Macrophage-derived chemokine gene transfer results in tumor regression in murine lung carcinoma model through efficient induction of antitumor immunity

Chemokine gene transfer represents a promising approach in the treatment of malignancies. Macrophage-derived chemokine (MDC) (CCL22) belongs to the CC chemokine family and is a strong chemoattractant for dendritic cells (DC), NK cells and T cells. Using adenoviral vectors, human MDC gene was transferred in vivo to investigate its efficacy to induce an antitumor response and to determine the immunologic mechanisms involved. We observed that intratumoral injection of recombinant adenovirus encoding human MDC (AdMDC) resulted in marked tumor regression in a murine model with pre-established subcutaneous 3LL lung carcinoma and induced significant CTL activity. The antitumor response was demonstrated to be CD4+ T cell- and CD8+ T cell-dependent. Administration of AdMDC induced chemoattraction of DC to the tumor site, facilitated DC migration to draining lymph nodes or spleen, and finally activated DC to produce high levels of IL-12. Furthermore, a significant increase of IL-4 production within the tumors was observed early after the AdMDC administration and was followed by the increase of IL-12 and IL-2 production. The levels of IL-2, IL-12 and IFN-gamma in serum, lymph nodes and spleen were also found to be higher in mice treated with AdMDC as compared with that in AdLacZ- or PBS-treated mice. The antitumor response induced by AdMDC was markedly impaired in IL-4 knockout mice, suggesting an important role of IL-4 in the induction of antitumor immunity by MDC. These results suggest that MDC gene transfer might elicit significant antitumor effects through efficient induction of antitumor immunity and might be of therapeutic potentials for cancer.     

A structure-activity relationship for induction of meningeal inflammation by muramyl peptides.

Components of bacterial peptidoglycans have potent biological activities, including adjuvant effects, cytotoxicity, and induction of sleep. Mixtures of peptidoglycan components also induce inflammation in the lung, subarachnoid space, and joint, but the structural requirements for activity are unknown. Using a rabbit model for meningitis, we determined the biological activities of 14 individual muramyl peptides constituting > 90% of the peptidoglycan of the gram-negative pediatric pathogen Haemophilus influenzae. Upon intracisternal inoculation, most of the muropeptides induced leukocytosis in cerebrospinal fluid (CSF), influx of protein into CSF, or brain edema, alone or in combination. The disaccharide-tetrapeptide, the major component of all gram-negative peptidoglycans, induced CSF leukocytosis and protein influx at doses as low as 0.4 microgram (0.42 nM). Modification of the N-acetyl muramic acid or substitution of the alanine at position four in the peptide side chain decreased leukocytosis but enhanced brain edema. As the size of the muropeptide increased, the inflammatory activity decreased. Muropeptide carrying the diaminopimelyl-diaminopimelic acid cross-link specifically induced cytotoxic brain edema. These findings significantly expand the spectrum of biological activities of natural muramyl peptides and provide the basis for a structure-activity relationship for the inflammatory properties of bacterial muropeptides.     

A novel approach using functional peptides for efficient intestinal absorption of insulin.

The aim of this study was to evaluate whether oligoarginine, a cell-penetrating peptide (CPP), can improve intestinal absorption of insulin in rats. Peptides composed of six (R(6)), eight (R(8)) and 10 (R(10)) residues of arginine were used as the CPP. No insulin absorption was observed following administration of insulin solution alone; however, insulin absorption increased dramatically after coadministration of the D-form of R(6) (D-R(6)) and the L-form of R(6) (L-R(6)) in a dose-dependent manner. The effects on insulin absorption were more pronounced for D-R(6) than for L-R(6). Among oligoarginines composed of six, eight, or 10 arginine residues, D-R(8) showed the strongest enhancing effects on insulin intestinal absorption. In contrast, intestinal absorption of other model hydrophilic macromolecules, interferon-beta and fluorescein isothiocyanate-labeled dextran 4400, was not affected by coadministration with oligoarginine. Pretreatment by the effective dose of L-R(6) did not induce lactate dehydrogenase leakage or histological damage, suggesting that oligoarginine has no untoward effect on the intestinal mucosa. Our data demonstrate that coadministration of oligoarginine increases intestinal insulin absorption markedly without causing detectable damage in cellular integrity and that the covalent binding between insulin and oligoarginine is not necessary for this effect. We conclude that oligoarginines are likely to become powerful tools for overcoming the low permeability of insulin through the epithelial cell membrane, the major barrier to oral insulin delivery.     

Current methods for loading dendritic cells with tumor antigen for the induction of antitumor immunity

The immunotherapy of cancer is predicated on the belief that it is possible to generate a clinically meaningful antitumor response that provides patient benefit, such as improvement in the time to progression or survival. Indeed, immunotherapeutics with dendritic cells (DC) as antigen-presenting delivery vehicles for cell-based vaccines have already improved patient outcome against a wide range of tumor types (1-9). This approach stimulates the patient's own antitumor immunity through the induction or enhancement of T-cell immunity. It is generally believed that the activity of cytotoxic T lymphocytes (CTL), the cells directly responsible for killing the tumor cells in vivo, are directed by DC. Therefore, the goal of many current designs for DC-based vaccines is to induce strong tumor-specific CTL responses in patients with cancer. In practice, most studies for DC-based cancer vaccine development have focused on the development of methods that can effectively deliver exogenous tumor antigens to DC for cross-priming of CD8+ T cells through the endogenous MHC class I processing and presentation pathway (10). To date, many methods have been developed or evaluated for the delivery of defined and undefined tumor antigens to DC. This review provides a brief summary on these methods, the techniques used in these methods, as well as the advantages and disadvantages of each method.     

Endotoxin induction of tumor necrosis factor is enhanced by acid-labile interferon-alpha in acquired immunodeficiency syndrome.

High levels of an acid-labile IFN-alpha have been demonstrated in the sera of patients with symptomatic HIV infection. IFNs have been shown to enhance the cytotoxic and antiproliferative actions of tumor necrosis factor (TNF), which is a potent mediator of inflammation and sepsis. We show that the acid-labile IFN-alpha present in AIDS sera can induce TNF synthesis and sensitize blood monocytes (BM) to endotoxin stimulation resulting in further synthesis of TNF in vitro. TNF production by BM from patients with HIV infections and normal controls was measured by a cytotoxicity assay on L929 cells using human TNF alpha as a standard. BM from AIDS patients spontaneously produce high levels of TNF and are hypersensitive to endotoxin stimulation, resulting in enhanced synthesis of TNF. In determining the mechanism involved, we demonstrated that treatment of normal BM with AIDS sera results in induction of TNF. Neutralization of the acid-labile IFN-alpha in AIDS sera with polyclonal anti-IFN-alpha antibodies results in diminution of TNF induction. In addition, pretreatment of normal BM with AIDS sera, IFN-alpha, or IFN-gamma renders the cells hypersensitive to endotoxin. Consequently, activation of the TNF system by the acid-labile IFN-alpha contributes to some of the physiological disturbances, such as the wasting syndrome, and to the pathophysiology of sepsis in AIDS patients.     

Lymphocytes genetically modified to express tumor antigens target DCs in vivo and induce antitumor immunity

The exploitation of the physiologic processing and presenting machinery of DCs by in vivo loading of tumor-associated antigens may improve the immunogenic potential and clinical efficacy of DC-based cancer vaccines. Here we show that lymphocytes genetically modified to express self/tumor antigens, acting as antigen carriers, efficiently target DCs in vivo in tumor-bearing mice. The infusion of tyrosinase-related protein 2-transduced (TRP-2-transduced) lymphocytes induced the establishment of protective immunity and long-term memory in tumor-bearing mice. Analysis of the mechanism responsible for the induction of such an immune response allowed us to demonstrate that cross-presentation of the antigen mediated by the CD11c(+)CD8alpha(+) DC subset had occurred. Furthermore, we demonstrated in vivo and in vitro that DCs had undergone activation upon phagocytosis of genetically modified lymphocytes, a process mediated by a cell-to-cell contact mechanism independent of CD40 triggering. Targeting and activation of secondary lymphoid organ-resident DCs endowed antigen-specific T cells with full effector functions, which ultimately increased tumor growth control and animal survival in a therapeutic tumor setting. We conclude that the use of transduced lymphocytes represents an efficient method for the in vivo loading of tumor-associated antigens on DCs.     

Anti-tumor immunity induced by in vivo adenovirus vector-mediated expression of CD40 ligand in tumor cells.

CD40 ligand (CD40L), the ligand for CD40 on antigen-presenting cells, is essential for the initiation of antigen-specific T cell responses, an important component of the immune response to tumors. This study is based on the hypothesis that in vivo genetic modification of tumor cells to express CD40L will trigger CD40 on local antigen-presenting cells to present tumor antigen to the cellular immune systems, thus eliciting anti-tumor immunity to suppress growth of the tumor. To examine this concept, subcutaneous tumors of three different murine tumor models in two strains of mice were infected with a recombinant adenovirus (Ad) vector expressing murine CD40L (AdmCD40L). In the B16 (H-2b, melanoma) and CT26 (H-2d, colon cancer) murine models, injection of AdmCD40L into established subcutaneous tumors resulted in sustained tumor regression and tumor-free status in >60% of animals. Intratumoral injection of AdmCD40L also significantly suppressed the growth of established, weakly immunogenic Lewis lung carcinoma (H-2b) tumors, but to a lesser extent. Ex vivo AdmCD40L-transduced tumor cells implanted in syngeneic hosts induced significant antitumor response against preexisting identical tumors at a distant site. Both in vivo and in vitro AdmCD40L modification of tumors to express CD40L elicited tumor-specific cytolytic T lymphocytes responses, and the transfer of spleen cells from treated mice efficiently protected naive mice against a subsequent tumor challenge. These results support the concept that transduction of tumors with a recombinant CD40L adenovirus vector may be a useful strategy for cancer immunotherapy.     

A comprehensive study of eriocitrin metabolism in vivo and in vitro based on an efficient UHPLC-Q-TOF-MS/MS strategy

Eriocitrin, a main flavonoid in lemons, possesses strong antioxidant, lipid-lowering and anticancer activities and has long been used in food, beverages and wine. However, its metabolism in vivo and in vitro is still unclear. In this study, an efficient strategy was developed to detect and identify metabolites of eriocitrin by using ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) based on online data acquisition and multiple data processing techniques. A total of 32 metabolites in vivo and 27 metabolites in vitro were obtained based on the above method. Furthermore, the main metabolic pathways of eriocitrin included reduction, hydrogenation, N-acetylation, ketone formation, oxidation, methylation, sulfate conjugation, glutamine conjugation, glycine conjugation, desaturation and demethylation to carboxylic acid. This study will lay a foundation for further studies on the metabolic mechanisms of eriocitrin.

41 metabolites of eriocitrin in vivo and in vitro was identified based on the efficient UHPLC-Q-TOF-MS/MS strategy.     

Sine-wave current for efficient and safe in vivo gene transfer.

Recently, electro-gene transfer has become a powerful tool to enhance the efficiency of gene expression in different organs and particularly in muscle, which provides efficient secretion of therapeutic proteins into the circulation. However, its toxicity, owing to the high field strengths of conventional direct current (DC) square-waves, should be taken into account and should be minimized if electroporation is to be used routinely for the treatment of human diseases. In this study, we demonstrate that efficient in vivo gene transfer could be safely achieved using pulses of alternating current sine-waves (ACSWs) with a frequency of 60 Hz. Importantly, the field strength was decreased to as low as 20 V/cm and the efficiency of muscle gene transfer increased more than tenfold and showed less toxicity than with conventional DC square-wave pulses. Using ACSW pulses to transfer human clotting factor IX (hFIX) plasmid into muscle, we observed significant phenotypic correction in mice with hemophilia B.     

Optimising histidine rich peptides for efficient DNA delivery in the presence of serum.

We recently showed that the antibacterial histidine rich amphipathic peptide LAH4 has significant DNA transfection capabilities in the absence of serum. To further understand the transfection process and to develop the peptides for future applications, we have combined a range of biochemical and biophysical techniques, including fluorescence assisted cell sorting and (2)H solid-state NMR, to characterise the initial binding of the peptide/DNA complexes to the cell surface and the subsequent release of the complexes from the endosome in the presence of serum. Our results show that both primary and secondary peptide structure play important roles in both of these processes. Specifically, we show that an ideal helix length and positioning of the histidine residues should be maintained to obtain optimal resistance to serum effects and release of DNA from the endosome. Inclusion of d-amino acids at the peptide termini does not reduce serum effects however further enrichment of the peptides with histidine residues can enhance transfection efficiency in the presence of serum. The detailed understanding of these two key stages in the transfection process shows that LAH4-L1 and its derivatives are likely to be highly efficient and robust vectors for a range of applications.     

Interleukin-18 gene transfer increases antitumor effects of suicide gene therapy through efficient induction of antitumor immunity

To increase the antitumor effects of cytosine deaminase (AdCD) gene therapy and induce more potent antitumor immunity, Th1 cytokine interleukin-18 encoded adenovirus (AdIL18) was combined with adenovirus encoding CD (AdCD) for the therapy of established murine B16 melanoma. Combination therapy of the tumor-bearing mice with AdIL 18 and AdCD/5FC inhibited the growth of the subcutaneous B16 tumors more significantly, compared with AdIL 18 or AdCD/5FC alone. In vivo depletion analysis with anti-CD4, anti-CD8 or anti-NK 1.1 McAb illustrated that both CD8+ T cells and CD4+ T cells played key roles in the augmented antitumor response of the combined therapy. Peptide/MHC tetramer represents a powerful and general tool for rapid, highly sensitive, and direct analysis of antigen-specific T cells. In this study, we prepared H-2Kb/TRP-2180-188 tetramer, which was demonstrated to bind H-2Kb-restricted, B16 melanoma-specific CD8+ T cells. B16 specific H-2Kb/TRP2180-188 tetramer was used to stain the tumor-specific CD8+ T cells and the results showed that CD8+ tetramer+ T cells were about 3-5% of the splenic CD8+ T cells derived from tumor-bearing mice after combined therapy. The CTL cytotoxicity was markedly induced in mice after combined therapy, suggesting efficient induction of tumor-specific CD8+ T cells after combined gene therapy with AdCD/5FC/AdIL18. IL-18 gene transfer could significantly augment the cytotoxicity of NK cells and macrophages, and increase the production of interleukin-2 and interferon-gamma, as compared with treatments with AdCD/5FC, AdlacZ/5FC or PBS. These data suggested that in vivo IL-18 gene transfer could augment the antitumor effects of CD suicide gene therapy through efficient induction of antitumor immunity.     

A novel general strategy for cloning tumor suppressor genes using radiation-reduced chromosomal superfragments.

To identify the tumor suppressor gene on human chromosome 11p15, we generated mouse microcell hybrids containing small transferable chromosome 11p15 fragments, which we have termed "DNA superfragments". These hybrids will be used to identify which fragments contain a tumor suppressor gene by direct transfer of the fragments to tumor cells via microcell fusion.     

The immunological basis of endotoxin-induced tumor regression. Requirement for T-cell-mediated immunity

It was shown that although intravenous administration of bacterial endotoxin caused extensive hemorrhagic necrosis of four different syngeneic murine tumors, only two of these tumors subsequently underwent complete regression: the two that were shown to be immunogeneic as classically defined. An immunologic basis for endotoxin- induced regression was further indicated by the additional findings that regression, but not hemorrhagic necrosis, of the two immunogenic tumors failed to occur in mice that were immunodepressed by whole-body gamma-irradiation, or that were made T-cell deficient by thymectomy and irradiation. That endotoxin-induced regression is T-cell mediated was suggested by the findings that tumor regression was followed by a state of long-lived immunity to a tumor cell challenge implant, and with the possession by the host of T cells that were capable of passively transferring this state of immunity to normal recipients. It is concluded that although parenteral injection of endotoxin causes hemorrhagic necrosis of most solid murine tumors, it is only those tumors that are immunogenic enough to evoke the generation of T-cell- mediated immunity which subsequently go on to completely regress.     

Augmentation of mouse natural killer activity and induction of interferon by tumor cells in vivo

Conventional and nude mice inoculated with syngeneic or allogenic tumor cells developed a rapid rise in serum interferon (IF) levels, peaking within 24 h. Within the same period, natural killer (NK) activity was readily boosted in the spleen. Both activities usually declined at 3 d. Cells that lacked the ability to augment NK activity also failed to induce detectable levels of IF. The boosting of IF and NK functions did not appear to be a result of contamination of the tumor lines by viruses because inoculation of several type C viruses into normal mice had no effect, and other viruses, like lymphocytic choriomeningitis virus and influenza, elevated IF and NK levels with a significantly later kinetics, peaking 3-4 d. The IF induced by tumor cells was heat and acid labile, species specific, and appeared to be in the type II class, although it was susceptible to antisera against Newcastle disease virus-induced IF. These data suggest that an early, nonthymus-dependent consequence of tumor-cell recognition is the production of IF, which, in turn, activates NK cells to lyse the tumor cells.     

逆转录病毒双顺反子策略有效转导和共表达ALDH1基因与mdr1基因的研究

目的研究含内在核糖体进入位点(IRES) 的逆转录病毒双顺反子载体介导的醛脱氢酶基因(ALDH1)与多药耐药基因(mdr1)转导和共表达.方法以携带ALDH1与mdr1基因的重组逆转录病毒双顺反子G1Na-ALDH1-IRES-mdr1(G1Na-AIM)为载体,电穿孔法转导双嗜型包装细胞PA317,长春新碱筛选;所得病毒生产细胞PA317/ AIM与单嗜型包装细胞GP+E86乒乓感染提高病毒滴度;以重组病毒上清转染K562细胞,应用聚合酶链反应(PCR)和Southern blot检测转移基因的整合,流式细胞术(FCM)及MTT分析基因表达,集落培养法测定转导效率.结果双嗜型病毒上清滴度达1.0×105cfu/ml.基因修饰细胞K562/ AIM经PCR 和Southern blot证实双基因稳定整合至基因组,对4-氢过氧化环磷酰胺(4-HC)及长春新碱(VCR)耐药 (提高3～10倍),FCM及集落法检测基因转导效率为62%～70%.结论逆转录病毒IRES双顺反子载体能引导不同类型的耐药基因有效共表达,可用于扩大耐药范围,进行体内显性选择.     

In vivo assessment of enhanced topical delivery of terbinafine to human stratum corneum.

PURPOSE: The objective of this study was to evaluate, using attenuated total reflectance Fourier transform infrared spectroscopy, the stratum corneum (SC) bioavailability of terbinafine (TBF) following topical treatment with four different formulations. METHODS: Four skin sites on the ventral forearms of five healthy volunteers were treated for 2 h using one of four formulations based on a vehicle consisting of 50% ethanol and 50% isopropyl myristate. Three of these formulations included a percutaneous penetration enhancer: either 5% oleic acid, 10% 2-pyrrolidone or 1% urea. The SC concentration profile of TBF was measured by repeated infrared spectroscopic measurements while sequentially stripping off the layers of this barrier membrane with adhesive tape. This method was validated by HPLC analysis of TBF extracted from the stripped tapes. Transepidermal water loss (TEWL) measurements were also performed, to permit facile estimation of SC thickness. RESULTS: The SC concentration profiles of TBF were fitted to the appropriate solution of Fick's second law of diffusion, thereby allowing determination of the characteristic diffusion and partitioning parameters of the permeating drug. This analysis enabled the efficacies of the different formulations tested to be compared to the no-enhancer control. While it was found that the formulation containing 5% oleic acid significantly enhanced the SC availability of TBF, the other formulations did not improve the apparent drug delivery. CONCLUSIONS: A facile and minimally invasive methodology to evaluate an important aspect of topical drug bioavailability has been described. The analytical methods used (infrared spectroscopy and HPLC) allow estimates of both relative and absolute drug bioavailability in the SC and may be useful, therefore, in the critical determination of bioequivalence between topical formulations.     

Conjugation of a self-antigen to papillomavirus-like particles allows for efficient induction of protective autoantibodies

High avidity and long-lasting autoantibodies to a self-polypeptide (TNF-alpha) were generated after parenteral vaccination of mice with low doses of virus-like particle-based (VLP-based) vaccines that were constructed by linking mouse TNF-alpha peptides to the surface of papillomavirus VLPs. High-titer autoantibodies were induced with or without coadministration of potent conventional adjuvants, but were enhanced by coadministration of CFA. Compared with immunization with the fusion protein alone, attachment to VLPs increased autoantibody titers 1,000-fold. A comparison of Ab responses against the self (TNF-alpha) and foreign components of the fusion protein showed that VLP conjugation abrogated the ability of the humoral immune system to distinguish between self and foreign. Similar levels of IgM were detected to self and foreign epitopes regardless of the assembly state of the antigen, suggesting that conjugation of self-peptides to VLPs promotes survival or expansion of mature autoreactive B cells. In a mouse model, vaccination with conjugated particles inhibited development of type II collagen-induced arthritis. Together, these results suggest a potentially flexible method to efficiently generate autoantibodies against specific self-proteins that mediate arthritis and other diseases.