ABSTRACT
Successful DNA vaccination generally requires the aid of either a viral vector within vaccine components or an electroporation device into the muscle or skin of the host. However, these systems come with certain obstacles, including limited transgene capacity, broad preexisting immunity in humans, and substantial cell death caused by high voltage pulses, respectively. In this study, we repurposed the use of an amphiphilic bioresorbable copolymer (ABC), called PLA-PEG, as a surface engineering agent that conciliates lipid nanoparticles (LNPs) between stability during preparation and biocompatibility post-vaccination. The LNP carrier can be loaded with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-specific DNA; in this form, the DNA-LNP is immunogenic in hamsters and elicits protective immunity following DNA-LNP vaccination against heterologous virus challenge or as a hybrid-type vaccine booster against SARS-CoV-2 variants. The data provide comprehensive information on the relationships between LNP composition, manufacturing process, and vaccine efficacy. The outcomes of this study offer new insights into designing next-generation LNP formulations and pave the way for boosting vaccine power to combat existing and possible emerging infectious diseases/pathogens.
ABSTRACT
DUSP22, an atypical dual-specificity phosphatase enzyme, plays a significant role in regulating multiple kinase signaling pathways by dephosphorylation. Our study demonstrated that decreased DUSP22 expression is associated with shorter disease-free survival, advanced TNM (tumor, lymph nodes, and metastasis), cancer stage, and higher tumor grade in lung adenocarcinoma (LUAD) patients. Exogenous DUSP22 expression reduces the colony-forming capacity of lung cancer cells and inhibits xenograft tumor growth primarily by targeting EGFR and suppressing its activity through dephosphorylation. Knockdown of DUSP22 using shRNA enhances EGFR dependency in HCC827 lung cancer cells and increases sensitivity to gefitinib, an EGFR inhibitor. Consistently, genetic deletion of DUSP22 enhances EGFRdel (exon 19 deletion)-driven lung tumorigenesis and elevates EGFR activity. Pharmacological inhibition of DUSP22 activates EGFR, ERK1/2, and upregulates downstream PD-L1 expression. Additionally, lentiviral deletion of DUSP22 by shRNA enhances lung cancer cell migration through EGFR/c-Met and PD-L1-dependent pathways. Gefitinib, an EGFR inhibitor, mechanistically suppresses migration induced by DUSP22 deletion and inhibits c-Met activity. Furthermore, cabozantinib, a c-Met inhibitor, reduces migration and attenuates EGFR activation caused by DUSP22 deletion. Collectively, our findings support the hypothesis that loss of DUSP22 function in lung cancer cells confers a survival advantage by augmenting EGFR signaling, leading to increased activation of downstream c-Met, ERK1/2, and PD-L1 axis, ultimately contributing to the progression of advanced lung cancer.
ABSTRACT
mRNA vaccines for cancer immunotherapy are commonly delivered using lipid nanoparticles (LNPs), which, when administered intravenously, may accumulate in the liver, potentially limiting their therapeutic efficacy. To overcome this challenge, the study introduces an oral mRNA vaccine formulation tailored for efficient uptake by immune cells in the gastrointestinal (GI) tract, known for its high concentration of immune cells, including dendritic cells (DCs). This formulation comprises mRNA complexed with ß-glucans (ßGlus), a potential adjuvant for vaccines, encapsulated within LNPs (ßGlus/mRNA@LNPs). The ßGlus/mRNA complexes within the small compartments of LNPs demonstrate a distinctive ability to partially dissociate and reassociate, responding to pH changes, effectively shielding mRNA from degradation in the harsh GI environment. Upon oral administration to tumor-bearing mice, ßGlus/mRNA@LNPs are effectively taken up by intestinal DCs and local nonimmune cells, bypassing potential liver accumulation. This initiates antigen-specific immune responses through successful mRNA translation, followed by drainage into the mesenteric lymph nodes to stimulate T cells and trigger specific adaptive immune responses, ultimately enhancing antitumor effects. Importantly, the vaccine demonstrates safety, with no significant inflammatory reactions observed. In conclusion, the potential of oral ßGlus/mRNA@LNPs delivery presents a promising avenue in cancer immunotherapy, offering needle-free and user-friendly administration for widespread adoption and self-administration.
Subject(s)
Cancer Vaccines , Immunotherapy , Nanoparticles , RNA, Messenger , beta-Glucans , Animals , beta-Glucans/chemistry , Administration, Oral , Hydrogen-Ion Concentration , Mice , Cancer Vaccines/chemistry , Cancer Vaccines/immunology , RNA, Messenger/genetics , Nanoparticles/chemistry , Dendritic Cells/immunology , Lipids/chemistry , mRNA Vaccines , Cell Line, Tumor , Neoplasms/therapy , Neoplasms/immunology , Mice, Inbred C57BL , Humans , LiposomesABSTRACT
DNA vaccines for infectious diseases and cancer have been explored for years. To date, only one DNA vaccine (ZyCoV-D) has been authorized for emergency use in India. DNA vaccines are inexpensive and long-term thermostable, however, limited by the low efficiency of intracellular delivery. The recent success of mRNA/lipid nanoparticle (LNP) technology in the coronavirus disease 2019 (COVID-19) pandemic has opened a new application for nucleic acid-based vaccines. Here, we report that plasmid encoding a trimeric spike protein with LNP delivery (pTS/LNP), similar to those in Moderna's COVID-19 vaccine, induced more effective humoral responses than naked pTS or pTS delivered via electroporation. Compared with TSmRNA/LNP, pTS/LNP immunization induced a comparable level of neutralizing antibody titers and significant T helper 1-biased immunity in mice; it also prolonged the maintenance of higher antigen-specific IgG and neutralizing antibody titers in hamsters. Importantly, pTS/LNP immunization exhibits enhanced cross-neutralizing activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and protects hamsters from the challenge of SARS-CoV-2 (Wuhan strain and the Omicron BA.1 variant). This study indicates that pDNA/LNPs as a promising platform could be a next-generation vaccine technology.
ABSTRACT
The major challenge in COVID-19 vaccine effectiveness is immune escape by SARS-CoV-2 variants. To overcome this, an Omicron-specific messenger RNA (mRNA) vaccine was designed. The extracellular domain of the spike of the Omicron variant was fused with a modified GCN4 trimerization domain with low immunogenicity (TSomi). After immunization with TSomi mRNA in hamsters, animals were challenged with SARS-CoV-2 virus. The raised nonneutralizing antibodies or cytokine secretion responses can recognize both Wuhan S and Omicron S. However, the raised antibodies neutralized SARS-CoV-2 Omicron virus infection but failed to generate Wuhan virus neutralizing antibodies. Surprisingly, TSomi mRNA immunization protected animals from Wuhan virus challenge. These data indicated that non-neutralizing antibodies or cellular immunity may play a more important role in vaccine-induced protection than previously believed. Next-generation COVID-19 vaccines using the Omicron S antigen may provide sufficient protection against ancestral or current SARS-CoV-2 variants.
Subject(s)
Blood Group Antigens , COVID-19 , Animals , Cricetinae , Humans , SARS-CoV-2/genetics , COVID-19 Vaccines , Antibodies, Neutralizing , COVID-19/prevention & control , RNA, Messenger/genetics , mRNA Vaccines , Antibodies, Viral , Spike Glycoprotein, Coronavirus/geneticsABSTRACT
Nasal spray vaccination is viewed as a promising strategy for inducing both mucosal and systemic protection against respiratory SARS-CoV-2 coronavirus. Toward this goal, a safe and efficacious mucosal adjuvant is necessary for the transportation of the antigen across the mucosal membrane and antigen recognition by the mucosal immune system to generate broad-spectrum immune responses. This study describes the immunological aspects of SARS-CoV-2 spike (S)-protein after being formulated with CpG oligodeoxynucleotides (ODNs) and squalene nanoparticles (termed PELC). Following intranasal delivery in mice, higher expression levels of major histocompatibility complex (MHC) class II and costimulatory molecules CD40 and CD86 on CD11c+ cells were observed at the draining superficial cervical lymph nodes in the CpG-formulated S protein group compared with those vaccinated with S protein alone. Subsequently, the activated antigen-presenting cells downstream modulated the cytokine secretion profiles and expanded the cytotoxic T lymphocyte activity of S protein-restimulated splenocytes. Interestingly, the presence of PELC synergistically enhanced cell-mediated immunity and diminished individual differences in S protein-specific immunogenicity. Regarding humoral responses, the mice vaccinated with the PELC:CpG-formulated S protein promoted the production of S protein-specific IgG in serum samples and IgA in nasal and bronchoalveolar lavage fluids. These results indicate that PELC:CpG is a potential mucosal adjuvant that promotes mucosal/systemic immune responses and cell-mediated immunity, a feature that has implications for the development of a nasal spray vaccine against COVID-19.
ABSTRACT
Clinical cases of allergic reaction that are due to excipients containing polyethylene glycol (PEG), a hydrophilic molecule commonly used in drug/vaccine formulations, has attracted much attention in recent years. In order to develop PEG-free adjuvants, we investigated the feasibility of natural ingredients in the human body such as hyaluronic acid in the form of hyaluronic acid-glycine cholesterol (HACH) conjugate as an excipient for vaccine formulation. Interestingly, HACH grafted with ~13 wt.% cholesterol has good water dispersity and can serve as an emulsifier to stabilize the squalene/water interfaces, yielding a milky white and isotropic emulsion (SQ@HACH) after being passed through a high-shear microfluidizer. Our results show that SQ@HACH particles possessed a unimodal average hydrodynamic diameter of approximately 190 nm measured by dynamic light scattering and exhibited good stability upon storage at 4 °C and 37 °C for over 20 weeks. The results of immunogenicity using a mouse model with ovalbumin (OVA) as the antigen revealed that SQ@HACH significantly enhanced antigen-specific immune responses, including the polarization of IgG antibodies, the cytokine secretions of T cells, and enhancement of cytotoxic T lymphocyte (CTL) activation. Moreover, SQ@HACH revealed lower local inflammation and rapidly absorbing properties compared with AlPO4 after intramuscular injection in vivo, indicating the potential functions of the HA-derived conjugate as an excipient in vaccine formulations for enhancement of T cell-mediated immunity.
ABSTRACT
Vaccination is regarded as the most effective intervention for controlling the coronavirus disease 2019 (COVID-19) pandemic. The objective of this study is to provide comprehensive information on lipid squalene nanoparticle (SQ@NP)-adjuvanted COVID-19 vaccines regarding modulating immune response and enhancing vaccine efficacy. After being adjuvanted with SQ@NP, the SARS-CoV-2 spike (S) subunit protein was intramuscularly (i.m.) administered to mice. Serum samples investigated by ELISA and virus neutralizing assay showed that a single-dose SQ@NP-adjuvanted S-protein vaccine can induce antigen-specific IgG and protective antibodies comparable with those induced by two doses of nonadjuvanted protein vaccine. When the mice received a boosting vaccine injection, anamnestic response was observed in the groups of adjuvanted vaccine. Furthermore, the secretion of cytokines in splenocytes, such as interferon (IFN)-γ, interleukin (IL)-5 and IL-10, was significantly enhanced after adjuvantation of S-protein vaccine with SQ@NP; however, this was not the case for the vaccine adjuvanted with conventional aluminum mineral salts. Histological examination of injection sites showed that the SQ@NP-adjuvanted vaccine was considerably well tolerated following i.m. injection in mice. These results pave the way for the performance tuning of optimal vaccine formulations against COVID-19.
Subject(s)
COVID-19 , Nanoparticles , Adjuvants, Immunologic , Animals , Antibodies, Viral , COVID-19 Vaccines , Humans , Lipids , Mice , SARS-CoV-2 , SqualeneABSTRACT
This study describes the assessment of mucosal adjuvant activity of a squalene-based nanoemulsion (SQ@NE) following intravaginal delivery in mice. After immunization, a high level of recruitment of CD11b/c+ granulocytes and F4/80+ macrophages was observed in the vaginal mucosal tissues of the mice immunized with a model protein ovalbumin (OVA) formulated with SQ@NE, and then downstream regulated the expression of MHC II and costimulatory molecules CD40 and CD86 on CD11c+ cells harvested from the associated draining lymph node. With respect to cytotoxic T lymphocyte immunity, the mice immunized with SQ@NE-formulated OVA elicited a high population of OVA-specific CD8+ cells in the spleen and increased the secretion of IFN-γ, IL-2 and IL-17 from OVA-restimulated splenocytes compared with those immunized with OVA alone. By studying in vivo fluorescence imaging and B-cell immunoassays, we discovered how SQ@NE prolongs the retention of antigen depots at the mucosal membrane of the immune inductive site and allows them to properly drive the production of antibodies. The data demonstrated that SQ@NE prolonged fluorescence-labeled OVA retention at the genital tract and augmented the production of OVA-specific IgG in sera and IgA in vaginal washes. These results indicate that SQ@NE is a promising vaginal adjuvant for the induction of both mucosal and systemic immune responses, a feature that provides implications for the development of a mucosal vaccine against genital infections and sexually transmitted diseases.
Subject(s)
Mucous Membrane/drug effects , Mucous Membrane/immunology , Nanoparticles/administration & dosage , Squalene/administration & dosage , Vagina/drug effects , Vagina/immunology , Adjuvants, Immunologic/administration & dosage , Administration, Intravaginal , Animals , Emulsions , Female , Mice , Mice, Inbred BALB C , Ovalbumin/administration & dosageABSTRACT
Aluminum-containing salts are commonly used as antacids and vaccine adjuvants; however, key features of functional activities remain unclear. Here, we characterized vaccine formulations based on aluminum phosphate and aluminum hydroxide and investigated the respective modes of action linking physicochemical properties and catalytic ability. TEM microscopy indicated that aluminum phosphate gel solutions are amorphous, whereas aluminum hydroxide gel solutions have a crystalline structure consistent with boehmite. At very low BSA concentrations, 100 % adsorption of the protein on aluminum hydroxide could be achieved. As the protein concentration increased, the amount of adsorbed BSA decreased as fewer vacant sites were available on the surface of the adjuvants. Notably, less than 20 % adsorption was observed in aluminum phosphate. The protein adsorption profiles should confront the requirements for vaccine immunoavailability. In terms of catalytic ability, the prepared aluminum salts were tested for their ability to drive the amphiphilic engineering of oligo(lactic acid) (OLA) onto methoxy poly(ethylene glycol). It was concluded that aluminum hydroxide, rather than aluminum phosphate, is suitable to be a vaccine adjuvant according to the morphology and antigen adsorption efficiency results; on the other hand, aluminum phosphate may be a more efficient catalyst for the synthesis of polymeric emulsifiers than aluminum hydroxide. The results provide critical mechanistic insight into aluminum-containing salts in vaccine formulations.
ABSTRACT
BACKGROUND: Emulsion adjuvants are a potent tool for effective vaccination; however, the size matters on mucosal signatures and the mechanism of action following intranasal vaccination remains unclear. Here, we launch a mechanistic study to address how mucosal membrane interacts with nanoemulsion of a well-defined size at cellular level and to elucidate the impact of size on tumor-associated antigen therapy. METHODS: The squalene-based emulsified particles at the submicron/nanoscale could be elaborated by homogenization/extrusion. The mucosal signatures following intranasal delivery in mice were evaluated by combining whole-mouse genome microarray and immunohistochemical analysis. The immunological signatures were tested by assessing their ability to influence the transportation of a model antigen ovalbumin (OVA) across nasal mucosal membranes and drive cellular immunity in vivo. Finally, the cancer immunotherapeutic efficacy is monitored by assessing tumor-associated antigen models consisting of OVA protein and tumor cells expressing OVA epitope. RESULTS: Uniform structures with ~200 nm in size induce the emergence of membranous epithelial cells and natural killer cells in nasal mucosal tissues, facilitate the delivery of protein antigen across the nasal mucosal membrane and drive broad-spectrum antigen-specific T-cell immunity in nasal mucosal tissues as well as in the spleen. Further, intranasal vaccination of the nanoemulsion could assist the antigen to generate potent antigen-specific CD8+ cytotoxic T-lymphocyte response. When combined with immunotherapeutic models, such an effective antigen-specific cytotoxic activity allowed the tumor-bearing mice to reach up to 50% survival 40 days after tumor inoculation; moreover, the optimal formulation significantly attenuated lung metastasis. CONCLUSIONS: In the absence of any immunostimulator, only 0.1% content of squalene-based nanoemulsion could rephrase the mucosal signatures following intranasal vaccination and induce broad-spectrum antigen-specific cellular immunity, thereby improving the efficacy of tumor-associated antigen therapy against in situ and metastatic tumors. These results provide critical mechanistic insights into the adjuvant activity of nanoemulsion and give directions for the design and optimization of mucosal delivery for vaccine and immunotherapy.
Subject(s)
Adjuvants, Immunologic/therapeutic use , Administration, Intranasal/methods , Immunomodulation/immunology , Immunotherapy/methods , Mucous Membrane/immunology , Nanoparticles/chemistry , Vaccination/methods , Adjuvants, Immunologic/pharmacology , Animals , Female , Humans , MiceABSTRACT
Dual-specificity phosphatases (DUSPs) regulate the activity of various downstream kinases through serine or threonine or tyrosine dephosphorylation. Loss of function and aberrant expression of DUSPs has been implicated in cancer progression and poor survival, yet the function of DUSP22 in prostate cancer (PCa) cells is not clear. Gene Expression Omnibus and cBioPortal microarray database analyses showed that DUSP22 expression was lower in PCa tissues than normal prostate tissues, and altered DUSP22 expression was associated with shorter progression-free and disease-free survival of patients with PCa. Exogenous DUSP22 expression in LNCaP, PC3, and C4-2B PCa cells inhibited cellular proliferation and colony formation, supporting a growth inhibitory role for DUSP22 in PCa cells. DUSP22 expression significantly attenuated epidermal growth factor (EGF) receptor (EGFR) and its downstream ERK1/2 signaling by dephosphorylation. However, DUSP22 failed to suppress the growth of CWR22Rv1 and DU145 cells with elevated phosphorylated (p-)ERK1/2 levels. A serine-to-alanine mutation at position 58, a potential ERK1/2-targeted phosphorylation site in DUSP22, was sufficient to suppress growth of CWR22Rv1 cells with elevated p-ERK1/2 levels, suggesting a mutually antagonistic relationship between DUSP22 and ERK1/2 dependent on phosphorylation status. We showed that DUSP22 can suppress prostate-specific antigen gene expression through phosphatase-dependent pathways, suggesting that DUSP22 is an important regulator of the androgen receptor (AR) in PCa cells. Mechanistically, DUSP22 can interact with AR as a regulatory partner and interfere with EGF-induced AR phosphorylation at Tyr534, suggesting that DUSP22 serves as a crucial suppressor of both EGFR and AR-dependent signaling in PCa cells via dephosphorylation. Our findings indicate that loss of function of DUSP22 in PCa cells leads to aberrant activation of both EGFR-ERKs and AR signaling and ultimately progression of PCa, supporting the potential for novel therapeutic design of harnessing DUSP22 in the treatment of PCa.-Lin, H.-P., Ho, H.-M., Chang, C.-W., Yeh, S.-D., Su, Y.-W., Tan, T.-H., Lin, W.-J. DUSP22 suppresses prostate cancer proliferation by targeting the EGFR-AR axis.
Subject(s)
Dual-Specificity Phosphatases/metabolism , ErbB Receptors/metabolism , Mitogen-Activated Protein Kinase Phosphatases/metabolism , Prostatic Neoplasms/metabolism , Receptors, Androgen/metabolism , Signal Transduction , Animals , Cell Line, Tumor , Cell Proliferation , Dual-Specificity Phosphatases/genetics , Humans , Male , Mice , Mice, Inbred C57BL , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Mitogen-Activated Protein Kinase Phosphatases/genetics , Phosphorylation , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Protein BindingABSTRACT
Prostate-specific antigen (PSA) is regarded as the most sensitive biomarker for prostate cancer. Although androgen/androgen receptor (AR) signaling promotes prostate cancer progression, suppression of AR signaling induces chemokine (CC motif) ligand 2 (CCL2), which enables prostate cancer cells to gain metastatic potential. AR-controlled PSA alone may be an unreliable biomarker for patients receiving androgen deprivation therapy. Therefore, we investigated the validity of CCL2 as a complementary biomarker to PSA for prostate cancer. Our in vitro approach of enriching for prostate cancer cells with higher migration potential showed that CCL2 activated cellular migration. Importantly, we found that CCL2 levels were significantly different between men (n = 379) with and without prostate cancer. Patients with CCL2 ≥ 320 pg/mL had worse overall survival and prostate cancer -specific survival than those with CCL2 < 320 pg/mL. A novel risk classification was developed according to the risk factors CCL2 ≥ 320 pg/mL and PSA ≥ 100 ng/mL, and scores of 2, 1, and 0 were defined as poor, intermediate, and good risk, respectively, and clearly distinguished patient outcomes. CCL2 may serve as a novel biomarker for prostate cancer. The novel risk classification based on combining CCL2 and PSA is more reliable than using either alone.
Subject(s)
Biomarkers, Tumor/blood , Chemokine CCL2/blood , Prostatic Neoplasms/pathology , Aged , Aged, 80 and over , Apoptosis , Biomarkers, Tumor/genetics , Cell Movement , Cell Proliferation , Chemokine CCL2/genetics , Combined Modality Therapy , Follow-Up Studies , Humans , Male , Middle Aged , Neoplasm Grading , Neoplasm Staging , Prognosis , Prostatic Neoplasms/blood , Prostatic Neoplasms/therapy , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Survival Rate , Tumor Cells, CulturedABSTRACT
Hand-foot-mouth diseases (HFMD) caused by enterovirus 71 (EV71) and coxsackievirus 16 (CVA16) in children have now become a severe public health issue in the Asian-Pacific region. Recently we have successfully developed transgenic mice expressing human scavenger receptor class B member 2 (hSCARB2, a receptor of EV71 and CVA16) as an animal model for evaluating the pathogenesis of enterovirus infections. In this study, hSCARB2-transgenic mice were used to investigate the efficacy conferred by a previously described EV71 neutralizing antibody, N3. A single injection of N3 effectively inhibited the HFMD-like skin scurfs in mice pre-infected with clinical isolate of EV71 E59 (B4 genotype) or prevented severe limb paralysis and death in mice pre-inoculated with 5746 (C2 genotype). This protection was correlated with remarkable reduction of viral loads in the brain, spinal cord and limb muscles. Accumulated viral loads and the associated pro-inflammatory cytokines were all reduced. The protective efficacy of N3 was not observed in animals challenged with CVA16. This could be due to dissimilarity sequences of the neutralizing epitope found in CVA16. These results indicate N3 could be useful in treating severe EV71 infections and the hSCARB2-transgenic mouse could be used to evaluate the protective efficacy of potential anti-enterovirus agent candidates.
Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Capsid Proteins/immunology , Cytokines/immunology , Enterovirus A, Human/immunology , Hand, Foot and Mouth Disease/immunology , Hand, Foot and Mouth Disease/virology , Viral Load , Animals , Antibodies, Monoclonal/immunology , Antibody Specificity/immunology , Capsid Proteins/genetics , Coxsackievirus Infections/immunology , Coxsackievirus Infections/prevention & control , Coxsackievirus Infections/virology , Cross Reactions/immunology , Cytokines/metabolism , Disease Models, Animal , Enterovirus A, Human/genetics , Gene Expression , Genotype , Hand, Foot and Mouth Disease/genetics , Hand, Foot and Mouth Disease/mortality , Hand, Foot and Mouth Disease/prevention & control , Humans , Inflammation Mediators/immunology , Inflammation Mediators/metabolism , Lysosomal Membrane Proteins/genetics , Mice , Mice, Transgenic , Receptors, Scavenger/geneticsABSTRACT
In the present study, a novel plant transformation system for Doritaenopsis and Phalaenopsis has been developed. The pollen-mediated activation tagging system was established by artificial pollination. The pollens, co-cultured with Agrobacterium tumefaciens strain EHA105 harbouring an activation tagging vector (pTAG-8), were used for pollination. In order to optimize the transformation efficiency, several factors (concentration of A. tumefaciens, concentration of acetosyringone during co-cultivation and the duration of co-cultivation) known to influence Agrobacterium-mediated DNA transfer were examined. A concentration of 0.5-1 x 10(8) CFU/ml for A. tumefaciens, 0.1 mM acetosyringone, and 6 hrs of co-culture period were found to be the optimal condition for high transformation efficiency. Integration of T-DNA into the genome of putative transgenic plants was confirmed by PCR and DNA blot analyses. Single copy of the transgene was observed in all transgenic plants analyzed. Most of the transgenic plants had a morphologically normal phenotype and the overall capsule formation efficiency was similar to control plant. Our results showed a new approach of genetic transformation in orchids and this method can be employed for genetic improvement of the orchids.
Subject(s)
Agrobacterium tumefaciens , Orchidaceae/genetics , Pollination , DNA Transposable Elements/genetics , Polymerase Chain Reaction , Transformation, GeneticABSTRACT
A live enterovirus 71 (EV71) isolate designated, EV71/E59, with genotype B4 produced in Vero cells and purified over a sucrose gradient was used as the immunogen to generate EV71-specific murine monoclonal antibodies. Four hybridoma clones derived from the fusion of splenocytes of EV71/E59-preimmunized BALB/c (H-2(d)) mice and the NS-1 myeloma cells that exhibit stable growth were selected for detailed characterization. The proof that the hybridomas produced are indeed true independent clones was based on the obervations that they expressed different complementarity-determining regions (CDRs) in their κ light chain genes. Purified ascitic fluids produced by the individual clones reacted against the viral capsid protein, VP1, in Western blot; and recognized distinct sites of a common epitope localized at the C-terminal half of VP1. Each of the monoclonal antibodies exhibited potent neutralizing activities against the immunizing virus strain, as well as two other isolates namely, N0781-TW-01, and N2838, of subgenogroups B4 and B5, respectively, that were found commonly in recent outbreaks in Taiwan. It was also observed the monoclonal antibodies acted cooperatively in neutralizing the EV71/E59 virus.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Animals , Antibodies, Monoclonal/isolation & purification , Antibodies, Neutralizing/isolation & purification , Chlorocebus aethiops , Cross Reactions , Enterovirus B, Human/immunology , Enterovirus Infections/virology , Female , Humans , Mice , Mice, Inbred BALB C , Neutralization Tests , Taiwan , Vero CellsABSTRACT
Interstitial flow in and around bone tissue is oscillatory in nature and affects the mechanical microenvironment for bone cell growth and formation. We investigated the role of oscillatory shear stress (OSS) in modulating the proliferation of human osteoblast-like MG63 cells and its underlying mechanisms. Application of OSS (0.5 +/- 4 dynes/cm(2)) to MG63 cells induced sustained activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR/p70S6K (p70S6 kinase) signaling cascades and hence cell proliferation, which was accompanied by increased expression of cyclins A and D1, cyclin-dependent protein kinases-2, -4, and -6, and bone formation-related genes (c-fos, Egr-1, and Cox-2) and decreased expression of p21(CIP1) and p27(KIP1). OSS-induced activation of PI3K/Akt/mTOR/p70S6K and cell proliferation were inhibited by specific antibodies or small interference RNAs of alpha(v)beta(3) and beta(1) integrins and by dominant-negative mutants of Shc (Shc-SH2) and focal adhesion kinase (FAK) (FAK(F397Y)). Co-immunoprecipitation assay showed that OSS induces sustained increases in association of Shc and FAK with alpha(v)beta(3) and beta(1) integrins and PI3K subunit p85, which were abolished by transfecting the cells with FAK(F397Y) or Shc-SH2. OSS also induced sustained activation of ERK, which was inhibited by the specific PI3K inhibitor LY294002 and was required for OSS-induced activation of mTOR/p70S6K and proliferation in MG63 cells. Our findings provide insights into the mechanisms by which OSS induces osteoblast-like cell proliferation through activation of alpha(v)beta(3) and beta(1) integrins and synergistic interactions of FAK and Shc with PI3K, leading to the modulation of downstream ERK and Akt/mTOR/p70S6K pathways.