treatment of various disorders. Environmental noise pollution is thought to be an important factor for many health problems and it causes immune abnormalities. In the present study immune-regulating potential of I. tinctoria and S. dulcis aqueous extracts on innate and adaptive immune system of wistar albino rats was evaluated during normal and chronic noise induced stress conditions. The results demonstrated that both I. tinctoria and S. dulcis aqueous extracts (200mg/kg b.w) showed immunostimulant effect on both innate and adaptive immune response of wistar albino rat compared to control group under normal condition. The noise stress (100dB for 1hour, 20 days) induced animals showed suppressive effects on immune response by decreasing macrophage phagocytosis, antibody secretion by spleen cells, humoral immune response, proliferation of lymphocytes, cytotoxicity, TNF α expression, granzyme B and perforin expression in splenic NK cells. Similarly, noise stress also caused DNA damage in tissues. However, the suppressed effects induced by noise stress on rat immune system were significantly prevented by oral administration of both I. tinctoria and S. dulcis aqueous extracts. Considering all these results it is suggested that the selected medicinal plant’s aqueous extracts have the potential to prevent the effects of noise stress induced rat immune system and explore a strong immunostimulant potential applicable to clinical practices.
... Cytokine-induced killer (CIK) cells are derived from peripheral blood mononuclear cells (PBMCs), which are usually obtained from the peripheral blood or cord blood. Three cytokines were added in the course of the cell culture: rh IFN-γ was used to induce some immune accessory molecules secretion and enhance the antitumor function of CIK cells [38]; CD3 monoclonal antibody was added into the culture liquid and induced higher level of cytotoxic activity and proliferation rate of human CIK cells [31]; added cytokine rhIL-2 can get a higher proliferation rate of CIK cells [32]. Schmidt-Wolf [13] et al. reported that the higher lytic activity of CIK cells as compared to LAK cells was mainly due to the higher proliferation of CD3 + CD56 + T cells. ...
How to realize targeted photoacoustic imaging, enhanced immunotherapy, and photothermal therapy of gastric cancer has become a great challenge. Herein, we reported for the first time that human cytokine-induced killer cells (CIK) loaded with gold nanorods were used for targeted photoacoustic imaging, enhanced immunotherapy, and photothermal therapy of gastric cancer. Silica-modified gold nanorods were prepared; then incubated with human cytokine-induced killer cells (CIK), resultant human CIK cells loaded with Au nanorods were evaluated for their cytotoxicity, targeted ability of gastric cancer in vitro and in vivo, immunotherapy, and photothermal therapy efficacy. In vitro cell experiment shows that human CIK cells labeled with gold nanorods actively target gastric cancer MGC803 cells, inhibit growth of MGC803 cells by inducing cell apoptosis, and kill MGC803 cells under low power density near-infrared (NIR) laser treatment (808-nm continuous wave laser, 1.5 W/cm(2), 3 min). In vivo experiment results showed that human CIK cells labeled with gold nanorods could target actively and image subcutaneous gastric cancer vessels via photoacoustic imaging at 4 h post-injection, could enhance immunotherapy efficacy by up-regulating cytokines such as IL-1, IL-12, IL-2, IL-4, IL-17, and IFN-γ, and kill gastric cancer tissues by photothermal therapy via direct injection into tumor site under near-infrared (NIR) laser irradiation. High-performance human CIK cells labeled with Au nanorods are a good novel theranostic platform to exhibit great potential in applications such as tumor-targeted photoacoustic imaging, enhanced immunotherapy, and photothermal therapy in the near future.
... Polysaccharides from natural sources have attracted increasing attention in recent years due to their potential biological functions, especially their antioxidant and immunomodulation activities, such as scavenging free radical, inhibiting lipid oxidation, promoting natural killer (NK) cell cytotoxicity, activating macrophages and potentiating interleukins (Song, Zhang, Zhang, & Wang, 2010;Yang, Zhao, & Lv, 2008;Zhu & Lin, 2006). The bioactivity of polysaccharides mainly depends on several structural parameters, including the sugar composition, molecular weight, type of glycoside bond, and degree of sulfation (Li, Huang, Lu, & Hou, 2011). ...
... Anti-Ulcerogenic Polysaccharides [5][6][7] Anti-oxidant Polysaccharides [85] Inhibition of angiogenesis induced by solid tumors; growth-inhibition of bladder cancer in rats; inhibition of iron-dependent lipidic peroxidation of membranes; reduction of pain associated to inflammation; reduction in the incidence of cardiovascular diseases and antimicrobial activity; Anti-complement activity against the classical pathway of the complement system ...
Ganoderma lucidum is a basidiomycetes white rot fungus belongs to the genus Ganoderma which has been used for medicinal purpose for centuries particularly in eastern countries like China, Japan & Korea. The basidiocarp, mycelia & spores of Ganoderma lucidum contain approximately 400 different bioactive compounds. The metabolites consist of mainly triterpenoids & polysaccharides & protiens. To date more than 140 triterpenoids & 200 polysaccharides have been isolated from different species Ganoderma. These compounds has been reported to have a number of pharmacological effects including immunomodulation, anti-atherosclerotic, anti-inflammatory, analgesic, chemopreventive, antitumor, chemo & radio protective, sleep promoting, antibacterial, antiviral (including anti-HIV), hypolipidemic, anti-fibrotic, hepatoprotective, anti-diabetic, anti-&rogenic, anti-angiogenic, anti-herpetic, antioxidative & radical-scavenging, anti-aging, hypoglycemic, estrogenic activity & anti-ulcer properties. The biological activities reported in Ganoderma lucidum, due to the wide range of structural diversity found in its metabolites. This review collates the publications which reported the different bioactive metabolites from this medicinal mushroom considering the most valid claim & research on biological activities of compounds.
... Currently, polysaccharides from natural sources have attracted increased attention due to their potential biological functions, especially antioxidant and immunomodulation activities such as scavenging free radicals, inhibiting lipid oxidation, promoting natural killer cells (NK) cytotoxicity, and activating macrophages and interleukins [1][2][3][4]. The bioactivity of polysaccharides mainly depends on OPEN ACCESS several structural parameters including sugar composition, molecular weight, type of glycosidic bond, and degree of sulfation [5]. ...
The water-extractable (QWP) and the alkali-extractable (QAP) polysaccharides from quinoa (named QWP and QAP, respectively) and their four polysaccharide sub-fractions (QWP-1, QWP-2, QAP-1 and QAP-2), were isolated and purified by anion-exchange and gel filtration chromatography. QWP-1 and QWP-2 were composed of Rha, Ara, Gal and GalA. QAP-1 and QAP-2 were composed of Rha, Ara, Man, Gal and GalA. Antioxidant and immunoregulatory activities of the polysaccharides were evaluated. The results showed that QWP-1, QWP-2, QAP-1 and QAP-2 had significant antioxidant and immunoregulatory activities. The results suggest that QWP-1, QWP-2, QAP-1 and QAP-2 could be used as potential antioxidants and immunomodulators.
... The FBGs are released from the cells and activate NK cells and granulocytes by binding to CR3 (Chan et al., 2009). These activated NK cells release perforins and granzymes, which make pores and disintegrate the DNA of tumor cells, respectively (Zhu and Lin, 2006). Macrophages and neutrophils are activated by IFN-g and IL-17A secreted by Th1 and Th17 lymphocytes, respectively, and provide protection against the infecting fungi (Fig. 1). ...
During the course of evolution, animals encountered the harmful effects of fungi, which are strong pathogens. Therefore, they have developed powerful mechanisms to protect themselves against these fungal invaders. β-Glucans are glucose polymers of a linear β(1,3)-glucan backbone with β(1,6)-linked side chains. The immunostimulatory and antitumor activities of β-glucans have been reported; however, their mechanisms have only begun to be elucidated. Fungal and particulate β-glucans, despite their large size, can be taken up by the M cells of Peyer's patches, and interact with macrophages or dendritic cells (DCs) and activate systemic immune responses to overcome the fungal infection. The sampled β-glucans function as pathogen-associated molecular patterns (PAMPs) and are recognized by pattern recognition receptors (PRRs) on innate immune cells. Dectin-1 receptor systems have been incorporated as the PRRs of β-glucans in the innate immune cells of higher animal systems, which function on the front line against fungal infection, and have been exploited in cancer treatments to enhance systemic immune function. Dectin-1 on macrophages and DCs performs dual functions: internalization of β-glucan-containing particles and transmittance of its signals into the nucleus. This review will depict in detail how the physicochemical nature of β-glucan contributes to its immunostimulating effect in hosts and the potential uses of β-glucan by elucidating the dectin-1 signal transduction pathway. The elucidation of β-glucan and its signaling pathway will undoubtedly open a new research area on its potential therapeutic applications, including as immunostimulants for antifungal and anti-cancer regimens.
... It has therefore stimulated increasing interest in biomedical applications. More interestingly, OCMCs can load hydrophobic anticancer drugs effectively [19][20][21] and also immobilize a targeting agent such as folic acid (Fol). Several studies have recently reported that OCMCs-Fol is a potential targeted drug delivery system [22][23][24][25]. ...
Curcumin (Cur) is a yellow compound isolated from rhizome of the herb curcuma longa. Curcumin possesses antioxidant, anti-inflammatory, anti-carcinogenic and antimicrobial properties, and suppresses proliferation of many tumor cells. However, the clinical application of curcumin in cancer treatment is considerably limited due to its serious poor delivery characteristics. In order to increase the hydrophilicity and drug delivery capability, we encapsulated curcumin into copolymer PLA-TPGS, 1,3-beta-glucan (Glu), O-carboxymethyl chitosan (OCMCs) and folate-conjugated OCMCs (OCMCs-Fol). These polymer-encapsulated curcumin nanoparticles (Cur-PLA-TPGS, Cur-Glu, Cur-OCMCs and Cur-OCMCs-Fol) were characterized by infrared (IR), fluorescence (FL), photoluminescence (PL) spectra, field emission scanning electron microscopy (FE-SEM), and found to be spherical particles with an average size of 50–100 nm, being suitable for drug delivery applications. They were much more soluble in water than not only free curcumin but also other biodegradable polymer-encapsulated curcumin nanoparticles. The anti-tumor promoting assay was carried out, showing the positive effects of Cur-Glu and Cur-PLA-TPGS on tumor promotion of Hep-G2 cell line in vitro. Confocal microscopy revealed that the nano-sized curcumin encapsulated by polymers OCMCs and OCMCs-Fol significantly enhanced the cellular uptake (cancer cell HT29 and HeLa).
... It has been shown that many natural products have immunomodulatory and antitumor effects (7)(8)(9)(10)(11)(12)(13)(14)(15), as well as Gl-PS. The multiple biological activities of Gl-PS include improvement of host immune function (16), prevention of oxidative damage (17), protection of liver and reduction of serum glucose levels, along with a lack of toxicity (18,19), modification of biological response and potentiation of immune effectiveness (20). Previous studies demonstrated the effects of Gl-PS on acceleration of wound repair in intestinal epithelial cells (21), antagonism against the tumor-induced immunosuppression (22), promotion of B16F10 cells to activate lymphocytes (23), induction of stronger cytotoxicity in cytotoxic lymphocytes (CTLs) with granzyme B and porforin by action on B16F10 cells (24), and enhancement of major histocompatability complex (MHC) class I and costimulatory molecules on B16F10 cells to induce stronger anti-B16F10 cytotoxicity in lymphocytes (25). ...
Immune responses to tumor-associated antigens are often detectable in tumor-bearing hosts, but they fail to eliminate malignant cells or prevent development of metastases. Tumor cells produce factors such as interleukin-10, transforming growth factor-β1 and vascular endothelial growth factor (VEGF) that suppress the function of immune cells or induce apoptosis of immune cells. Culture supernatant of tumor cells may contain these immunosuppressive factors which suppress lymphocyte activation. CD71 and FasL are two important molecules that are expressed upon lymphocyte activation. Counteraction against suppression CD71 and FasL expression upon lymphocyte activation may benefit tumor control. A potential component with this effect is Ganoderma lucidum polysaccharides (Gl-PS). In this study, Gl-PS was used on lymphocytes incubating with culture supernatant of B16F10 melanoma cells (B16F10-CS) in the presence of phytohemagglutinin. Following induction with phytohemagglutinin, B16F10-CS suppressed CD71 expression in lymphocytes (as detected by immunofluorescence and flow cytometry), proliferation in lymphocytes (as detected by MTT assay), and FasL expression in lymphocytes (as detected by immunocytochemistry and western blot analysis), while Gl-PS fully or partially counteracted these suppressions. Gl-PS showed counteractive effects against suppression induced by B16F10-CS on CD71 and FasL expression upon lymphocyte activation, suggesting the potential of Gl-PS to facilitate cancer immunotherapy.
Electrospun biopolymer fibers are utilized in a wide variety of industries such as tissue engineering, sensors, drug delivery, membrane filtration, and protective membranes. The biopolymer chitosan, the partially N ‐deacetylated derivative of chitin, which has been the focus of many studies, contains amine or hydroxyl functionalities that may be substituted with a number of chemistries such as carboxylate, benzene, or cyano groups. Modified chitosan solutions are often challenging to electrospin, as an entirely new set of solution and operating conditions must be developed for each modification. In this study, a facile post‐modification processing method for chitosan is introduced that circumvents the need to perform bulk modification prior to electrospinning, and therefore new spinning conditions. The chitosan mats were solution‐phase post‐processed by chemically functionalizing the mats with carboxylate, benzene and cyano groups. Scanning electron microscopy and Fourier‐transform infrared have been performed to determine fiber morphology retention and chemical interactions, respectively. Post‐modification retained the fibrous structure of the white‐colored, round and smooth mats with spectral changes indicating changes in the chitosan mat. Mean fiber diameters were 131 ± 75 nm (~31% smaller), 210 ± 81 nm (46% larger), and 85 ± 29 nm (~11% smaller) for carboxymethylchitosan, benzylidenechitosan, and cyanochitosan, respectively.
In recent decades the traditional Chinese medicinal mushroom Ganoderma lucidum (GL), a fungal specie widely consumed homoeopathically in the Eastern Hemisphere, has been studied particularly with respect to antitumour and immunoenhancing effects. Research into the various claims however remains limited owing to the lack of quality and consistency across investigations. As such, efficacy and feasibility of scaleup has not been evaluated in a way that allows widespread consumption or approved treatment. This project tackles three aspects of drug development from Ganoderma lucidum: Biocompound extraction, healthcare evaluation via in-vitro testing, and encapsulation for smart delivery. These avenues are brought together for the first time to evaluate the prospects of developing GL for effective and safe healthcare. This research investigates the parameters that would influence the extractability of a biocompound from the spores of Ganoderma lucidum (GLS), via two conventional methods: Hot Water Extraction (HWE) and Ultrasound-Assisted Extraction (UAE). They are evaluated with respect to their crude water-soluble polysaccharide yield (GLPS). Solvent polarity and process duration were statistically significant factors affecting extract yield, with both extraction methods showing considerable gains over similar setups in literature, recovering over 6% crude GLPS using shorter durations and lower temperatures than other published investigations. This investigation highlighted the importance of solvent viscosity on specific DGlucan extraction in the GLPS yield. Bioactive effects of the extract were evaluated via cytotoxicity toward Human Osteosarcoma (HOS) cells in-vitro, achieving over 40% cell growth inhibition. Cytotoxicity however was only achieved when water-insoluble fractions were administered – suggesting cytotoxicity was a result of the unextracted crude triterpenoids (GLTP) containing Ganoderic Acids. Therefore, HOS-inhibitory capabilities are then compared to a GLPS extract containing Ganoderic Acids (in this work termed “PSGA”), extracted using HWE subject to supervised machine learning optimisation. As well as determining that this yield was maximised at the longest HWE duration and smallest solvent volume, it was observed to inhibit HOS growth by nearly 58% after 24 hours. Low doses and shorter incubation were most effective - suggesting concepts such as resistance (clonal selectivity) and delayed apoptosis, but further work will verify the reported effects of PSGA dosage and exposure time on cancer proliferation. Lastly, research effort is devoted to creating an alginate matrix for the controllable delivery of GLS using Electrohydrodynamic Atomisation (EHDA). Significant effects of the system’s process parameters on particle morphology are observed, in particular EHDA voltage. The carrier’s size, shape and surface features are correlated with its release profile. Importantly, GLS content (something traditionally compromised to maintain particle integrity) was maximised at 50 wt% whilst maintaining a controlled and spherical shape and size – making this study novel and extremely important. It is established that GLS-Alginate particles could offer controlled release over a 2-week administration in pH-neutral conditions; an environment not yet established as “stable” for alginate, yet reflective of physiological passage. Thus, for the first time sodium alginate is proven to be a real contender in controlling the delivery of GLS biomolecules. The reconciliation of these essential stages of drug development highlights some crucial points of focus as GL continues to undergo rigorous development in the realm of drug discovery.
Ganoderma (also known as Lingzhi) is an important source of natural medicines. It contains many well-known chemical components such as triterpenes, terpenes, alkaloids, steroids, nucleotides, nucleobases, polysaccharides, and so on. Lingzhi exhibits broad-spectrum biological activities and has been utilized as a traditional drug and functional food for the treatment of various diseases for over two thousand years in China. Particularly, Lingzhi has shown significant efficacy as an antitumor agent, which has immunomodulatory, anti-inflammatory, anticancer, and antioxidant activities both in preclinical and clinical studies. However, few studies have focused on the usage of Lingzhi in cancer immunotherapy. Here, we reported a comprehensive view of the active chemical compositions and immunomodulatory actions of the Lingzhi for treating various cancers and the main signaling pathways of immune cells in response to Lingzhi treatment. Additionally, we demonstrated that polysaccharides and immunomodulatory proteins of Lingzhi represent the core chemical compositions underlying the cancer immunotherapeutic activities. In the meantime, the NF-κB and MAPK pathways are the primary pathways related to the effects of Lingzhi. Moreover, the toxicology and clinical studies of Lingzhi are also summarized in this chapter. The results imply that Lingzhi have a wide range of applications for cancer treatment through regulating the immune system. The literature review offers valuable and informative references that warrant further investigation of Ganoderma's potential cancer immunotherapy applications.
Ganoderma lucidum spore oil (GLSO) has been long practiced and widely recognized as a healthy food composite performing potential anticancer and antioxidant activity. However, its further application might be limited by the native poorly soluble and instable properties. Herein, size-tunable GLSO nanosystems have been designed and tailored to three sizes of particles at 20 nm, 40 nm and 80 nm by high-pressure homogenization, for achieving robust therapeutic effect against cancer cells. As expected, toxicity of biocompatible different-sized [email protected] on MGC803 cancer cells significantly enhanced, thanks to the improved cellular uptake and retention efficacy of [email protected] In addition, middle-sized 40 [email protected] performed superior permeability across Caco-2 intestinal absorption barrier to improve internalized quantity of GLSO. More importantly, [email protected] could effectively prevent cell migration and invasion, simultaneously activating caspase activity to trigger cell death. Interestingly, middle size of 40 [email protected] performed superior accumulation efficacy in cells. Ultimately, 40 [email protected] showed potent suppression effect on tumors in vivo, effectively reversing liver and kidney damage induced by cancer, which was accompanied with no obvious toxicity and side effects during treatment. Therefore, this study not only provides a simple method for preparation of GLSO-like traditionally functional composite by high-pressure homogenization, but also sheds light on the application in future clinical cancer therapy.
The antitumor effect of Ganoderma (Lingzhi) is closely related to immunoregulation. Based on our research and other references, this article discussed the antitumor effect of Ganoderma mediated by immunological mechanism, including promoting the function of mononuclear-macrophages and natural killers; promoting M1-type macrophage polarization vs M2-type; promoting maturation and differentiation of dendritic cells, increasing its antigen presentation, activating lymphocytes and increasing cytotoxicity of cytotoxin T lymphocyte; promoting production of cytokines; and inhibiting tumor escape from immune surveillance. Also, clinical studies with immunological indexes were reviewed.
Traditionally, Mushrooms are considered as an important natural source of food and
medicines. Ganoderma lucidum, (Ganodermataceae) an oriental fungus, has a long history of use for
promoting health and longevity in China, Japan, Malasiya, Japan and other Asian countries. G.
lucidum may be Reishi (Japanese) and Lingzhi (Chienese) type. Reishi mushroom has also been
commonly referred to as the immortality or Ten thousand year or Spiritual potency or spirit plant
mushroom. Various polysaccharides (i.e., β-D-glucans and glycoproteins) and triterpenoids are the
major active constituents present in Ganoderma. Ganoderma is an Adaptogen and contains up to
400 different nutrients. Nutritionally G. lucidum provide folin-positive materia, glucose, protein and
metals like K, Mg Ca, Se, Fe, Zn and Cu. G. lucidum therapeutically used for cancer, hypertension,
Diabetes, Tumors, antiaging, Infections and Immunity disorder. Biologically, G. lucidum behaves as
antioxidative, radical-scavenging effects, enhancement of host immune function, induction of cellcycle
arrest and apoptosis. A variety of commercial G. lucidum products are available in various
forms, such as powders, dietary supplements, and tea. Globally with aid of ornamental, nutritional
and therapeutic values, G. lucidum are cultivated in challenging manner with high economic and
commercialize values.
Background
The global interest in edible medicinal herbs for healthcare has significantly increased during the last few years.Ganoderma lucidum is a medicinal mushroom which is known to be a potential source of many therapeutic and pharmaceutical products with significant health importance.
Methodology
The available literature using PubMed, Scopus and Google Scholar database was thoroughly reviewed using the keywords Natural Products, Ganoderma, secondary metabolites and therapeutics. This narrative review of all the relevant papers with significant citations leads the authors to greater insight into the potential therapeutic significance of Ganoderma lucidum.
Results
The presence of a wide array of secondary metabolites in this herb contributes to its pharmaceutical uses.G. lucidum is rich in polysaccharides (β-glucan,mannitol), alkaloids, and a group oftriterpenes (ganoderic acid). Many cellular mechanisms have been proposed to explain the mode of action of its active metabolites and their healthcare attributes including anticancer, antiviral, antioxidant and protective effects on liver and other secondary lymphoid organs.
Conclusion
This review illustrates the broad spectrum therapeutic potential of secondary metabolites derived fromGanoderma and supports our understanding of the main pharmacologically active compounds present in this fungus. Insight into the actions of its secondary metabolites could further pave a way for establishing G. lucidum, as a pharmacologically important product.
Ganoderma lucidum is traditionally applied in various countries for treatment of variety of diseases. It is a prime example of an ancient remedy being of immense relevance to the modern era and known as mushroom of immortality as well as spirit mushroom. It has been used as a health promoting agent due to its wide pharmacological and nutraceutical activity and availability. Increasing awareness about fitness and health-promoting capability of G. lucidum pull the majority of people to get healthier lifestyles, propelling expansion of its market globally. The importance of Ganoderma lucidum has fascinated scientific community to carry out extensive phytochemical and pharmacological investigations. Different studies have been carried out, and a broad spectrum of its pharmacological actions has been established which include immunomodulatory, anticancerous, antioxidant, antidiabetic, hepatoprotective, neuroprotective, cardioprotective, antimicrobial, anti-inflammatory, anti-allergic, antiosteoporotic, suppression of angiogenesis, and antinociceptive properties. The current review is an attempt to explore the reported chemical composition and pharmacological activity. It will act as a reference for all researchers, scientists, and other health professionals who are working on this mushroom.
Traditionally, Mushrooms are considered as an important natural source of food and
medicines. Ganoderma lucidum, (Ganodermataceae) an oriental fungus, has a long history of use for
promoting health and longevity in China, Japan, Malasiya, Japan and other Asian countries. G.
lucidum may be Reishi (Japanese) and Lingzhi (Chienese) type. Reishi mushroom has also been
commonly referred to as the immortality or Ten thousand year or Spiritual potency or spirit plant
mushroom. Various polysaccharides (i.e., β-D-glucans and glycoproteins) and triterpenoids are the
major active constituents present in Ganoderma. Ganoderma is an Adaptogen and contains up to
400 different nutrients. Nutritionally G. lucidum provide folin-positive materia, glucose, protein and
metals like K, Mg Ca, Se, Fe, Zn and Cu. G. lucidum therapeutically used for cancer, hypertension,
Diabetes, Tumors, antiaging, Infections and Immunity disorder. Biologically, G. lucidum behaves as
antioxidative, radical-scavenging effects, enhancement of host immune function, induction of cellcycle
arrest and apoptosis. A variety of commercial G. lucidum products are available in various
forms, such as powders, dietary supplements, and tea. Globally with aid of ornamental, nutritional
and therapeutic values, G. lucidum are cultivated in challenging manner with high economic and
commercialize values.
Traditionally, Mushrooms are considered as an important natural source of food and
medicines. Ganoderma lucidum, (Ganodermataceae) an oriental fungus, has a long history of use for
promoting health and longevity in China, Japan, Malasiya, Japan and other Asian countries. G.
lucidum may be Reishi (Japanese) and Lingzhi (Chienese) type. Reishi mushroom has also been
commonly referred to as the immortality or Ten thousand year or Spiritual potency or spirit plant
mushroom. Various polysaccharides (i.e., β-D-glucans and glycoproteins) and triterpenoids are the
major active constituents present in Ganoderma. Ganoderma is an Adaptogen and contains up to
400 different nutrients. Nutritionally G. lucidum provide folin-positive materia, glucose, protein and
metals like K, Mg Ca, Se, Fe, Zn and Cu. G. lucidum therapeutically used for cancer, hypertension,
Diabetes, Tumors, antiaging, Infections and Immunity disorder. Biologically, G. lucidum behaves as
antioxidative, radical-scavenging effects, enhancement of host immune function, induction of cellcycle
arrest and apoptosis. A variety of commercial G. lucidum products are available in various
forms, such as powders, dietary supplements, and tea. Globally with aid of ornamental, nutritional
and therapeutic values, G. lucidum are cultivated in challenging manner with high economic and
commercialize values.
The chemical modification of chitosan via polymer-analogous reactions and grafting and block copolymerizations with vinyl monomers in liquid media and in the solid phase is considered. The main attention is given to factors influencing the efficiency of copolymerization processes, such as the nature of a monomer and initiator, the molecular mass, the degree of deacetylation, and the conformation of chitosan macromolecules (pH of a medium), and temperature. The effects of composition and structure of the copolymers on their properties are ascertained. Some prospects for the application of chitosan derivatives are analyzed.
ABSTRACT
As functional materials, chitin and chitosan offer a unique set of characteristics: biocompatibility, biodegradability to harmless products, nontoxicity, physiological inertness, antibacterial properties, heavy metal ions chelation, gel forming properties and hydrophilicity, and remarkable affi nity to proteins. In this article, an effort has been made to review the available literature information on chitin and chitosan. The purpose of this chapter is to present a review of about 30 years of research in our team in the context of a precise scientifi c strategy. Thus, these years were devoted to improve the production of chitin and chitosan, produce series of co-polymers and co-oligomers, improve their characterizations, reveal a general law of behavior, generate nano-particles, physical gels and derived forms, show a continuum of structure from solutions to other physical states, propose the concept of materials decoys of biological media, etc., The chapter ends with a review of the applications of chitin and chitosan in medicine, pharmacy, agriculture, the food industry, cosmetics, among others.
Ultrasonic treatment was performed on water-extractable polysaccharides from the seed of mung beans. Purified by anion-exchange and gel filtration chromatography, MWP-1′ and MWP-2′ were obtained. Average molecular weights (Mws) of MWP-1′ and MWP-2′ were 68.4 kDa, and 52.4 kDa, respectively. Monosaccharides components analysis indicated that MWP-1′ was composed of Rha, Ara, Man and Gal in a molar percent of 0.4:2.6:5.3:0.7. MWP-2′ was composed of Ara, Man, Gal and Glc in a molar percent of 0.5:1.4:2.1:0.4. In vitro study showed that both polysaccharides samples were able to stimulate the production of secretory molecules (NO, TNF-α and IL-6) of RAW264.7 murine macrophages in a dosage dependent manner. MWP-2′ seemed to be the most potent and induced significantly higher the NO production. These findings suggest that the ultrasonic treatment polysaccharides isolated in our study have immune potentiation effects on macrophages.
Cytotoxic T lymphocytes (CTLs) exert cytotoxicity against tumor cells with granzyme B and porforin (two important components for cytotoxicity). One main reason for the limitation of clinical success in tumor immunotherapy is tumor cell inefficacy in inducing sufficient immune responses, such as efficient CTLs, and subsequently sufficient granzyme B and porforin activity because of weak immunogenicity of the tumor cells. It is therefore important to boost tumor cells to induce efficient CTLs and sufficient granzyme B and porforin. We suggest that Ganoderma lucidum polysaccharides (Gl-PS), with multiple bioactivities have this potential. We have shown that after incubation with Gl-PS, B16F10 melanoma cells, which are deficient in antigen presentation, promoted cytotoxicity of CTLs against B16F10 cells, induced more granzyme B and porforin in CTLs, decreased the in vivo incidence of tumorigenesis 15 days after inoculation and prolonged the latency of tumorigenesis 21 days after inoculation, demonstrating that the effects of Gl-PS on B16F10 cells induced stronger immune responses against tumor cells.
Terpenes are one of the most abundant groups of natural products that occur mostly in plants, being identified more than 30,000 different compounds. Chemically, terpene group is one of the most diverse in structure, sharing isoprene units as a common structural motif. The number of isoprene units that defined each terpene compound classifies them into several groups including hemiterpenes, monoterpenes, sesquiterpenes, diterpenes, sesterpenes, triterpenes, tetraterpenes and politerpenes. Terpenes, as a natural product group, have sparked a great interest in research because of their relevant and broad spectrum of health-promoting effects such as anti-inflammatory, antimicrobial, antitumor and antioxidant, among others. Nowadays, new findings in pharmacological area are in constant growing. Since oxidative stress has been involved in the pathophysiological development of approximately one hundred of different diseases including cancer, autoimmune diseases, heart attack and neurodegenerative diseases, there have been numerous attempts to cope with this redox imbalance. Exogenous compounds with antioxidant capacity provide a protective effect against free radical overproduction and its consequence cellular damage, being this one the most promising therapeutic strategy. Over the last recent years, there is an increasing interest in the search of antioxidant compounds among terpenes, existing emerging number of reports focused on this issue. This chapter will cover the chemistry, the natural occurrence and the most recent findings in antioxidant activity of terpenes.
The chapter Higher Fungi for the production of pharmaceuticals provides an overview of wood degrading basidiomycetes with pharmacological effects. Since these mushrooms are rare in nature, artificial cultivation of fruit bodies has been established, as well as a biotechnological cultivation of fungal biomass in bioreactors, both on solid substrates and in liquid media. Cultivation technologies are presented, including traditional cultivation of fruit bodies on wood logs and on sawdust-based substrates, and modern biotechnological cultivation of mycelia in bioreactors by submerged and solid-state cultivation technologies. Bioreactor design and process parameters are presented and discussed. An industrially important medicinal mushroom Ganoderma lucidum - Ling zhi or Reishi is described as an outstanding example, with emphasis on the cultivation possibilities, structure and properties of pharmacologically active compounds, pharmacological effects, and medicinal applications.
N-carboxymethyl chitosan (N-CMCS) and O-carboxymethyl chitosan (O-CMCS) based amphoteric or pH-responsive charged membranes were prepared for protein separations. Both membranes (O-CMCS and N-CMCS) exhibited different charged nature and acidic/alkaline ion-exchange capacities along with 16-19 kDa molecular weight cut-off (MWCO) corresponding to 0.53 nm and 0.48 nm pore radius at pH: 7.0, for O-CMCS, and N-CMCS respectively. Water permeability for O-CMCS and N-CMCS membrane was varied between 5.0-6.0×10-8 N-1 m3 h-1 (ultra-filter range) and increased with pH of the equilibrating medium. Dual charged nature of these membranes (presence of –COOH and –N+H3 groups) was used with advantage to achieve their antifouling nature for bio-molecule separation. N-CMCS membrane exhibited positive charged nature in 2.0-12.0 pH-range. Thus, Donnan exclusion due to mutual electrostatic attraction between membrane and protein was insignificant to accelerate the mobility of a protein molecule thus achieve their separation. While O-CMCS membrane showed pH-tuneable charged nature and suitable separation performance for β-Casein (β-Cas) and Lysozyme (Lys) across in alkaline media, as a representative case. Furthermore, no membrane fouling was detected after its 50 days operation under protein environment.
The preparation of 10-(2-[18F]fluoroethoxy)-20(S)-camptothecin, a potential positron emission tomography tracer for the imaging of topoisomerase I in cancers, is described. 10-(2-[18F]Fluoroethoxy)-20(S)-camptothecin was synthesized by the [18F]fluoroalkylation of the corresponding hydroxy precursor molecule with 2-[18F]fluoroethyl bromide ([18F]FEtBr) in dimethylsulfoxide (DMSO) at 55 °C for 20 min; this was followed by purification using high performance liquid chromatography (HPLC) with a total preparation time of 60 min. The overall radiochemical yield was approximately 5.4–12 % (uncorrected), and the radiochemical purity was above 96 %.
Four sulfated heteropolysaccharide fractions (OJP-1, OJP-2, OJP-3 and OJP-4) were isolated and purified from the tuber of Ophiopogon japonicus by DEAE-Sepharose Fast Flow and Sepharose 6 Fast Flow column chromatography. OJP-1, the least sulfated product, was sulfated synthetically to OJP-1S. Their chemical–physical properties were determined by chemical methods, GC, FT-IR spectroscopy, and HPLC. The antioxidant and immunomodulation activities of these fractions were also investigated. The content of hexuronic acid and sulfate were decreased in the order OJP-4>OJP-3>OJP-2>OJP-1 and OJP-1S>OJP-4>OJP-3>OJP-2>OJP-1, respectively. In comparison with OJP-1, other polysaccharides showed stronger 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and hydroxyl radical scavenging activity. Five polysaccharides also exhibited remarkable macrophage-activating capability by the promotion of phagocytic capacity, energy metabolism rate, NO and interleukin-1 production. It was significantly different between five polysaccharides (POJP-4>OJP-3>OJP-2>OJP-1. Taken together, our results suggested that hexuronic acid and sulfate were effective indicators of antioxidant and immunomodulation activity of polysaccharide.
Of late, the most bountiful natural biopolymer chitin and chitosan have become cynosure of all party because of an unusual combination of biological activities plus mechanical and physical properties. However applications of chitin are limited due to its inherent insoluble and intractable nature. Chitosan, alkaline hydrolytic derivative of chitin has better solubility profile, less crystallinity and is amenable to chemical modifications due to presence of functional groups as hydroxyl, acetamido, and amine. The chemical modification of chitosan is of interest because the modification would not change the fundamental skeleton of chitosan, would keep the original physicochemical and biochemical properties and finally would bring new or improved properties. In view of rapidly growing interest in chitosan its chemical aspects and chemical modification studies is reviewed. The several chemical modifications such as oligomerization, alkylation, acylation, quternization, hydroxyalkylation, carboxyalkylation, thiolation, sulfation, phosphorylation, enzymatic modifications and graft copolymerization along with many assorted modifications have been carried out. The chemical modification affords a wide range of derivatives with modified properties for specific end use applications in diversified areas mainly of pharmaceutical, biomedical and biotechnological fields. Assorted modifications including chitosan hybrids with sugars, cyclodextrin, dendrimers, and crown ethers have also emerged as interesting multifunctional macromolecules. The versatility in possible modifications and applications of chitosan derivatives presents a great challenge to scientific community and to industry. The successful acceptance of this challenge will change the role of chitosan from being a molecule in waiting to a lead player.
Yield of polysaccharides from Phascolosoma esulenta obtained by phosphate buffer extraction through an orthogonal experiment (L9(3)(4)) were investigated to get the best extraction conditions. The results showed that extraction temperature, ratio of phosphate buffer to raw material, extraction time, and ratio of trypsinase to raw material were the main four variables that influenced the yields of extracts. The highest yield was obtained when extraction temperature, ratio of phosphate buffer to raw material, extraction time and ratio of trypsinase to raw material were 40°C, 2, 5.5h and 1.6, respectively. The immunity-stimulating method showed that polysaccharides from P. esulenta could significantly raise liver, spleen and thymus index of mice and enhance Con A-stimulated mouse spleen cells proliferation. These results indicate that polysaccharides from P. esulenta had significantly higher immunity-stimulating activities.
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Objective:
The aim of the present investigation was to evaluate the use of spray-dried O-carboxymethyl chitosan (OCMCS) as potential hydrophilic matrix excipient for sustained release of drug.
Methods:
The polymer was synthesized from chitosan, then spray-dried and characterized. Tablets with different OCMCS concentrations (80, 50, 30, 5 and 2% w/w), containing diltiazem (DTZ) as model drug, were prepared for direct compression (DC) and after the wet granulation method (WG).
Results:
The spray-dried OCMCS powder was spherical, with a smooth surface and an average size of 2.2 µm. The tablets prepared for WG disintegrated in time less than 30 min. The tablets obtained for DC presented high retention of the drug, with zero order or Higuchi release kinetic. There was a direct relationship between the OCMCS concentration and the release ratio, swelling degree and water uptake behavior. DC tablets containing 80% OCMCS presented behavior as an effective swelling-control system. The DC tablets with 5% OCMCS showed a similar release profile at formulations with 30% HPMC.
Conclusion:
Spray-dried OCMCS showed great potential as hydrophilic matrices for drug-sustained release.
Based on the analysis of more than 270 patents and scientific articles, this state-of-the-art review presents Ganoderma lucidum, a medicinal basidiomycete mushroomwithimmunomodulatory and anti-cancer effects. Cultivation methods for the commercial production of G. lucidum fruit bodies and mycelia are summarized, with main active compounds of triterpenoids, polysaccharides, and proteins, often found in forms of proteoglycans or glycopeptides. Pharmacological effects with emphasis on anti-cancer and immunomodulatory functions are presented, separately for spores and dry mycelia, and for the groups of triterpenoids, polysaccharides, proteins and glycoproteins. Patents disclosing preparation methods of extracts and purified pharmaceutical isolates are reviewed, and examples of anti-cancer formulations, used as pharmaceuticals or nutraceuticals, are given. The review suggests that according to the present understanding, the anti-cancer activity of G. lucidum may be attributed to at least five groups of mechanisms: (1) activation/modulation of the immune response of the host, (2) direct cytotoxicity to cancer cells, (3) inhibition of tumor-induced angiogenesis, (4) inhibition of cancer cells proliferation and invasive metastasis behaviour, and (5) carcinogens deactivation with protection of cells. Although the data from recent in vitro and in vivo studies demonstrate promising anti-cancer effects, a need is identified for further (1) isolation and purification of compounds, with deeper understanding of their individual and synergistic pharmacological effects, (2)molecular level studies of the antitumor and immuno-supportive mechanisms, (3) well designed in vivo tests and controlled clinical studies, and (4)standardisation and quality control for G. lucidum strains, cultivation processes, extracts and commercial formulations.
Ionic hydrogels are attractive for the protection, delivery and controlled release of charged biomacromolecules such as proteins, growth factors or DNA. We have preparted and characterized a series of photocrosslinked anionic hydrogels based on water soluble methacrylated (MA) O-carboxymethylchitosan (OCMCS) and polyethylene glycol (PEG) diacrylate. OCMCS samples with varying degree of substitution of carboxymethyl group ranging from 0.69 to 1.86 were prepared by reacting native chitosan with different amounts of monochloroacetic acid. The OCMCS products demonstrated differences in solubility, zeta potential (-52.7 to -12.8 mV) and thermal decomposition temperature (260 to 283 degrees C). The OCMCS products were then reacted with glycidyl methacrylate to make ultra-violet (UV) crosslinkable OCMCS-MAs which were blended with PEG diacrylate, a photoinitiator and water and successfully photocrosslinked to create OCMCS-MA/PEG hydrogels. Water uptake of the hydrogels varied between 226 % to 358 % and the porous structures of the prepared OCMCS-MA/PEG hydrogels could be modulated by the degree of methacrylation. All the OCMCS-MA/PEG hydrogel substrates similarly supported attachment and proliferation of Smooth Muscle Cells (SMCs). The in vitro biodegradation of these hydrogels, in the presence of SMCs, could be controlled by the degree of methacrylation; weight loss after 9-week was (15+/-1) % and (19+/-2) % using OCMCS 4-MA (12.7 % MA) and OCMCS 1-MA (4.6 % MA), respectively. In addition, the hydrogel based on the most anionic OCMCS 1 showed higher adsorption of basic TGF-beta(1) than similarly modified -agarose, -dextran, and -chondroitin sulfate hydrogels.
C.B-17 severe combined immune deficient (SCID) mice, which lack functional B and T lymphocytes, allow xenografts and, therefore, can be used to study the biology of human malignancies. Two different human B cell lymphoma cell lines, SU-DHL-4 and OCI-Ly8, which both harbor the t(14;18) chromosomal translocation, were injected into C.B-17 SCID mice. Mice injected intravenously or intraperitoneally developed tumors and died in a dose-dependent manner. The presence of tumor cells in various murine tissues could be demonstrated by a clonogenic tumor assay, staining of frozen sections with a monoclonal antibody (mAb) against a human B cell antigen (CD19), and with the polymerase chain reaction technique. A protocol using cytotoxic effector cells was developed and used to selectively deplete the tumor cells from bone marrow. These cells were developed by growing peripheral blood mononuclear cells in the presence of interferon gamma (IFN-gamma), anti-CD3 mAb, and interleukin 2 (IL-2). The timing of IFN-gamma treatment was critical and optimal if IFN-gamma was added before IL-2 treatment. The cells that were stimulated by IFN-gamma, followed by IL-2, could be expanded by treatment with a mAb directed against CD3. These cells could be further activated by IL-1, but not by tumor necrosis factor alpha. With this protocol, a tumor cell kill of 3 logs was obtained as measured by a clonogenic assay. Interestingly, despite their high cytotoxic activity against lymphoma cells, these cells had little toxicity against a subset of normal human hematopoietic precursor cells (granulocyte/macrophage colony-forming units). These cells were further tested by treating murine bone marrow contaminated with the human lymphoma cell line SU-DHL-4, and injecting these cells into SCID mice to assay for tumor growth in vivo. The animals injected with bone marrow contaminated with SU-DHL-4 cells had enhanced survival if the bone marrow was treated with the cytokine-induced killer cells before infusion. The SCID mouse provides a useful in vivo model for evaluation of new therapeutic approaches for lymphoma treatment. The cytokine-induced killer cells generated as described here could have an important impact on bone marrow purging for autologous bone marrow transplantation as well as for adoptive immunotherapy.
We studied the 5' untranslated regions (UTRs) of the mouse lymphocyte pore-forming protein (PFP, perforin, and cytolysin). 5' UTRs were determined by primer extension analysis, sequencing PFP cDNA clone PFP-7, ribonuclease protection assays, and amplification of poly(A)+ RNA of cytolytic T lymphocyte using polymerase chain reaction (PCR). Two alternatively spliced 5' UTRs, designated type I and type II, of 222 and 115 bp, respectively, were found associated with PFP. Type II is identical to type I, except for being 107 bp shorter in the second exon. This deletion was generated by the use of alternative acceptor splice sites. The mouse PFP gene (Pfp) encodes three exons, is separated by two small introns, and spans a chromosomal region of approximately 7 kb. The first exon contains 79 bp of 5' UTR, the second exon contains 143 or 36 bp of 5' UTR (type I or type II UTR, respectively) plus the NH2-terminal region of the mouse PFP, and the third exon contains the rest of the COOH-terminal mouse PFP. The organization of the mouse Pfp is similar to that of the human gene. Moreover, the 5' flanking sequence of the mouse Pfp is highly homologous to that of the human Pfp. In contrast to the human sequence, the more immediate 5' flanking sequence of mouse Pfp contains two tandem "TATA" box-related elements and a GC box, but lacks a typical CAAT box-related sequence. Several other enhancer elements were found further upstream, including cAMP-, phorbol ester-, interferon-gamma-, and UV-responsive elements, and PU box-like and NFkB binding site-like elements. In addition, we found a nuclear inhibitory protein-like element, a transcriptional silencer, and a pair of purine-rich sequence motifs that were found in other T cell-specific genes, and three repeats of GGCCTG that may be a variation of a highly repetitious GCCCTG consensus sequence found in human Pfp.
Polymerase chain reaction-assisted reverse transcription was used to study the temporal course of rejection in unmodified recipients of murine pancreatic islet cell allografts (DBA/2-->B6AF1) by using syngeneic tissues as controls. The histologic appearance of the grafts was analyzed in parallel. Preproinsulin and constant region of the TCR-beta chain transcripts were studied as markers of graft integrity and infiltrating T cell mass, respectively. The participation of certain cytokines and CTL were analyzed by the detection of IL-2, IFN-gamma, IL-4, and CTL-specific serine protease (granzyme B) transcripts. The time-related disappearance of intragraft preproinsulin transcripts correlated with graft destruction, whereas the intensity of intragraft TCR-beta chain transcript levels correlated with the magnitude of mononuclear leukocyte infiltration in allografts. In unmodified allografts, the magnitude of IL-2 and IFN-gamma intragraft mRNA levels correlated with the intense mononuclear leukocyte infiltrate found on histologic examination at day 8. Only after stable IL-2 gene transcription on day 8 does evidence of graft destruction become apparent, indicating that IL-2 gene activation is closely related to and probably required for expression of alloimmune cytopathic processes. In contrast, IL-4 transcripts were absent or detected in low copy number throughout this time course. Intragraft expression of granzyme B mRNA, a CTL-specific transcript, peaked from day 8 to day 12 in allografts compared with syngeneic grafts or normal tissue. In syngeneic grafts IL-2 and/or IL-4 mRNA was essentially not detected. Although IFN-gamma and granzyme B transcripts were detected in syngeneic grafts, after 4 days the levels of detected transcripts were far less than those noted in allografts. In vivo detection of intragraft IL-2 transcripts in the relative absence of detectable IL-4 transcripts strongly suggests IL-2-dependent immune effector mechanisms are associated with, and perhaps responsible, for allograft rejection. Apparently IL-4-dependent effector mechanisms are not necessary for allograft rejection.
During the past two decades, nitric oxide (NO) has been recognized as one of the most versatile players in the immune system. It is involved in the pathogenesis and control of infectious diseases, tumors, autoimmune processes and chronic degenerative diseases. Because of its variety of reaction partners (DNA, proteins, low-molecular weight thiols, prosthetic groups, reactive oxygen intermediates), its widespread production (by three different NO synthases (NOS) and the fact that its activity is strongly influenced by its concentration, NO continues to surprise and perplex immunologists. Today, there is no simple, uniform picture of the function of NO in the immune system. Protective and toxic effects of NO are frequently seen in parallel. Its striking inter- and intracellular signaling capacity makes it extremely difficult to predict the effect of NOS inhibitors and NO donors, which still hampers therapeutic applications.
There are numerous reports of in vitro and in vivo usage of dendritic cells (DC) pulsed with idiotype, the tumor-specific antigen of multiple myeloma (MM), for immunotherapy of MM. Data suggest that not only T-cells, but also the innate immune system reacts against MM. Here, we examined the cytotoxic activity of cytokine-induced killer (CIK) cells against myeloma cells. This heterogeneous effector population consists of T-, NK- and NKT-cells.
CIK cells generated from buffy coats or blood from patients with MM were co-cultured with autologous idiotype-pulsed DC. The cytotoxic activity was investigated in lactate dehydrogenase release assays against cell lines or autologous CD138 positive cells from bone marrow.
CIK cells were able to lyse MM cells at low effector to target ratios. This effect was significantly enhanced by co-culturing with specifically pulsed DC (83.8% lysis at an effector to target ratio of 16:1). Using an interferon-g secreting MACS separation assay, the cytotoxic activity of CIK cells was enhanced to maximal lysis at the lower effector to target ratio of 5:1. High cytotoxic activity was also shown in a completely autologous setting against enriched CD138+ cells from a patient with MM (54.4% lysis at an effector to target ratio of 6:1). Interestingly, there was no cytotoxic activity against the CD138- fraction of the bone-marrow.
Using a heterogeneous population of effector cells, we were able to activate the innate and the adoptive immune-system against myeloma cells. CIK cells showed high lytic activity against MM cells, which could be enhanced by co-culturing with antigen-specific pulsed DC.
To examine the effect of ganoderma lucidum polysaccharide (GLP) on the immune liver injury induced by BCG infection, and investigate the relationship between degrees of hepatic damage and NO production in mice.
Immune hepatic injury was markedly induced by BCG-pretreatment (125 mg.kg(-1), 2-week, iv) or by BCG-pretreatment plus lipopolysaccharide (LPS, 125 microg.kg(-1), 12-hour,iv) in mice in vivo. Hepatocellular damage induced by BCG-pretreated plus inflammatory cytokines mixture (CM), which was included TNF-alpha, IL-1beta, IFN-gamma and LPS in culture medium in vitro. Administration of GLP was performed by oral or incubating with culture medium at immune stimuli simultaneity. Liver damage was determined by activity of alanine aminotransferase (ALT) in serum and in hepatocytes cultured supernatant, by liver weight changes and histopathological examination. NO production in the cultured supernatant was determined by the Griess reaction. Moreover, inducible nitric oxide synthase (iNOS) protein expression was also examinated by immunohistochemical method.
Immune hepatic injury was markedly induced by BCG or BCG plus inflammatory cytokines in BALB/c mice in vivo and in vitro. Under BCG-stimulated condition, augment of the liver weight and increase of the serum/supernatant ALT level were observed, as well as granuloma forming and inflammatory cells soakage were observed by microscopic analysis within liver tissues. Moreover, NO production was also increased by BCG or/and CM stimuli in the culture supernatant, and a lot of iNOS positive staining was observed in BCG-prestimulated hepatic sections. Application of GLP significantly mitigated hepatic tumefaction, decreased ALT enzyme release and NO production in serum/supernatant, improved the pathological changes of chronic and acute inflammation induced by BCG-stimuli in mice. Moreover, the immunohistochemical result showed that GLP inhibited iNOS protein expression in BCG-immune hepatic damage model.
The present study indicates that NO participates in immune liver injury induced by Mycobacterium bovis BCG infection. The mechanisms of protective roles by GLP for BCG-induced immune liver injury may be due to influence NO production in mice.
Ganoderma lucidum (G lucidum) is a medicinal fungus with a variety of biological activities. It has long been used as a folk remedy for promotion of health and longevity in China and other oriental countries. The most attractive character of this kind of medicinal fungus is its immunomodulatory and anti-tumor activities. Large numbers of studies have shown that G lucidum modulate many components of the immune system such as the antigen-presenting cells, NK cells, T and B lymphocytes. The water extract and the polysaccharides fraction of G lucidum exhibited significant anti-tumor effect in several tumor-bearing animals mainly through its immunoenhancing activity. Recent studies also showed that the alcohol extract or the triterpene fraction of G lucidum possessed anti-tumor effect, which seemed to be related to the cytotoxic activity against tumor cells directly. Preliminary study indicated that antiangiogenic effect may be involved antitumor activity of G lucidum.
A cytolytic pore-forming protein (PFP, perforin) was purified from isolated granules of cloned NK-like cytolytic cells, which showed an apparent Mr of 70–75 kd (reduced) and 62–66 kd (nonreduced). Cytolysis produced by this protein occurred only in the presence of Ca2+ and was accompanied by the formation of membrane lesions of 160 Å diameter. The purified protein depolarized cells and made lipid vesicles leaky to monovalent and divalent ions. This protein formed large, voltage insensitive and nonselective ion channels in planar bilayers that remained preferentially in the open state. The channels were heterogeneous in size distribution averaging 400 pS/U in 0.1 M NaCl. The membrane lesions formed by PFP were morphologically and functionally similar to those formed by intact NK-like cells and their granules. This PFP could be released from granules during cell killing, followed by its polymerization on target membranes to form large transmembrane pores.
We have investigated the role of interleukin-6 (IL-6) in the induction of major histocompatibility complex (MHC)-unrestricted cytotoxicity, as well as granzyme B, perforin, and Fas ligand gene expression, following mouse T lymphocyte activation with anti-CD3 monoclonal antibody (mAb), The generation of anti-CD3-activated killer-T (AK-T) cells was inhibited when anti-IL-6 neutralizing mAb was added at initiation of culture but not 24 h later, indicating that IL-6 is involved at an early stage of AK-T cell development, However, AK-T cell induction in the presence of exogenous IL-6 did not result in enhanced cytotoxicity, suggesting that saturating levels of IL-6 are normally synthesized in AK-T cell cultures, The inhibitory effect of IL-6 neutralization on AK-T cell generation could not be attributed to a defect in AK-T cell proliferation or to an inability of AK-T cells to recognize and adhere to P815 tumor target cells, However, IL-2 synthesis and CD25 expression were downregulated in AK-T cell cultures performed in the presence of anti-IL-6 mAb, In addition, IL-6 neutralization resulted in decreased expression of granzyme B and perforin, but not Fas ligand, mRNA, Exogenous IL-2 (50 U/ml) added at initiation of culture completely reversed the inhibitory effect of anti-IL-6 mAb on AK-T cell development, restoring CD25 expression and tumoricidal activity, as well as granzyme B and perforin mRNA expression, to control levels, We conclude that IL-6 modulates AK T cell induction through an IL-2-dependent mechanism.
The present study was to ascertain the immunomodulating and anti-tumor effects of Ganoderma (G.) lucidum. Polysaccharides (PS) from fresh fruiting bodies of G. lucidum (PS-G) were isolated and used to potentiate cytokine production by human monocytes-macrophages and T lymphocytes. Our results had shown that the levels of interleukin (IL)-Iβ, tumor necrosis factor (TNF)-, and IL-6 in macrophage cultures treated with PS-G (100 μg/ml) were 5.1-, 9.8- and 29-fold higher, respectively, than those of untreated controls. In addition, the release of interferon (IFN)- from T lymphocytes was also greatly promoted in the presence of PS-G (25–100 μ/ml). Furthermore, these cytokine-containing mononuclear cell-conditioned media (PSG-MNC-CM) were found to suppress the proliferation and clonogenicity of both the HL-60 and the U937 leukemic cell lines. DNA labeling and gel electrophoresis showed that treatment with PSG-MNC-CM markedly induced leukemic-cell apoptosis. Flow-cytometric analysis revealed that few (2.3 ± 0.8%) apoptotic cells were seen in the control cultures, while PSG-MNC-CM treatment resulted in a significant increase in the apoptotic population both in the HL-60 (38.3 ± 4.5%) and in the U937 (44.5 ± 3.8%) cells. In addition, 40 to 45% of the treated leukemic cells were triggered to differentiate into mature monocytic cells expressing CD14 and CD68 surface antigens. However, PS-G alone had no such effects even at a higher dose of 400 μg/ml. Since untreated macrophages and T lymphocytes produced little or no cytokine, and normal MNC-CM did not suppress leukemic cell growth, it was suggestive that the anti-tumor activity of PSG-MNC-CM was derived from the elevated levels of cytokines. Antibody-neutralization studies further revealed that the anti-tumor cytokines in the PSG-MNC-CM were mainly of TNF- and IFN-, and these 2 cytokines acted synergistically on the inhibition of leukemic-cell growth. Int. J. Cancer 70:699–705, 1997. © 1997 Wiley-Liss, Inc.
Mice immunized with attenuated Salmonella typhimurium, strain SL3235, while protected against virulent challenge, are unable to mount in vivo and in vitro antibody responses to non-Salmonella antigens, such as tetanus toxoid and sheep red blood cells, and exhibit profoundly suppressed responses to B and T cell mitogens. Suppression of antibody responses is mediated by macrophage (M phi)-released soluble factors, and is completely reversed by treatment with interleukin (IL)-4. The present report identifies the suppressor factor as nitric oxide (NO), and provides evidence for a mechanism by which IL-4 abrogates suppression. Suppressed antibody responses correlated with high levels of NO secretion by splenocytes of SL3235-immunized mice. NO production was observed only in cultures consisting of the adherent cell fraction of immune splenocytes. Further, immunosuppression was reversed by NG-monomethyl-L-arginine (NMLA), a competitive inhibitor of NO synthesis, and was completely blocked by the addition of excess L-arginine. Treatment with IL-4, or anti-interferon (IFN)-gamma monoclonal antibody (mAb), also abrogated suppression. Optimal reversal of suppression was observed only when NMLA, IL-4, or anti-IFN-gamma mAb, was added at day 0 of the 5-day plaque-forming cell assay. Treatment with either IL-4 or anti-IFN-gamma mAb also lead to a sharp inhibition of NO production by immune spleen cells. Moreover, the addition of IL-4 to splenic adherent M phi inhibited their ability to generate NO. Our data characterize an immunoregulatory pathway, involving IFN-gamma and NO, by which M phi mediate immunosuppression and identify IL-4 as a potent inhibitor of this pathway.
We have investigated the functional interaction between IL-2 and TNF on the generation of alloreactive CTL. The study was performed by using primary mixed cultures of lymphocytes from a MHC-recombinant sibling identical for MHC class II Ag (DR, DP, DQ) and displaying MHC class I disparity. Our data show that MHC class I disparity can trigger the induction of TNF receptor without promoting significant TNF production. Addition of exogenous TNF at the sensitizing phase of the primary mixed lymphocyte reaction did not result in CTL activation. However, when simultaneously added with IL-2, TNF could promote an optimal induction of cytotoxic T cell generation. The enhanced lytic ability of MHC class-I primed CTL by TNF was associated with a selective up-regulation of Tac Ag and subsequent amplification of cell proliferation. Furthermore, TNF was also found to induce a considerable increase in IL-2-induced intracellular benzyloxycarbonyl-L-lysine thiobenzylester-esterase activity by MHC class I-primed cells. TNF did not affect the expression of LFA1, CD2, CD4, and CD8, molecules that are associated with CTL-target interactions, on responder cells. These results extend our earlier observations on the role of class I MHC molecules that may function to transduce activation signals and suggest that TNF may be a potent mediator involved in the IL-2-induced acquisition of optimal lytic competence by precursor cytotoxic T cells.
Evidence has accumulated that T cell-mediated autoimmunity plays an important role in the pathogenesis of viral myocarditis. T lymphocytes are known to recognize antigen-presenting cells, such as virus-infected cells, being restricted by syngeneic major histocompatibility complex (MHC) antigens. To clarify in more detail the immunological mechanisms involved, we induced acute viral myocarditis in C3H/He mouse ventricles with coxsackievirus B3 (CVB3) and examined, by immunofluorescence, the expression of MHC class I and II antigens, previously reported not to be expressed by normal cardiac myocytes. Furthermore, to confirm the expression of MHC class I (H-2Kk) antigens at the cellular level, we treated cultured cardiac myocytes with interferon gamma and examined the antigen expression by immunofluorescence and Northern blot hybridization, using an antisense RNA probe for MHC messenger RNA. Our observations demonstrated 1) CVB3-induced myocarditis resulted in the enhanced expression of MHC class I (H-2Kk) gene product on the surface of cardiac myocytes but low or undetectable levels of MHC class II or H-2Dk gene products, and moderate focal transient (days 5-7) expression of both MHC class I (Kk + Dk) region gene products and MHC class II antigens were induced on capillary endothelial cells; 2) murine fetal cardiac myocytes cultured in vitro in the presence of interferon gamma similarly were shown to express marked levels of MHC class I (H-2Kk) but low to undetectable levels of the H-2Dk gene product; however, weak to moderate MHC Class II antigens were expressed by these cultured myocytes; and 3) the expression of MHC antigens in cardiac myocytes was modulated at the transcriptional level.(ABSTRACT TRUNCATED AT 250 WORDS)
The expression of perforin and serine esterase (SE) activities and genes was examined in a murine cytotoxic T lymphocyte line (R8i) that does not require exogenous IL-2 for proliferation. Although perforin (hemolytic) activity was detected in unstimulated R8i, it was induced 2- to 14-fold in the presence of IL-2, IL-3, IL-4, and IL-6, and to a lesser degree (less than 4-fold) by TNF and IFN-gamma. A transient induction was also observed at the mRNA level. Peak perforin protein and mRNA levels were reached within 24 h and started to decline 48 h after stimulation. A trypsinlike SE activity which cleaves the chromogenic substrate N, alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester was also induced 2- to 4-fold in the presence of the various IL tested. At the mRNA level, the message for SE SE1/granzyme A/Hanukah factor was absent from R8i whereas SE2/granzyme B/CTLA-1 increased by greater than 3-fold in the presence of IL-2, IL-3, IL-4, and IL-6 and occurred with the same kinetics and pattern as perforin. The induction response occurred without any enhancement of cell proliferation, suggesting that the cytokines tested may provide a direct differentiation signal to CTL. The induction response was abrogated effectively by inhibitors of protein (cycloheximide or emetine) and RNA (actinomycin D) syntheses. These findings suggest that the various IL may provide both a growth signal and a differentiation signal to CTL, resulting in the direct activation of perforin and SE genes.
A cytolytic pore-forming protein (PFP, perforin) was purified from isolated granules of cloned NK-like cytolytic cells, which showed an apparent Mr of 70-75 kd (reduced) and 62-66 kd (nonreduced). Cytolysis produced by this protein occurred only in the presence of Ca2+ and was accompanied by the formation of membrane lesions of 160 A diameter. The purified protein depolarized cells and made lipid vesicles leaky to monovalent and divalent ions. This protein formed large, voltage insensitive and nonselective ion channels in planar bilayers that remained preferentially in the open state. The channels were heterogeneous in size distribution averaging 400 pS/U in 0.1 M NaCl. The membrane lesions formed by PFP were morphologically and functionally similar to those formed by intact NK-like cells and their granules. This PFP could be released from granules during cell killing, followed by its polymerization on target membranes to form large transmembrane pores.
Peripheral blood lymphocytes (PBL) cultured in interleukin 2 (IL 2)-containing medium in conventional tissue culture develop the ability to lyse fresh tumor cells; such cells are referred to as lymphokine-activated killer (LAK) cells. LAK activity peaks by day 5 of culture and declines rapidly thereafter. We studied culture conditions and signals that allow for long-term culture and expansion of cells with LAK activity. By culturing cells at relatively low densities and regularly replenishing medium and recombinant IL 2 (r-IL 2), LAK function is significantly higher as compared with short-term cultures, and remains present for at least 21 days while cell numbers undergo an average 100-fold expansion. By activating these cultures with anti-CD3 (OKT3) monoclonal antibody and r-IL 2, an approximately 1000-fold expansion in the cell number is obtained with maintenance of comparable levels of LAK activity. The exogenous addition of beta interleukin 1 (beta-IL 1), interferon-beta (IFN-beta) or interferon-gamma (IFN-gamma) can augment the lytic activity of cell populations expanded by anti-CD3 plus r-IL 2. These approaches may enable the in vitro generation from individual donors of much greater numbers of LAK cells for adoptive immunotherapy than can now be obtained with the 3 to 5 day in vitro culture systems.
This study showed that a mAb (145-2C11) against the T3 epsilon-chain of the TCR complex augmented the cytotoxic activity of the lymphokine-activated killer (LAK) effectors. The LAK cells were induced by culturing normal spleen cells with purified human rIL-2. Adding alpha T3 at the effector phase of the cytotoxic reactions augmented the LAK-mediated cytotoxicity. The alpha T3-augmented LAK killing was seen only with tumor targets, and there was no increase of killing against Con A-induced lymphoblasts. The augmentation effect was dose dependent on both the amounts of alpha T3 and the number of LAK cells added. A very low concentration of alpha T3 (1/10,000 dilution of culture supernatants) was sufficient to induce alpha T3-augmented LAK-mediated cytotoxicity. Human rIL-2 at 10 to 30 U/ml was sufficient to generate LAK cells for maximal alpha T3 augmentation, whereas 300 to 1000 U/ml of IL-2 were needed to generate maximal LAK activity when tested in the absence of alpha T3. LAK cells generated for longer periods of time showed a progressive increase of alpha T3-augmented cytotoxicity. For some targets, the alpha T3-augmented LAK killing was FcR dependent as evidenced by the ability of alpha FcR mAb 2.4G2 to inhibit, and for others it was not inhibited. The alpha T3-augmented killing did not correlate with the FcR expression on target cells as defined by 2.4G2. The LAK cells were both Lyt-2+ and Lyt-2-, but the LAK cells involved in alpha T3-augmented killing were exclusively Lyt-2+. Preincubation of LAK cells with alpha T3, but not preincubation of targets with alpha T3, resulted in augmented killing suggesting that the alpha T3 effect was unrelated to an antibody-dependent cell-mediated cytotoxicity. Our findings indicate that alpha T3 is a potent reagent to augment the cytotoxic reaction of LAK cells. These results suggested that a relationship might exist between the T3 complex and the cytotoxic activity of a subpopulation of Lyt-2+ LAK cells.
Cytokine-induced killer (CIK) cells are non-major histocompatibility complex-restricted cytotoxic cells generated by incubation of peripheral blood lymphocytes with anti-CD3 monoclonal antibody (MoAb), interleukin-2 (IL-2), IL-1, and interferon-gamma. Cells with the greatest effector function in CIK cultures coexpress CD3 and CD56 surface molecules. CIK cell cytotoxicity can be blocked by MoAbs directed against the cell surface protein leukocyte function associated antigen-1 but not by anti-CD3 MoAbs. CIK cells undergo release of cytoplasmic cytotoxic granule contents to the extracellular space upon stimulation with anti-CD3 MoAbs or susceptible target cells. Maximal granule release was observed from the CD3+ CD56+ subset of effector cells. The cytoplasmic granule contents are lytic to target cells. Treatment of the effector cells with a cell-permeable analog of cyclic adenosine monophosphate (cAMP) inhibited anti-CD3 MoAb and target cell-induced degranulation and cytotoxicity of CIK cells. The immunosuppressive drugs cyclosporin (CsA) and FK506 inhibited anti-CD3-mediated degranulation, but did not affect cytotoxicity of CIK cells against tumor target cells. In addition, degranulation induced by target cells was unaffected by CsA and FK506. Our results indicate that two mechanisms of cytoplasmic granule release are operative in the CD3+ CD56+ killer cells; however, cytotoxicity proceeds through a cAMP-sensitive, CsA- and FK506-insensitive pathway triggered by yet-to-be-identified target cell surface molecules.
Perforin is a cytolytic mediator produced by killer lymphocytes, and is stored in and released by cytoplasmic granules. The protein is partially homologous to the terminal components of the membrane attack complex of complement and produces pores of up to 20 nm in diameter on target membranes. Its genomic and protein structures have recently been unraveled, and its function elucidated through the availability of genetically engineered, perforin-deficient mice. Here Chau-Ching Liu, Craig M. Walsh and John Ding-E Young briefly outline certain biochemical and molecular features of perforin, and discuss the still-evolving issues concerning the relevance of perforin and Fas in cell killing.
CD8+ cytotoxic T lymphocyte (CTL) clones begin to synthesize the lytic proteins granzyme A, granzyme B and perforin after stimulation with allogeneic target cells. The lytic proteins are stored in the secretory granules which are released after cross-linking of the T cell receptor (TcR) upon target cell recognition. During lytic granule biogenesis granzyme A protein synthesis can be detected between 2 and 10 days after allogeneic stimulation of the CTL. Although granzyme A is stored in the lytic granules over this period, the majority of granzyme A synthesized is secreted directly from the CTL.
TcR triggering of degranulation also results in new synthesis of the lytic proteins, which can be inhibited by cycloheximide (CHX). Some of the newly synthesized lytic proteins can be stored in the cell and refill the granules. But up to one third of granzymes A and B can be secreted directly from the CTL via the constitutive secretory pathway as shown by granzyme A enzymatic activity and immunoblots of secreted granzyme B, where one third of the protein fails to acquire the granule targeting signal. Perforin is also secreted via the constitutive pathway, both from the natural killer cell line, YT, and from CTL clones after TcR cross-linking. Constitutive secretion of the lytic proteins can be blocked by both CHX and brefeldin A (BFA). While BFA does not affect the directional killing of recognized targets, it abrogates bystander killing, indicating that bystander killing arises from newly synthesized lytic proteins delivered via a non-granule route.
These results demonstrate that the perforin/granzyme-mediated lytic pathway can be maintained while CTL kill multiple targets. We show that CTL not only re-fill their granules during killing, but also secrete lytic proteins via a non-granule-mediated pathway.
Accumulating evidence suggests that the functional properties of alloactivated T cells may depend upon the microenvironment in which the T cells reside. For instance, we showed previously that heparan sulfate, a biologically active polysaccharide present on cell surfaces and extracellular matrices, modulates the proliferative responses of splenocytes through enhancement of cytokine and prostaglandin production by macrophages. Here we report that under conditions of suboptimal stimulation, heparan sulfate causes discrete alterations in the functional responses of murine cytolytic T cells. When present in a 5-day mixed leukocyte culture (MLC), heparan sulfate mediates an increase, from 3- to 10-fold, in T cell-mediated cytotoxicity. This increase is dose dependent and most pronounced when heparan sulfate is present in the highest concentration during the first 24 hr of the culture period. On the other hand, when added during the last 48-72 hr of an MLC, heparan sulfate decreases cytotoxicity by 3- to 30-fold. Neutralizing antibodies against IL-1 alpha, but not antibodies against IL1 beta, IL-6, or TNF alpha/beta, abrogate the heparan sulfate-mediated increase in cytotoxicity, suggesting that the increase depended in part upon the production of IL-1 alpha. However, studies in which exogenous IL-1 was added to MLC showed that increased cytotoxicity was not due only to increased cytokine production. Augmentation of cytotoxicity was in part independent of T cell help, as depletion of CD4+ cells from the responder population before MLC, or addition of neutralizing anti-murine IL-2 antibodies plus human IL-2 to the MLC, did not abrogate the stimulatory effect of heparan sulfate. Heparan sulfate-treated CD8+ lymphoblasts isolated after 7 days in MLC demonstrated an increased cytotoxicity, elevated intracellular serine esterase, and perforin levels compared with lymphoblasts from control MLC. The decrease in cytotoxicity observed when heparan sulfate was present during the last several days of an MLC was likely mediated by PGE2, as elevated levels of PGE2 were detected in MLC supernatants of heparan sulfate-treated cultures, and because the decrease was not observed in the presence of indomethacin. Our results are consistent with the idea that the metabolism of heparan sulfate, an endogenous component of parenchymal tissues, may regulate the tempo and magnitude of alloreactive cytotoxic T cell responses.
We have investigated the effect of interferon-gamma (IFN-gamma) and interleukin (IL)-10 on granzyme B expression and the induction of major histocompatibility complex (MHC)-unrestricted cytotoxic activity in mouse T cell cultures following activation with anti-CD3 monoclonal antibody (mAb). First, metabolic inhibitors of granule-dependent and granule-independent cytolytic pathways were used to show that anti-CD3-activated killer T (AK-T) cells kill allogeneic P815 mastocytoma target cells primarily by the granule-dependent granzyme/perforin pathway. In comparison to control AK-T cells, lower levels of cytolytic activity were evident when AK-T cells were generated in the presence of anti-IFN-gamma neutralizing mAb or exogenous IL-10, whereas enhanced cytotoxicity was observed when AK-T cell cultures contained anti-IL-10 neutralizing mAb or exogenous IFN-gamma. In addition, granzyme B mRNA expression by AK-T cells was diminished when IFN-gamma bioactivity was neutralized or exogenous IL-10 was present in AK-T cell-cultures, whereas neutralization of IL-10 bioactivity or the addition of exogenous IFN-gamma resulted in increased expression of granzyme B mRNA. Similar results were obtained when granzyme B enzymatic activity in AK-T cell lysates was quantified using a colorimetric granzyme B assay. Altered cytotoxic potential, granzyme B mRNA expression, and granzyme B enzymatic activity following T cell activation in the presence of anti-IFN-gamma or anti-IL-10 neutralizing mAb or exogenous IFN-gamma or IL-10 could not be attributed to gross changes in T cell activation status or to altered percentages of CD4+ and CD8+ T cells in AK-T cell cultures. We conclude that IFN-gamma and IL-10 cross-regulate the induction of the granule-dependent cytolytic machinery of AK-T cells.
Pentoxifylline (PTX), a methylxanthine derivative, is known to inhibit the production of the TH1 cytokines interleukin-2, tumour necrosis factor-alpha and interferon-gamma. Because these cytokines play an important role in promoting the development of cell-mediated immunity, we hypothesized that PTX would also interfere with the generation of cytotoxic effector cells in response to an immunological stimulus. In this study we used a mouse model system to investigate the effect of PTX on the induction of non-specific killer lymphocytes by anti-CD3 monoclonal antibody. Anti-CD3-induced T-cell proliferation, and the generation of anti-CD3-activated killer (AK) cells was inhibited in a dose-dependent fashion by PTX (25-100 micrograms/ml). The inhibitory effect of PTX could not be attributed to a defect in the recognition/adhesion phase of cytolysis because AK cells generated in the presence of PTX conjugated normally with P815 tumour target cells. However, AK cell expression of the cytoplasmic granule-associated cytolytic effector molecules granzyme B and perforin was markedly reduced when AK cells were induced in the presence of PTX. In eontrast, PTX had no effect on AK cell expression of Fas ligand, a cell-surface cytolytic effector molecule which is involved in granule-independent cytotoxicity. PTX thus has a profound inhibitory effect in vitro on the induction of granule-dependent cytolytic effector mechanisms in a mouse model system.
The present study was to ascertain the immunomodulating and anti-tumor effects of Ganoderma (G.) lucidum. Polysaccharides (PS) from fresh fruiting bodies of G. lucidum (PS-G) were isolated and used to potentiate cytokine production by human monocytes-macrophages and T lymphocytes. Our results had shown that the levels of interleukin (IL)-1 beta, tumor necrosis factor (TNF)- alpha, and IL-6 in macrophage cultures treated with PS-G (100 micrograms/ml) were 5.1-, 9.8- and 29-fold higher, respectively, than those of untreated controls. In addition, the release of interferon (IFN)- gamma from T lymphocytes was also greatly promoted in the presence of PS-G (25-100 micrograms/ml). Furthermore, these cytokine-containing mononuclear cell-conditioned media (PSG-MNC-CM) were found to suppress the proliferation and clonogenicity of both the HL-60 and the U937 leukemic cell lines. DNA labeling and gel electrophoresis showed that treatment with PSG-MNC-CM markedly induced leukemic-cell apoptosis. Flow-cytometric analysis revealed that few (2.3 +/- 0.8%) apoptotic cells were seen in the control cultures, while PSG-MNC-CM treatment resulted in a significant increase in the apoptotic population both in the HL-60 (38.3 +/- 4.5%) and in the U937 (44.5 +/- 3.8%) cells. In addition, 40 to 45% of the treated leukemic cells were triggered to differentiate into mature monocytic cells expressing CD14 and CD68 surface antigens. However, PS-G alone had no such effects even at a higher dose of 400 micrograms/ml. Since untreated macrophages and T lymphocytes produced little or no cytokine, and normal MNC-CM did not suppress leukemic cell growth, it was suggestive that the anti-tumor activity of PSG-MNC-CM was derived from the elevated levels of cytokines. Antibody-neutralization studies further revealed that the anti-tumor cytokines in the PSG-MNC-CM were mainly of TNF- alpha and IFN- gamma, and these 2 cytokines acted synergistically on the inhibition of leukemic-cell growth.
Immunologic effector cells termed cytokine-induced killer (CIK) cells are generated in vitro from peripheral blood lymphocytes by addition of interferon-gamma, interleukin (IL)-2, IL-1 and an antibody against CD3. CIK cells have been shown to eradicate established tumors in a SCID mouse/human lymphoma model. CIK cells are dependent on exogenous cytokines such as IL-2, IL-7, or IL-12. We studied the effect of these cytokines in detail. Cellular proliferation was analyzed using an MTT proliferation assay, surface antigen expression via flow cytometry, cytotoxic activity using an LDH release assay, and apoptosis via flow cytometric analysis. IL-2, IL-7 and IL-12 led to significant growth of lymphocytes. Cells grown in IL-2 and IL-7 showed higher proliferation rates than cells grown in IL-12 according to the MTT assay. Concerning surface antigen expression, exogenous IL-7 led to a decrease in IL-7 receptor expression (4.8% from 60.4%) and exogenous IL-2 to a decrease in IL-2 receptor expression (61.2% from 73.2%). CD28 expression was higher in cells grown in IL-7 (77.3%) than in cells grown in IL-2 (62.5%). IL-12 led to a decrease in ICAM-1 adhesion molecule expression (57.7% from 76.7%) and an increase in CD56 expression compared with exogenous IL-7. IL-7 led to higher number of CD4-positive cells than IL-2 (53.0% vs 49.5%). No significant difference was found between IL-2, IL-7 and IL-12 in cytotoxic activity measured in an LDH release assay. Small amounts of apoptotic cells were found with all cytokines. However, the percentage of necrotic cells was higher with exogenous IL-12 than with IL-2 or IL-7. In summary, CIK cells can be generated using exogenous IL-2, IL-7 or IL-12. No difference in cytotoxic activity was found. However, significant differences were found in cell proliferation rates, antigen expression and percentage of necrotic cells.
Alcohol consumption in mice suppresses the cytolytic activity of natural killer (NK) and lymphokine-activated killer (LAK) cells through unknown mechanisms. Herein, we found that alcohol consumption decreased target cell-induced release of granzyme A activity in freshly isolated splenic NK cells, in NK cells stimulated for 18 h with 1000 IU/ml of interleukin 2, and in LAK cells. The total activity and protein expression of granzymes A and B also were lower in these cells than in cells isolated from water-drinking mice. Interleukin 2 increased granzyme A protein expression independent of alcohol consumption; however, this increase was associated with decreased enzyme activity. In contrast, granzyme B protein expression and enzymatic activity increased in response to interleukin 2. Perforin activity and protein expression were reduced in LAK cells generated from alcohol-consuming mice. We conclude that the mechanism underlying the suppression of NK and LAK cytolytic activity by alcohol consumption involves the collective reduction of target-induced release, activity, and expression of perforin and granular proteases.
A variety of malignancies express Fas ligand (FasL), which can induce apoptosis in effector lymphocytes and may limit the success of cellular immunotherapy. Our laboratory has been investigating a population of ex vivo activated T cells, termed cytokine-induced killer (CIK) cells. These cells share functional and phenotypic properties with natural killer cells and a subset of cytolytic cells have the phenotype CD3+CD56+. CIK cells expand in culture, have significant antitumor activity and are presently being tested in phase I/II clinical trials. In this study, we investigated the sensitivity of CIK cells to Fas-mediated apoptosis. Fas engagement leads to apoptosis in small numbers of CIK cells and does not significantly influence antitumor cytotoxicity. CIK cells will undergo apoptosis following Fas engagement when protein synthesis is inhibited, suggesting the expression of antiapoptotic genes. Evaluation of antiapoptotic gene transcripts shows an upregulation in the expression of cFLIP, Bcl-2, Bcl-xL, DAD1 and survivin. Resistance to Fas-mediated apoptosis may come about through an in vitro selection for Fas resistance, since CIK cells synthesize FasL and supernatant from CIK cultures contains biologically active soluble FasL, which can be inhibited with Fas:Fc. These results indicate that CIK cells are a suitable form of immunotherapy against FasL-positive tumors.
A fucose-containing glycoprotein fraction which stimulates spleen cell proliferation and cytokine expression has been identified from the water-soluble extract of Ganoderma lucidum. Proteomic analysis of mouse spleen cells treated with this glycoprotein fraction showed approximately 50% change of the proteome. Further studies on the activities of this glycoprotein fraction through selective proteolysis and glycosidic cleavage indicate that a fucose containing polysaccharide fraction is responsible for stimulating the expression of cytokines, especially IL-1, IL-2 and INF-gamma.
Carrageenan is a high molecular weight polysaccharide and is widely used as a food additive for the solidification of plant oils and the thickening of many beverages. It is known that acute toxicity of carrageenan is possibly induced by the activation of phagocytic cells. We investigated other effects of carrageenan on lymphocytes in this study.
Carrageenan was intraperitoneally injected once into mice and phenotypic and functional characterizations were conducted in various immune organs.
Natural killer (NK) cells were prominently activated in the liver, lungs, and spleen. A time-kinetic study showed sequential activation of NK and natural killer T (NKT) cells in the liver on days 3-10 after the injection. In parallel with the activation of NK and NKT cells in number, NK and NKT cytotoxicities were augmented. At this time, liver injury was induced, accompanied by massive hepatic necrosis and the elevation of transaminases. The in vivo elimination of NK cells reduced the liver injury induced by carrageenan. Direct binding of carrageenan onto NK cells was also demonstrated. Such a binding then induced a subsequent production of IFN gamma. Perforin molecules of NK cells were responsible for this liver injury.
These results suggest that not only phagocytic cells but also primitive lymphocyte (mainly NK cells) subsets might be important targets for the acute toxicity of carrageenan.
To study the protective effects of Ganoderma lucidum polysaccharides peptide (GLPP) on the mice peritoneal macrophages injured by reactive oxygen species (ROS), derived from tert-butylhydroperoxide (tBOOH) in vitro and in vivo.
Mice peritoneal macrophages were injured by ROS, derived from tBOOH. The survival rate of macrophages was measured by MTT assay, and the morphological changes of macrophages were observed under light and electron microscopes.
GLPP (50, 100, 200 mg/kg, ip for 5 d) could inhibit the foam cell formation and necrosis of macrophages. The survival rate of macrophages was increased. GLPP (3.125, 12.5, 50, 200 mg/L) given to the cultured macrophages brought the same protective effects. Under the electron microscope it was found that GLPP (100 mg/kg, ip, for 5 d) could protect the organelle such as mitochondria against injury by tBOOH.
GLPP had significant scavenging ROS and antioxidant effects.
This study was undertaken to investigate the protective effect against alloxan-induced pancreatic islets damage by Ganoderma lucidum Polysaccharides (Gl-PS) isolated from the fruiting body of Ganoderma lucidum (Leyss. ex Fr.) Karst. In vitro, alloxan caused dose-dependent toxicity on the isolated pancreatic islets. Pre-treatment of islets with Gl-PS for 12 h and 24 h significantly reversed alloxan-induced islets viability loss. Gl-PS was also found to inhibit the free radicals production induced by alloxan in the isolated pancreatic islets using confocal microscopy. Gl-PS dose-dependently increased serum insulin and reduced serum glucose levels when pretreated intragastrically for 10 days in alloxan-induced diabetic mice. It was found that the pancreas homogenates had higher lipid peroxidation products in alloxan-treated mice than in the Gl-PS-treated animals. Aldehyde fuchsin staining revealed that alloxan caused nearly all the beta cells disappearing from the pancreatic islets, while Gl-PS partly protected the beta cells from necrosis. Alloxan (60 mg/kg) induced NF-kappa B activation in the pancreas at 30 min after injection, pretreatment with Gl-PS inhibited alloxan-induced activation of NF-kappa B. These results suggest that Gl-PS was useful in protecting against alloxan-induced pancreatic islets damage in vitro and in vivo; one of the mechanisms is through its scavenging ability to protect the pancreatic islets from free radicals-damage induced by alloxan.
To study the effects (and the mechanisms thereof) of Ganoderma lucidum polysaccharides (Gl-PS) on the proliferation and the anti-tumor activity of cytokine-induced killer (CIK) cells, and to make use of CIK cells as a means to investigate the interactions between Gl-PS and cytokines.
CIK cells were prepared by using the standard protocol as a positive control. Experimental groups also underwent the standard protocol, except that Gl-PS (400 mg/L or 100 mg/L) was added and the dose of anti-CD3 and interleukin-2 they received was reduced by 50% and 75%, respectively. For negative controls, Gl-PS in the experimental protocol was replaced with soluble starch or methylcellulose (400 mg/L or 100 mg/L). CIK cell proliferation, cytotoxicity, and phenotype were determined by using the Trypan blue exclusion method, MTT assay, and flow cytometry.
By synergizing cytokines, Gl-PS (400 mg/L or 100 mg/L) could decrease the amount of cytokine in lymphokine activated killer (LAK) cells and CIK cells culture, but had no significant effect on the proliferation, cytotoxicity, or phenotype of LAK cells, or CIK cells induced by cytokines at higher doses alone, in which CIK cells expanded about 80-fold and the main effectors, CD3+NK1.1+ cells, expanded by more than 15%. The cytotoxicity of CIK cells in experimental groups was 79.3%+/-4.7%, 76.9%+/-6.8% versus the positive control 80.7%+/-6.8% against P815 (P>0.05) and 88.9%+/-5.5%, 84.7%+/-7.9% versus the positive control 89.8%+/-4.5% against YAC-1 (P>0.05). The activity of Gl-PS could mostly be blocked by anti-CR3.
Gl-PS was shown to be a promising biological response modifier and immune potentiator. The effect of Gl-PS on CIK cells is possibly mediated primarily through complement receptor type 3.
Liver injury due to sequential activation of natural killer cells and natural killer T cells by carrageenan Immunosuppression induced by nitric oxide and its inhibition by interleukin-4
- T Abe
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- S Kawabe
- H Watanabe
- F Gejyo
- T Abo
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Generation of cytokine-induced killer (CIK) cells using exogenous interleukin-2 (IL-2), -7 (IL-7) or -12 (IL-12) Cancer Immunology Immunotherapy
- B Zoll
- P Lefterova
- S Finke
- B Trojaneck
- O Ebert
- B Micka
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