Adenoviral-vector mediated transfer of HBV-targeted ribonuclease can inhibit HBV replication in vivo

Hepatitis B virus (HBV)-targeted ribonuclease (HBV-TR) is a fused protein of HBV core protein and a ribonuclease, human eosinophil-derived neurotoxin (hEDN). Our previous results showed that HBV-TR could effectively inhibit HBV replication in vitro. To test whether HBV-TR can inhibit HBV replication in vivo, we constructed a recombinant adenoviral vector expressing HBV-TR (Ad-TR) and used it to treat HBV-transgenic mice. Immunohistochemical staining showed that TR was expressed at varied levels in different tissues of Ad-TR-treated mice. Serum HBsAg concentration was decreased by 64.8% for the Ad-TR-treated mice compared with empty adenoviral vector-treated control mice. The amount of HBV-DNA in the livers of the Ad-TR-treated mice was 0.74 × 10⁷ copies/μg of genomic DNA while the amount of HBV-DNA in the livers of the empty adenoviral vector-treated control mice was 2.86 × 10⁷ copies/μg of genomic DNA. Serum HBV-DNA of Ad-TR-treated mice was also decreased by 71.4% compared with empty adenoviral vector-treated control mice. In addition, for some Ad-TR-treated mice, the expression of HBsAg in the liver cells turned negative. No discernible adverse effects were observed for Ad-TR-treated mice. Taken together, our results indicated that adenovirus mediated transfer of HBV-TR can inhibit HBV replication in vivo.     

Recruited cells can become transformed and overtake PDGF-induced murine gliomas in vivo during tumor progression

Background

Gliomas are thought to form by clonal expansion from a single cell-of-origin, and progression-associated mutations to occur in its progeny cells. Glioma progression is associated with elevated growth factor signaling and loss of function of tumor suppressors Ink4a, Arf and Pten. Yet, gliomas are cellularly heterogeneous; they recruit and trap normal cells during infiltration.

Methodology/Principal Findings

We performed lineage tracing in a retrovirally mediated, molecularly and histologically accurate mouse model of hPDGFb-driven gliomagenesis. We were able to distinguish cells in the tumor that were derived from the cell-of-origin from those that were not. Phenotypic, tumorigenic and expression analyses were performed on both populations of these cells. Here we show that during progression of hPDGFb-induced murine gliomas, tumor suppressor loss can expand the recruited cell population not derived from the cell-of-origin within glioma microenvironment to dominate regions of the tumor, with essentially no contribution from the progeny of glioma cell-of-origin. Moreover, the recruited cells can give rise to gliomas upon transplantation and passaging, acquire polysomal expression profiles and genetic aberrations typically present in glioma cells rather than normal progenitors, aid progeny cells in glioma initiation upon transplantation, and become independent of PDGFR signaling.

Conclusions/Significance

These results indicate that non-cell-of-origin derived cells within glioma environment in the mouse can be corrupted to become bona fide tumor, and deviate from the generally established view of gliomagenesis.     

Intercellular transfer of carcinoembryonic antigen from tumor cells to NK cells

The inhibition of NK cell killing is mainly mediated via the interaction of NK inhibitory receptors with MHC class I proteins. In addition, we have previously demonstrated that NK cells are inhibited in a class I MHC-independent manner via homophilic carcinoembryonic Ag (CEA) cell adhesion molecules (CEACAM1)-CEACAM1 and heterophilic CEACAM1-CEA interactions. However, the cross-talk between immune effector cells and their target cells is not limited to cell interactions per se, but also involves a specific exchange of proteins. The reasons for these molecular exchanges and the functional outcome of this phenomenon are still mostly unknown. In this study, we show that NK cells rapidly and specifically acquire CEA molecules from target cells. We evaluated the role of cytotoxicity in the acquisition of CEA and demonstrated it to be mostly killing independent. We further demonstrate that CEA transfer requires a specific interaction with an unknown putative NK cell receptor and that carbohydrates are probably involved in CEA recognition and acquisition by NK cells. Functionally, the killing of bulk NK cultures was inhibited by CEA-expressing cells, suggesting that this putative receptor is an inhibitory receptor.     

Adoptive transfer of dying cells causes bystander-induced apoptosis

The anti-apoptotic Bcl-2 protein has the remarkable ability to prevent cell death from several noxious stimuli. Intriguingly, Bcl-2 overexpression in one cell type has been reported to protect against cell death in neighboring non-Bcl-2 overexpressing cell types. The mechanism of this "trans" protection has been speculated to be secondary to the release of a cytoprotective factor by Bcl-2 overexpressing cells. We employed a series of adoptive transfer experiments in which lymphocytes that overexpress Bcl-2 were administered to either wild type mice or mice lacking mature T and B cells (Rag-1-/-) to detect the presence or absence of the putative protective factor. We were unable to demonstrate "trans" protection. However, adoptive transfer of apoptotic or necrotic cells exacerbated the degree of apoptotic death in neighboring non-Bcl-2 overexpressing cells (p < or= 0.05). Therefore, this data suggests that dying cells emit signals triggering cell death in neighboring non-Bcl-2 overexpressing cells, i.e., a "trans" destructive effect.     

G-rich oligonucleotides alter cell cycle progression and induce apoptosis specifically in OE19 esophageal tumor cells

Short synthetic oligonucleotides (ODNs) can be used to block cellular processes involved in cell growth and proliferation. Often acting as aptamers, these molecules interact with critical proteins that regulate the induction of apoptosis or necrosis. We have used a specialized class of ODNs that contain a monomeric sequence of guanosine to induce apoptosis specifically in the malignant esophageal cell line, OE19, in cell culture, and in a NODscid mouse model. OE19 cells were grown in culture and treated with a stable G-rich oligonucleotide (GRO). Cells were processed and apoptosis was measured by FACS analyses, caspase activity, and Hoescht staining. Circular dichroism (CD) was used to define the structure and stability of various GROs. The GRO works by first inducing retardation in the progression of the cell cycle and then by creating a sub-G1 population of apoptotic cells. The reaction is dose dependent, and appears to rely on the capacity of the G-rich ODN to adopt a G-quartet conformation. Apoptosis was measured by determining caspase 3/7 levels and by staining for nuclear fragmentation using the Hoechst dye. Importantly, nonmalignant esophageal cells or normal human lung fibroblasts are not impeded in their cell cycle progression when incubated with the G-rich ODNs. These results suggest that a selective killing of esophageal tumor cells is directed by G-rich ODNs. Selective killing was demonstrated in the unique activity of the GRO compared to other ODNs of different sequences as well as the response of oncogenic cells compared to nononcogenic cells.     

Adoptive immunotherapy of breast cancer with lymph node cells primed by cryoablation of the primary tumor

Cryoablation of cancer leaves tumor-associated antigens intact in an inflammatory microenvironment that can stimulate a regional anti-tumor immune response. We examined whether cryoablated tumor draining lymph nodes (CTDLN) as adoptive immunotherapy may be an effective immunotherapeutic approach in the adjuvant treatment of breast cancer. BALB/c mice with MT-901 mammary adenocarcinoma tumors underwent cryoablation, resection or no treatment and tumor draining lymph nodes were harvested. Cryoablation resulted in only a mild increase in the absolute number of T-cells but a significant increase in the fraction of tumor-specific T-cells as evidenced on IFN-gamma release assay. FACS analysis demonstrated no significant relative shift in the proportion of CD4(+) or CD8(+) cells. The adoptive transfer of CTDLN resulted in a significant reduction of pulmonary metastases as compared to TDLN from either tumor-bearing mice or mice who underwent surgical excision. Cryoablation prior to surgical resection of breast cancer can be used as a method to generate effector T-cells for adjuvant adoptive cellular immunotherapy.     

NSAIDs induce apoptosis in nonproliferating ovarian cancer cells and inhibit tumor growth in vivo

Ovarian cancer (OC) is one of the most lethal gynaecological cancers, which usually has a poor prognosis due to late diagnosis. A large percentage of the OC cell population is in a nonproliferating and quiescent stage, which poses a barrier to success when using most chemotherapeutic agents. Recent studies have shown that several nonsteroidal anti-inflammatory drugs (NSAIDs) are effective in the treatment of OC. Furthermore, we have previously described the molecular mechanisms of NSAIDs' induction of cancer apoptosis. In this report, we evaluated various structurally distinct NSAIDs for their efficacies in inducing apoptosis in nonproliferating OC cells. Although several NSAIDs-induced apoptosis, Flufenamic Acid, Flurbiprofen, Finasteride, Celocoxib, and Ibuprofen were the most potent NSAIDs inducing apoptosis. A combination of these agents resulted in an enhanced effect. Furthermore, we demonstrate that the combination of Flurbiprofen, which targets nonproliferative cells, and Sulindac Sulfide, that affects proliferative cells, strongly reduced tumor growth when compared with a single agent treatment. Our data strongly support the hypothesis that drug treatment regimens that target nonproliferating and proliferating cells may have significant efficacy against OC. These results also provide a rationale for employing compounds or even chemically modified NSAIDs, which selectively and efficiently induce apoptosis in cells during different stages of the cell cycle, to design more potent anticancer drugs.     

Human K/natural killer cells targeted with hetero-cross-linked antibodies specifically lyse tumor cells in vitro and prevent tumor growth in vivo

We have induced fresh peripheral blood K/natural killer cells to lyse a variety of target cells by coating them with anti-Fc gamma receptor (anti-Fc gamma R) (CD16) antibody hetero-cross-linked with anti-target cell antibody. The cytotoxic cell mediating this activity is different, as judged by depletion studies, from the CD3+, CD8+ T cell which is targeted by anti-CD3 cross-linked to anti-target cell antibody. Targeted K cell activity from some donors is enhanced by exposure to interleukin 2 but not interferon-gamma; other donors exhibit high amounts of this activity without stimulation. Specificity of lysis mediated by targeted K cells is dictated by the specificity of the anti-target cell antibody within the heteroconjugate, and bystander cells are not lysed by targeted K cells. Hetero-cross-linked antibodies containing anti-histocompatibility leukocyte antigen class I instead of anti-Fc gamma R (CD16) do not promote lysis, suggesting that the bridging of the target cell to Fc gamma R on the K cell is required to activate the lytic process. Lysis mediated by targeted K cells is much less inhibitable by polymerized IgG than is classical antibody-dependent cellular cytotoxicity. Fresh human melanoma cells are lysed specifically by K cells coated with anti-Fc gamma R (CD16) cross-linked to the 96.5 anti-melanoma antibody. In vivo, targeted K cells prevent tumor growth at low effector to target ratios in Winn-type tumor neutralization assays. Targeted K cells may therefore provide a new immunotherapeutic approach for the destruction of detrimental cells, such as tumor and virally infected cells.     

Luteinizing hormone releasing hormone-RNase A conjugates specifically inhibit the proliferation of LHRH-receptor-positive human prostate and breast tumor cells

Human prostate and breast tumor cells produce luteinizing hormone-releasing hormone (LHRH) receptors on their cell surface even when they have lost dependency on sex steroid hormones for growth. To investigate whether LHRH can be used as a cell-binding moiety to deliver toxin molecules into prostate and breast tumor cells, LHRH-bovine RNase A conjugates were constructed using the chemical cross-linking method. The treatment of the LHRH receptor-positive cells such as prostate LNCapFGC and breast MCF7 tumor cells with LHRH-RNase A conjugates resulted in a dose-dependent inhibition of growth. The cytotoxic activities of these conjugates were effectively reduced by the presence of exogenous LHRH. Either free RNase A or LHRH alone did not affect the proliferation of these cells. The LHRH-RNase A conjugates did not show cytotoxicity against FRTL5 and TM4 cells which do not express the LHRH receptors. These results suggest that LHRH can be used as a cell-binding molecule for the specific delivery of toxin molecules into the cells which express LHRH receptors on their surface. Thus, a new class of biomedicines that act as fusion proteins between LHRH and toxins will give us a new avenue for the treatment of human prostate and breast cancers, regardless of their steroid hormone dependency.     

GD3-reactive antibodies can inhibit the lysis of autologous tumor cells by tumor-infiltrating lymphocytes

Summary GD3 is expressed in high concentrations on melanoma cells and may serve as a useful target antigen for mAb-mediated immunotherapy. Monoclonal antibodies (mAbs) against GD3 stimulate cell-mediated immune responses against tumor cells in vitro and this activity may contribute to antitumor effects in patients with melanoma treated with GD3-reactive mAbs. In the present study the effects of GD3-reactive mAbs on autologous tumor cell lysis by a human melanoma-derived tumor-infiltrating lymphocyte (TIL) population were examined. Unlike results reported for other GD3⁺ T cells isolated from melanoma patients, the tumor-specific lytic activity of the TIL line was inhibited by incubation with mAbs against GD3. Other melanoma-reactive mAbs, including those against GD2 and the high-molecular-weight melanoma-associated Ag, had no effect on the TIL lytic activity. Overall, these results indicate that mAbs against GD3 may have different effects on T cell/tumor cell interactions.     

NK cells rapidly remove B16F10 tumor cells in a perforin and interferon-gamma independent manner in vivo

Natural killer (NK) cells have been shown critical in reducing tumor lung metastasis in various murine cancer models. Effector molecules such as perforin and IFN-γ may play important roles in inhibition of metastasis. However, most of these conclusions were based on experiments that involved quantitation of metastatic colonies several weeks after tumor challenge. The roles of NK cells and their effector molecules (perforin and IFN-γ) in the initial immune responses against tumor metastasis in lungs are still unknown. By using the B16F10 melanoma tumor model combined with confocal microscopy, we observed an increase in numbers of B16F10 cells in NK-depleted mice at 60 min post tumor inoculation, but this effect was independent of perforin or IFN-γ. In addition, NK cell numbers in lungs after tumor injection rapidly increased suggesting a redistribution of NK cells in the lungs. However, NK cells were not found in contact with tumor cells until day 6 or later. Our data indicate that during early responses against B16F10 cells, NK cells use another mechanism(s) besides perforin and IFN-γ to prevent tumor metastasis.     

IFN-gamma-dependent delay of in vivo tumor progression by Fas overexpression on murine renal cancer cells

The role of Fas in the regulation of solid tumor growth was investigated. Murine renal carcinoma (Renca) cells were constitutively resistant to Fas-mediated killing in vitro, but exhibited increased expression of Fas and sensitivity to Fas-mediated killing after exposure to IFN-gamma and TNF. Transfected Renca cells overexpressing Fas were efficiently killed in vitro upon exposure to anti-Fas Ab (Jo2). When Fas-overexpressing Renca cells were injected into syngenic BALB/c mice, there was a consistent and significant delay in tumor progression, reduced metastasis, and prolonged survival that was not observed for Renca cells that overexpressed a truncated nonfunctional Fas receptor. The delay of in vivo tumor growth induced by Fas overexpression was not observed in IFN-gamma-/- mice, indicating that IFN-gamma is required for the delay of in vivo tumor growth. However, there was a significant increase of infiltrated T cells and in vivo apoptosis in Fas-overexpressing Renca tumors, and Fas-overexpressing Renca cells were also efficiently killed in vitro by T cells. In addition, a strong therapeutic effect was observed on Fas-overexpressing tumor cells by in vivo administration of anti-Fas Ab, confirming that overexpressed Fas provides a functional target in vivo for Fas-specific ligands. Therefore, our findings demonstrate that Fas overexpression on solid tumor cells can delay tumor growth and provides a rationale for therapeutic manipulation of Fas expression as a means of inducing tumor regression in vivo.     

Possible involvement of regulatory T cells in tumor onset and progression in primary breast cancer

Background  The FOXP3 mRNA expression and the other regulatory T cell-related molecules were investigated and compared with clinicopathological parameters in human primary breast cancer. Method  This study included 136 breast cancer patients operated in our department from 2003 to 2006. Total RNA was extracted from frozen normal breast and breast cancer tissues, and the expression of FOXP3, IL-10, TGFβ1 and CCL22 mRNA was evaluated using quantitative real-time RT-PCR. Result   FOXP3, IL-10, TGFβ1 and CCL22 mRNA expressions were significantly higher in cancer tissue than in normal tissue, not only at pT1, 2, and 3 stages but also at the DCIS stage. There were positive correlations between FOXP3 and IL-10, FOXP3 and TGFβ1, as well as FOXP3 and CCL22 mRNA expressions, respectively. FOXP3 and IL-10 mRNA expressions were significantly upregulated in PgR-negative or HER2-positive tumors. Conclusion  These results suggest that regulatory T cells are involved in tumor onset and progression in human primary breast cancer, possibly contributing to poor prognosis of patients with breast cancer.     

In vivo regulation of experimental autoimmune encephalomyelitis by NK cells: alteration of primary adaptive responses

Innate immune responses provide the host with its first line of defense against infections. Signals generated by subsets of lymphocytes, including NK cells, NKT cells, and APC during this early host response determine the nature of downstream adaptive immune responses. In the present study, we have examined the role of innate NK cells in an autoimmune model through the use of primary immunization with the myelin oligodendrocyte glycoprotein peptide to induce experimental autoimmune encephalomyelitis (EAE). Our studies have shown that in vivo depletion of NK cells can affect the adaptive immune responses, because NK cells were found to regulate the degree of clinical paralysis and to alter immune adaptive responses to the myelin oligodendrocyte glycoprotein peptide. The requirement for NK cells was reflected by changes in the T cell responses and diminished clinical disease seen in mice treated with anti-NK1.1, anti-asialo GM1, and selected Ly49 subtype-depleted mice. In addition to alteration in T cell responses, the maturational status of dendritic cells in lymph nodes was altered both quantitatively and qualitatively. Finally, examination of TCR Vbeta usage of the brain lymphocytes from EAE mice indicated a spectra-type change in receptor expression in NK- depleted mice as compared with non-NK-depleted EAE mice. These findings further establish a recently postulated link between NK cells and the generation of autoreactive T cells.     

Structural analogues of pyrroline 5-carboxylate specifically inhibit its uptake into cells

Summary Pyrroline 5-carboxylate, a naturally occurring intermediate, is a potent activator of redox-dependent metabolic pathways. This effect of pyrroline 5-carboxylate is due, at least in part, to the special mechanism mediating its entry into cells. Using Chinese hamster ovary cells we recently characterized the cellular uptake of pyrroline 5-carboxylate as a process transferring oxidizing potentialpari passu with cell entry, a process consistent with group translocation. We sought to identify specific inhibitors to probe this unique uptake mechanism, to blockade the metabolic effects of pyrroline 5-carboxylate, and to provide strategies to identify the putative carrier protein. Because pyrroline 5-carboxylate, a ring structure with a tertiary nitrogen, is in spontaneous equilibrium with glutamic--semialdehyde, an openchain structure, we tested analogues of both. Most open-chain aldehydes at 10mm had little effect on the uptake of pyrroline 5-carboxylate. Although succinic semialdehyde did inhibit, its effect was nonspecific in that the uptake of (methylamino) isobutyric acid was inhibited as much as the uptake of pyrroline 5-carboxylate. In contrast, pyrroline 2-carboxylate and other cyclic compounds with teriary nitrogens, e.g., pyridines, were specific inhibitors of pyrroline 5-carboxylate uptake. Respective potencies of pyridine derivatives depended on the nature and location of constituent groups. Kinetics studies showed that these inhibitors were competitive with pyrroline 5-carboxylate and the most potent inhibitor, 2,6-pyridinedicarboxaldehyde, exhibited aK ₁₂ of 0.27±0.05mm. In the face of their effect on P5C uptake, the most potent of thse analogues, 2-pyridinecarboxaldehyde and 2,6-pyridinedicarboxaldehyde, did not inhibit the activity of pyrroline 5-carboxylate to proline. Nevertheless, the analogues markedly inhibited the stimulatory effect of P5C on the pentose phosphate shunt. Importantly, not only did 2-pyridinecarboxaldehyde protect the pyrroline 5-carboxylate uptake mechanism from the inhibitory effects of a sulfhydryl-reactive agent, but also its inhibitory effect became irreversible in the presence of sodium cyanoborohydride. These inhibitors may help discriminate events mediated by the transport carrier from those mediated by intracellular metabolism and may provide a method for identifying and characterizing the putative carrier for P5C.     

Dynamic compression can inhibit chondrogenesis of mesenchymal stem cells

The objective of this study was to investigate the influence of dynamic compressive loading on chondrogenesis of mesenchymal stem cells (MSCs) in the presence of TGF-β3. Isolated porcine MSCs were suspended in 2% agarose and subjected to intermittent dynamic compression (10% strain) for a period of 42 days in a dynamic compression bioreactor. After 42 days in culture, the free-swelling specimens exhibited more intense alcian blue staining for proteoglycans, while immunohistochemical analysis revealed increased collagen type II immunoreactivity. Glycosaminoglycan (GAG) content increased with time for both free-swelling and dynamically loaded constructs, and by day 42 it was significantly higher in both the core (2.5 ± 0.21%w/w vs. 0.94 ± 0.03%w/w) and annulus (1.09 ± 0.09%w/w vs. 0.59 ± 0.08%w/w) of free-swelling constructs compared to dynamically loaded constructs. This result suggests that further optimization is required in controlling the biomechanical and/or the biochemical environment if such stimuli are to have beneficial effects in generating functional cartilaginous tissue.     

Adoptive transfer of induced-Treg cells effectively attenuates murine airway allergic inflammation

Both nature and induced regulatory T (Treg) lymphocytes are potent regulators of autoimmune and allergic disorders. Defects in endogenous Treg cells have been reported in patients with allergic asthma, suggesting that disrupted Treg cell-mediated immunological regulation may play an important role in airway allergic inflammation. In order to determine whether adoptive transfer of induced Treg cells generated in vitro can be used as an effective therapeutic approach to suppress airway allergic inflammation, exogenously induced Treg cells were infused into ovalbumin-sensitized mice prior to or during intranasal ovalbumin challenge. The results showed that adoptive transfer of induced Treg cells prior to allergen challenge markedly reduced airway hyperresponsiveness, eosinophil recruitment, mucus hyper-production, airway remodeling, and IgE levels. This effect was associated with increase of Treg cells (CD4(+)FoxP3(+)) and decrease of dendritic cells in the draining lymph nodes, and with reduction of Th1, Th2, and Th17 cell response as compared to the controls. Moreover, adoptive transfer of induced Treg cells during allergen challenge also effectively attenuate airway inflammation and improve airway function, which are comparable to those by natural Treg cell infusion. Therefore, adoptive transfer of in vitro induced Treg cells may be a promising therapeutic approach to prevent and treat severe asthma.     

Th17 cells are involved in the local control of tumor progression in primary intraocular lymphoma

Background

Th17 cells play an important role in the pathogenesis of many autoimmune diseases, but despite some reports of their antitumor properties, too little is known about their presence and role in cancers. Specifically, knowledge is sparse about the relation of Th17 to lymphoma microenvironments and, more particularly, to the microenvironment of primary intraocular B-cell lymphoma (PIOL), an aggressive lymphoma with a poor prognosis.

Methods and Principal Findings

In this work, we investigated the presence of Th17 cells and their related cytokines in a syngeneic model of PIOL, a subtype of non-Hodgkin lymphoma. The very small number of lymphocytes trafficking in normal eyes, which represent a low background as compared to tumor-bearing eyes, allows us to develop the present model to characterize the different lymphocyte subsets present when a tumor is developing. IL-21 mRNA was expressed concomitantly with IL-17 mRNA in tumor-bearing eyes and intracellular expression of IL-17A and IL-21 in infiltrating CD4⁺ T lymphocytes. Interestingly, IL-17A production by T cells was negatively correlated with tumor burden. We also showed that IL-21 but not IL-17 inhibits tumor cell proliferation in vitro.

Conclusions

These data demonstrate that IL-17A and IL-21-producing CD4⁺ T cells, referred as Th17 cells, infiltrate this tumor locally and suggest that Th17-related cytokines may counteract tumor progression via IL-21 production. Thus, Th17 cells or their related cytokines could be considered to be a new therapeutic approach for non-Hodgkin B-cell lymphomas, particularly those with an ocular localization.