Bacteria colonization in tumor microenvironment creates a favorable niche for immunogenic chemotherapy.

The tumor microenvironment (TME) presents differential selective pressure (DSP) that favors the growth of cancer cells, and monovalent therapy is often inadequate in reversing the cancer cell dominance in the TME. In this work, we introduce bacteria as a foreign species to the TME and explore combinatorial treatment strategies to alter DSP for tumor eradication. We show that cancer-selective chemotherapeutic agents and fasting can provide a strong selection pressure against tumor growth in the presence of bacteria. Moreover, we show that an immunogenic drug (oxaliplatin), but not a non-immunogenic one (5-FU), synergizes with the bacteria to activate both the innate and adaptive immunity in the TME, resulting in complete tumor remission and a sustained anti-tumor immunological memory in mice. The combination of oxaliplatin and bacteria greatly enhances the co-stimulatory and antigen-presenting molecules on antigen-presenting cells, which in turn bridge the cytotoxic T cells for cancer-cell killing. Our findings indicate that rational combination of bacterial therapy and immunogenic chemotherapy can promote anticancer immunity against the immunosuppressive TME.

High-Affinity Superantigen-Based Trifunctional Immune Cell Engager Synergizes NK and T Cell Activation for Tumor Suppression.

The development of immune cell engagers (ICEs) can be limited by logistical and functional restrictions associated with fusion protein designs, thus limiting immune cell recruitment to solid tumors. Herein, a high affinity superantigen-based multivalent ICE is developed for simultaneous activation and recruitment of NK and T cells for tumor treatment. Yeast library-based directed evolution is adopted to identify superantigen variants possessing enhanced binding affinity to immunoreceptors expressed on human T cells and NK cells. High-affinity superantigens exhibiting improved immune-stimulatory activities are then incorporated into a superantigen-based tri-functional yeast-display-enhanced multivalent immune cell engager (STYMIE), which is functionalized with a nanobody, a Neo-2/15 cytokine, and an Fc domain for tumor targeting, immune stimulation, and prolonged circulation, respectively. Intravenous administration of STYMIE enhances NK and T cell recruitment into solid tumors, leading to enhanced inhibition in multiple tumor models. The study offers design principles for multifunctional ICEs.

Fragment-Directed Random Mutagenesis by the Reverse Kunkel Method.

Two fundamentally different approaches are routinely used for protein engineering: user-defined mutagenesis and random mutagenesis, each with its own strengths and weaknesses. Here, we invent a unique mutagenesis protocol, which combines the advantages of user-defined mutagenesis and random mutagenesis. The new method, termed the reverse Kunkel method, allows the user to create random mutations at multiple specified regions in a one-pot reaction. We demonstrated the reverse Kunkel method by mimicking the somatic hypermutation in antibodies that introduces random mutations concentrated in complementarity-determining regions. Coupling with the phage display and yeast display selections, we successfully generated dramatically improved antibodies against a model protein and a neurotransmitter peptide in terms of affinity and immunostaining performance. The reverse Kunkel method is especially suitable for engineering proteins whose activities are determined by multiple variable regions, such as antibodies and adeno-associated virus capsids, or whose functional domains are composed of several discontinuous sequences, such as Cas9 and Cas12a.

Identification and characterization of a cytochrome b559 Synechocystis 6803 mutant spontaneously generated from DCMU-inhibited photoheterotrophical growth conditions.

We identified a spontaneously generated mutant from Synechocystis sp. PCC6803 wild-type cells grown in BG-11 agar plates containing 5 mM Glu and 10 microM DCMU. This mutant carries an R7L mutation on the alpha-subunit of cyt b559 in photosystem II (PSII). In the recent 2.9 A PSII crystal structural model, the side chain of this arginine residue is in close contact with the heme propionates of cyt b559. We called this mutant WR7Lalpha cyt b559. This mutant grew at about the same rate as wild-type cells under photoautotrophical conditions but grew faster than wild-type cells under photoheterotrophical conditions. In addition, 77 K fluorescence and 295 K chlorophyll a fluorescence spectral results indicated that the energy delivery from phycobilisomes to PSII reaction centers was partially inhibited or uncoupled in this mutant. Moreover, WR7Lalpha cyt b559 mutant cells were more susceptible to photoinhibition than wild-type cells under high light conditions. Furthermore, our EPR results indicated that in a significant fraction of mutant reaction centers, the R7Lalpha cyt b559 mutation induced the displacement of one of the axial histidine ligands to the heme of cyt b559. On the basis of these results, we propose that the Arg7Leu mutation on the alpha-subunit of cyt b559 alters the interaction between the APC core complex and PSII reaction centers, which reduces energy delivery from the antenna to the reaction center and thus protects mutant cells from DCMU-induced photo-oxidative stress.

SLC38A2 Overexpression Induces a Cancer-like Metabolic Profile and Cooperates with SLC1A5 in Pan-cancer Prognosis.

Cancer cells have dramatically increased demands for energy as well as biosynthetic precursors to fuel their restless growth. Enhanced glutaminolysis is a hallmark of cancer metabolism which fulfills these needs. Two glutamine transporters, SLC1A5 and SLC38A2, have been previously reported to promote glutaminolysis in cancer with controversial perspectives. In this study, we harnessed the proximity labeling reaction to map the protein interactome using mass spectrometry-based proteomics and discovered a potential protein-protein interaction between SLC1A5 and SLC38A2. The SLC1A5/SLC38A2 interaction was further confirmed by bimolecular fluorescence complementation assay. We further investigated the metabolic influence of SLC1A5 and SLC38A2 overexpression in human cells, respectively, and found that only SLC38A2, but not SLC1A5, resulted in a cancer-like metabolic profile, where the intracellular concentrations of essential amino acids and lactate were significantly increased as quantified by nuclear magnetic resonance spectroscopy. Finally, we analyzed the 5-year survival rates in a large pan-cancer cohort and found that the SLC1A5hi /SLC38A2lo group did not relate to a poor survival rate, whereas the SLC1A5lo /SLC38A2hi group significantly aggravated the lethality. Intriguingly, the SLC1A5hi /SLC38A2hi group resulted in an even worse prognosis, suggesting a cooperative effect between SLC1A5 and SCL38A2. Our data suggest that SLC38A2 plays a dominant role in reprogramming the cancer-like metabolism and promoting the cancer progression, whereas SLC1A5 may augment this effect when co-overexpressed with SLC38A2. We propose a model to explain the relationship between SLC1A5, SLC38A2 and SCL7A5, and discuss their impact on glutaminolysis and mTOR signaling.

The cysteine-free single mutant C32S of APEX2 is a highly expressed and active fusion tag for proximity labeling applications.

APEX2, an engineered ascorbate peroxidase for high activity, is a powerful tool for proximity labeling applications. Owing to its lack of disulfides and the calcium-independent activity, APEX2 can be applied intracellularly for targeted electron microscopy imaging or interactome mapping when fusing to a protein of interest. However, APEX2 fusion is often deleterious to the protein expression, which seriously hampers its wide utility. This problem is especially compelling when APEX2 is fused to structurally delicate proteins, such as multi-pass membrane proteins. In this study, we found that a cysteine-free single mutant C32S of APEX2 dramatically improved the expression of fusion proteins in mammalian cells without compromising the enzyme activity. We fused APEX2 and APEX2C32S to four multi-transmembrane solute carriers (SLCs), SLC1A5, SLC6A5, SLC6A14, and SLC7A1, and compared their expressions in stable HEK293T cell lines. Except the SLC6A5 fusions expressing at decent levels for both APEX2 (70%) and APEX2C32S (73%), other three SLC proteins showed significantly better expression when fusing to APEX2C32S (69 ± 13%) than APEX2 (29 ± 15%). Immunofluorescence and western blot experiments showed correct plasma membrane localization and strong proximity labeling efficiency in all four SLC-APEX2C32S cells. Enzyme kinetic experiments revealed that APEX2 and APEX2C32S have comparable activities in terms of oxidizing guaiacol. Overall, we believe APEX2C32S is a superior fusion tag to APEX2 for proximity labeling applications, especially when mismatched disulfide bonding or poor expression is a concern.

Construction of antithrombogenic polyelectrolyte multilayer on thermoplastic polyurethane via layer-by-layer self-assembly technique.

The improvement of hydrophilicity and hemocompatibility of thermoplastic polyurethane (TPU) film was developed using surface modification of polyelectrolyte multilayers (PEMs) deposition. The polysaccharide PEMs included chitosan (CS, as a positive-charged agent) and dextran sulfate (DS, as a negative-charged and an antiadhesive agent) that were successfully prepared on the aminolyzed TPU film in a layer-by-layer (LBL) self-assembly manner. X-ray photoelectron spectroscopy (XPS), field-emission scanning electronic microscopy (FE-SEM), and atomic force microscopy (AFM) data will verify the progressive buildup of the PEMs film. The obtained results showed that the contact angle and Zeta-potential reached the steady value after four bilayers of coating, hence proving that the full coverage of coating with PEM layers was achieved. It could be found that the PEMs-deposited TPU films with DS as the outmost layer could resist the platelet adhesion and human plasma fibrinogen (HPF) adsorption, thereby prolonging effectively the blood coagulation times. Besides, the results of growth inhibition index (GI) of L929 fibroblast proliferation suggested that the as-fabricated TPU films were noncytotoxic. Overall results demonstrated that such an easy, valid, shape-independent, and noncytotoxic processing should be potential for the ion of TPU substrate in the application of hemodialysis or cardiovascular devices.

Surface modification of poly(tetramethylene adipate-co-terephthalate) membrane via layer-by-layer assembly of chitosan and dextran sulfate polyelectrolyte multiplayer.

The improvement of hydrophilicity and hemocompatibility of poly(tetramethylene adipate-co-terephthalate) (PTAT) membrane was developed via polyelectrolyte multilayers (PEMs) immobilization. The polysaccharide PEMs included chitosan (CS, as a positive-charged and antibacterial agent) and dextran sulfate (DS, as a negative-charged and anti-adhesive agent) were successfully prepared using the aminolyzed PTAT membrane in a layer-by-layer (LBL) self-assembly manner. The obtained results showed that the contact angle of as-modified PTAT membranes reached to the steady value after four bilayers of coating, hence suggesting that the full coverage was achieved. It could be found that the PTAT-PEMs membranes with DS as the outmost layer could resist the platelet adhesion and human plasma fibrinogen (HPF) adsorption, thereby prolonging effectively the blood coagulation times. According to L929 fibroblast cell growth inhibition index, the as-prepared PTAT membranes exhibited non-cytotoxic. Overall results demonstrated that such an easy, valid and shape-independent processing should be potential for surface modification of PTAT membrane in the application of hemodialysis devices.

Chronic renal failure rats are highly sensitive to aristolochic acids, which are nephrotoxic and carcinogenic agents.

Aristolochic acid (AA), a component of some Chinese herbal medicines, may cause Chinese Herbs Nephropathy (CHN) and multi-systemic tumors by the formation of AA-DNA adducts. In this study, we established an animal model to further characterize the mechanisms of AA-induced diseases. Our results indicated that AA significantly inhibited rat growth in terms of weight gain. By measuring the serum creatinine levels, AA resulted in considerable damage to the rat renal system, not only for those in which chronic renal failure (CRF) was induced but also for normal healthy rats. Mutation-specific polymerase chain reaction (PCR) and XbaI restriction fragment length polymorphism (RFLP) revealed the CAA-->CTA transversion mutation at codon 61 of the H-ras proto-oncogene from the stomach tissues of CRF rats fed with AA, but not from other tissues of rats in the same experimental group. In addition, no such mutations were found in the tissues of CRF rats without AA treatment or healthy rats fed with AA. Our results strongly demonstrated that AA was in fact nephrotoxic and carcinogenic, especially to those CRF rats.

Cytocompatibility and antibacterial activity of a PHBV membrane with surface-immobilized water-soluble chitosan and chondroitin-6-sulfate.

A water-soluble chitosan (WSC)/chondroitin-6-sulfate (ChS) polyelectrolyte complex (PEC) is covalently immobilized onto the surface of poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) membranes via ozone-induced oxidation and poly(acrylic acid) (PAA) graft polymerization. To characterize the modified membranes, X-ray photoelectron spectroscopy (XPS) and water contact angle measurements are performed. It is shown that by coupling WSC as a spacer, the amount of ChS immobilized can be significantly increased. The water contact angle decreases with the amount of PAA, WSC, and ChS immobilized, which indicates the improving hydrophilicity. After WSC- and PEC-immobilization modification, the PHBV membranes possess antibacterial activity against S. aureus, E. coli, P. aeruginosa, and Methicilin resistant Staphylococus aureus (MRSA). According to the L929 fibroblast cell growth inhibition index, the as-prepared PHBV membranes are non-cytotoxic. In addition, the in-vitro evaluation of L929 fibroblast attachment, proliferation, and viability of PEC-immobilized PHBV membranes are ascertained to be superior to those of immobilized WSC or ChS alone. The overall results demonstrate that WSC/ChS PEC immobilization can not only improve the hydrophilicity and cytocompatibility of the PHBV membrane, but also endows antibacterial activity. [GRAPH: SEE TEXT] The bacterial survival ratio of as-prepared PHBV membranes (n=3).

Blood compatibility of novel poly(gamma-glutamic acid)/polyvinyl alcohol hydrogels.

Sodium poly(gamma-glutamic acid) (PGA), a water-soluble and biodegradable polypeptide, was reacted with polyvinyl alcohol (PVA) to form hydrogel without any chemical treatment. The gelation occurred probably due to physical cross-linking of polymer chains by interpenetrating hydrogen bonding. From the results of thermal analysis, PGA/PVA exhibited better thermal stability than native PVA. Although the swelling ratio decreased with the increase of PGA content, however, the water resistance and retention were improved. The tensile strength of the PGA/PVA hydrogel membranes was about 15-30% lower than that of the native PVA, whereas the elongation was increased 2.0-2.6 times. The amount of protein adsorbed and platelets adhered on the PGA/PVA membranes were significantly curtailed with increasing PGA content, thereby showing improved blood compatibility. The as-fabricated hydrogels were proven to be non-cytotoxic evaluated in vitro by L-929 fibroblast incubation. Overall results demonstrate that the non-cytotoxic PGA/PVA hydrogels, due to better water resistance, mechanical properties and blood compatibility could be very promising candidates for blood-contacting medical devices.

Hemocompatibility and anaphylatoxin formation of protein-immobilizing polyacrylonitrile hemodialysis membrane.

Plasma proteins were covalently immobilized onto polyacrylonitrile (PAN) membrane to evaluate the hemocompatibility and anaphylatoxin formation. This is used as a model to study the effect of protein-adsorption on the blood-contacting response of hemodializing membranes. The proteins used were either platelet-adhesion-promoting collagen (COL) or platelet-adhesion-inhibiting human serum albumin (HSA). The microstructure and characterization of the protein-immobilizing PAN membranes were evaluated by Coomassie dye assay, atomic force microscopy, X-ray photoelectron spectroscopy and water contact angle measurement. PAN-HSA membrane improved not only hemocompatibility including less platelet adhesion, longer blood coagulation times, and higher thrombin inactivity level, but also induced lower complement activation. On the other hand, PAN-COL membrane exhibited blood incompatibility, although induced less increase of C3, C4 antigens of serum. Overall results of this study demonstrated that the immobilization of HSA onto the surface of PAN membrane would be beneficial to improve the hemocompatibility and to reduce the anaphylatoxin formation during hemodialysis.

In-vitro hemocompatibility evaluation of a thermoplastic polyurethane membrane with surface-immobilized water-soluble chitosan and heparin.

The surface of a thermoplastic polyurethane (TPU) membrane was treated with low temperature plasma (LTP) and was then grafted with poly(acrylic acid) (PAA), followed by the grafting of water-soluble chitosan (WSC) and heparin (HEP). The surface was characterized with static contact-angle and X-ray photoelectron spectroscopy (XPS). The results showed that the surface densities of peroxides and PAA reached a maximum when treated with LTP for 90 s. A higher pH of the reacting solution led to higher graft densities of WSC and HEP. After WSC and HEP grafting, the hydrophilicity of the TPU membrane was increased. The adsorption of proteins on HEP-grafted TPU membranes was effectively curtailed. In addition, HEP grafting also reduced platelet adhesion, elevated thrombin inactivation, and prolonged the blood coagulation time. According to the L929 fibroblast cell growth inhibition index, the HEP-grafted TPU membranes exhibited non-cytotoxicity. Overall results demonstrated that the HEP immobilization could not only improve the hydrophilicity but also the hemocompatibility of the TPU membrane, while maintaining the ascendant biocompatibility.

Blood compatibility of thermoplastic polyurethane membrane immobilized with water-soluble chitosan/dextran sulfate.

Water-soluble chitosan (WSC)/dextran sulfate (DS) was immobilized onto the surface of thermoplastic polyurethane (TPU) membrane after ozone-induced graft polymerization of poly(acrylic acid) (PAA). The surface was characterized with contact angle measurement and X-ray photoelectron spectroscopy (XPS). The adsorption of human plasma fibrinogen (HPF) followed the Langmuir adsorption isotherm. The results showed that the surface density of peroxides generated and poly(acrylic acid) (PAA) grafted reached the maximum value at 20 min of ozone treatment. It was found that the WSC- and DS-immobilized amount increased with pH and the molecular weight of WSC. The membrane/water interfacial free energy increased with PAA-grafting and WSC/DS-immobilization, indicating the increasing wettability of TPU membrane. The adsorption of HPF on TPU-WSC/DS membranes could be effectively curtailed and exhibited unfavorable adsorption. Moreover, WSC/DS immobilization could effectively reduce platelet adhesion and prolong the blood coagulation time, thereby membrane improving blood compatibility of TPU membrane. In addition, the in vitro cytotoxicity test of PEC modification was non-cytotoxic according to much low growth inhibition of L929 fibroblasts. Furthermore, TPU-WSC/DS membranes exhibited higher cell viability than native TPU membrane.

pH-sensitive polyelectrolyte complex gel microspheres composed of chitosan/sodium tripolyphosphate/dextran sulfate: swelling kinetics and drug delivery properties.

Porous chitosan (CS) polyelectrolyte complex (PEC) hydrogel microspheres were prepared via either wet phase-inversion or ionotropic crosslinking with sodium tripolyphosphate (Na+ - TPP) and dextran sulfate (DS). The resulting microspheres were characterized using scanning electron microscopy (SEM) and elemental analysis (EA). The controlled release behavior of ibuprofen (IBU) from these microspheres was investigated. The PEC microspheres were about 700-950 microm in diameter with large pores and open porous structure. The CS/TPP/DS microspheres resisted hydrolysis in strong acid and biodegradation in enzymatic surroundings. The swelling kinetics for CS microspheres was close to Fickian diffusion, whereas those for CS/TPP and CS/TPP/DS were non-Fickian. Furthermore, the equilibrium water content (EWC) and water diffusion coefficient (D) increased with the pH of the media. The release profiles of IBU from CS/TPP/DS microspheres were slow in simulated gastric fluid (SGF, pH 1.4) over 3 h, but nearly all of the initial drug content was released in simulated intestinal fluid (SIF, pH 6.8) within 6 h after changing media. Overall the results demonstrated that CS/TPP/DS microspheres could successfully deliver a hydrophobic drug to the intestine without losing the drug in the stomach, and hence could be potential candidates as an orally administered drug delivery system.

Integrative approach to analyze biodiversity and anti-inflammatory bioactivity of Wedelia medicinal plants.

For the development of "medical foods" and/or botanical drugs as defined USA FDA, clear and systemic characterizations of the taxonomy, index phytochemical components, and the functional or medicinal bioactivities of the reputed or candidate medicinal plant are needed. In this study, we used an integrative approach, including macroscopic and microscopic examination, marker gene analysis, and chemical fingerprinting, to authenticate and validate various species/varieties of Wedelia, a reputed medicinal plant that grows naturally and commonly used in Asian countries. The anti-inflammatory bioactivities of Wedelia extracts were then evaluated in a DSS-induced murine colitis model. Different species/varieties of Wedelia exhibited distinguishable morphology and histological structures. Analysis of the ribosomal DNA internal transcribed spacer (ITS) region revealed significant differences among these plants. Chemical profiling of test Wedelia species demonstrated candidate index compounds and distinguishable secondary metabolites, such as caffeic acid derivatives, which may serve as phytochemical markers or index for quality control and identification of specific Wedelia species. In assessing their effect on treating DSS induced-murine colitis, we observed that only the phytoextract from W. chinensis species exhibited significant anti-inflammatory bioactivity on DSS-induced murine colitis among the various Wedelia species commonly found in Taiwan. Our results provide a translational research approach that may serve as a useful reference platform for biotechnological applications of traditional phytomedicines. Our findings indicate that specific Wedelia species warrant further investigation for potential treatment of human inflammatory bowel disease.

Hemocompatibility of polyacrylonitrile dialysis membrane immobilized with chitosan and heparin conjugate.

Chitosan (CS)/heparin (HEP) polyelectrolyte complex (PEC) was covalently immobilized onto the surface of polyacrylonitrile (PAN) membrane. The effect of surface modification on the protein adsorption and platelet adhesion, metabolites permeation and anticoagulation activity of the resulting membrane was investigated. Surface characterization such as water contact angle, and X-ray photoelectron spectroscope were performed. The immobilization of PEC caused the water contact angle to reduce, thereby indicating the increase in the hydrophilicity. Protein adsorption, platelet adhesion, and thrombus formation were all reduced by the immobilization of HEP. Anticoagulant activity was evaluated with activated partial thrombin time (APTT), prothrombin time (PT), fibrinogen time, and thrombin time (TT). The results revealed that PEC-immobilizing membrane can improve antithrombogenicity of PAN membrane. In addition, the PEC-immobilized membranes can suppress the proliferation of Pseudomonas aeruginosa. In vitro cytotoxicity test showed leachable substance released was below cytotoxic level. The pure water permeability results show little variation due to PEC-immobilization. Thus PEC-immobilization can endow the PAN membrane hemocompatibility and antibacterial activity while retaining the original permeability.

Fractal dynamics and synchronization of rhythms in urodynamics of female Wistar rats.

Two phenomena are ubiquitous in physiological signals. First, they have temporal fractal structure. Secondly, they are rhythmic but with fluctuations. The fractal structure and rhythms can alter from normal to abnormal with disease or injury. Here, we demonstrate that a temporal "coherent" fractal structure and synchronization of rhythms are two essential features for concerted systemic functions in urodynamics of female Wistar rats. We report evidence on the dynamic interaction in the micturition reflex of lower urinary tract: the rhythms of detrusor of the bladder and external urethral sphincter are synchronized around 7 Hz and their fractal dimensions as functions of time are temporally "coherent" under 1.5 during voiding in order to facilitate the concerted process. On the other hand, in the spinal cord injured rats, either the temporal coherent structure of fractal or the underlying synchronization of rhythms is impaired, weakened, or absent. These results have potential implications in the diagnosis of the so-called "dynamical" diseases.

Novel silicone hydrogel based on PDMS and PEGMA for contact lens application.

A silicone-based hydrogel was synthesized from poly(dimethylsiloxane) dialkanol (PDMS), isophorone diisocyanate (IPDI), 2-hydroxyethyl methacrylate (HEMA) and poly(ethylene glycol) methacrylate (PEGMA). The hydrophilicity of the resulting block copolymer was adjustable by manipulating the ratio of PDMS and PEGMA. The results showed that higher PEGMA content led to a lower water contact angle, higher water content, lower elastic modulus and higher glucose permeability. At a PEGMA content of 20%, the protein adsorption decreased to 23% and 18% for lysozyme and human serum albumin (HSA), respectively, of those of the control (PDMS-PU). This indicated that the PDMS-PU-PEGMA hydrogels exhibited an ability to resist protein adsorption. The oxygen permeability (Dk) was 92 barrers for the hydrogel with 20% PEGMA. Furthermore, these hydrogels were non-cytotoxic according to an in vitro L929 fibroblast assay. Overall, the results demonstrated that the PDMS-PU-PEGMA hydrogels exhibited not only relatively high oxygen permeability and relative optical transparency, but also hydrophilicity and anti-protein adsorption; therefore, they would be applicable as a contact lens material. Furthermore, this study demonstrated a new approach to controlling the performance of silicone hydrogels.

Dietary uptake of Wedelia chinensis extract attenuates dextran sulfate sodium-induced colitis in mice.

SCOPE: Traditional medicinal herbs are increasingly used as alternative therapies in patients with inflammatory diseases. Here we evaluated the effect of Wedelia chinensis, a medicinal herb commonly used in Asia, on the prevention of dextran sulfate sodium (DSS)-induced acute colitis in mice. General safety and the effect of different extraction methods on the bioactivity of W. chinensis were also explored. METHODS AND RESULTS: C57BL/6 mice were administrated hot water extract of fresh W. chinensis (WCHF) orally for one week followed by drinking water containing 2% DSS for nine days. WCHF significantly attenuated the symptoms of colitis including diarrhea, rectal bleeding and loss of body weight; it also reduced the shortening of colon length and histopathological damage caused by colonic inflammation. Among four W. chinensis extracts prepared using different extraction techniques, WCHF showed the highest anti-colitis efficacy. Analyses of specific T-cell regulatory cytokines (TNF-α, IL-4, IFN-γ, IL-17, TGF-ß, IL-12) revealed that WCHF treatment can suppress the Th1 and Th17, but not Th2, responses in colon tissues and dendritic cells of DSS-induced colitis mice. A 28-day subacute toxicity study showed that daily oral administration of WCHF (100, 500, 1000 mg/kg body weight) was not toxic to mice. CONCLUSION: Together, our findings suggest that specific extracts of W. chinensis have nutritional potential for future development into nutraceuticals or dietary supplements for treatment of inflammatory bowel disease.