[Paeoniflorin induces apoptosis and cycle arrest in B-cell acute lymphoblastic leukemia cells by inhibiting SENP1/c-Myc signaling pathway].

The effect of paeoniflorin on apoptosis and cell cycle in human B-cell acute lymphoblastic leukemia(B-ALL) and its underlying mechanism were investigated in this study. Nalm-6 and SUP-B15 cells were cultured in vitro and divided into control group(0 µg·mL~(-1)) and experimental groups(200, 400, and 800 µg·mL~(-1) paeoniflorin). Cell counting kit-8(CCK-8) was used to measure the viability of Nalm-6 and SUP-B15 cells, and cell apoptosis and cell cycle distribution were analyzed by flow cytometry. Western blot was used to detect the protein levels of cleaved caspase-3, cleaved poly(ADP-ribose) polymerase(cleaved PARP), c-Myc, and small ubiquitin-like modifier-specific protease 1(SENP1). The mRNA levels of c-Myc and SENP1 in acute lymphoblastic leukemia(ALL) patients were analyzed based on the Oncomine database. AutoDock was used for molecular docking to analyze the interaction of paeoniflorin with c-Myc and SENP1 proteins. RESULTS:: showed that paeoniflorin inhibited the viability of Nalm-6 and SUP-B15 cells in concentration and time-dependent manners. Compared with the control group, paeoniflorin significantly up-regulated the expression of apoptosis-related proteins cleaved caspase-3 and cleaved PARP to induce apoptosis, evidently increased the proportion of G_2/M phase cells and induced G_2/M phase arrest, and obviously down-regulated the expression of c-Myc and SENP1 proteins in Nalm-6 and SUP-B15 cells. The mRNA levels of c-Myc and SENP1 in ALL patients were higher than those in the normal cell. Molecular docking demonstrated that paeoniflorin had good binding to c-Myc and SENP1 proteins. In summary, paeoniflorin inhibits the proliferation of Nalm-6 and SUP-B15 cells by inducing apoptosis and G_2/M phase arrest, which may be related to the down-regulation of c-Myc and SENP1 proteins.

Synergistic anti-proliferative activity of JQ1 and GSK2801 in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) constitutes 10-20% of breast cancers and is challenging to treat due to a lack of effective targeted therapies. Previous studies in TNBC cell lines showed in vitro growth inhibition when JQ1 or GSK2801 were administered alone, and enhanced activity when co-administered. Given their respective mechanisms of actions, we hypothesized the combinatorial effect could be due to the target genes affected. Hence the target genes were characterized for their expression in the TNBC cell lines to prove the combinatorial effect of JQ1 and GSK2801. METHODS: RNASeq data sets of TNBC cell lines (MDA-MB-231, HCC-1806 and SUM-159) were analyzed to identify the differentially expressed genes in single and combined treatments. The topmost downregulated genes were characterized for their downregulated expression in the TNBC cell lines treated with JQ1 and GSK2801 under different dose concentrations and combinations. The optimal lethal doses were determined by cytotoxicity assays. The inhibitory activity of the drugs was further characterized by molecular modelling studies. RESULTS: Global expression profiling of TNBC cell lines using RNASeq revealed different expression patterns when JQ1 and GSK2801 were co-administered. Functional enrichment analyses identified several metabolic pathways (i.e., systemic lupus erythematosus, PI3K-Akt, TNF, JAK-STAT, IL-17, MAPK, Rap1 and signaling pathways) enriched with upregulated and downregulated genes when combined JQ1 and GSK2801 treatment was administered. RNASeq identified downregulation of PTPRC, MUC19, RNA5-8S5, KCNB1, RMRP, KISS1 and TAGLN (validated by RT-qPCR) and upregulation of GPR146, SCARA5, HIST2H4A, CDRT4, AQP3, MSH5-SAPCD1, SENP3-EIF4A1, CTAGE4 and RNASEK-C17orf49 when cells received both drugs. In addition to differential gene regulation, molecular modelling predicted binding of JQ1 and GSK2801 with PTPRC, MUC19, KCNB1, TAGLN and KISS1 proteins, adding another mechanism by which JQ1 and GSK2801 could elicit changes in metabolism and proliferation. CONCLUSION: JQ1-GSK2801 synergistically inhibits proliferation and results in selective gene regulation. Besides suggesting that combinatorial use could be useful therapeutics for the treatment of TNBC, the findings provide a glimpse into potential mechanisms of action for this combination therapy approach.

Human Amnion Chorion Membrane Allografts in the Treatment of Chronic Diabetic Foot Ulcers: A Literature Review.

OBJECTIVE: To discuss human amnion chorion (placental) membrane allograft (HACMA) use for the treatment of chronic diabetic foot ulcers (DFUs) and to evaluate the effectiveness, cost, and product waste of this therapy. DATA SOURCES: PubMed, Cochrane, and OVID databases. STUDY SELECTION: Twenty-four articles pertaining to HACMA and DFUs published from 2016 to 2020 were selected. DATA EXTRACTION: The data collected included type of wound care product, study design, study size, baseline size of DFU, cost, product wastage, number of applications, and wound healing outcomes. DATA SYNTHESIS: Human amnion chorion membrane allografts in the treatment of chronic DFUs have led to a reduction in healing time and increased the overall percentage of healing, making them more effective in treating DFUs compared with standard of care. These products are offered in multiple sizes with various shelf lives and methods of storage, making them accessible, easy to use, less wasteful, and lower in cost compared with other commercially available products. Promising evidence demonstrates that HACMAs are beneficial in treating complex, high-grade DFUs with exposed tendon or bone. CONCLUSIONS: Human amnion chorion membrane allografts are effective in treating chronic DFUs with a greater percentage of complete wound closure and a reduction in healing time versus standard of care.

Legumain promotes tubular ferroptosis by facilitating chaperone-mediated autophagy of GPX4 in AKI.

Legumain is required for maintenance of normal kidney homeostasis. However, its role in acute kidney injury (AKI) is still unclear. Here, we induced AKI by bilateral ischemia-reperfusion injury (IRI) of renal arteries or folic acid in lgmnWT and lgmnKO mice. We assessed serum creatinine, blood urea nitrogen, histological indexes of tubular injury, and expression of KIM-1 and NGAL. Inflammatory infiltration was evaluated by immunohistological staining of CD3 and F4/80, and expression of TNF-α, CCL-2, IL-33, and IL-1α. Ferroptosis was evaluated by Acsl4, Cox-2, reactive oxygen species (ROS) indexes H2DCFDA and DHE, MDA and glutathione peroxidase 4 (GPX4). We induced ferroptosis by hypoxia or erastin in primary mouse renal tubular epithelial cells (mRTECs). Cellular survival, Acsl4, Cox-2, LDH release, ROS, and MDA levels were measured. We analyzed the degradation of GPX4 through inhibition of proteasomes or autophagy. Lysosomal GPX4 was assessed to determine GPX4 degradation pathway. Immunoprecipitation (IP) was used to determine the interactions between legumain, GPX4, HSC70, and HSP90. For tentative treatment, RR-11a was administrated intraperitoneally to a mouse model of IRI-induced AKI. Our results showed that legumain deficiency attenuated acute tubular injury, inflammation, and ferroptosis in either IRI or folic acid-induced AKI model. Ferroptosis induced by hypoxia or erastin was dampened in lgmnKO mRTECs compared with lgmnWT control. Deficiency of legumain prevented chaperone-mediated autophagy of GPX4. Results of IP suggested interactions between legumain, HSC70, HSP90, and GPX4. Administration of RR-11a ameliorated ferroptosis and renal injury in the AKI model. Together, our data indicate that legumain promotes chaperone-mediated autophagy of GPX4 therefore facilitates tubular ferroptosis in AKI.

Update on relevant trypanosome peptidases: Validated targets and future challenges.

Trypanosoma cruzi, the agent of the American Trypanosomiasis, Chagas disease, and Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, the agents of Sleeping sickness (Human African Trypanosomiasis, HAT), as well as Trypanosoma brucei brucei, the agent of the cattle disease nagana, contain cysteine, serine, threonine, aspartyl and metallo peptidases. The most abundant among these enzymes are the cysteine proteases from the Clan CA, the Cathepsin L-like cruzipain and rhodesain, and the Cathepsin B-like enzymes, which have essential roles in the parasites and thus are potential targets for chemotherapy. In addition, several other proteases, present in one or both parasites, have been characterized, and some of them are also promising candidates for the developing of new drugs. Recently, new inhibitors, with good selectivity for the parasite proteasomes, have been described and are very promising as lead compounds for the development of new therapies for these neglected diseases. This article is part of a Special Issue entitled: "Play and interplay of proteases in health and disease".

Differential DNA methylation profiles of peripheral blood mononuclear cells in allergic asthmatic children following dust mite immunotherapy.

BACKGROUND/PURPOSE: Allergen-specific immunotherapy (SIT) is now considered curative to allergic diseases such as asthma. Mechanistically, our previous work showed DNA hypermethylation of cytokine genes, in T-helper cells, in allergic asthmatic children treated with allergen-SIT. In this study, we extended to work to assess possible changes in the DNA methylomes of peripheral blood mononuclear cells (PBMCs), isolated from mite allergen-SIT asthmatic children, to explore further the underlying methylation changes. METHODS: Thirteen allergic asthmatic children who received Der p-SIT, 12 non-SIT allergic asthmatic controls, and 12 healthy controls were enrolled. Bisulfite-converted DNA from Der p-stimulated PBMCs was analyzed using Human Methylation 450 k BeadChip. Pyrosequencing and quantitative real-time PCR were used to validate the DNA methylation levels and the gene expression of individual samples. RESULTS: We identified 108 significantly differentially methylated regions (DMRs) unique to Der p-treated PBMCs, with 53 probes linked to demethylated DMRs, and 55 probes linked to methylated DMRs. Three associated genes (BCL6, HSPG2, and HSP90AA1), of selected DMRs, were subjected to bisulfite pyrosequencing. Of these, BCL6 showed significant hypomethylation, while HSPG2 and HSP90AA1 were hypermethylated in SIT group, compared to the AA group. Furthermore, SIT group had significantly higher gene expression of BCL6 and lower gene expression of HSPG2. KEGG pathway analysis further revealed DMR genes involved in ECM-receptor interactions, asthma, and antigen processing and presentation pathways. CONCLUSIONS: Several DNA regions showed DNA methylation altered by Der p specific immunotherapy, indicating desensitization-associated methylomes. Genes belonging to these SIT-altered pathways may represent therapeutic targets for better clinical management of asthma.

Effects of Native Fucosylated Glycosaminoglycan, Its Depolymerized Derivatives on Intrinsic Factor Xase, Coagulation, Thrombosis, and Hemorrhagic Risk.

A native fucosylated glycosaminoglycan from sea cucumber Holothuria fuscopunctata (nHG), mainly branched with Fuc3S4S, exhibited potent anticoagulant activity by intrinsic tenase iXase (FIXa-FVIIIa complex) and antithrombin-dependent factor IIa (FIIa) inhibition, but also had the effects of FXII activation and platelet aggregation. For screening a selective iXase inhibitor, depolymerized nHG (dHG-1 ∼ -6) and a pure octasaccharide (oHG-8) were prepared. Like nHG, dHG-1 ∼ -6 and oHG-8 could potently inhibit iXase, and competitive binding assay indicated that dHG-5 and oHG-8 could bind to FIXa. Nevertheless, dHG-5 and oHG-8 had no effects on FXII and platelet activation. nHG, dHG-5, and oHG-8 could significantly prolong the activated partial thromboplastin time of human, rat, and rabbit plasma. In the rat deep venous thrombosis model, dHG-5 and oHG-8 showed potent antithrombotic effects in a dose-dependent manner, while the thrombus inhibition rate of nHG at high dose was markedly reduced. Additionally, dHG-5 and oHG-8 did not increase bleeding at the doses up to 10-fold of the effectively antithrombotic doses compared with nHG and low molecular weight heparin in the mice tail-cut model. Considering that dHG-5 possesses strong anti-iXase and antithrombotic activities, and its preparation process is simpler and its yield is higher compared with oHG-8, it might be a promising antithrombotic candidate.

Protective Cellular Immune Response Induction for Cutaneous Leishmaniasis by a New Immunochemotherapy Schedule.

The palladacycle complex DPPE 1.2 was previously shown to inhibit Leishmania (Leishmania) amazonensis infection in vitro and in vivo. The present study aimed to evaluate the effect of DPPE 1.2 associated with a recombinant cysteine proteinase, rLdccys1, and the adjuvant Propionibacterium acnes on L. (L.) amazonensis infection in two mouse strains, BALB/c, and C57BL/6. Treatment with this association potentiated the leishmanicidal effect of DPPE 1.2 resulting in a reduction of parasite load in both strains of mice which was higher compared to that found in groups treated with either DPPE 1.2 alone or associated with P. acnes or rLdccys1. The reduction of parasite load in both mice strains was followed by immunomodulation mediated by an increase of memory CD4+ and CD8+ T lymphocytes, IFN-γ levels and reduction of active TGF-ß in treated animals. No infection relapse was observed 1 month after the end of treatment in mice which received DPPE 1.2 associated with rLdccys1 or rLdccys1 plus P. acnes in comparison to that exhibited by animals treated with DPPE 1.2 alone. Evaluation of serum levels of AST, ALT, urea, and creatinine showed no alterations among treated groups, indicating that this treatment schedule did not induce hepato or nephrotoxicity. These data indicate the potential use of this association as a therapeutic alternative for cutaneous leishmaniasis caused by L. (L) amazonensis.

Legumain Promotes Gastric Cancer Progression Through Tumor-associated Macrophages In vitro and In vivo.

Tumor-associated macrophages (TAMs) play a crucial role in the tumor microenvironment. Legumain (LGMN) has been shown to be a tumor-promoting protein, but the effect of LGMN on TAMs in the progression of gastric cancer (GC) is under exploration. Our studies included the construction of LGMN-knockdown and LGMN-overexpressing TAMs induced from the human cell line THP-1 (PMA/IL-4/IL-13) and murine cell line Raw264.7 (IL-4/IL-13). A CCK-8 assay and transwell migration assay indicated that upregulation of LGMN expression in TAMs stimulated cell proliferation, migration and invasion in vitro, while downregulation of LGMN expression reduced cell proliferation, migration and invasion. In vivo experiments revealed slower growth, less angiogenesis, and less Ki67 expression in LGMN-knockdown TAMs injected with gastric cancer cells compared to control TAMs injected with GC cells. Together, these study results suggested that LGMN+ TAMs, which may serve as a potential target for GC treatment, promoted gastric cancer cell proliferation and angiogenesis in vitro and in vivo.

[Experimental research on Arginine-gingipain A gene vaccine from Porphyromonas gingivalis that prevents peri-implantitis in Beagle dogs].

OBJECTIVE: This study aims to use Arginine-gingipain A gene vaccine (pVAX1-rgpA) to immunize adult Beagle dogs and to evaluate its effect during peri-implantitis progression and development. METHODS: Plasmid pVAX1-rgpA was constructed. The second and third bilateral mandible premolars of 15 adult Beagle dogs were extracted, and the implants were placed immediately. After 3 months, the animals were randomly divided into groups A, B, and C. Afterward, the animals were immunized thrice with plasmid pVAX1-rgpA, with heat-killed Porphyromonas gingivalis, or pVAX1, respectively. IgG in the serum and secretory IgA (sIgA) in saliva were quantitatively analyzed by enzyme-linked immunosorbent assay before and after 2 weeks of immunization. Peri-implantitis was induced with cotton ligatures fixed around the neck of implants. Probing depth (PD) and bleeding on probing were recorded. All animals were sacrificed after ligaturation for 6 weeks. Decalcified sections with thickness of 50 µm were prepared and dyed with methylene blue to observe the bone phenotype around implants. RESULTS: Levels of serum IgG and sIgA in saliva were higher in groups A and B after immunization than before the process (P<0.05) and higher than those in group C (P<0.05). However, no difference was observed between groups A and B (P>0.05). At 4 and 6 weeks after ligaturation, PD of the ligatured side in group C was higher than that in groups A and B (P<0.05). On the other hand, no difference was identified between groups A and B (P>0.05). Bone loss in group A was significantly lower than that of the other groups (P<0.05). Abundant inflammatory cells and bacteria were present in the bone loss area around the implants in the three groups, as identified through hard tissue section observation. However, group C presented the most number of inflammatory cells and bacteria in the bone loss area around the implants. CONCLUSIONS: IgG and sIgA can be generated by immunity with rgpA DNA vaccine, which can significantly slow down bone loss during experimental peri-implantitis in dogs.

Commercial Applications for Enzyme-Mediated Protein Conjugation: New Developments in Enzymatic Processes to Deliver Functionalized Proteins on the Commercial Scale.

The field of protein conjugation most commonly refers to the chemical, enzymatic, or chemoenzymatic formation of new covalent bonds between two polypeptides, or between a single polypeptide and a new molecule (polymer, small molecule, nucleic acid, carbohydrate, etc.). Due to the modest selectivity of chemical methods for protein conjugation, there are increased efforts to develop biocatalysts that confer regioselectivity for site-specific modification, thereby complementing the existing toolbox of chemical conjugation strategies. This review summarizes key advances in the use of enzymes to functionalize proteins with commercial relevance. The examples put forth have demonstrated value at the industrial level or show promising industrial potential in the laboratory.

Inflammatory Monocytes Loading Protease-Sensitive Nanoparticles Enable Lung Metastasis Targeting and Intelligent Drug Release for Anti-Metastasis Therapy.

Metastasis causes high mortality of breast cancer, and the inability of drug delivery to metastatic sites remains a crucial challenge for antimetastasis therapy. Herein, we report that inflammatory monocytes loading legumain-activated nanoparticles can actively target lung metastases and initiate metastasis-specific intelligent drug release for antimetastasis therapy. The cytotoxic mertansine is conjugated to poly(styrene-co-maleic anhydride) with a legumain-sensitive peptide and self-assembled into nanoparticles (SMNs), and then loaded into inflammatory monocytes to prepare the SMNs-loaded monocytes delivery system (M-SMNs). M-SMNs would be in living state in circulation to ensure their active targeting to lung metastases, and responsively damaged at the metastatic sites upon the differentiation of monocytes into macrophages. The anticancer drugs are intelligently released from M-SMNs as free drug molecules and drug-loaded microvesicles, resulting in considerable inhibition on the proliferation, migration, and invasion activities of metastatic 4T1 breast cancer cells. Moreover, M-SMNs significantly improve the delivery to lung metastases and penetrate the metastatic tumors, thus producing a 77.8% inhibition of lung metastases. Taken together, our findings provide an intelligent biomimetic drug delivery strategy via the biological properties of inflammatory monocytes for effective antimetastasis therapy.

IgG Endopeptidase in Highly Sensitized Patients Undergoing Transplantation.

BACKGROUND: Donor-specific antibodies create an immunologic barrier to transplantation. Current therapies to modify donor-specific antibodies are limited and ineffective in the most highly HLA-sensitized patients. The IgG-degrading enzyme derived from Streptococcus pyogenes (IdeS), an endopeptidase, cleaves human IgG into F(ab')2 and Fc fragments inhibiting complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity, which suggests that IdeS might be useful for desensitization. We report on the combined experience of two independently performed open-label, phase 1-2 trials (conducted in Sweden and the United States) that assessed the efficacy of IdeS with regard to desensitization and transplantation of a kidney from an HLA-incompatible donor. METHODS: We administered IdeS to 25 highly HLA-sensitized patients (11 patients in Uppsala or Stockholm, Sweden, and 14 in Los Angeles) before the transplantation of a kidney from an HLA-incompatible donor. Frequent monitoring for adverse events, outcomes, donor-specific antibodies, and renal function was performed, as were renal biopsies. Immunosuppression after transplantation consisted of tacrolimus, mycophenolate mofetil, and glucocorticoids. Patients in the U.S. study also received intravenous immune globulin and rituximab after transplantation to prevent antibody rebound. RESULTS: Recipients in the U.S. study had a significantly longer cold ischemia time (the time elapsed between procurement of the organ and transplantation), a significantly higher rate of delayed graft function, and significantly higher levels of class I donor-specific antibodies than those in the Swedish study. A total of 38 serious adverse events occurred in 15 patients (5 events were adjudicated as being possibly related to IdeS). At transplantation, total IgG and HLA antibodies were eliminated. A total of 24 of 25 patients had perfusion of allografts after transplantation. Antibody-mediated rejection occurred in 10 patients (7 patients in the U.S. study and 3 in the Swedish study) at 2 weeks to 5 months after transplantation; all these patients had a response to treatment. One graft loss, mediated by non-HLA IgM and IgA antibodies, occurred. CONCLUSIONS: IdeS reduced or eliminated donor-specific antibodies and permitted HLA-incompatible transplantation in 24 of 25 patients. (Funded by Hansa Medical; ClinicalTrials.gov numbers, NCT02224820 , NCT02426684 , and NCT02475551 .).

Coadministration of cruzipain and GM-CSF DNAs, a new immunotherapeutic vaccine against Trypanosoma cruzi infection.

Therapeutic vaccine research and development are especially important in Chagas disease considering the characteristics of the chronic infection and the number of people in the Americas living with a parasite infection for decades. We have previously reported the efficacy of attenuated Salmonella enterica (S) carrying plasmid encoding cruzipain (SCz) to protect against Trypanosoma cruzi infection. In the present work we investigated whether Cz DNA vaccine immunotherapy could be effective in controlling an ongoing T. cruzi infection in mice. We here report the intramuscular administration of naked Cz DNA or the oral administration of Salmonella as Cz DNA delivery system as therapeutic vaccines in mice during acute or chronic infection. The coadministration of a plasmid encoding GM-CSF improved vaccine performance, indicating that the stimulation of innate immune cells is needed in the event of an ongoing infection. These therapeutic vaccines were able to address the response to a protective and sustained Th1 biased profile not only against Cz but also against a variety of parasite antigens. The combined therapeutic vaccine during the chronic phase of infection prevents tissue pathology as shown by a reduced level of enzyme activity characteristic of tissue damage and a tissue status compatible with normal tissue. The obtained results suggest that immunotherapy with Cz and GM-CSF DNAs, either alone or in combination with other drug treatments, may represent a promising alternative for Chagas disease therapy.

Clinical and cytokine responses to house dust mite sublingual immunotherapy.

BACKGROUND: Cytokine responses accompanying sublingual immunotherapy (SLIT) responder phenotypes have not previously been reported. OBJECTIVE: To investigate clinical and cytokine responses of house dust mite (HDM) sensitive patients with allergic rhinitis receiving HDM SLIT or placebo for 2 years. METHODS: Sixty adults were randomized to receive SLIT or placebo. Clinical symptoms were measured using the Total 5 Symptom Score (TSS5) and Juniper Rhinitis Quality of Life Questionnaire. HDM specific IgE, IgG, skin prick tests, and HDM-stimulated release of interleukin (IL) 5 and interferon γ (IFN-γ) in peripheral blood mononuclear cells was studied at 0, 6, 12, and 24 months and IL-13, IL-4, and IL-10 at 0 and 24 months. RESULTS: A total of 32 of 39 SLIT and 16 of 21 placebo patients completed the study. There was significant clinical improvement in both the SLIT and placebo groups. Median T5SS decreased from 14.75 to 5.25 in the SLIT group (P < .001) and 12.7 to 6.0 in the placebo group (P = .003). The median quality-of-life score also decreased in the SLIT group (P < .001) and the placebo group (P < .001). A subgroup analysis of patients found a 60% or greater improvement (on the T5SS and the Juniper Rhinitis Quality of Life Questionnaire) in the good responders group and a 30% to 59% improvement or no improvement in the intermediate responders group. This subgroup analysis also found more good responders in the SLIT group (47%) compared with the placebo group (25%; P = .07). Significant decreases in the IL-5/IFN-γ (P < .001), IL-13/IFN-γ (P < .001), and IL-4/IFN-γ (P = .03) ratios were found in the combined good clinical improvement group at 24 months. CONCLUSION: A good clinical response (≥60% improvement in both TSS5 and quality of life) is associated with significant decreases in IL-5, IL-13, and IL-4 relative to IFN-γ during 2 years of SLIT therapy for HDMs.

[Experimental study on the Der f 1 mRNA molecules derived from dermatophagoides farinae for specific immunotherapy on murine model of asthma].

OBJECTIVE: To investigate the effect of Der f 1 mRNA molecules for specific immunotherapy on murine model of asthma. METHODS: Fifty BALB/c mice were randomly divided into 5 groups: PBS group, Der f 1 sensitization group, Der f 1 specific immunotherapy (SIT) group, beta-actin mRNA SIT group, and Derf 1 mRNA SIT group. On days 0, 7 and 14, mice in PBS group received PBS injection; mice in the other groups were intraperitoneally injected with 10 microg Derf 1. At day 21, the mice in the 4 experimental groups were challenged with a 30-min inhaled dose of Der f 1 (100 microg/ml) for 7 successive days. Two weeks after the final sensitization, the mice in the above five groups were im- munized by intradermal injection with PBS, 1 microg Der f 1, 10 microg Der f 1, 2 microg beta-actin mRNA, and 2 microg Der f 1 mRNA, respectively for 3 times at one-week intervals. Two weeks after the last intradermal injection, all mice were sacrificed and bronchoalveolar lavage fluid (BALF) was collected. ELISA was performed to detect the levels of IFN-gamma and IL-13 in BALF, the number of eosinophils in the BALF was recorded. Splenocytes were prepared, and cultured with Der f 1 al- lergen (10 Jg/ml) for 72 h. Splenocytes of PBS group was cultured without Derf 1 allergen. The levels of IFN-gamma and IL-13 in splenocyte culture supernatant were measured by ELISA, as well as serum antibody levels of total IgE, allergen- specific IgE (sIgE), sIgG1, and sIgG2a. Lung sections were stained in hematoxylin and eosin, and observed under the microsope. RESULTS: Except for PBS group, mice in the other 4 group showed symptoms of acute asthma attack. Com- pared with Derf 1 sensitization group [(897.56 +/- 105.73) pg/ml] and beta-actin mRNA SIT group [(219.47 +/- 64.72) pg/ml], the level of IFN-gamma in BALF from Der f 1 mRNA SIT group [(897.56 +/- 105.73) pg/ml] and Derfl SIT group [(864.48 +/- 70.62)pg/ml] significantly increased (P<0.01). However, the level of IL-13 in BALF from Derf 1 mRNA SIT group [(241.64 +/- 31.41) pg/ml] and Derf 1 SIT group [(321.94 +/- 41.07)pg/ml] was significantly lower than that of Der f 1 sensitization group [(520.62 +/- 43.77) pg/ml] and beta-actin mRNA SIT group [(507.22 +/- 42.26) pg/ml](P<0.01). The number of eosinophils in Der f 1 mRNA SIT group [(1.33 +/- 0.44) x 10(5)/ml] and Der f 1 SIT group [(1.48 +/- 0.39) x 10(5)/ml] was also lower than that of Der f 1 sensitization group [(3.54 +/- 0.52)x10(5)/ml] and beta-actin mRNA SIT group [(2.98-0.53) x 10(5)/ml] (P<0.01). The levels of IFN-GAMMA and IL-13 in splenocyte culture supernatant showed that IFN-gamma level in Der f 1 mRNA SIT group [(420.91+69.92) pg/ml] and Der f 1 SIT group [(334.92 +/- 43.72) pg/ml] was significantly higher than that of Der f 1 sensitization group[(123.75 +/- 5.48) pg/ml] and beta-actin mRNA SIT group[(128.84 +/- 59.00) pg/ml] (P<0.01). However, IL-13 level of Der f 1 mRNA SIT group [(268.51 +/- 40.42) pg/ml] and Der f 1 SIT group [(285.26 +/- 62.21) pg/ml] was significantly lower than that of Derf 1 sensitization group [(613.89 +/- 51.54) pg/ml] and beta-actin mRNA SIT group [(524.05 +/- 39.12) pg/ml] (P<0.01). Compared with Der f 1 sensitization group [total IgE: (94.34 +/- 11.66) ng/ml, sIgE: (65.67 +/- 9.47) ng/ml, sIgG1: (75.18 +/- 9.52) ng/ml, sIgG2a: (2.81 +/- 1.17) ng/ml] and beta-actin mRNA SIT group[total IgE: (86.48 +/- 10.26) ng/ml, sIgE: (62.36 +/- 8.35) ng/ml, sIgG1: (69.51 +/- 8.98) ng/ml, IgG2a: (1.06 +/- 0.11) ng/ml], the serum antibody levels of total IgE [(33.72 +/- 9.78) ng/ml], sIgE [(22.76 +/- 8.09) ng/ml], sIgG1 [(17.87 +/- 7.59) ng/ml] of Der f 1 mRNA SIT group decreased significantly (P<0.01), whereas the level of IgG% [(7.74 +/- 0.88) ng/ml] increased (P<0.01). Compared with Der f 1 sensitization group, the asthmatic symptoms were relieved after immunization with Der f 1 mRNA for specific immunotherapy, including intact structure of respiratory and alveolar epithelial cells, decreased inflammatory cell infiltration, and similar to those in Der f 1 SIT group. However, the breakage and detachment of bronchial epithelial cells occurred in beta-actin mRNA SIT group. CONCLUSION: Derf 1 mRNA vaccine can correct Th1 and Th2 imbalance.

Design of a ProDer f 1 vaccine delivered by the MHC class II pathway of antigen presentation and analysis of the effectiveness for specific immunotherapy.

Dermatophagoides farinae (Der f 1) is one of leading cause for allergic asthma, and allergen-specific immunotherapy (SIT) is currently recognized as the only etiological therapy to ameliorate asthmatic symptom. The current study was designed on the major histocompatibility complex (MHC) class II pathway, invariant chain (Ii)-segment hybrids as vaccine basis to explore the efficacy of Der f 1 hybrid vaccine by virtue of Ii as carrier in enhancing the protective immune response to asthma. Initially, we engineered a fused molecule, DCP-IhC-ProDer f 1, to deliver ProDer f 1 antigen via specific dendritic cell-targeting peptides to dendritic cells (DCs). Then the DCP-IhC-ProDer f 1 was immunized to the asthmatic models of murine induced by ProDer f 1 allergen. The findings showed that the cytokine repertoire in the murine model was shifted after SIT, including stronger secretion of IFN-γ and IL-10, and a decreased production of IL-4 and IL-17. ELISA determination revealed that the hybrid displayed weak IgE and IgG1 reactivities, and IgG2a levels were elevated. Furthermore, DCP-IhC-ProDer f 1 treatment inhibited inflammatory cell infiltration in the lung tissues. Our results suggest that the DCP-Ihc-ProDer f 1 may be used as a candidate SIT against asthma.

[[Expression of house dust mite allergens Der f1 and Der f2 in Nicotiana benthamiana leaves].

The class E immunoglobulins (IgE) is known to recognize conformational epitopes and therefore the native conformation of recombinant allergens is essential for their using in test-systems. Recombinant Dermatophagoides farinae house dust mite (HDM) allergens Der f1 and Der f2 were expressed in bacteria Escherichia coli and Nicotiana benthamiana plants. It has been shown that IgE in sera from children allergic to HDM recognizes Der f2 expressed both in E. coli and N. benthamiana. Mature form of Der f1 expressed in E. coli does not interact with IgE while the protein purified from N. benthamiana is able to recognize IgE as a native allergen.

Effectiveness of cysteine proteases on protein/pigment film removal.

OBJECTIVE: Theaflavin (TF) from the black tea can react to human salivary proline-rich proteins (PRPs) to form stains on exposed dental surfaces. Here, we employed a model of protein/pigment film using TF and dephosphorylated bovine ß-casein (Dß-CN), which has an extended conformation, similar to that of salivary PRPs, on a sensor surface to assess the efficacy of cysteine proteases (CPs) including papain, stem bromelain, and ficin, on removing TF bound to Dß-CN and the control TF readsorption on the residual substrate surfaces was also measured. METHODS: The protein/pigment complex film was built by using a quartz crystal microbalance with dissipation (QCM-D). The efficacies of CPs were assessed by Boltzman equation model. The surface details were detected by grazing angle infrared spectroscopy spectra, atomic force microscopy images, and contact angles. RESULTS: The efficacy order of CPs on hydrolyzing protein/pigment complex film is ficin>papain>bromelain. The results from grazing angle infrared spectroscopy spectra, atomic force microscopy images, and contact angles demonstrated that TF bound on the Dß-CN was effectively removed by the CPs, and the amount of TF readsorption on both the residual film of the Dß-CN/TF and the Dß-CN was markedly decreased after hydrolysis. CONCLUSION: This study indicates the potential application of the CPs for tooth stain removal and suggests that these enzymes are worthy of further investigation for use in oral healthcare.

Sublingual immunotherapy for experimental allergic conjunctivitis in a murine model induced by Dermatophagoides farinae allergen.

BACKGROUND: Sublingual immunotherapy (SLIT) is a clinically effective treatment in allergic conjunctivitis (AC); however, the mechanism of the underlying pharmacodynamics remains unclear. Here, we investigate the efficacy and the mechanism of a sublingually administered Dermatophagoides farinae (Der f) vaccine in a murine AC model. METHODS: A murine model of AC caused by Der f extract was developed in BALB/c mice by repeated application of allergen. Sensitized mice were SLIT treated by Der f drops and subsequently analyzed for AC symptoms, histopathological and immunological parameters. RESULTS: In this study, Der f extract successfully induced the symptoms of AC in BALB/c mice. In these sensitized mice, clinical symptoms (scratching behavior, lacrimation, conjunctival hyperemia and edema), immunological and histopathological findings (inflammatory cell infiltration) were very similar to those in human AC. SLIT treatment of sensitized mice markedly reduced the clinical and histopathological symptoms and decreased the expression levels of total immunoglobulin E (IgE), Der f-specific IgE and T helper cell 2 (Th2) cytokine interleukin-4, with a significant increase in Der f-specific IgG4 and Th1 cytokine interferon-γ. CONCLUSIONS: SLIT with Der f drops is a potentially effective means of immunotherapy for Der f-induced AC by modulating the Th2-biased allergic immune response.