Sonoporation-mediated anti-angiogenic gene transfer into muscle effectively regresses distant orthotopic tumors.

Ultrasound (US) is an effective tool for local delivery of genes into target tumors or organs. In combination with microbubbles, US can temporarily change the permeability of cell membranes by cavitation and facilitate entry of plasmid DNA into cells. Here, we demonstrate that repeated US-mediated delivery of anti-angiogenic genes, endostatin or calreticulin, into muscle significantly inhibits the growth of orthotopic tumors in the liver, brain or lung. US-mediated anti-angiogenic gene therapy also seems to function as an adjuvant therapy that significantly enhances the antitumor effects of the chemotherapeutic drug doxorubicin and adenovirus-mediated cytokine gene therapy. Significantly higher levels of tumor apoptosis or tumor-infiltrating lymphocytes were observed after combined therapy consisting of either anti-angiogenic therapy and chemotherapy, or anti-angiogenic therapy and immunotherapy. Taken together, our experiments demonstrate that intramuscular delivery of anti-angiogenic genes by US exposure can effectively treat distant orthotopic tumors, and thus has great therapeutic potential in terms of clinical treatment.

T-cell antigenic determinants within hepatitis C virus nonstructural protein 3 and cytokine production profiles in hepatitis C.

The aim of this study was to further investigate the role of T-helper cells in hepatitis C virus (HCV) infection, focusing on the T-cell antigenic determinants and cytokine profiles of nonstructural 3 (NS3) protein-stimulated peripheral blood mononuclear cells (PBMCs) of HCV patients. A total of 12 recombinant proteins of theNS3 region were purified and used to test T-cell proliferative response and antigenic determinants of HCV-seropositive patients. In addition, cytokines produced by antigen stimulated PBMCs were measured. Our data showed that PBMCs from 55.7% (34/61) of HCV patients proliferated to at least one antigen, but PBMCs of HCV seronegative patients did not. In addition, PBMCs from about 82.0% (32/39) HCV-seropositive patients produced significant amounts of cytokines (10 pg/mL). Interestingly, PBMCs from 66% of patients produced TH2-related cytokines such as interleukin (IL)-4 and IL-5. In mappingexperiments, the data showed multiple T-cell antigenic determinants. Our data demonstrated that NS3 antigen-stimulated PBMCs of HCV patients recognized multiple T-cell antigenic determinants and produced significant amounts of TH0 or TH2-related cytokines, which might play a critical role in the chronicity of HCV infection.

Ribavirin enhances interferon-gamma levels in patients with chronic hepatitis C treated with interferon-alpha.

Some patients with chronic hepatitis C respond to interferon (IFN)-alpha treatment, and the efficiency can be improved by combining it with ribavirin. The mechanism of this improvement is unknown. To investigate the effects of these two regimens on the immune responses in 51 patients with chronic hepatitis C, we examined the hepatitis C core antigen-specific proliferative response and cytokine production profiles, natural killer (NK) cell cytotoxicity and cytotoxic T cell function during treatment. The results are as follows: (1) both viral clearance and biochemical normalization occurred more frequently in patients receiving combination therapy; (2) the function of NK cells increased after treatment in the responders of both groups (p < 0.05); (3) the level of IFN-gamma produced by hepatitis C core antigen-stimulated peripheral blood mononuclear cells was higher in patients receiving combination therapy, especially in responders; (4) the core antigen-specific proliferative response decreased after treatment, and (5) in addition, the core-specific cytotoxic T cell activities of five responder patients also increased significantly after therapy. In conclusion, enhancement of immune responses, especially those related to type-1 T helper cell activity, may contribute to better efficacy in combining ribavirin with IFN-alpha for treatment of chronic hepatitis C.

Functional measurement of hepatitis C virus core-specific CD8(+) T-cell responses in the livers or peripheral blood of patients by using autologous peripheral blood mononuclear cells as targets or stimulators.

As is widely recognized, CD8(+) cytotoxic T lymphocytes (CTLs) play a crucial role in hepatitis C virus (HCV) infection, both in pathogenesis of liver injury and in clearing the virus. CTL studies with HCV-infected patients have been difficult because of the relatively low frequency of CTL precursors in the peripheral blood and because the targeted epitopes vary depending on the human leukocyte antigen (HLA) types of the individuals. This study attempts to overcome these problems by assessing the feasibility of using autologous peripheral blood mononuclear cells (PBMCs) expressing viral antigens as stimulators or targets in order to monitor the CTL responses. Primary PBMCs were transduced using a retroviral vector pseudotyped with a vesicular stomatitis virus G glycoprotein expressing the HCV core gene. Additionally, the vector-transduced PBMCs were used as targets of CTL assays to measure the HCV core-specific CTL activities from the liver-infiltrating lymphocytes of six different HLA-type patients with chronic HCV infection. The core-expressing PBMCs also served as stimulators, allowing us to measure core-specific CD8(+) T-cell responses by intracellular gamma interferon staining of the peripheral blood of hepatitis C patients who had received treatment with alpha interferon plus ribavirin. This approach provides an efficient means of measuring antigen-specific CTL responses without HLA constraints.

Structure-based mutational analysis of the hepatitis C virus NS3 helicase.

The carboxyl terminus of the hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses ATP-dependent RNA helicase activity. Based on the conserved sequence motifs and the crystal structures of the helicase domain, 17 mutants of the HCV NS3 helicase were generated. The ATP hydrolysis, RNA binding, and RNA unwinding activities of the mutant proteins were examined in vitro to determine the functional role of the mutated residues. The data revealed that Lys-210 in the Walker A motif and Asp-290, Glu-291, and His-293 in the Walker B motif were crucial to ATPase activity and that Thr-322 and Thr-324 in motif III and Arg-461 in motif VI significantly influenced ATPase activity. When the pairing between His-293 and Gln-460, referred to as gatekeepers, was replaced with the Asp-293/His-460 pair, which makes the NS3 helicase more like the DEAD helicase subgroup, ATPase activity was not restored. It thus indicated that the whole microenvironment surrounding the gatekeepers, rather than the residues per se, was important to the enzymatic activities. Arg-461 and Trp-501 are important residues for RNA binding, while Val-432 may only play a coadjutant role. The data demonstrated that RNA helicase activity was possibly abolished by the loss of ATPase activity or by reduced RNA binding activity. Nevertheless, a low threshold level of ATPase activity was found sufficient for helicase activity. Results in this study provide a valuable reference for efforts under way to develop anti-HCV therapeutic drugs targeting NS3.

Induction of antitumor immunity with combination of HER2/neu DNA vaccine and interleukin 2 gene-modified tumor vaccine.

The therapeutic effects of both cytokine-secreting tumor vaccine and DNA vaccine were studied using mouse MBT-2 bladder cancer cells as a model. Cytokine-secreting MBT-2 cells were obtained by infecting cells with retroviral particles containing interleukin (IL) 2-, IL-4-, or granulocyte-macrophage colony-stimulating factor (GM-CSF)-expression vector. The MBT-2-IL-2 cells were not tumorigenic in syngenic C3H mice at all. Tumor formation decreased significantly for the MBT-2-GM-CSF cells. MBT-2-IL-2, -IL-4, and -GM-CSF cells were killed by irradiation and tested as tumor vaccines. The irradiated MBT2-IL-2 cells could complete protect mice from the growth of the preexisting tumor cells, and the immune memory lasted for 8 months. On the other hand, irradiated MBT-2-IL-4 and MBT-2-GM-CSF cells were less effective. When the loading tumor mass increased, all tumor vaccines lost protective effects. DNA vaccine encoding the tumor antigen neu was additionally tested to improve the therapeutic efficacy. Coinjection of 60 microg pSV-neu DNA was effective in enhancing the antitumor effects of MBT2-IL-2; however, DNA vaccine alone cannot prevent the progression of the preexisting tumor. Immunohistochemical analysis of tumor infiltrate revealed massive increase of CD4+ lymphoid cells in the group of mice treated with both DNA vaccine and IL-2-secreted tumor vaccine. Western blotting demonstrated the presence of anti-neu antibody in the serum from immunized mice. In contrast, combination of DNA vaccine and MBT-2-GM-CSF has no additive effect. The results indicate the combination of DNA vaccine and IL-2-secreting tumor vaccine can additionally improve therapeutic efficacy, and the efficacy is correlated with the increase of CD4+ T lymphocytes and anti-neu antibody.

Ribavirin enhancement of hepatitis C virus core antigen-specific type 1 T helper cell response correlates with the increased IL-12 level.

BACKGROUNDS/AIMS: Combination IFN-a and ribavirin therapy for hepatitis C virus-infected patients has been reported to improve the response rate up to 50%. In this study, we aimed to study further the role of ribavirin in hepatitis C virus-specific immune responses. METHODS: We immunized mice with hepatitis C virus core protein with or without different concentrations of ribavirin. Forty days after immunization, hepatitis C virus-specific immune responses were followed in these mice. RESULTS: We found that the mice immunized with core antigen once every 2 weeks and 0.5 mg ribavirin every day showed higher levels of core-specific IgG2 compared with those mice immunized with core antigen only. In addition, core antigen-stimulated spleen cells produced higher levels of T helper type 1 cytokines and the core-specific cytotoxic T cell activity also increased significantly. Furthermore, lipopolysaccharide-stimulated peritoneal cells produced higher levels of IL-12 in ribavirin-treated mice, and peritoneal cells isolated from naive mice also produced significantly higher level of IL-12 when cultured with ribavirin. CONCLUSIONS: Ribavirin may significantly promote the T helper type 1 immune response in vivo, and, furthermore, the effect of ribavirin on IL-12 level produced by accessory cells may contribute to the T helper type 1 enhancing effect.

ATP-binding domain of NTPase/helicase as a target for hepatitis C antiviral therapy.

To enhance the inhibitory potential of 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin) vs hepatitis C virus (HCV) NTPase/helicase, ribavirin-5'-triphosphate (ribavirin-TP) was synthesized and investigated. Ribavirin-TP was prepared with the use of modified Yoshikawa-Ludwig-Mishra-Broom procedure (cf. Mishra & Broom, 1991, J. Chem. Soc., Chem. Commun, 1276-1277) involving phosphorylation of unprotected nucleoside. Kinetic analysis revealed enhanced inhibitory potential of ribavirin-TP (IC50=40 microM) as compared to ribavirin (IC50 > 500 microM). Analysis of the inhibition type by means of graphical methods showed a competitive type of inhibition with respect to ATP. In view of the relatively low specificity towards nucleoside-5'-triphosphates (NTP) of the viral NTPase/helicases, it could not be ruled out that the investigated enzyme hydrolyzed the ribavirin-TP to less potent products. Investigations on non- hydrolysable analogs of ribavirin-TP or ribavirin-5'-diphosphate (ribavirin-DP) are currently under way.

Tumor-induced immunosuppression: a barrier to immunotherapy of large tumors by cytokine-secreting tumor vaccine.

An active immunotherapy strategy with cytokine-assisted tumor vaccine, although often effective for small tumor burdens, is much less so for large tumor burdens. This study examines how large tumors might suppress the T cell functions and escape from the immune responses elicited by a granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor vaccine. According to our results, the T cells isolated from the tumor-bearing mice treated late with the vaccine failed to confer protective activity on naive mice against a wild-type tumor challenge, unlike those isolated from the early-treated group. Nevertheless, the antitumor activity of the inactive T cells could be restored on in vitro stimulation. Expression of transforming growth factor beta (TGF-beta) and interleukin 10 (IL-10), the potent immunosuppressive factors, was detected in the parental tumor cell line RLmale 1 (a murine T leukemia cell line), as well as in the tumor region, the levels of which correlated with tumor progression. An in vitro assay of T cell functions revealed that the TGF-beta in the conditioned medium of RLmale 1 cells mainly affected the activation, whereas the IL-1male affected the activation to a lesser extent, but significantly affected the cytolytic activity, of tumor-specific T cells. The immunosuppressive activity of IL-10 was also signified by the findings that administration of the conditioned medium of RLmale 1 cultured in a serum-free medium, in which the TGF-beta activity was then lost while the IL-10 activity still remained, or of recombinant IL-10 to the early-treated group of mice abrogated the known efficacy of tumor vaccine on the small tumors. These data suggested that the efficacy of cytokine-secreting tumor vaccine was blocked by the immunosuppressive factors secreted from the large tumors. The results have important implications for the clinical design of immunotherapeutic strategies for advanced cancer patients.

Biochemical properties of a minimal functional domain with ATP-binding activity of the NTPase/helicase of hepatitis C virus.

The RNA-stimulated nucleoside triphosphatase (NTPase) and helicase of hepatitis C virus (HCV) consists of three domains with highly conserved NTP binding motifs located in the first domain. The ATP-binding domain was obtained by limited proteolysis of a greater fragment of the HCV polyprotein, and it was purified to homogenity by column chromatography. The identity of the domain, comprising amino acids 1203 to 1364 of the HCV polyprotein, was confirmed by N- and C-terminal sequencing and by its capability to bind 5'-fluorosulfonylbenzoyladenosine (FSBA). The analyses of the kinetics of ATP binding revealed a single class of binding site with the Kd of 43.6 microM. The binding is saturable and dependent on Mn2+ or Mg2+ ions. Poly(A) and poly(dA) show interesting properties as regulators of the ATP-binding capacity of the domain. Polynucleotides bind to the domain and enhance its affinity for ATP. In addition, ATP enhances the affinity of the domain for the polynucleotides. Different compounds, which are known to interact with nucleotide binding sites of various classes of enzymes, were tested for their ability to inhibit the binding of ATP to the domain. Of the compounds tested, two agents behaved as inhibitors: paclitaxel, which inhibits the ATP binding competitively (IC50 = 22 microM), and trifluoperazine, which inhibits the ATP binding by a noncompetitive mechanism (IC50 = 98 microM). Kinetic experiments with the NTPase/helicase indicate that both compounds inhibit the NTPase activity of the holoenzyme by interacting with its ATP-binding domain.

Regression of orthotopic brain tumors by cytokine-assisted tumor vaccines primed in the brain.

This study investigated the therapeutic effects of a rat glioma cell line, C6, that was engineered to secrete mouse GM-CSF (mGM-CSF) on intracerebral (i.c.) brain tumors. Significant antitumor immunity was induced in rats when the live or irradiated mGM-CSF-secreting tumor vaccine was implanted i.c. The antitumor activity was effective on small tumors and, to a lesser extent, on large tumors or tumors existing in vivo for a longer duration. Immunohistochemical analysis revealed cellular infiltrates (granulocytes, macrophages, and CD4+ and CD8+ T cells) at both the vaccine site and the tumor site, indicating that immune responses were similarly activated when tumor vaccine was inoculated in the brain, as at the subcutis. Additional studies demonstrated that the therapeutic effects of tumor vaccines on the large tumors or the long-existing tumors were enhanced by strategies such as increasing the dosage of tumor vaccines, using combined vaccines consisting of mGM-CSF and human interleukin-2, or combining tumor vaccine with herpes simplex virus thymidine kinase/ganciclovir treatment. All of the modified strategies yielded synergistic therapeutic effects on the large tumor burdens. The data presented herein suggest that cytokine gene therapy is highly promising for the treatment of i.c. gliomas.

Characterization of monoclonal antibodies against hepatitis C virus nonstructural protein 3: different antigenic determinants from human B cells.

The nonstructural (NS3) region protein of hepatitis C virus (HCV) possesses major B-cell epitopes that induce antibodies after infection. To elucidate further the characteristics of these B cells and their role in the immune regulation of HCV infection, T9 (portion of NS3 region, amino acids [a.a.] 1188-1493)-specific monoclonal antibodies were derived and mapped for B-cell antigenic determinants with recombinant proteins. A total of 10 T9-specific hybridomas were generated and tested for B-cell antigenic determinants. To analyze the B-cell antigenic determinants, eight recombinant proteins including NS3-e (a.a. 1175-1334), NS3-a' (a.a. 1175-1250), NS3-a (a.a. 1251-1334), NS3-b (a.a. 1323-1412), NS3-c (a.a. 1407-1499), NS3-a/b (a.a. 1251-1412), NS3-bc (a.a. 1323-1499), and NS3-abc (a.a. 1251-1499) encoded by NS3-region internal clones were expressed and tested for immunoblotting. The data suggested IgG hybridomas recognized NS3-a, NS3-a', or NS3-b protein by immunoblotting. By contrast, the NS3-e protein bears the major antigenic determinant recognized by human sera. Half of the hybridomas were found to react with protein NS3-a', which is not a major B-cell antigenic determinant in humans. These data suggested that conformational epitopes in vivo may be important for B-cell recognition.

Involvement of the 5' proximal coding sequences of hepatitis C virus with internal initiation of viral translation.

The 5' nontranslated region (NTR) of hepatitis C virus (HCV) consists of 341 nucleotides (nt). This region comprises the majority of the internal ribosome entry site (IRES) which controls the efficiency of viral translation. Previous studies of the 3' boundary of the HCV IRES yielded conflicting data regarding the involvement of viral coding sequences in IRES activity. We therefore studied the functional significance of the 5' proximal coding sequences of the HCV core gene on IRES activity. We constructed monocistronic and bicistronic DNAs that contained either a chloramphenicol acetyl transferase (CAT) gene or a luciferase (Luc) gene as the reporter. Results from both in vitro and in vivo experiments indicated that the optimal IRES ranged within nt 1-371. Further mutational analyses of sequences surrounding the initiation codon revealed that primary sequences downstream of the AUG initiator rather than the secondary structure are important in regulating optimal IRES function. We are also able to demonstrate that a non-AUG codon could be used to initiate the synthesis of a reporter protein, albeit with lower efficiency. These findings bear important implications for the HCV IRES secondary structures.

Establishment and characterization of NS3 protein-specific T-cell clones from a patient with chronic hepatitis C.

Our previous study showed dominant proliferative response of peripheral mononuclear cells to hepatitis C virus (HCV) nonstructural (NS-3) (T9, from aa 1188 to 1493) in chronically infected patients. Six T9-specific T-cell clones derived in an HCV patient were established and studied for the antigen specificity and the ability of augmentation of in vitro antibody production. All these cloned T-cell lines responded exclusively to T9 antigen and could help autologous B cells in producing anti-T9 antibody in vitro. Cytokine mRNAs of these T cells was detected by polymerase chain reaction and predominant IL-2 and IFN-gamma production was noted. In addition, further elucidation of T-cell antigenic determinant and MHC restriction suggested that these T-cell clones recognized at least two different T-cell antigenic determinants within the NS-3 region in an HLA DQ2-restricted manner. We believe characterization of HCV-specific T-cell responses, especially T-cell epitope mapping and cytokine production pattern, may shed light on further understanding the pathogenic mechanism and designing therapy for HCV infection.

Budding yeast Cdc20: a target of the spindle checkpoint.

The spindle checkpoint regulates the cell division cycle by keeping cells with defective spindles from leaving mitosis. In the two-hybrid system, three proteins that are components of the checkpoint, Mad1, Mad2, and Mad3, were shown to interact with Cdc20, a protein required for exit from mitosis. Mad2 and Mad3 coprecipitated with Cdc20 at all stages of the cell cycle. The binding of Mad2 depended on Mad1 and that of Mad3 on Mad1 and Mad2. Overexpression of Cdc20 allowed cells with a depolymerized spindle or damaged DNA to leave mitosis but did not overcome the arrest caused by unreplicated DNA. Mutants in Cdc20 that were resistant to the spindle checkpoint no longer bound Mad proteins, suggesting that Cdc20 is the target of the spindle checkpoint.

An ELISA for RNA helicase activity: application as an assay of the NS3 helicase of hepatitis C virus.

A convenient enzyme-linked immunosorbent assay (ELISA) for RNA helicase activity was developed with principles similar to the standard assay. The helicase ELISA utilizes a non-radioactive double-stranded substrate with a biotin-labeled template (long) strand hybridized to a digoxigenin (DIG)-labeled release (short) strand. The template strand binds to the wells of streptavidin-coated microtiter plates (SA-MTP) where the helicase catalyzes the unwinding reaction. Substrate not unwound retains the DIG-labeled release strand and is detected using anti-DIG coupled to horseradish peroxidase. Chromogenic detection follows. Absorbance measurement allows determination of unwinding efficiency of reactions. To demonstrate effectiveness, the ELISA-based assay was used to study the unwinding activity of the hepatitis C virus (HCV) NS3 helicase. Using a known inhibitor of NS3 helicase activity and two mutant HCV helicases, the ability of the assay to screen potential anti-helicase drugs and putative helicases is illustrated. The helicase ELISA is more convenient than the standard helicase assay and is especially suited for the testing of large numbers of samples.

A novel yeast screen for mitotic arrest mutants identifies DOC1, a new gene involved in cyclin proteolysis.

B-type cyclins are rapidly degraded at the transition between metaphase and anaphase and their ubiquitin-mediated proteolysis is required for cells to exit mitosis. We used a novel enrichment to isolate new budding mutants that arrest the cell cycle in mitosis. Most of these mutants lie in the CDC16, CDC23, and CDC27 genes, which have already been shown to play a role in cyclin proteolysis and encode components of a 20S complex (called the cyclosome or anaphase promoting complex) that ubiquitinates mitotic cyclins. We show that mutations in CDC26 and a novel gene, DOC1, also prevent mitotic cyclin proteolysis. Mutants in either gene arrest as large budded cells with high levels of the major mitotic cyclin (Clb2) protein at 37 degrees C and cannot degrade Clb2 in G1-arrested cells. Cdc26 associates in vivo with Doc1, Cdc16, Cdc23, and Cdc27. In addition, the majority of Doc1 cosediments at 20S with Cdc27 in a sucrose gradient, indicating that Cdc26 and Doc1 are components of the anaphase promoting complex.

Assembly of hepatitis delta virus-like empty particles in yeast.

Large delta antigen (L-HDAg) of hepatitis delta virus (HDV) and small-form hepatitis B surface antigen (HBsAg) of helper hepatitis B virus have previously been shown to be the minimum components for the assembly of HDV-like particles in mammalian cells. Extending from this finding, we coexpressed L-HDAg and small HBsAg in Saccharomyces cerevisiae to study their assembly in yeast cells. The assembly of virus particles from L-HDAg and HBsAg in yeast was demonstrated by their coexistence in the same isopycnic fractions and by the coimmunoprecipitation of L-HDAg with HBsAg using an antibody against HBsAg (anti-HBs). Furthermore, after purification by affinity chromatography with anti-HBs, HDV-like particles with size and morphology similar to those derived from mammalian cells could be visualized by electron microscopy. Mice immunized with yeast-derived HDV-like particles simultaneously acquired antibodies against HBsAg and HDAg, indicating that both viral proteins are antigenic. The results indicated that S. cerevisiae could serve as a host for the assembly of HDV-like empty particles. This system may be useful in investigating cellular processes involved in HDV assembly and in producing ample amount of HDV-like particles for structural and immunological studies.

Direct interaction of hepatitis C virus core protein with the cellular lymphotoxin-beta receptor modulates the signal pathway of the lymphotoxin-beta receptor.

Previous studies suggest that the core protein of hepatitis C virus (HCV) has a pleiotropic function in the replication cycle of the virus. To understand the role of this protein in HCV pathogenesis, we used a yeast two-hybrid protein interaction cloning system to search for cellular proteins physically interacting with the HCV core protein. One such cellular gene was isolated and characterized as the gene encoding the lymphotoxin-beta receptor (LT-betaR). In vitro binding analysis demonstrated that the HCV core protein binds to the C-terminal 98 amino acids within the intracellular domain of the LT-betaR that is involved in signal transduction, although the binding affinity of the full-length HCV core protein was weaker than that of its C-terminally truncated form. Our results also indicated that the N-terminal 40-amino-acid segment of the HCV core protein was sufficient for interaction with LT-betaR and that the core protein could form complexes with the oligomeric form of the intracellular domain of LT-betaR, which is a prerequisite for downstream signaling of this receptor. Similar to other members of the tumor necrosis factor (TNF) receptor superfamily, LT-betaR is involved in the cytotoxic effect of the signaling pathway, and thus we have elucidated the biological consequence of interaction between the HCV core protein and LT-betaR. Our results indicated that in the presence of the synergizing agent gamma interferon, the HCV core protein enhances the cytotoxic effects of recombinant forms of LT-betaR ligand in HeLa cells but not in hepatoma cells. Furthermore, this enhancement of the cytolytic activity was cytokine specific, since in the presence of cycloheximide, the expression of the HCV core protein did not elicit an increase in the cytolytic activity of TNF in both HeLa and hepatoma cells. In summary, the HCV core protein can associate with LT-betaR, and this protein-protein interaction has a modulatory effect on the signaling pathway of LT-betaR in certain cell types. Given the known roles of LT-betaR/LT-alpha1,beta2 receptor-ligand interactions in the normal development of peripheral lymphoid organs and in triggering cytolytic activity and NF-kappaB activation in certain cell types, our finding implies that the HCV core protein may aggravate these biological functions of LT-betaR, resulting in pathogenesis in HCV-infected cells.

Induction of antitumor immunity by intracerebrally implanted rat C6 glioma cells genetically engineered to secrete cytokines.

To test whether cytokine gene therapy can be applied to an immunologically privileged site, such as the brain, we investigated antitumor immunity in the brain induced by cytokine-secreting glioma cells. Three cytokine genes, interleukin-2 (IL-2), interleukin-4 (IL-4), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were transduced into a rat C6 glioma cell line via a retroviral vector, S2. Rats intracerebrally (IC) implanted with the C6 cells genetically engineered to secrete the cytokines, especially GM-CSF, manifested significantly higher survival rates than those with C6 cells or with C6 cells bearing the control vector (p < 0.002). In vivo, C6 tumors bearing the cytokine genes grew more slowly than wild-type tumors at any time point, and eventually diminished within 6 weeks after tumor cell implantation. Histopathological and immunohistochemical studies revealed that different cytokines induced diverse immune reactions. In the IL-2 group, CD4+ and CD8+ T cells dominated from day 3 to week 4, but disappeared at week 6. Some granulocytes were noted between weeks 2 and 4. In the IL-4 group, eosinophils were noted from day 3 to week 4, and CD4+ and CD8+ T cells, as well as macrophages at week 2. At week 6, only residual levels of macrophages and CD8+ T cells remained. In the GM-CSF group, granulocytes appeared as early as day 1 post-IC tumor implantation, and macrophages at day 2. CD4+ and CD8+ T cells were found from day 3 to week 4. At week 6, only residual CD4+ T cells and macrophages remained. Long-lasting antitumor immunity was confirmed in all groups by rechallenging surviving rats with wild-type C6 cells in the brain 100 days after implanting cytokine gene-bearing C6 cells. In vivo depletion of GM-CSF by anti-GM-CSF antibody further confirmed that the immune reaction induced by GM-CSF-secreting tumor cells were mainly from the action of GM-CSF, rather than the immunogenicity of C6 cells.