The purpose of this study was to further characterize cell growth-inhibitory effects of a recently identified androgen receptor (AR) signaling inhibitor 6-amino-2-[2-(4-tert-butyl-pnenoxy)-ethylsulfanyl]-1H-pyrimidin-4-one (DL3)5 and antiandrogen bicalutamide (Bic). DL3 was more potent than Bic in induction of G1 arrest and reduction of G1-related cell cycle protein expression in AR-positive LNCaP cells. DL3, but not Bic, moderately inhibited growth of AR-negative PC-3 cells independent of G1 arrest. The data indicated that DL3 inhibit cell growth in both AR-dependent and -independent manners and is potentially a potent therapeutic agent for the management of advanced human prostate cancer.
androgen receptor; antagonist; prostate cancer; cell cycle
Heat shock protein (hsp) 70-1 (hsp70-1) is overexpressed in human prostate cancer cells and may play important roles in prostate cancer resistance to conventional therapies. The purpose of this study was to investigate whether androgen receptor (AR) and its signaling regulate hsp70-1 expression. Several lines of AR-positive (LNCaP, LAPC-4, and 22Rv1) and -negative (PC-3, DU145, WPE1-NB14 and WPE1-NB-26) human prostatic cells were used in the study. Dihydrotestosterone (DHT) enhanced hsp70-1 expression in LNCaP cells. Expression of hsp70-1 in LNCaP cells was downregulated by the anti-androgens bicalutamide (Bic), and flutamide (Flut), and a newly identified AR signaling antagonist DL3. The downregulation of hsp70-1 by DL3 was also observed in LAPC-4 and 22Rv1 cells, but not in the four lines of AR-negative cells examined. Expression of hsp70-1 was also reduced by DL3 in PC-3 cells engineered with AR. On the other hand, knocking down AR in LNCaP cells by siRNA moderately reduced hsp70-1 level and abolished effects of DL3 on hsp70-1 expression. DL3 reduced hsp70-1 mRNA synthesis in cells and its in vitro gene transcription but did not significantly alter the stabilities of hsp70-1 mRNA and protein. Chromatin-immunoprecipitation (ChIP) assay showed that AR bound to the promoter region of HSPA1B gene, which was reduced in cells treated with DL3 or Bic. These data suggest that AR and its signaling regulate hsp70-1 expression in prostate cancer cells and that HSPA1B could be an AR target gene.
androgen receptor; heat shock protein 70-1; antagonist; prostate cancer
Our previous study revealed that Vav3 oncogene is overexpressed in human prostate cancer, activates androgen receptor (AR), and stimulates growth in prostate cancer cells. The purpose of this study is to further determine the potential role of Vav3 in prostate cancer development in genetically engineered mouse model. We generated Vav3 transgenic mice by targeted overexpression of a constitutive active Vav3 in the prostatic epithelium. We found that overexpression of Vav3 led to development of mouse prostatic intraepithelial neoplasia and prostate cancer at the age of as early as 3 months. The AR signaling axis and phosphatidylinositol 3-kinase-Akt signaling were elevated in the prostate glands of Vav3 transgenic mice. In addition to prostate cancer, Vav3 transgenic mice developed significant nonbacterial chronic prostatitis in the prostate gland with notable infiltration of lymphomononuclear cells (monocytes, lymphocytes, and plasma cells), which was associated with elevated incidence of prostate cancer. DNA microarray and signaling pathway analysis revealed that the top diseases and disorders were inflammatory diseases and cancer of the prostate gland in Vav3 transgenic mice. In vitro analysis showed that overexpression of Vav3 in prostate cancer cells enhanced nuclear factor-κB (NF-κB) activity, implicating an underlying mechanism of innate inflammatory response induced by elevated Vav3 activity. These data showed that Vav3 overexpression in the prostate epithelium enhanced both the AR signaling axis and NF-κB–mediated pathway, which potentially contributed to the development of nonbacterial prostatitis and prostate cancer.
Dozens of broadly neutralizing HIV-1 antibodies have been isolated in the last few years from the sera of HIV-1-infected individuals. Only a limited number of regions on the HIV-1 spike, however, are recognized by these antibodies. One of these regions (N332) is characterized by an N-linked glycan at residue 332 on HIV-1 gp120 and is recognized by antibody 2G12 and by the recently reported antibodies PGT121-137, the latter isolated from three donors. To investigate the diversity in mode of antibody recognition at the N332 site, we used functional complementation between antibody heavy and light chains as a means of assessing similarity in mode of recognition. We examined a matrix of 12 PGT-heavy chains with each of 12 PGT-light chains. Expression in 96-well format for the 144 antibodies (132 chimeric and 12 wild-type) was generally consistent (58±10 µg/ml). In contrast, recognition of HIV-1 gp120 was bimodal: when the source of heavy and light chains was from the same donor, recognition was good; when sources of heavy and light chains were from different donors, recognition was poor. Moreover, neutralization of HIV-1 strains SF162.LS and TRO.11 generally followed patterns of gp120 recognition. These results are consistent with published sequence, mutational, and structural findings, all of which indicate that N332-directed neutralizing antibodies from different donors utilize different modes of recognition, and provide support for a correlation between functional complementation of antibody heavy and light chains and similarity in antibody mode of recognition. Overall, our results add to the growing body of evidence that the human immune system is capable of recognizing the N332-region of HIV-1 gp120 in diverse ways.
This study aimed to evaluate the feasibility of intraarterial (IA) delivery and in vivo MR imaging of superparamagnetic iron oxide (SPIO)-labeled mesenchymal stem cells (MSCs) in a canine stroke model.
MSCs harvested from beagles’ bone marrow were labeled with home-synthesized SPIO. Adult beagle dogs (n = 12) were subjected to left proximal middle cerebral artery (MCA) occlusion by autologous thrombus, followed by two-hour left internal carotid artery (ICA) occlusion with 5 French vertebral catheter. One week later, dogs were classified as three groups before transplantation: group A: complete MCA recanalization, group B: incomplete MCA recanalization, group C: no MCA recanalization. 3×106 labeled-MSCs were delivered through left ICA. Series in vivo MRI images were obtained before cell grafting, one and 24 hours after transplantation and weekly thereafter until four weeks. MRI findings were compared with histological studies at the time point of 24 hours and four weeks.
Home-synthesized SPIO was useful to label MSCs without cell viability compromise. MSCs scattered widely in the left cerebral hemisphere in group A, while fewer grafted cells were observed in group B and no cell was detected in group C at one hour after transplantation. A larger infarction on the day of cell transplantation was associated with more grafted cells in the brain. Grafted MSCs could be tracked effectively by MRI within four weeks and were found in peri-infarction area by Prussian blue staining.
It is feasible of IA MSCs transplantation in a canine stroke model. Both the ipsilateral MCA condition and infarction volume before transplantation may affect the amount of grafted cells in target brain. In vivo MR imaging is useful for tracking IA delivered MSCs after SPIO labeling.
A problem for the generation of polyclonal antibodies is the potential difficulties for obtaining a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar.
Whole-virus vaccines, including inactivated or live-attenuated influenza vaccines, have been conventionally developed and supported as a prophylaxis. These currently available virus-based influenza vaccines are widely used in the clinic, but the vaccine production takes a long time and a huge number of embryonated chicken eggs. To overcome the imperfection of egg-based influenza vaccines, epitope-based peptide vaccines have been studied as an alternative approach. Here, we formulated an efficacious peptide vaccine without carriers using phosphodiester CpG-DNA and a special liposome complex. Potential epitope peptides predicted from the hemagglutinin (HA) protein of the H5N1 A/Viet Nam/1203/2004 strain (NCBI database, AAW80717) were used to immunize mice along with phosphodiester CpG-DNA co-encapsulated in a phosphatidyl-β-oleoyl-γ-palmitoyl ethanolamine (DOPE):cholesterol hemisuccinate (CHEMS) complex (Lipoplex(O)) without carriers. We identified a B cell epitope peptide (hH5N1 HA233 epitope, 14 amino acids) that can potently induce epitope-specific antibodies. Furthermore, immunization with a complex of the B cell epitope and Lipoplex(O) completely protects mice challenged with a lethal dose of recombinant H5N1 virus. These results suggest that our improved peptide vaccine technology can be promptly applied to vaccine development against pandemic influenza. Furthermore our results suggest that potent epitopes, which cannot be easily found using proteins or a virus as an antigen, can be screened when we use a complex of peptide epitopes and Lipoplex(O).
Viral drug toxicity, resistance, and an increasing immunosuppressed population warrant continued research into new avenues for limiting diseases associated with human cytomegalovirus (HCMV). In this study, a small interfering RNA (siRNA), siX3, was designed to target coding sequences within shared exon 3 of UL123 and UL122 transcripts encoding IE1 and IE2 immediate-early proteins of HCMV. Pretreatment of cells with siX3 reduced the levels of viral protein expression, DNA replication, and progeny virus production compared to control siRNA. Two siRNAs against UL54 and overlapping transcripts (UL55-57) were compared to siX3 in HCMV infection and were also found to be effective at inhibiting HCMV replication. Further investigation into the effects of the siRNAs on viral replication showed that pretreatment with each of the siRNAs resulted in an inhibition in the formation of mature replication compartments. The ability of these siRNAs to prevent or reduce certain cytopathic effects associated with HCMV infection was also examined. Infected cells pretreated with siX3, but not siUL54, retained promyelocytic leukemia (PML) protein in cellular PML bodies, an essential component of this host intrinsic antiviral defense. DNA damage response proteins, which are localized in nuclear viral replication compartments, were reduced in the siX3- and siUL54-treated cells. siX3, but not siUL54, prevented DNA damage response signaling early after infection. Therapeutic efficacy was demonstrated by treating cells with siRNAs after HCMV replication had commenced. Together, these findings suggest that siRNAs targeting exon 3 of the major IE genes or the UL54-57 transcripts be further studied for their potential development into anti-HCMV therapeutics.
Peripheral arterial disease (PAD) is a common disease accounting for about 12% of the adult population, and causes significant morbidity and mortality. Therapeutic angiogenesis using angiogenic factors has been considered to be a potential treatment option for PAD patients. In this study, we assessed the potential of a new angiogenic factor AGGF1 for therapeutic angiogenesis in a critical limb ischemia model in mice for PAD.
Methods and Results
We generated a unilateral hindlimb ischemia model in mice by ligation of the right common iliac artery and femoral artery. Ischemic mice with intrasmuscular administration of DNA for an expression plasmid for human AGGF1 (AGGF1 group) resulted in increased expression of both AGGF1 mRNA and protein after the administration compared with control mice with injection of the empty vector (control group). Color PW Doppler echocardiography showed that the blood flow in ischemic hindlimbs was significantly increased in the AGGF1 group compared to control mice at time points of 7, 14, and 28 days after DNA administration (n = 9/group, P = 0.049, 0.001, and 0.001, respectively). Increased blood flow in the AGGF1 group was correlated to increased density of CD31-positive vessels and decreased necrosis in muscle tissues injected with AGGF1 DNA compared with the control tissue injected with the empty vector. Ambulatory impairment was significantly reduced in the AGGF1 group compared to the control group (P = 0.004). The effect of AGGF1 was dose-dependent. At day 28 after gene transfer, AGGF1 was significantly better in increasing blood flow than FGF-2 (P = 0.034), although no difference was found for tissue necrosis and ambulatory impairment.
These data establish AGGF1 as a candidate therapeutic agent for therapeutic angiogenesis to treat PAD.
Hydrogen sulfide (H2S), produced by cystanthionine-γ-lysase (CSE) in the cardiovascular system, has been suggested to be the third gasotransmitter in addition to nitric oxide (NO) and carbon monoxide (CO). The present study aimed to investigate the role of H2S in ischemic postconditioning (IPO) during the early period of reperfusion. IPO with 6 episodes of 10 sec reperfusion followed by 6 episodes of 10 sec ischemia (IPO 2’) was administered when reperfusion was initiated. Cardiodynamics and the concentration of H2S were measured at 1, 2, 3, 4, 5, 10, 20, 30, 60, 90 and 120 min of reperfusion. Lactate dehydrogenase (LDH) levels and infarct size were determined at the end of the reperfusion. The concentration of H2S was stable during the whole experiment in the control group, whereas it reached a peak at the first minute of reperfusion in the ischemia-reperfusion (IR) group. The concentration of H2S at the first minute of reperfusion in the IPO 2’ group was higher compared to that of the IR group, which correlated with cardioprotection including improved heart contractile function and reduced infarct size and LDH levels. However, the above effects of IPO 2’ were attenuated by pre-treatment with blockade of endogenous H2S production with DL-propargylglycine for 20 min prior to global ischemia. Furthermore, we found that other forms of IPO, IPO commencing at 1 min after reperfusion (delayed IPO) or lasting only for 1 min (IPO 1’), failed to increase the concentration of H2S and protect the myocardium. We conclude that the peak of endogenous H2S in the early reperfusion phase is the key to cardioprotection induced by IPO.
hydrogen sulfide; postconditioning; ischemia-reperfusion injury; cardioprotection
Yersinia pestis (Y. pestis) is the causative pathogen of plague, a highly fatal disease for which an effective vaccine, especially against mucosal transmission, is still not available. Like many bacterial infections, antigen-specific antibody responses have been traditionally considered critical, if not solely responsible, for vaccine-induced protection against Y. pestis. Studies in recent years have suggested the importance of T cell immune responses against Y. pestis infection but information is still limited about the details of Y. pestis antigen-specific T cell immune responses. In current report, studies are conducted to identify the presence of CD8+ T cell epitopes in LcrV protein, the leading antigen of plague vaccine development. Furthermore, depletion of CD8+ T cells in LcrV DNA vaccinated Balb/C mice led to reduced protection against lethal intranasal challenge of Y. pestis. These findings establish that an LcrV DNA vaccine is able to elicit CD8+ T cell immune responses against specific epitopes of this key plague antigen and that a CD8+ T cell immune response is involved in LcrV DNA vaccine-elicited protection. Future studies in plague vaccine development will need to examine if the presence of detectable T cell immune responses, in particular CD8+ T-cell immune responses, will enhance the protection against Y. pestis in higher animal species or humans.
Y. pestis; CD8+ T-cell immune responses; LcrV DNA vaccine-elicited protection
Failure to elicit broadly neutralizing (bNt) antibodies (Abs) against the membrane-proximal external region of HIV-1 gp41 (MPER) reflects the difficulty of mimicking its neutralization-competent structure (NCS). Here, we analyzed MPER antigenicity in the context of the plasma membrane and identified a role for the gp41 transmembrane domain (TM) in exposing the epitopes of three bNt monoclonal Abs (MAbs) (2F5, 4E10, and Z13e1). We transiently expressed DNA constructs encoding gp41 ectodomain fragments fused to either the TM of the platelet-derived growth factor receptor (PDGFR) or the gp41 TM and cytoplasmic tail domain (CT). Constructs encoding the MPER tethered to the gp41 TM followed by a 27-residue CT fragment (MPER-TM1) produced optimal MAb binding. Critical binding residues for the three Nt MAbs were identified using a panel of 24 MPER-TM1 mutants bearing single amino acid substitutions in the MPER; many were previously shown to affect MAb-mediated viral neutralization. Moreover, non-Nt mutants of MAbs 2F5 and 4E10 exhibited a reduction in binding to MPER-TM1 and yet maintained binding to synthetic MPER peptides, indicating that MPER-TM1 better approximates the MPER NCS than peptides. Replacement of the gp41 TM and CT of MPER-TM1 with the PDGFR TM reduced binding by MAb 4E10, but not 2F5, indicating that the gp41 TM plays a pivotal role in orienting the 4E10 epitope, and more globally, in affecting MPER exposure.
Schistosomiasis japonica remains a real threat to public health in China. The currently used immunodiagnostic assays are sensitive and have a certain degree of specificity, however, they all use complex crude antigens, are based on detection of schistosome-specific antibodies, and have been shown to cross-react with other parasitic diseases. Therefore, these assays cannot be used to evaluate chemotherapy efficacy. The development of highly sensitive and highly specific immunodiagnostic techniques that can monitor the decline of antibodies specific for S. japonica will be extremely valuable as part of the ongoing strategy to control schistosomiasis in endemic areas. Here we report on the identification of unique fraction antigens of soluble egg antigen (SEA) to which the antibodies disappear 7 weeks after effective treatment. Furthermore, we use these SEA fractions to develop a modified assay with both high sensitivity and specificity.
SEA of S. japonicum was fractionated by electrophoresis using 7.5% SDS-PAGE under non-reducing conditions. The SEA fraction antigens to which antibodies were decreased soon after treatment were collected and used as the detection antigens to establish the FA-ELISA. Sera from patients with acute and chronic schistosomiasis infection, healthy people, and those with other parasitic diseases, were used to evaluate their sensitivity and specificity. Furthermore, sera from patients with chronic schistosomiasis infection were evaluated before and after treatment at different time points to evaluate their chemotherapeutic efficacy.
We demonstrated that this novel FA-ELISA provided high sensitivity and specificity, with very low cross-reactivity, and can serve as an effective tool to determine the efficacy of chemotherapy against S. japonicum.
Recently, a new class of broadly neutralizing anti-influenza virus antibodies that target the stalk domain of the viral hemagglutinin was discovered. As such, induction, isolation, characterization, and quantification of these novel antibodies has become an area of intense research and great interest. Since most of these antibodies bind to conformational epitopes, the structural integrity of hemagglutinin substrates for the detection and quantification of these antibodies is of high importance. Here we evaluate the binding of these antibodies to soluble, secreted hemagglutinins with or without a carboxy-terminal trimerization domain based on the natural trimerization domain of T4 phage fibritin. The lack of such a domain completely abolishes binding to group 1 hemagglutinins and also affects binding to group 2 hemagglutinins. Additionally, the presence of a trimerization domain positively influences soluble hemagglutinin stability during expression and purification. Our findings suggest that a carboxy-terminal trimerization domain is a necessary requirement for the structural integrity of stalk epitopes on recombinant soluble influenza virus hemagglutinin.
The RV144 clinical trial showed for the first time that vaccination could provide modest but significant protection from HIV-1 infection. To understand the protective response, and to improve upon the vaccine's efficacy, it is important to define the structure of the immunogens used in the prime/boost regimen. Here we examined the heterogeneity in net charge, attributable to glycoform variation, of the gp120 immunogens contained in the AIDSVAX B/E vaccine.
Isoelectric focusing and glycosidase digestion were used to assess variation in net charge of the gp120s contained in the AIDSVAX B/E vaccine used in the RV144 trial. We observed 16 variants of MN-rgp120 and 24 variants of A244-rgp120. Glycoform variation in gp120 produced in Chinese hamster ovary cells was compared to glycoform variation in gp120 produced in the 293F human embryonic kidney cell line, often used for neutralization assays. We found that gp120 variants produced in CHO cells were distinctly more acidic than gp120 variants produced in 293 cells. The effect of glycoform heterogeneity on antigenicity was assessed using monoclonal antibodies. The broadly neutralizing PG9 MAb bound to A244-rgp120, but not to MN-rgp120, whether produced in CHO or in 293. However, PG9 was able to bind with high affinity to MN-rgp120 and A244-rgp120 produced in 293 cells deficient in N-acetylglucosaminyltransferase I.
MN- and A244-rgp120 used in the RV144 trial exhibited extensive heterogeneity in net charge due to variation in sialic acid-containing glycoforms. These differences were cell line-dependent, affected the antigenicity of recombinant envelope proteins, and may affect assays used to measure neutralization. These studies, together with recent reports documenting broadly neutralizing antibodies directed against carbohydrate epitopes of gp120, suggest that glycoform variation is a key variable to be considered in the production and evaluation of subunit vaccines designed to prevent HIV infection.
Existing technologies allow isolating antigen-specific monoclonal antibodies (mAbs) from B cells. We devised a direct approach to isolate mAbs with predetermined conformational epitope specificity, using epitope mimetics (mimotopes) that reflect the three-dimensional structure of given antigen subdomains. We performed differential biopanning using bacteriophages encoding random peptide libraries and polyclonal antibodies (Abs) that had been affinity-purified with either native or denatured antigen. This strategy yielded conformational mimotopes. We then generated mimotope-fluorescent protein fusions, which were used as baits to isolate single memory B cells from rhesus monkeys (RMs). To amplify RM immunoglobulin variable regions, we developed RM-specific PCR primers and generated chimeric simian-human mAbs with predicted epitope specificity. We established proof-of-concept of our strategy by isolating mAbs targeting the conformational V3 loop crown of HIV Env; the new mAbs cross-neutralized viruses of different clades. The novel technology allows isolating mAbs from RMs or other hosts given experimental immunogens or infectious agents.
HIV-1 variants that show unusual sensitivity to autologous antibodies due to presence of critical neutralization signatures would likely contribute towards rational envelope based HIV-1 vaccine design. In the present study, we found that presence of a naturally occurring H681 in gp41 membrane proximal external region (MPER) of a clade C envelope (Env) obtained from a recently infected Indian patient conferred increased sensitivity to autologous and heterologous plasma antibodies. Furthermore, Env-pseudotyped viruses expressing H681 showed increased sensitivity to soluble CD4, b12 and 4E10 monoclonal antibodies both in related and unrelated Envs and was corroborated with increased Env susceptibility and binding to cellular CD4 as well as with prolonged exposure of MPER epitopes. The increased gp120-CD4 interaction was further associated with relative exposure of CD4-induced epitopes and macrophage infectivity. In summary, our data indicate that Y681H substitution exposes neutralizing epitopes in CD4bs and MPER towards comprehensive interference in HIV-1 entry.
The V3 epitope is a known target for HIV-1 neutralizing antibodies (NAbs), and V3-scaffold fusion proteins used as boosting immunogens after gp120 DNA priming were previously shown to induce NAbs in rabbits. Here, we evaluated whether the breadth and potency of the NAb response could be improved when boosted with rationally designed V3-scaffold immunogens. Rabbits were primed with codon-optimized clade C gp120 DNA and boosted with one of five V3-cholera toxin B fusion proteins (V3-CTBs) or with double combinations of these. The inserts in these immunogens were designed to display V3 epitopes shared by the majority of global HIV-1 isolates. Double combinations of V3-CTB immunogens generally induced more broad and potent NAbs than did boosts with single V3-CTB immunogens, with the most potent and broad NAbs elicited with the V3-CTB carrying the consensus V3 of clade C (V3C-CTB), or with double combinations of V3-CTB immunogens that included V3C-CTB. Neutralization of tier 1 and 2 pseudoviruses from clades AG, B, and C and of peripheral blood mononuclear cell (PBMC)-grown primary viruses from clades A, AG, and B was achieved, demonstrating that priming with gp120 DNA followed by boosts with V3-scaffold immunogens effectively elicits cross-clade NAbs. Focusing on the V3 region is a first step in designing a vaccine targeting protective epitopes, a strategy with potential advantages over the use of Env, a molecule that evolved to protect the virus by poorly inducing NAbs and by shielding the epitopes that are most critical for infectivity.
Cytochomosome P450 enzymes (CYP) are heme-containing monooxygenases responsible for oxidative metabolism of many exogenous and endogenous compounds including drugs. The species difference of CYP limits the extent to which data obtained from animals can be translated to humans in pharmacodynamics or pharmacokinetics studies. Transgenic expression of human CYP in animals lacking or with largely reduced endogenous CYP counterparts is recognized as an ideal strategy to correct CYP species difference. CYP3A is the most abundant CYP subfamily both in human and mammals. In this study, we designed a microRNA-based shRNA (miR-shRNA) simultaneously targeting four members of mouse CYP3A subfamily (CYP3A11, CYP3A16, CYP3A41 and CYP3A44), and transgenic mice expressing the designed miR-shRNA were generated by lentiviral transgenesis. Results showed that the CYP3A expression level in transgenic mice was markedly reduced compared to that in wild type or unrelated miR-shRNA transgenic mice, and was inversely correlated to the miR-shRNA expression level. The CYP3A expression levels in transgenic offspring of different generations were also remarkably lower compared to those of controls, and moreover the inhibition rate of CYP3A expression remained comparable over generations. The ratio of the targeted CYP3A transcriptional levels was comparable between knockdown and control mice of the same gender as detected by RT-PCR DGGE analysis. These data suggested that transgenic miR-shRNA suppressed CYP3A expression in a dose-dependent and inheritable manner, and transcriptional levels of the targeted CYP3As were suppressed to a similar extent. The observed knockdown efficacy was further confirmed by enzymatic activity analysis, and data showed that CYP3A activities in transgenic mice were markedly reduced compared to those in wild-type or unrelated miR-shRNA transgenic controls (1.11±0.71 vs 5.85±1.74, 5.9±2.4; P<0.01). This work laid down a foundation to further knock down the remaining murine CYP3As or CYPs of other subfamilies, and a basis to generate CYP knockdown animals of other species.
Vaccinia virus (VACV), the vaccine for smallpox, induces an antibody response that is largely responsible for conferring protection. Here, we studied the antibody response to VACV by generating and characterizing B cell hybridomas from a mouse immunized with VACV. Antibodies from 66 hybridomas were found to recognize 11 VACV antigens (D8, A14, WR148, D13, H3, A56, A33, C3, B5, A10 and F13), 10 of which were previously recognized as major antigens in smallpox vaccine by a microarray of VACV proteins produced with a prokaryotic expression system. VACV C3 protein, which was not detected as a target of antibody response by the proteome array, was recognized by two hybridomas, suggesting that selection of hybridomas based on immune recognition of infected cells has the advantage of detecting additional antibody response to native VACV antigens. In addition, these monoclonal antibodies are valuable reagents for studying poxvirus biology and protective mechanism of smallpox vaccine.
A novel HIV-1 Env expression vector (SF162-Z) was developed by introducing two new cloning sites on the backbone of an existing vector that produces a full length Env from HIV-1 SF162 isolate. These sites facilitate the swapping of the gp120 portion of the SF162 Env with matching gp120 antigens from HIV-1 isolates of different genetic clades. Final production of functional pseudotyped viruses will express chimeric Env antigens, including gp41 of the parental SF162 and gp120 from other primary isolates. This system is useful for testing the neutralizing sensitivity of partial env gene products frequently identified in viral quasi species in patients infected with HIV or when only partial gp120 gene products are available.
HIV-1; Envelope protein; gp120; pseudotyped virus; neutralizing antibody
Highly pathogenic avian influenza A (HPAI) H5N1 viruses are circulating among poultry populations in parts of Asia, Africa, and the Middle East, and have caused human infections with a high mortality rate. H5 subtype hemagglutinin (HA) has evolved into phylogenetically distinct clades and subclades based on viruses isolated from various avian species. Since 1997, humans have been infected by HPAI H5N1 viruses from several clades. It is, therefore, important to develop strategies to produce protective antibody responses against H5N1 viruses from multiple clades or antigenic groups. In the current study, we optimized the signal peptide design of DNA vaccines expressing HA antigens from H5N1 viruses. Cross reactivity analysis using sera from immunized rabbits showed that antibody responses elicited by a polyvalent formulation, including HA antigens from different clades, was able to elicit broad protective antibody responses against multiple key representative H5N1 viruses across different clades. Data presented in this report support the development of a polyvalent DNA vaccine strategy against the threat of a potential H5N1 influenza pandemic.
Five-year survival for lung cancer has remained at 16% over last several decades largely due to the fact that over 50% of patients are diagnosed with locally-advanced or metastatic disease. Diagnosis at an earlier and potentially curable stage is crucial. Solitary pulmonary nodules (SPNs) are common, but the difficulty lies in the determination of which SPN is malignant. Currently, there is no convenient and reliable biomarker effective for early diagnosis. Secretory phospholipase A2-IIa (sPLA2-IIa) is secreted into the circulation by cancer cells and may allow for an early detection of lung cancer.
Plasma samples from healthy donors, patients with only benign SPN, and patients with lung cancer were analyzed. Expression of sPLA2-IIa protein in lung cancer tissues was also determined.
We found that the levels of plasma sPLA2-IIa were significantly elevated in lung cancer patients. The receiver operating characteristic curve analysis, comparing lung cancer patients to patients with benign nodules, revealed an optimum cutoff value for plasma sPLA2-IIa of 2.4 ng/ml to predict an early stage cancer with 48% sensitivity and 86% specificity and up to 67% sensitivity for T2 stage lung cancer. Combined sPLA2-IIa, CEA, and Cyfra21.1 tests increased the sensitivity for lung cancer prediction. High level of plasma sPLA2-IIa was associated with a decreased overall cancer survival. sPLA2-IIa was overexpressed in almost all non-small cell lung cancer and in the majority of small cell lung cancer by immunohistochemistry analysis.
Our finding strongly suggests that plasma sPLA2-IIa is a potential lung biomarker to distinguish benign nodules from lung cancer and to aid lung cancer diagnosis in patients with SPNs.
The majority of prostate cancers are indolent, whereas a significant portion of patients will require systemic treatment during the course of their disease. To date, only high Gleason scores are best associated with a poor prognosis in prostate cancer. No validated serum biomarker has been identified with prognostic power. Previous studies showed that secretory phospholipase A2-IIa (sPLA2-IIa) is overexpressed in almost all human prostate cancer specimens and its elevated levels are correlated with high tumor grade. Here, we found that sPLA2-IIa is overexpressed in androgen-independent prostate cancer LNCaP-AI cells relative to their androgen-dependent LNCaP cell counterparts. LNCaP-AI cells also secrete significantly higher levels of sPLA2-IIa. Blocking sPLA2-IIa function compromises androgen-independent cell growth. Inhibition of the ligand-induced signaling output of the HER network, by blocking PI3K-Akt signaling and the nuclear factor-kappaB (NF-κB)-mediated pathway, compromises both sPLA2-IIa protein expression and secretion, as a result of downregulation of sPLA2-IIa promoter activity. More importantly, we demonstrated elevated serum sPLA2-IIa levels in prostate cancer patients. High serum sPLA2-IIa levels are associated significantly with high Gleason score and advanced disease stage. Increased sPLA2-IIa expression was confirmed in prostate cancer cells, but not in normal epithelium and stroma by immunohistochemistry analysis. We showed that elevated signaling of the HER/HER2-PI3K-Akt-NF-κB pathway contributes to sPLA2-IIa overexpression and secretion by prostate cancer cells. Given that sPLA2-IIa overexpression is associated with prostate development and progression, serum sPLA2-IIa may serve as a prognostic biomarker for prostate cancer and a potential surrogate prostate biomarker indicative of tumor burden.
Identification of immunogens capable of eliciting broadly neutralizing antibody (NAb) responses against HIV-1 is a major goal toward the development of an AIDS vaccine. Despite significant progress in understanding the structural features of the HIV-1 envelope glycoprotein (Env) and the discovery of multiple broadly neutralizing monoclonal antibodies with defined antigenic structures, the design of optimal Env immunogens to elicit broad NAbs remains a major challenge. As the structural determinants of Env immunogenicity remain unclear, we assessed two closely related Env antigens isolated from the same HIV-1-infected patient with different phenotypic features to identify what may result in a favorable immunogenic profile. One Env, B33, isolated from brain, was highly macrophage tropic with a high CD4 affinity, while the other, LN40, isolated from the lymph nodes, was poorly macrophage tropic with a low CD4 affinity. Using a DNA prime-protein boost approach, rabbits primed with LN40 Env antigen had a NAb response against heterologous primary isolates, while B33 Env antigens were capable of eliciting NAbs against only homologous and sensitive viral isolates. Further analysis revealed that the specificity of NAbs elicited by the LN40 antigen mapped to limited residues within or flanking the CD4 binding site. Certain key structural determinants were identified that could differentiate primary Env immunogens based on their potential to elicit broader NAbs. This progress will facilitate the rational design of effective HIV-1 vaccine formulations with optimal Env antigens.