Conditional deletion of PTEN in peripheral T cells augments TCR-mediated activation but does not abrogate CD28 dependency or prevent anergy induction.

PTEN is thought to play a critical role in T cell activation by negatively regulating the PI3K signaling pathway important for cellular activation, growth, and proliferation. To directly eliminate PTEN in postthymic T cells for studies of functional effects, we used CAR transgenic × PTEN(flox/flox) mice, which enabled gene deletion using a Cre adenovirus in vitro. These mice were also immunized to generate stable Th1 clones that could have PTEN deleted when desired. PTEN-deleted T cells exhibited enhanced IL-2 production, proliferation, and Akt phosphorylation upon TCR/CD28 engagement, whereas T cell survival was not potentiated. Gene expression profiling revealed a small subset of induced genes that were augmented upon PTEN deletion. However, PTEN-deficient T cells still required CD28 costimulation for IL-2 production and remained susceptible to anti-CD3-induced anergy. The absence of PTEN within the CD8 T cell compartment led to markedly increased cytolytic activity following an allogeneic MLR in vitro, without increasing autologous MLR activity. Our results indicate that deletion of PTEN can augment the activation of postthymic T cells but does not mediate CD28 independence or anergy resistance. Nonetheless, PTEN inhibition may be a viable target for immune potentiation owing to increased cytokine production by activated CD4(+) cells and increased cytotoxicity by CD8(+) T cells.

Concanavalin A-conjugated poly(ethylene glycol)-poly(lactic acid) nanoparticles for intranasal drug delivery to the cervical lymph nodes.

Concanavalin A (ConA)-conjugated poly(ethylene glycol)-poly(lactic acid) nanoparticles (ConA-NPs) were prepared for targeted drug delivery to the cervical lymph nodes after intranasal administration. ConA, a lectin specifically binding to α-mannose and α-glucose, was covalently conjugated on NPs without loss of its carbohydrates binding bioactivity. In vitro cellular uptake experiment demonstrated that NPs could be uptaken by Calu-3 cells in a time- and concentration-dependent manner, and conjugation of ConA on NPs could significantly increase the rate and amount of cellular uptake. ConA-NP showed no obvious toxicity to Calu-3 cells in vitro or to the nasal cilia of rats in vivo. Compared with NPs without ConA, ConA-NP is more effective in targeting drugs to the deep cervical lymph nodes, as evidenced by 1.36-2.52 times increase of targeting efficiency, demonstrating that ConA-NP is a potential carrier for targeted drug delivery to the cervical lymph nodes via nasal route.

PEGylated poly(2-(dimethylamino) ethyl methacrylate)/DNA polyplex micelles decorated with phage-displayed TGN peptide for brain-targeted gene delivery.

Phage-displayed TGN peptide-decorated polymeric micelle-like polyplexes based on pegylated poly(2-(dimethylamino) ethyl methacrylate) (PEG-PDMAEMA) were prepared for efficient brain-targeted gene delivery. The diblock copolymers Methoxy-PEG-PDMAEMA and Maleimide-PEG-PDMAEMA were synthesized by the atom transfer radical polymerization method. The TGN ligand, a 12-amino acid peptide that could facilitate blood-brain barrier (BBB) targeting, was conjugated to the PEG terminus of the copolymer via a maleimide-mediated covalent binding procedure. TGN-PEG-PDMAEMA was complexed with plasmid DNA to yield polyplexes. The physiochemical properties of the polyplexes, such as morphology, particle size, zeta potential, cytotoxicity and DNA complex formation ability, were studied prior to the successful in vitro and in vivo transfection. The TGN-PEG-PDMAEMA/DNA polyplexes maintained their stable nano-size, were characterized by good condensation capacity and low toxicity and even provided higher cellular uptake than the unmodified polyplexes (PEG-PDMAEMA/DNA polyplexes). Confocal microscopy studies showed that the DNA of TGN-PEG-PDMAEMA/DNA polyplexes entered into the nuclei through the endosome/lysosome pathway. The transfection efficiency of TGN-modified polyplexes was higher than that of unmodified polyplexes both in vitro and in vivo. The results obtained from frozen sections indicated the widespread expression of an exogenous gene in the mouse brain after intravenous injection. Therefore, the results demonstrate that the TGN-decorated PEG-PDMAEMA developed in this study could be utilized as a potential vehicle for gene delivery to the brain.

Enhancement of nose-to-brain delivery of basic fibroblast growth factor for improving rat memory impairments induced by co-injection of ß-amyloid and ibotenic acid into the bilateral hippocampus.

Basic fibroblast growth factor (bFGF) delivery to the brain of animals appears to be an emerging potential therapeutic approach to neurodegenerative diseases, such as Alzheimer's disease (AD). The intranasal route of administration could provide an alternative to intracerebroventricular infusion. A nasal spray of bFGF had been developed previously and the objective of the present study was to investigate whether bFGF nasal spray could enhance brain uptake of bFGF and ameliorate memory impairment induced by co-injection of ß-amyloid(25-35) and ibotenic acid into bilateral hippocampus of rats. The results of brain uptake study showed that the AUC(0-12h) of bFGF nasal spray in olfactory bulb, cerebrum, cerebellum and hippocampus was respectively 2.47, 2.38, 2.56 and 2.19 times that of intravenous bFGF solution, and 1.11, 1.95, 1.40 and 1.93 times that of intranasal bFGF solution, indicating that intranasal administration of bFGF nasal spray was an effective means of delivering bFGF to the brain, especially to cerebrum and hippocampus. In Morris water maze tasks, intravenous administration of bFGF solution at high dose (40 µg/kg) showed little improvement on spatial memory impairment. In contrast, bFGF solution of the same dose following intranasal administration could significantly ameliorate spatial memory impairment. bFGF nasal spray obviously improved spatial memory impairment even at a dose half (20 µg/kg) of bFGF solution, recovered their acetylcholinesterase and choline acetyltransferase activity to the sham control level, and alleviated neuronal degeneration in rat hippocampus, indicating neuroprotective effects on the central nerve system. In a word, bFGF nasal spray may be a new formulation of great potential for treating AD.

Targeting the brain with PEG-PLGA nanoparticles modified with phage-displayed peptides.

The relative impermeability of the blood-brain barrier (BBB) results from tight junctions and efflux transport systems limits drug delivery to the central nervous system (CNS), and thus severely restricts the therapy of many central nervous system diseases. In order to enhance the brain-specific drug delivery, we employed a 12-mer phage display peptide library to isolate peptides that could target the drug delivery system to the brain. A 12-amino-acid-peptide (denoted as Pep TGN) which was displayed by bacteriophage Clone 12-2 was finally selected by rounds of in vivo screening. Pep TGN was covalently conjugated onto the surface of poly (ethyleneglycol)-poly (lactic-co-glycolic acid) (PEG-PLGA) based nanoparticles (NPs). The cellular uptake of Pep TGN decorated nanoparticles was significantly higher than that of unmodified nanoparticles when incubated with bEnd.3 cells. Enhanced brain accumulation efficiency together with lower accumulation in liver and spleen was observed in the nude mice intravenously injected with Pep TGN conjugated nanoparticles compared with those injected with plain nanoparticles, showing powerful brain selectivity of Pep TGN. Coumarin 6 was used as a fluorescent probe for the evaluation of brain delivery properties. The brain Drug Targeting Index (DTI) of coumarin 6 incorporated in targeted nanoparticles was significantly higher than that of coumarin 6 incorporated in plain nanoparticles. In conclusion, the Pep TGN is a motif never been reported before and Pep TGN modified nanoparticles showed great potential in targeted drug delivery across the blood brain barrier.

Homeostatic proliferation plus regulatory T-cell depletion promotes potent rejection of B16 melanoma.

PURPOSE: To investigate the antitumor efficacy of T-cell anergy reversal through homeostatic proliferation and regulatory T-cell (Treg) depletion in a clinically relevant murine adoptive immunotherapy model. EXPERIMENTAL DESIGN: B16 melanoma cells were engineered to express the model SIYRYYGL (SIY) antigen to enable immune monitoring. Tumor-specific T cells expanded in tumor-challenged wild-type hosts but became hyporesponsive. To examine whether lymphopenia-induced homeostatic proliferation could reverse tumor-induced T-cell anergy, total splenic T cells were transferred into lymphopenic RAG2-/- mice or control P14/RAG2-/- mice. Tumor growth was measured, and SIY-specific immune responses were monitored using ELISPOT and SIY/K(b) tetramers. To determine whether Treg depletion could synergize with homeostatic proliferation, RAG2-/- mice received total or CD25-depleted T cells, followed or preceded by B16.SIY challenge. This approach was further investigated in wild-type mice lymphodepleted with sublethal total body irradiation. RESULTS: Adoptive transfer of total splenic T cells into RAG2-/- mice moderately affected the growth rate of B16.SIY. As Treg expansion occurred in tumor-bearing mice, CD25+ T cells were depleted from total T cells before adoptive transfer. Interestingly, transfer of CD25-depleted T cells into RAG2-/- mice resulted in potent rejection of B16 melanoma in both prophylactic and short-term preimplanted tumor settings and was associated with maintained T-cell effector function. Using a clinically applicable approach, wild-type mice were lymphodepleted using sublethal total body irradiation, which similarly supported tumor rejection upon transfer of CD25-depleted T cells. CONCLUSIONS: Our results indicate that combined CD25 depletion and homeostatic proliferation support a potent antitumor immune response--an approach with potential for clinical translation.

Amelioration of rat adjuvant-induced arthritis by Met-RANTES.

OBJECTIVE: CC chemokines and their receptors play a fundamental role in trafficking and activation of leukocytes at sites of inflammation, contributing to joint damage in rheumatoid arthritis. Met-RANTES, an amino-terminal-modified methionylated form of RANTES (CCL5), antagonizes the binding of the chemokines RANTES and macrophage inflammatory protein 1alpha (MIP-1alpha; CCL3) to their receptors CCR1 and CCR5, respectively. The aim of this study was to investigate whether Met-RANTES could ameliorate adjuvant-induced arthritis (AIA) in the rat. METHODS: Using immunohistochemistry, enzyme-linked immunosorbent assay, real-time reverse transcription-polymerase chain reaction, Western blot analysis, adoptive transfer, and chemotaxis, we defined joint inflammation, bony destruction, neutrophil and macrophage migration, Met-RANTES binding affinity to rat receptors, proinflammatory cytokine and bone marker levels, CCR1 and CCR5 expression and activation, and macrophage homing into joints with AIA. RESULTS: Administration of Met-RANTES as a preventative reduced the severity of joint inflammation. Administration of Met-RANTES to ankles with AIA showed decreases in inflammation, radiographic soft tissue swelling, and bone erosion. Met-RANTES significantly reduced the number of neutrophils and macrophages at the peak of arthritis compared with saline-injected controls. Competitive chemotaxis in peripheral blood mononuclear cells demonstrated that Met-RANTES inhibited MIP-1alpha and MIP-1beta at 50% inhibition concentrations of 5 nM and 2 nM, respectively. Furthermore, levels of tumor necrosis factor alpha, interleukin-1beta, macrophage colony-stimulating factor, and RANKL were decreased in joints with AIA in the Met-RANTES group compared with the control group. Interestingly, the expression and activation of CCR1 and CCR5 in the joint were down-regulated in the Met-RANTES group compared with the control group. Functionally, Met-RANTES administration decreased adoptively transferred peritoneal macrophage homing into the joint. CONCLUSION: The data suggest that the targeting of Th1-associated chemokine receptors reduce joint inflammation, bone destruction, and cell recruitment into joints with AIA.

Negative regulation of T-cell function by PD-1.

PD-1 is a receptor inducibly expressed on CD4+ and CD8+ T cells following activation. PD-1-deficient mice develop signs of autoimmunity, suggesting a negative regulatory role for PD-1 in dampening lymphocyte responses. The expression of one ligand for PD-1, designated PD-L1 or B7-H1, on tumor cells of a variety of histologies has suggested a potential mechanism for tumor escape from immune destruction. This review summarizes data regarding PD-1 and related negative regulatory receptors, focusing on implications for potentiating antitumor immunity in vivo.

[Development and characterization of a gemcitabine-resistant variant of human lung adenocarcinoma cell line A549].

BACKGROUND & OBJECTIVE: Gemcitabine (2',2-difluorodeo- xycytide) has antitumor activity in both experimental and clinical treatment of solid tumors. Although resistance to gemcitabine in ovarian cancer cell line and erythroleukemic cell line was described, there was no report on the lung cancer resistant variant. In order to elucidate the mechanism by which gemcitabine induce resistance in lung cancer, we have established the resistance to gemcitabine in human lung adenocarcinoma cell line A549 and described the characteristics of its resistant variant. METHODS: Resistance to gemcitabine was established by exposing A549 cells to increasing concentration of gemcitabine, which was designated as A549-Gem. The IC(50) and resistance index(RI) were tested by MTT assay and colony formation test. The growth curve and cell cycle of A549 and A549-Gem were compared. The cross-resistance profile of A549-Gem was also tested. RESULTS: The IC(50) increased from 6.56+/-1.19 micromol/L in A549 to 921.09+/-225.27 micromol/L in A549-Gem as tested by MTT assay at 72h exposure, the RI was 140.52 (P=0.019 5). The RI of colony formation test was 132.95. Double time of A549 and A549-Gem were 29.7 h and 36.4 h, respectively, as evaluated by the growth curve. A549-Gem was cross-resistant to vincristine and etoposide(54.38-fold and 6.18-fold)(P< 0.01), but not to adriamycin, cisplatin, cytarabine, and paclitaxel. CONCLUSION: A549-Gem, the gemcitabine resistant phenotype, is stable and suitable for the study of gemcitabine resistance in lung cancer. A549-Gem is cross-resistant to vincristine and etoposide, but not resistant to adriamycin, cisplatin, cytarabine and paclitaxel.

[Preparation and characterization of rabbit antibody against human selenoprotein P].

AIM: To prepare rabbit antibody against human selenoprotein P (HSelP) by using HSelP expressed in the prokaryotic expression system and identify its specificity. METHODS: HSelP gene fragment was expressed in E.Coli via IPTG induction and purified through DEAE and Ni-NTA columns sequentially. The purified HSelP as immunogen was used to immunize rabbit. The specificity of rabbit anti-HSelP antibody was analyzed by Western blot. RESULTS: HSelP protein had been expressed and purified successfully. The polyclonal anti-HSelP antibody could specifically recognize natural HSelP from human serum. CONCLUSION: Polyclonal anti-HSelP antibody with high specificity and purity has been prepared by using purified HSelP as immunogen, which lays the foundation for further research on the function and distribution of HSelP, as well as developing a simple method for detecting HSelP.

[Mechanisms of the drug resistance of a 2', 2-difluorodeoxycytide (gemcitabine)-resistant variant of the human lung adenocarcinoma cell line].

OBJECTIVE: To investigate the mechanisms of the drug resistance of gemcitabine resistance variant of the human lung adenocarcinoma cell line A549-Gem. METHOD: Immunohistochemistry and RT-PCR were used to tested the expression of P-glycoprotein and transcription of mRNA of multidrug resistance gene and deoxycytidine kinase. Using a cDNA microarray compared the expression profiles between the resistant A549-Gem and the parent cell line A549. RESULTS: The A549-Gem shown slight positive expression of P-glycoprotein compared with positive control by immunohistochemistry, but A549 was negative for P-gp. RT-PCR amplified mRNA, using specific dCK and multidrug resistant gene 1 (mdr1) primers, demonstrated that A549-Gem express amplicon of mdr1 but no products of mdr1 was detected in A549. In the parent cell line, dCK mRNA amplication product could be detected, but did not found in resistant cells. About 18.8% of the total DNA elements had substantially altered level of expression in resistant A549-Gem compared with A549 by cDNA microarray. The biochemical functions of the different expressed genes are diverse and include oncogene and tumor suppressor gene, cell cycle regulator, heat shock protein, apoptotic and antiapoptotic factors, DNA transcription factors, DNA repair and recombination factors, signal transduction genes, protein translation genes, as well as a large number of metabolic genes. Several cluster genes overexpressed in resistant cell line, including ubiquitin-proteasome system, zinc finger protein, glutaredoxin and heat shock protein, may be related to the mechanisms of gemcitabine resistance. CONCLUSION: The mechanisms of resistance to gemcitabine are multifactorial, may include multidrug resistance and dCK deficiency. Differential expression genes monitored by cDNA microarray between A549-Gem and A549 may be related to the mechanisms of gemcitabine resistance in human lung cancer and potentially suggest the prevalence of expression of these genes in drug resistance. The use of cDNA microarray has provided a global view of the response of lung cancer cells to gemcitabine at the genomic level, it should be suitable for examining the development of drug resistance in cancer.

[The apoptosis-inducing activity of human selenoprotein P shorter isoform].

OBJECTIVE: Human selenoprotein P (HSelP) is unique protein that contains 10 selenocysteines encoded by 10 inframe UGA, which typically function as stop codon. The function of HSelP remains unclear, in part due to the inability to express it by gene recombinant technique. This study is to investigate expression and purification of recombinant HSelP in prokaryotic expression system, and its activity to induce apoptosis in vitro. METHODS: The shorter HSelP isoform was cloned. After the selenocysteine (SeCys) at 40th position from N terminus of the HSelP shorter isoform was mutated into cysteine by PCR, it was expressed in E. coli. The expressed product was purified with DEAE column and identified by Western blot. Subsequently, its function on induction of mitochondrial apoptotic activity was studied. RESULTS: The mutant HSelP shorter isoform expressed in prokaryotic system was purified by DEAE column to 90% homogeneity. The purified product, HSelP280m, induced the opening of mitochondrial permeability transition pore (PTP) and decreased the transmembrane potential in a dose-dependent manner. These events could be abolished by PTP specific inhibitors. CONCLUSION: HSelP280m can induce the opening of mitochondrial PTP, which provides a basis for investigating the structure and function of recombinant HSelP.

[Antitumor effect of gene gun-mediated DNA vaccine pWRG-neu immunization in C57BL/6 mice].

OBJECTIVE: To investigate the inhibitory effect of DNA vaccine immunization on neu-overexpressed melanoma growth in prophylactic treatment and anti-lung-metastasis experiments in C57BL/6 mice. METHODS: pcDNA-neu transfected into B16F10 with transfection reagent Fugene 6, neu-overexpressed cell clone B16F10-neu was selected with limited dilution method. The growth curve was drawn to analyse its proliferating character in vitro. With Helios gene gun system, DNA vaccine pWRG-neu was immunized to 8-week-old C57BL/6 mice in the shaved abdominal skin for 3 times at two-weekly interval. After immunization, the life span was analyzed. Using MTT assay, the cytolysis activity of the DNA immunized mice spleen cells was compared. RESULTS: One clone of neu-overexpressed B16F10-neu was selected and its proliferating character was the same as B16F10 and B16F10-pcDNA. In prophylactic, treatment and anti-lung-metastasis experiments, gene gun-mediated pWRG-neu immunization could exhibit antitumor effects. The growth and metastasis of neu-overexpressed melanoma was reduced dramatically. The spleen cells of the immuned mice showed cytotoxic T lymphocyte (CTL) activity. CONCLUSION: Gene gun-mediated gene transfer is effective and practicable. DNA vaccine pWRG-neu is potent in preventing subsequent tumor cells challenge, inhibiting the tumor growth and metastasis.

Effect of estrogen and progestogen on nitric oxide content in rat endometrium.

OBJECTIVE: To observe the effect of estrogen and progestogen on the content of nitric oxide (NO) in the endometrium of SD rats. METHOD: Fifteen SD rats were divided into control, estrogen and levonorgestrel groups (5 in each group), with corresponding treatment indicated by the designation of the groups. The NO contents in the endometrium were determined by Griess method. RESULTS: he NO content of estrogen group was significantly higher, while that of levonorgestrel group significantly lower, than that of the control group (P < 0.01 for both comparisons). CONCLUSIONS: Estrogen and levonorgestrel may regulate NO synthesis in the endometrium.

[Redox reactions of Sep15 and its relationship with tumor development].

BACKGROUND & OBJECTIVE: Sep15 is a selenium-containing protein identified in 1998. This protein may be involved in cancer etiology and it may have redox function. The objective of this study was to investigate the relationship between the redox function of Sep15 and tumor development. METHODS: The full-length DNA sequence of Sep15 was obtained by RT-PCR and then recombined to eukaryotic expression vector pcDNA3.1(+). The BEL-7402- Sep15 cell line, which expressed the high levels of Sep15 by transfecting the cultured hepatocarcinoma cell line BEL-7402 with pcDNA3.1-Sep15 was generated. From morphologic investigation, cell growth curve, clone formation and nude mice tumor growth curve, the relationship between Sep15 and hepatocarcinoma cell line BEL-7402 was determined. Furthermore, the redox reaction of sep15 was detected by MTT assay. RESULTS: There was no distinct effect of transfection of Sep15 gene on BEL-7402-Sep15 cell. The cell survival rate was drastically different between BEL-7402-Sep15 cell and both BEL-7402- pcDNA cell and BEL-7402-Sep15 cell after foreign H2O2 reactive oxygen stress (P<0.05). CONCLUSION: Transfecting Sep15 gene did not influence the growth characteristics of BEL-7402 cell line and Sep15 may have redox function.

[Research of the anti-tumor effect of human endostatin mediated by recombinant adeno-associated virus].

BACKGROUND & OBJECTIVE: It was reported that endostatin could inhibit tumor angiogenesis, then inhibit the growth and metastasis of tumor. However, there was few report about the treatment usage of endostatin. This study was designed to explore the effect of endostatin mediated with recombinant adeno-associated virus(rAAV) on tumor growth and metastasis. METHODS: To obtain the endostatin gene complete cDNA by RT-PCR, and clone it into the plasmid pSNAV and package the recombinant rAAV-SS-endostatin; to analyze its anti-tumor effect through animal experiments. RESULTS: In the B16F10 melanoma tumor model, C57BL/6 mice were inoculated with 10(11)TU rAAV-SS-endostatin i.m. injection, which the inhibition rate was 57.1%; In the lung metastatic model, the inhibition rate of metastases was 70.7%. CONCLUSIONS: rAAV-SS-endostatin can effectively inhibit tumor growth and metastasis.