RNA editing of SLC22A3 drives early tumor invasion and metastasis in familial esophageal cancer.

Like many complex human diseases, esophageal squamous cell carcinoma (ESCC) is known to cluster in families. Familial ESCC cases often show early onset and worse prognosis than the sporadic cases. However, the molecular genetic basis underlying the development of familial ESCC is mostly unknown. We reported that SLC22A3 is significantly down-regulated in nontumor esophageal tissues from patients with familial ESCC compared with tissues from patients with sporadic ESCCs. A-to-I RNA editing of the SLC22A3 gene results in its reduced expression in the nontumor esophageal tissues of familial ESCCs and is significantly correlated with lymph node metastasis. The RNA-editing enzyme ADAR2, a familial ESCC susceptibility gene identified by our post hoc genome-wide association study, is positively correlated with the editing level of SLC22A3 Moreover, functional studies showed that SLC22A3 is a metastasis suppressor in ESCC, and deregulation of SLC22A3 facilitates cell invasion and filopodia formation by reducing its direct association with α-actinin-4 (ACTN4), leading to the increased actin-binding activity of ACTN4 in normal esophageal cells. Collectively, we now show that A-to-I RNA editing of SLC22A3 contributes to the early development and progression of familial esophageal cancer in high-risk individuals.

Knockdown of Notch1 inhibits nasopharyngeal carcinoma cell growth and metastasis via downregulation of CCL2, CXCL16, and uPA.

Notch pathway is a highly conserved cell signaling system that plays very important roles in controlling multiple cell differentiation processes during embryonic and adult life. Multiple lines of evidence support the oncogenic role of Notch signaling in several human solid cancers; however, the pleiotropic effects and molecular mechanisms of Notch signaling inhibition on nasopharyngeal carcinoma (NPC) remain unclear. In this study, we evaluated Notch1 expression in NPC cell lines (CNE1, CNE2, SUNE1, HONE1, and HK1) by real-time quantitative PCR and Western blot analysis, and we found that CNE1 and CNE2 cells expressed a higher level of Notch1 compared with HONE1, SUNE1, and HK1 cells. Then Notch1 expression was specifically knocked down in CNE1 and CNE2 cells by Notch1 short hairpin RNA (shRNA). In Notch1 knockdown cells, cell proliferation, migration, and invasion were significantly inhibited. The epithelial-mesenchymal transition of tumor cells was reversed in Notch1-shRNA-transfected cells, accompanied by epithelioid-like morphology changes, increased protein levels of E-cadherin, and decreased expression of vimentin. In addition, knockdown of Notch1 markedly inhibited the expression of urokinase plasminogen activator (uPA) and its receptor uPAR, and chemokines C-C motif chemokine ligand 2 and C-X-C motif chemokine ligand 16, indicating that these factors are downstream targets of Notch1. Furthermore, deleting uPA expression had similar effects as Notch1. Finally, knockdown of Notch1 significantly diminished CNE1 cell growth in a murine model concomitant with inhibition of cell proliferation and induction of apoptosis. These results suggest that Notch1 may become a novel therapeutic target for the clinical treatment of NPC.

Gastric cancer vaccines synthesized using a TLR7 agonist and their synergistic antitumor effects with 5-fluorouracil.

BACKGROUND: Vaccines play increasingly important roles in cancer treatment due to their advantages of effective targeting and few side effects. Our laboratory has attempted to construct vaccines by conjugating TLR7 agonists with tumor-associated antigens. Furthermore, immunochemotherapy has recently become an appealing approach to cancer therapy. 5-fluorouracil (5-FU), a commonly used chemotherapeutic agent, can reportedly potently and selectively kill tumor-associated MDSCs in vivo. METHODS: Gastric cancer vaccines were synthesized by the covalent attachment of our TLR7 agonist with the gastric cancer antigen MG7-Ag tetra-epitope, leading to T7 - ML (linear tetra-epitope) and T7 - MB (branched tetra-epitope). Cytokines induced by the vaccines in vitro were assessed by ELISA. A tumor challenge model was created by treating BALB/c mice on either a prophylactic or therapeutic vaccination schedule. 5-FU was simultaneously applied to mice in the combination treatment group. CTL and ADCC activities were determined by the LDH method, while CD3+/CD8+, CD3+/CD4+ T cells and MDSCs were evaluated by flow cytometry. RESULTS: In vitro, rapid TNF-α and IL-12 inductions occurred in BMDCs treated with the vaccines. In vivo, among all the vaccines tested, T7 - MB most effectively reduced EAC tumor burdens and induced CTLs, antibodies and ADCC activity in BALB/c mice. Immunization with T7 - MB in combination with 5-FU chemotherapy reduced tumor sizes and extended long-term survival rates, mainly by improving T cell responses, including CTLs, CD3+/CD8+ and CD3+/CD4+ T cells. 5-FU also enhanced the T7 - MB efficiency by reversing immunosuppressive factors, i.e., MDSCs, which could not be validly inhibited by the vaccines alone. In addition, T7 - MB repressed tumor growth and immune tolerance when the therapeutic schedule was used, although the effects were weaker than those achieved with either T7 - MB alone or in combination with 5-FU on the prophylactic schedule. CONCLUSIONS: A novel effective gastric cancer vaccine was constructed, and the importance of branched multiple antigen peptides and chemical conjugation to vaccine design were confirmed. The synergistic effects and mechanisms of T7 - MB and 5-FU were also established, observing mainly T cell activation and MDSC inhibition.

A conjugate of octamer-binding transcription factor 4 and toll-like receptor 7 agonist prevents the growth and metastasis of testis embryonic carcinoma.

BACKGROUND: The immune non-recognition is often the underlying cause of failure in tumor immunotherapeutic. This is because most tumor-related antigens are poorly immunogenic, and fail to arouse an efficient immune response against cancers. Here we synthesized a novel TLR7 agonist, and developed a safe and effective immunotherapeutic vaccine by conjugating this TLR7 agonist with the pluripotency antigen OCT4. METHODS: Purified recombinant OCT4 protein was covalently linked with a novel TLR7 agonist to form a TLR7-OCT4 conjugate (T7-OCT4). After conjugation, the in vitro release of IL-12 and IFN-γ was observed in spleen lymphocytes. Mice were immunized with TLR7-OCT4, and the release of IFN-γ, the percentages of CD3+/CD8+ T cells and the OCT4-specific cytotoxicity rates were measured. The immunized mice were challenged with mouse embryonic carcinoma (EC), and the tumor volume and tumor weight were determined. Blood routine examination was performed to evaluate the biosafety of TLR7 agonist and TLR7-OCT4 conjugate in mice. RESULTS: T7-OCT4 conjugate significantly increased the in vitro release of IL-12 and IFN-γ by mouse spleen lymphocytes. In addition, the release of IFN-γ, the percentages of CD3+/CD8+ T cells and the tumor-specific cytotoxicity rates in immunized mice were significantly higher. Importantly, in EC xenografted mice, immunization with T7-OCT4 conjugate decreased the growth of the tumor dramatically up to 90 %, as compared to mice immunized with OCT4 protein or TLR7 agonist alone. Furthermore, blood routine examination demonstrated that no abnormalities of the blood cells and components in the blood fluids were detected by T7-OCT4 and TLR7 agonist injections. CONCLUSIONS: Our results showed that conjugating OCT4 protein to the novel TLR7 agonist produced a vaccine which is effective and safe in preventing tumor growth in mice. Our results suggest that this type of vaccine formulation has great potentiality in preventive vaccines against OCT4 expressing tumors.

Synthesis and immunoregulatory activities of conjugates of a Toll-like receptor 7 inert ligand.

In the synthesis and modification of the analogs of an adenine type of Toll-like receptor (TLR) 7 agonists, we found a special compound, 9-propionyloxy-8-hydroxy-2-(2-methoxyethoxy)-adenine (6). It is a synthesized TLR7 inert ligand, which does not respond to TLR7 itself. However, it can be coupled with protein or peptide antigens via propionyloxy functional group to promote their immunogenicity significantly. The compound was covalently coupled to protein and peptide to get the conjugates. The inductivity of cytokine production by the conjugates was 872.4-fold compared with the unconjugated antigens in vitro by mouse splenocyte. These data show that the immunostimulatory activity of inert TLR7 ligand can be endowed, and the activity of antigens can be amplified by conjugation with various proteins and peptides, thus broadening the potential therapeutic application and reducing the risk of TLR7 agonists' side effects.

Enhanced binding to and killing of hepatocellular carcinoma cells in vitro by melittin when linked with a novel targeting peptide screened from phage display.

A random phage 12-mer peptide library and a whole-cell subtractive biopanning protocol against HepG2 cells were used to select a novel peptide-specific binding to hepatocellular carcinoma cells. As a result, peptide SLSLITMLKISR (AM-2) was screened as a novel homing peptide to hepatocellular carcinoma cells, tested by immunofluorescence and immunochemistry assays. Subsequently, peptide AM-2 was linked to melittin by A(EAAAK)2A, and the antitumor effect of this ligation product was detected by MTT assay, fluorescence-activated cell sorting, and scanning electron microscopy methods. Results of cell growth inhibition tests confirmed that the affinity of melittin was increased after being incorporated into AM-2, and AM-2-melittin specifically targeted and killed HepG2 cells in vitro. Thus, AM-2 is a valuable ligand for tumor targeting, which leads to increased binding and killing effect of hepatocellular carcinoma cells in vitro when ligated to melittin, and AM-2-melittin has a clinical potential application as target agents for the treatment of human hepatocellular carcinoma.

Designed synthetic analogs of the α-helical peptide temporin-La with improved antitumor efficacies via charge modification and incorporation of the integrin αvß3 homing domain.

How to target cancer cells with high specificity and kill cancer cells with high efficiency remains an urgent demand for anticancer drugs. Temporin-La, which belongs to the family of temporins, presents antitumor activity against many cancer cell lines. We first used a whole bioinformatic analysis method as a platform to identify new anticancer antimicrobial peptides (AMPs). On the basis of these results, we designed a temporin-La analog (temporin-Las) and related constructs containing the Arg-Gly-Asp (RGD) tripeptide, the integrin αvß3 homing domain (RGD-La and RGD-Las). We detected a link between the net charges and integrin αvß3 expression of cancer cell lines and the antitumor activities of these peptides. Temporin-La and its synthetic analogs inhibited cancer cell proliferation in a dose-dependent manner. Evidence was provided that the affinity between RGD-Las and tumor cell membranes was stronger than other tested peptides using a pull-down assay. Morphological changes on the cell membrane induced by temporin-La and RDG-Las, respectively, were examined by scanning electron microscopy. Additionally, time-dependent morphological changes were detected by confocal microscopy, where the binding process of RGD-Las to the cell membrane could be monitored. The results indicate that the electrostatic interaction between these cationic peptides and the anionic cell membrane is a major determinant of selective cell killing. Thus, the RGD tripeptide is a valuable ligand motif for tumor targeting, which leads to an increased anticancer efficiency by RGD-Las. These AMP-derived peptides have clinical potential as specifically targeting agents for the treatment of αvß3 positive tumors.

LysGH15B, the SH3b domain of staphylococcal phage endolysin LysGH15, retains high affinity to staphylococci.

LysGH15, a phage endolysin, exhibits a particularly broad lytic spectrum against Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA). Sequence analysis reveals that this endolysin contains a C-terminal cell wall binding domain (SH3b), which causes the endolysin to bind to host strains. In this study, the substrate binding affinity of the SH3b domain (LysGH15B) was evaluated. A fusion protein of LysGH15B and green fluorescent protein (LysGH15B-GFP) were cloned and expressed in Escherichia coli. Laser scanning confocal microscopy was used to detect the fluorescence of the treated cells irradiated at different excitation wavelengths and to determine the binding activity of LysGH15B-GFP and GFP. We found that LysGH15B-GFP not only generated green fluorescence, but, more importantly, also displayed specific affinity to staphylococcal isolates, especially MRSA. In contrast, the single GFP did not display any binding activity. The high affinity was attributed to the portion of LysGH15B and the binding activity of the fusion protein was specific to staphylococci. This study provides an insight into the SH3b domain of LysGH15. The specific binding activity may cause LysGH15B to serve as an anchoring device, and offer an alternative approach for cell surface attachment onto staphylococci.

Synthetic MUC1 breast cancer vaccine containing a Toll-like receptor 7 agonist exerts antitumor effects.

Adjuvant immunotherapy has recently emerged as a potential treatment strategy for breast cancer. The tumor-associated protein mucin 1 (MUC1) has received increasing attention due to its high expression in numerous types of common tumors, in which MUC1 acts as a cancer antigen. However, the simple mixed composition of an adjuvant and a peptide is not a sufficient rationale for a MUC1 peptide-based vaccine. The present study developed a novel Toll-like receptor 7 (TLR7) agonist-conjugated MUC1 peptide vaccine (T7-MUC1), which elicited an effective immune response and a robust antitumor effect in a mouse breast cancer model. In vitro, T7-MUC1 significantly increased the release of cytokines in mouse bone marrow dendritic cells and spleen lymphocytes, and induced the dendritic cell-cytokine-induced killer response against tumor cells with high MUC1 expression. In vivo, it was observed that the 4T1 tumor weights in mice immunized with the T7-MUC1 conjugate were reduced by ≥70% compared with those in the control group. Furthermore, the therapeutic responses in vivo were attributed to the increase in specific humoral and cellular immunity, including high antibody titers, antibody-dependent cell-mediated cytotoxicity and cytotoxic T-lymphocyte activity. The percentages of CD3+/CD8+ T-cells were significantly higher in the T7-MUC1 treatment group compared with those in the control group. Therefore, the results of the present study suggested that the T7-MUC1 vaccine inhibited tumor growth in mice and thus may have potential as a therapeutic candidate in clinical trials for breast cancer immunotherapy.

Synthetic Toll-like receptor 7 ligand inhibits porcine reproductive and respiratory syndrome virus infection in primary porcine alveolar macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV), a common viral pathogen, causes huge annual economic losses to the swine industry worldwide. After triggering by specific ligands, the Toll-like receptor 7 (TLR7), a type of pattern-recognition receptor (PRR), induces antiviral cytokines production. Previously, we synthesized an adenine analog, designated SZU101, a TLR7-specific ligand. In this study, we assessed the inhibitory effect of SZU101 on PRRSV infection in vitro. SZU101 significantly suppressed PRRSV infection in primary porcine alveolar macrophages (PAMs) in a dose-dependent manner. Moreover, SZU101-induced inhibition involved NF-κB pathway activation in PAMs to initiate expression of TLR7-mediated cytokines and induce expression of downstream signaling IFN-stimulated genes (ISGs). Chloroquine, a TLR7 inhibitor, and BAY 11-7082, an NF-κB inhibitor, reversed both the SZU101-induced antiviral effect and induction of cytokine genes and ISGs expression. Therefore, SZU101 antiviral effects depend at least in part on TLR7-NF-κB signaling pathway. Additionally, administration of SZU101 enhanced the humoral and cell-mediated immune responses against PRRSV antigens in mice. Given these results, SZU101 holds promise as an antiviral agent and a vaccine adjuvant to prevent PRRSV infection in pigs.

Immunomodulatory and Antitumor Effects of a Novel TLR7 Agonist Combined with Lapatinib.

As new treatment approaches, both immunotherapy and targeted treatments have been used in the clinical treatment of cancers. These therapies are different from traditional surgery, chemotherapy and radiotherapy. Use of a combination of immunotherapy and targeted treatments may improve tumor clearance. We investigated the feasibility of combining tyrosine kinase inhibitors (TKIs, targeted drugs) and SZU-101 (a novel TLR7 agonist synthesized by our laboratory). Thirteen different TKIs were combined with or without SZU-101 and studied to determine their effects on immunocytes. On the basis of the distinctive results, lapatinib and sunitinib were selected for further tumor-inhibition investigation and determination of the underlying mechanism. Interestingly, we found lapatinib to work better with SZU-101, enhancing tumor clearance in vivo, without affecting the TLR7-NF-κB pathway activated by the TLR7 agonist in mouse spleen lymphocytes and bone marrow dendritic cells (BMDCs).

Conjugation of weak ligands with weak antigens to activate TLR-7: A step toward better vaccine adjuvants.

To study the structure-activity relationship (SAR) of Toll-like receptor 7 (TLR-7) agonists based on 8-oxoadenines, a novel subset of C9-substituted 8-hydroxy-2-(2-methoxyethoxy)-adenines and their antigen conjugates were synthesized. In vitro, the ability of cytokines (IL-12p70 and IFN-γ) induction of ligands with alkyl acid at C9-position were very weak compared with benzoic acid counter parts. Unexpectedly, its antigen conjugates that conjugated with proteins or peptides with weak immunogenicity, showed enhanced activity of cytokines induction. After administered systemically in mice in vivo, all conjugates induced prolonged increase in pro-inflammatory cytokines and antigen-specific IgG levels in serum compared with free compounds. Results from molecular dynamics (MD) simulations further confirmed the conclusion and provided the details of interaction to explain the phenomenon of experiment. In conclusion, we discovered that TLR-7 could be activated via some conjugates of weak ligand and weak antigen, which could be safer adjuvant candidates for vaccines in the future.

Antitumor activity of a novel small molecule TLR7 agonist via immune response induction and tumor microenvironment modulation.

Immunotherapy is emerging as a powerful and active tumor-specific approach against cancer via triggering the immune system. Toll-like receptors (TLRs) are fundamental elements of the immune system, which facilitate our understanding of the innate and adaptive immune pathways. TLR agonists used as single agents can effectively eradicate tumors due to their potent stimulation of innate and adaptive immunity. We examined the effects of a novel adenine type of TLR7 agonists on both innate and adaptive immune activation in vitro and in vivo. We established the local and distant tumor­bearing mice derived from murine mammary carcinoma cell line (4T1) to model metastatic disease. Our data demonstrated that SZU101 was able to stimulate innate immune cells to release cytokines at the very high level compared with LPS at the same or lower concentration. Locally intratumoral SZU101 injection can elicit a systemic antitumor effect on murine breast tumor model. SZU101 affected the frequency of intratumoral immune cell infiltration, including the percentage of CD4+ and CD8+ increase, and the ratio of Tregs decrease. Our data reveal that the antitumor effect of SZU101 is associated with multiple mechanisms, inducing tumor­specific immune response, activation of innate immune cells and modulation of the tumor microenvironment.

Toll-like receptor 7 inactive ligands enhanced cytokine induction by conjugation to weak antigens.

Toll-like receptors (TLRs) 7/8 are key targets in the design and development of small-molecule drugs serving as anticancer/antiviral agents and vaccine adjuvants. Clinical trials of imiquimod were discontinued owing to its serious adverse side effects. Herein we report the synthesis and biological evaluation of a series of 8-hydroxy-2-(2-methoxyethoxy)adenine derivatives that cannot induce cytokine production and that lack activity toward TLR 7/8. Their ability to triggering remarkable levels of cytokine production were revealed upon their conjugation with antigens that have weak immunogenicity. This discovery demonstrated that TLR 7 can be activated by coupling an antigen to the terminal carboxyl group at N9 of the inactive ligand adenine analogues. These inactive analogues may be well suited as new adjuvants with superior activity after conjugation, effectively decreasing the side effects caused by conventional adjuvants.

Synergistic apoptosis-inducing effect of aspirin and isosorbide mononitrate on human colon cancer cells.

Aspirin and isosorbide mononitrate (ISMN) are two commonly used drugs, which are clinically applied for the treatment of inflammatory and cardiovascular diseases, respectively. Recently, aspirin has attracted interest due to its potential application for the treatment of cancer, particularly colon cancer. NO-aspirin, an aspirin derivative containing a covalently bound NO-donating moiety, has been proven to be an effective anti­tumor agent with apoptosis-inducing ability. In the present study, ISMN was used as an NO donor and its synergic effect with aspirin was assessed in human colon cancer cells. In vitro, an MTT assay demonstrated that ISMN had a synergistic effect on the growth inhibitory effects of aspirin on HCT116 and SW620 colon cancer cells, while the growth of EA.hy926 normal endothelial cells was unaffected. This synergistic anti­tumor effect was further validated in vivo using nude mouse HCT116 cell xenograft model. Observation of nuclear morphology, Annexin V-fluorescein isothiocyanate/propidium iodide double staining and a caspase-3 activity assay suggested that the combination of the two drugs induced apoptosis in HCT116 cells. Furthermore, the molecular mechanisms of the apoptotic effect of the drugs was assessed using an NO release assay, reverse transcription quantitative polymerase chain reaction analysis, western blot analysis and a luciferase reporter assay. It was certified that the increase in the amount of NO release, the decrease in the luciferase promoter activity and the expression of cyclin D1 and c-myc in HCT116 cells were affected by aspirin and ISMN in a synergistic manner. In conclusion, the present study was the first, to the best of our knowledge, to report on the synergistic apoptosis-inducing effects of aspirin and ISMN in human colon cancer cells, which were mediated via Wnt and NO signaling pathways. The results of the present study will facilitate the development of future therapeutic strategies.

Synthesis and Evaluation of Conjugates of Novel TLR7 Inert Ligands as Self-Adjuvanting Immunopotentiators.

During the design and synthesis of a series of 8-hydroxy-2-(2-methoxyethoxy)-adenine derivatives bearing various substituted -RCOOH groups at the 9-position, we identified a TLR7-inert ligand, which does not activate TLR7 signaling pathway. Of interest, the coupling of weakly immunogenic antigens via the -RCOOH group was able to significantly enhance the immunogenicity of the antigens. Herein, an inert ligand, 9-(3-carboxypropyl)-8-hydroxy-2-(2-methoxyethoxy)-adenine (5, GD2), was synthesized and conjugated to 5 different weakly immunogenic antigens (BSA, OVA, MSA, MG7, and thymosin). Compared with the GD2 and the potent agonist UC-1 V150, all conjugates demonstrated potent immunogenicity in vitro and in vivo. All conjugates induced prolonged increases, while UC-1 V150 showed a rapid decline in the levels of proinflammatory cytokines following initial increases. These data indicate that the immunostimulatory activity of TLR7-inert ligands could be amplified and prolonged by conjugation to antigens, thus broadening the potential therapeutic application of these agents.

Conjugation of toll-like receptor-7 agonist to gastric cancer antigen MG7-Ag exerts antitumor effects.

AIM: To investigate the effects of our tumor vaccines on reversing immune tolerance and generating therapeutic response. METHODS: Vaccines were synthesized by solid phase using an Fmoc strategy, where a small molecule toll-like receptor-7 agonist (T7) was conjugated to a monoclonal gastric cancer 7 antigen mono-epitope (T7-MG1) or tri-epitope (T7-MG3). Cytokines were measured in both mouse bone marrow dendritic cells and mouse spleen lymphocytes after exposed to the vaccines. BALB/c mice were intraperitoneally immunized with the vaccines every 2 wk for a total of three times, and then subcutaneously challenged with Ehrlich ascites carcinoma (EAC) cells. Three weeks later, the mice were killed, and the tumors were surgically removed and weighed. Serum samples were collected from the mice, and antibody titers were determined by ELISA using an alkaline phosphate-conjugated detection antibody for total IgG. Antibody-dependent cell-mediated cytotoxicity was detected by the lactate dehydrogenase method using natural killer cells as effectors and antibody-labeled EAC cells as targets. Cytotoxic T lymphocyte activities were also detected by the lactate dehydrogenase method using lymphocytes as effectors and EAC cells as targets. RESULTS: Vaccines were successfully synthesized and validated by analytical high performance liquid chromatography and electrospray mass spectrometry, including T7, T7-MG1, and T7-MG3. Rapid inductions of tumor necrosis factor-α and interleukin-12 in bone marrow dendritic cells and interferon γ and interleukin-12 in lymphocytes occurred in vitro after T7, T7-MG1, and T7-MG3 treatment. Immunization with T7-MG3 reduced the EAC tumor burden in BALB/c mice to 62.64% ± 5.55% compared with PBS control (P < 0.01). Six or nine weeks after the first immunization, the monoclonal gastric cancer 7 antigen antibody increased significantly in the T7-MG3 group compared with the PBS control (P < 0.01). As for antibody-dependent cell-mediated cytotoxicity, antisera obtained by immunization with T7-MG3 were able to markedly enhance cell lysis compared to PBS control (31.58% ± 2.94% vs 18.02% ± 2.26%; P < 0.01). As for cytotoxic T lymphocytes, T7-MG3 exhibited obviously greater cytotoxicity compared with PBS control (40.92% ± 4.38% vs 16.29% ± 1.90%; P < 0.01). CONCLUSION: A successful method is confirmed for the design of gastric cancer vaccines by chemical conjugation of T7 and multi-repeat-epitope of monoclonal gastric cancer 7 antigen.

Development and characterization of a mouse monoclonal antibody against antimicrobial peptide tachyplesin I.

Monoclonal antibodies against tachyplesin I (TP I) were developed to study its mechanisms of activity, a kind of cationic antimicrobial peptides (AMPs), in vivo or in vitro, and to purify TP I from expression products. The synthesized TP I was chemically conjugated with the carrier protein BSA and then injected into BALB/c mice. Positive hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) using TP I and subcloned three times with limiting dilution. Five MAbs effective in detecting the native TP I (named 2D8, 3B8, 5H2, 6B12, and 8F5) were obtained. Isotyping of all obtained MAbs indicated that MAbs 2D8, 3B8, 5H2, and 8F5 belong to IgG1, and MAb 6B12 belongs to IgG2a. Specificity assay showed that MAb 8F5 had almost the same level of specificity to natural TP I, recombinant TP I, and synthesized TP I and TP II, but did not cross-react with control peptides. These results suggest that the synthetic AMP conjugates can elicit antibodies against native AMPs and can be used to detect antimicrobial peptides.

Subtractive screening with the Mycobacterium tuberculosis surface protein phage display library.

Surface proteins consist of secreted and membrane proteins and play a central role in the interaction of the pathogen with its environment, especially in the pathogenicity of Mycobacterium tuberculosis (MTB). Research on surface proteins in MTB has focused on 2D electrophoresis of culture filtrate proteins (CFP), extraction of transmembrane proteins with detergent and predicting their properties with a range of available algorithms. However, functional analysis of these secretomes is possible only if many proteins are expressed and purified individually, which limits a large number of studies to the function of the proteome. Here, we utilized a phage display system to construct a whole genomic surface protein phage display library of MTB, which can complete direct selection, identification, expression, purification and functional research of surface proteins of MTB. With this system we made a new serological approach involving iterative subtraction screening. Cross-reactivity of antibodies was reduced by preadsorption of the surface protein phage display library with the sera of healthy BCG-vaccinated individuals prior to studying their reactivity against the sera of tuberculosis (TB) patients. As a result six antigens were identified, three of which have not previously been reported as diagnosis antigens. The surface protein phage display library shows great promise in the study of MTB.