Advancement in the development of single chain antibodies using phage display technology.

Phage display technology has become an important research tool in biological research, fundamentally changing the traditional monoclonal antibody preparation process, and has been widely used in the establishment of antigen-antibody libraries, drug design, vaccine research, pathogen detection, gene therapy, antigenic epitope research, and cellular signal transduction research.The phage display is a powerful platform for technology development. Using phage display technology, single chain fragment variable (scFv) can be screened, replacing the disadvantage of the large size of traditional antibodies. Phage display single chain antibody libraries have significant biological implications. Here we describe the types of antibodies, including chimeric antibodies, bispecific antibodies, and scFvs. In addition, we describe the phage display system, phage display single chain antibody libraries, screening of specific antibodies by phage libraries and the application of phage libraries.

Research Progress on Immunomodulatory Effects of Poly (Lactic-co-Glycolic Acid) Nanoparticles Loaded with Traditional Chinese Medicine Monomers.

Immunomodulatory mechanisms are indispensable and key factors in maintaining the balance of the environment in humans. When the immune function of the immune system is impaired, autoimmune diseases occur. Excessive body fatigue, natural aging of the human body, malnutrition, genetic factors and other reasons cause low immune function, due to which the body is prone to being infected by bacteria or cancer. Clinically, the existing therapeutic drugs still have problems such as high toxicity, long treatment cycle, drug resistance and high price, so we still need to explore and develop a high efficiency and low toxicity drug. Poly(lactic-co-glycolic acid) (PLGA) refers to a non-toxic polymer compound that exhibits excellent biocompatibility. Traditional Chinese medicine (TCM) monomers come from natural plants, and have the characteristics of high efficiency and low toxicity. Applying PLGA to TCM monomers can make up for the defects of traditional dosage forms, improve bioavailability, reduce the frequency and dosage of drug use, and reduce toxicity and side effects, thus having the characteristics of sustained release and targeting. Accordingly, PLGA nanoparticles loaded with TCM monomers have been the focus of development. The previous research on drug loading advantages, preparation methods, and immune regulation of TCM PLGA nanoparticles is summarized in the following sections.

Review of knockout technology approaches in bacterial drug resistance research.

Gene knockout is a widely used method in biology for investigating gene function. Several technologies are available for gene knockout, including zinc-finger nuclease technology (ZFN), suicide plasmid vector systems, transcription activator-like effector protein nuclease technology (TALEN), Red homologous recombination technology, CRISPR/Cas, and others. Of these, Red homologous recombination technology, CRISPR/Cas9 technology, and suicide plasmid vector systems have been the most extensively used for knocking out bacterial drug resistance genes. These three technologies have been shown to yield significant results in researching bacterial gene functions in numerous studies. This study provides an overview of current gene knockout methods that are effective for genetic drug resistance testing in bacteria. The study aims to serve as a reference for selecting appropriate techniques.

Research Progress of Dendritic Cell Surface Receptors and Targeting.

Dendritic cells are the only antigen-presenting cells capable of activating naive T cells in humans and mammals and are the most effective antigen-presenting cells. With deepening research, it has been found that dendritic cells have many subsets, and the surface receptors of each subset are different. Specific receptors targeting different subsets of DCs will cause different immune responses. At present, DC-targeted research plays an important role in the treatment and prevention of dozens of related diseases in the clinic. This article focuses on the current status of DC surface receptors and targeted applications.

Gossypin: A flavonoid with diverse pharmacological effects.

Gossypin is a flavonoid compound prepared from chinese medicine Hibiscus, which not only has significant pharmacological activities in antioxidant, anti-inflammatory, neuroprotective, anti-cancer, anti-tumor, and anti-diabetic aspects, but also has the advantages of small side effects and easy preparation because it is extracted from traditional chinese medicine, so it has received widespread attention from scholars and researchers. This paper reviews the pharmacological effects and mechanisms of gossypin in recent years, and hopes to provide a theoretical basis for its clinical application.

Functional Roles of Exosomes in Allergic Contact Dermatitis.

Allergic contact dermatitis (ACD) is an allergen-specific T-cell-mediated inflammatory response, albeit with unclear pathogenesis. Exosomes are nanoscale extracellular vesicles secreted by several cell types and widely distributed in various biological fluids. Exosomes affect the occurrence and development of ACD through immunoregulation among other ways. Nevertheless, the role of exosomes in ACD warrants further clarification. This review examines the progress of research into exosomes and their involvement in the pathogenesis, diagnosis, and treatment of ACD and provides ideas for exploring new diagnostic and treatment methods for this disease.

Effects of Fisetin on Allergic Contact Dermatitis via Regulating the Balance of Th17/Treg in Mice.

Background: Allergic contact dermatitis (ACD) is a form of chronic cutaneous inflammatory disease of immunological origin that has adverse impacts on patient quality of life, underscoring the need for the development of safe and effective therapeutic agents to treat affected individuals. Fisetin is a Chinese herbal preparation that reportedly exhibits antitumor, antioxidant, antimicrobial, anticoagulatory, and antimalarial activity. In the current report, the immunomodulatory activity of fisetin was appraised by assessing its impact on balance between regulatory T (Treg) and Th17 cells in an ACD model. Methods: BALB/c mice (n = 60) were randomized into control, ACD model, CTX positive control (20 mg/kg), and fisetin treatment groups (three dose levels: 2, 4, or 8 mg/kg). ACD induction was achieved by sensitizing mice on the shaved ventral abdomen via the application of 5% DNFB (50 µL) on days 1 and 2, followed by rechallenge in the right ear with 5% DNFB (20 µL) on day 5. Beginning on day 1, immunized mice were intraperitoneally injected with the appropriate fisetin dose (in saline) once per day for 7 days. On day 7, ear swelling, transcription factor expression, Th17/Treg cell populations, and cytokine production were assessed in vivo. Results: Fisetin treatment significantly suppressed ear swelling and associated inflammatory cell infiltration, besides reducing the production of Th17 cytokines (IL-17, TNF-α, and IL-6) and the expression of the Th17 lineage transcription factor RORγt while simultaneously enhancing Treg-specific cytokine production (TGF-ß and IL-10) and the expression of the Treg lineage transcription factor Foxp3, thereby restoring the Th17/Treg cell in ACD mice. Conclusions: These data indicate that fisetin exhibits immunomodulatory activity and can alter the Th17/Treg cell balance, highlighting its potential value as a treatment drug for ACD.

Immunosuppressive activity of daphnetin on the humoral immune responses in ovalbumin-sensitized BALB/c mice.

INTRODUCTION: Most of the immunosuppressive drugs are used for the treatment of autoimmune disease, allergic diseases, and transplant rejection, but toxicity is the major obstacle for the potent drugs in the wide use of these immunosuppressive drugs. Daphnetin, a Chinese herbal product, has been reported that daphnetin possesses antimicrobial, anticoagulation, antimalarial, anticancer, and antioxidant activity. In a previous study, we found that daphnetin exhibited a potential immunosuppressive effect on LPS-induced B lymphocyte cells in vitro, therefore, in this research, we investigated the immunosuppressive effects of daphnetin in BALB/c mice use OVA as a prototype antigen. METHODS: Sixty BALB/c mice were divided into six groups. The emulsion (100 µL containing 100 µg OVA) was injected subcutaneously with OVA + CFA into the shaved backs of the BALB/c mice on day 1, and a boosting injection was administered in OVA + IFA 2 weeks later. Beginning on the day of immunization, the immunized mice were administered intraperitoneally with daphnetin at a dose of 5, 10, and 20 mg/kg in saline solution for 28 consecutive days. We measured the effect of daphnetin on OVA-specific antibody, cytokine production, and Splenocyte proliferation in vivo. RESULTS: The results revealed that daphnetin significantly suppressed serum immunoglobulin G levels (IgG), and the OVA-specific IgG subclasses IgG1 and IgG2b, daphnetin was also significantly decreased the Th1 and Th2 cytokine productions, inhibited the splenocytes proliferation rate in vivo. CONCLUSIONS: It proved that daphnetin could suppress humoral response activity on OVA-sensitized mice, suggesting a potential role on daphnetin as a new immunosuppressive drug.

Anti-cervical carcinoma effect of Portulaca oleracea L. polysaccharides by oral administration on intestinal dendritic cells.

BACKGROUND: Cervical cancer is the second most prevalent cancer worldwide. Portulaca oleracea L. polysaccharide (POL-P3b) has been found to have enhancing immune and anti-cervical cancer activity by oral administration. Dendritic cells (DC) play a key roles in regulating intestinal immune homeostasis. In this study, we analyzed the inhibition apoptosis effects of POL-P3b on intestinal DC and relevant mechanisms. METHODS: Intestinal DC was isolated from U14-bearing mice treated with POL-P3b (50 mg/kg, 100 mg/kg and 200 mg/kg, respectively). The effects of POL-P3b on proliferation and inhibiting apoptosis in intestinal DC were evaluated by MTT assay, Hoechst 33342 and Annexin V-FITC/PI staining. Mitochondrial Ca2+ was analyzed using flow cytometry instrument. The potential mechanisms underlying POL-P3b-induced protection of intestinal DC from cervical cancer-induced apoptosis were detected with Western blotting evaluation of expression levels of TLR4 and relevant proteins for apoptotic signaling pathway. RESULTS: We found that a large number of intestinal DC were apoptosis in U14-bearing mice. Treatment with POL-P3b in U14-bearing mice at different doses for 12 d resulted in a significant increase in intestinal DC survival, and the mechanisms were related to inhibiting DC apoptosis. CONCLUSION: Our results suggested that POL-P3b-induced protection against tumor-induced intestinal DC apoptosis through stimulating the TLR4-PI3K/AKT-NF-κB signaling pathway. This study enhanced understanding of the oral administration with POL-P3b exerted on anti-tumor activity and its action mechanism.

Oral pre-administration of Purslane polysaccharides enhance immune responses to inactivated foot-and-mouth disease vaccine in mice.

BACKGROUND: Foot-and-mouth disease (FMD) is one of the greatest disease threats to animal husbandry worldwide. Though various vaccines against foot-and-mouth disease virus (FMDV) have been developed, vaccine effectiveness is still not satisfactory. In this work, we studied the potential ability of Purslane polysaccharide (POL-P3b) as a nutrient food additive to enhance immune responses to FMD vaccination in mice. RESULTS: Our results demonstrated that oral administration of POL-P3b at mid- and high-doses significantly enhanced the FMDV-specific cellular and humoral immune responses in mice and increased the concentration of Ca2+ in lymphocytes. Importantly, POL-P3b could promote intestinal DC maturation and stimulate the secretion of intestinal SIgA in a dose-dependent manner. Moreover, the acute toxicity study showed that POL-P3b was non-toxic and safe in mice. CONCLUSION: Our findings provided solid evidence that POL-P3b might be a novel immunostimulator and a boosting agent for increasing the efficacy of FMD vaccination, and the mechanism was related to stimulating the intestinal mucosal immune function that subsequently enhanced the efficacy of FMD vaccination through pre-administration of oral POL-P3b.

Immunosuppressive activity of alpinetin on activation and cytokines secretion of murine T lymphocytes.

Abstract Alpinetin, a flavonoid compound extracted from the seeds of Alpinia katsumadai Hayata, has been known to possess antibacterial, anti-inflammatory and other important therapeutic activities. In the current study, we investigated alpinetin for its immunosuppressive effect on activation and cytokines secretion of murine T lymphocytes. The data showed that alpinetin markedly suppressed ConA-induced murine splenocyte proliferation, Th1/Th2 cytokines production, CD4(+) T-cell populations and ratio of CD4(+)/CD8(+). This inspired us to further study the effects of alpinetin in vivo. The results showed that administration of alpinetin suppressed T-cell-mediated delayed-type hypersensitivity reaction in mice. In addition, we studied signal transduction pathways about T-cell activation on puried murine T lymphocytes by Western-blot assay. The data revealed that alpinetin could shock the activation of NF-κB, NFAT2 signal transduction pathways. These observations indicated that alpinetin have potential effects in downregulating the immune system and might be developed as a useful immunosuppressive agent in treating undesired immune responses.

Immunosuppressive activity of daphnetin, one of coumarin derivatives, is mediated through suppression of NF-κB and NFAT signaling pathways in mouse T cells.

Daphnetin, a plant-derived dihydroxylated derivative of coumarin, is an effective compound extracted from a plant called Daphne Korean Nakai. Coumarin derivates were known for their antithrombotic, anti-inflammatory, and antioxidant activities. The present study was aimed to determine the immunosuppressive effects and the underlying mechanisms of daphnetin on concanavalin A (ConA) induced T lymphocytes in mice. We showed that, in vitro, daphnetin suppressed ConA-induced splenocyte proliferation, influenced production of the cytokines and inhibited cell cycle progression through the G0/G1 transition. The data also revealed that daphnetin could down-regulate activation of ConA induced NF-κB and NFAT signal transduction pathways in mouse T lymphocyte. In vivo, daphnetin treatment significantly inhibited the 2, 4- dinitrofluorobenzene (DNFB) -induced delayed type hypersensitivity (DTH) reactions in mice. Collectively, daphnetin had strong immunosuppressive activity both in vitro and in vivo, suggesting a potential role for daphnetin as an immunosuppressive agent, and established the groundwork for further research on daphnetin.

Inhibitory effects of salidroside on nitric oxide and prostaglandin E2 production in lipopolysaccharide-stimulated RAW 264.7 macrophages.

The aim of this study was to evaluate the effect of salidroside on lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in RAW 264.7 macrophages and related anti-inflammatory mechanism. PGE2 production was measured by enzyme-linked immunosorbent assay (ELISA); NO production was tested by Griess reagent. Inducible nitric oxidesynthase (iNOS) and COX-2 were determined by RT-PCR and Western blot analysis; IκB and P-IκB protein express were detected by Western blot analysis; cytosolic free Ca²âº ([Ca²âº](i)) was measured by a fluorescent microscope. The data showed salidroside inhibited LPS-induced NO and PGE2 production and reduced iNOS and COX-2 protein expression in RAW 264.7 macrophages. Consistent with these observations, salidroside inhibited LPS-induced cytosolic free Ca²âº concentration ([Ca²âº](i)) elevation. In addition, we further investigated signal transduction mechanisms and found that the activation of NF-κB was suppressed by salidroside in a dose-dependent manner. These results suggest that salidroside suppresses NO and PGE2 production by inhibiting iNOS and COX-2 protein expression, level of [Ca²âº](i), and activation of NF-κB signal transduction pathway.

Tyrosol exhibits negative regulatory effects on LPS response and endotoxemia.

Tyrosol, a phenolic compound, was isolated from wine, olive oil and other plant-derived products. In the present study, we first investigated the negative regulatory effects of tyrosol on cytokine production by lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages in vitro, and the results showed that tyrosol reduced tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) secretion. This inspired us to further study the effects of tyrosol in vivo. Tyrosol significantly attenuated TNF-α, IL-1ß and IL-6 production in serum from mice challenged with LPS, and consistent with the results in vitro. In the murine model of endotoxemia, mice were treated with tyrosol prior to or after LPS challenge. The results showed that tyrosol significantly increased mice survival. We further investigated signal transduction ways to determine how tyrosol works. The data revealed that tyrosol shocked LPS-induced mitogen activated protein kinases (MAPKs) and nuclear transcription factor-κB (NF-κB) signal transduction pathways in RAW 264.7 macrophages. These observations indicated that tyrosol exerted negative regulatory effects on LPS response in vitro and in vivo through suppressing NF-κB and p38/ERK MAPK signaling pathways.

Suppressive effects of fisetin on mice T lymphocytes in vitro and in vivo.

BACKGROUND: Most of the immunosuppressive drugs have satisfactory therapeutic effects on organ transplantation and autoimmune disease. However, their clinical application is limited by side effects. Therefore, new and safe immunosuppressive drugs against acute and chronic rejections are eagerly awaited. Fisetin, a flavonoid present in various types of vegetables and fruits, has few side effects and low level of toxicity, which would be a desirable clinical feature. In the present study, we investigated the immunosuppressive effects and underlying mechanisms of fisetin against T-cell activation in vitro and in vivo. METHODS: We measured the effect of fisetin on T-lymphocyte proliferation, T-cell subsets, cell cycle progression, cytokine production, and nuclear factor activation in vitro, as well as its influence on T cell-mediated delayed-type hypersensitivity reaction in vivo. RESULTS: In vitro, the results showed that fisetin significantly suppressed mouse splenocytes proliferation, Th1 and Th2 cytokine production, cell cycle and the ratio of CD4(+)/CD8(+) T cells. Furthermore, fisetin exerts an immunosuppressive effect in mouse T lymphocytes through the suppression of nuclear factor kappa B activation and nuclear factor of activated T cells signaling in a dose-dependent manner. In vivo, fisetin treatment also significantly inhibited the dinitrofluorobenzene-induced delayed-type hypersensitivity reactions in mice. CONCLUSIONS: Fisetin had strong immunosuppressive activity in vitro and in vivo, suggesting a potential role for fisetin as an immunosuppressive agent.

Suppression of T-cell activation in vitro and in vivo by cordycepin from Cordyceps militaris.

BACKGROUND: In addition to achieving a balance between the positive (controlling rejection) and the negative (infection and malignancy) aspects of drug-induced immunodeficiency, new immunosuppressive combinations must address the issue of nonimmune drug toxicity that may be dose limiting. Cordycepin is a type of adenosine analog extracted from Cordyceps militaris. In the present study, we investigated its immunosuppressive effect on T cell both in vitro and in vivo. METHODS: We evaluated the effects of cordycepin on concanavalin A-induced production of immune mediators in mouse splenocyte by enzyme-linked immunosorbent assay and flow cytometry. Furthermore, using Western blotting, we studied signal transduction mechanisms to determine how cordycepin inhibited T-cell activation in purified mouse T lymphocytes. To confirm the immunosuppressive activity of cordycepin in vivo, we induced the T cell-mediated delayed-type hypersensitivity reaction in a 2,4-dinitro-1-fluorobenzene-induced mouse model. RESULTS: The in vitro results showed that cordycepin markedly suppressed concanavalin A-induced splenocyte proliferation, Th1 and Th2 cytokine production, and the ratio of CD4(+)-to-CD8(+) T cells. The administration of cordycepin in vivo markedly suppressed the T cell-mediated delayed-type hypersensitivity reaction. The data revealed that cordycepin effectively shocked the nuclear factor kappa B and nuclear factor of activated T cells 2 signal transduction pathways but had no effect on the mitogen activated protein kinase signal transduction pathway. CONCLUSIONS: These observations indicated that cordycepin has a potential role in downregulating the immune system and could be developed as a useful immunosuppressive agent for treating undesired immune responses.

Gossypol suppresses mouse T lymphocytes via inhibition of NFκB, NFAT and AP-1 pathways.

Gossypol is a kind of yellow polyphenolic compounds extracted from root stem and seed of the cotton plant. In the present study, we investigated its immunosuppressive mechanism by using BALB/c mouse T lymphocytes in vitro. When mouse splenocytes was incubated with gossypol, the extract effectively suppress the overproduction of the cell stimulated by concanavalin A (ConA) in a dose manner. This inhibitive activity was mainly due to interfering Th1 and Th2 cytokines production and decreasing CD4(+) T cell populations and ratio of CD4(+)/CD8(+). Furthermore, we also showed that signal transduction via NF-κB, NFAT and AP-1 are critical to the ConA-induced T cell activation in mice. The data revealed that gossypol could down-regulate activation of ConA-induced NF-κB, NFAT and AP-1 signal transduction pathways in mouse T lymphocyte. These observations indicated that gossypol exhibited immunosuppressive effects by inhibition T lymphocyte activation in vitro.

The inhibition of 2,3-dichloro-1-propanol on T cell in vitro and in vivo.

2,3-Dichloro-1-propanol (2,3-DCP) is a member of a group of chemicals known as chloropropanols. Currently, immunotoxicity of 2,3-DCP has not been reported. In the present study, we studied its inhibitory effects on T cell both in vivo and in vitro. The results showed that 2,3-DCP markedly inhibited ConA-induced splenocyte proliferation, Th1 and Th2 cytokine production, CD4(+) T cell populations, and the ratio of CD4(+)/CD8(+) T cells and cell cycle arrest in vitro. In addition, 2,3-DCP markedly suppressed DNFB-induced T-cell-mediated delayed-type hypersensitivity (DTH) reaction in mice. Furthermore, Western blot was used to study how 2,3-DCP affects signal transduction mechanisms. The data revealed that 2,3-DCP could down regulate activation of ConA-induced NF-κB and NFAT signal transduction pathways. These observations indicated that 2,3-DCP exhibited negative regulatory effects by directly suppressing T-cell-mediated immune responses in vitro and in vivo.

Investigation of effects of farrerol on suppression of murine T lymphocyte activation in vitro and in vivo.

Farrerol, a new type of 2,3-dihydro-flavonoid, has been isolated from the leaves of Rhododendron dauricum L. In the present study, we found that farrerol exerted potent immunosuppressive effects on murine T cells both in vitro and in vivo. In vitro, farrerol markedly suppressed concanavalin A (ConA)-induced lymphocyte proliferation, Th1 and Th2 cytokine production, cluster of differentiation 4-positive (CD4(+)) T cell populations, and the ratio of CD4(+)/cluster of differentiation 8-positive (CD8(+)) T cells. Moreover, farrerol significantly inhibited the T cell-mediated delayed-type hypersensitivity (DTH) reaction in vivo. In addition, we investigated signal transduction mechanisms to determine the effects of farrerol by Western blotting. The data revealed that farrerol could downregulate the activation of the nuclear factor κB (NF-қB) and nuclear factor of activated T cells 2 (NFAT2) signal transduction pathways. These findings suggested that farrerol has potential effects on the regulation of the immune system and could be developed as a practicable immunosuppressive compound.

Protective effects of salidroside from Rhodiola rosea on LPS-induced acute lung injury in mice.

Salidroside is a major component extracted from Rhodiola rosea. In this study, we investigated protective effects of salidroside on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. In the mouse model, we found that pretreatment with a single 120 mg/kg dose of salidroside prior to the administration of intratracheal LPS induced a significant decrease in the W/D ratio and mouse myeloperoxidase activity of lung, reduction protein concentration, the number of total cells, neutrophils and macrophages in the bronchoalveolar lavage fluid. In addition, salidroside also inhibited the production of several inflammatory cytokines, including tumor necrosis factor-α, interleukin-6 (IL-6) and IL-1ß, and the NF-κB DNA-binding activation after LPS challenge. These results indicated that salidroside possess a protective effect on LPS-induced ALI in mice.