Recombinant GM-CSF Research Articles

Microwave ablation combined with intra-tumoral inoculation of dendritic cells induces specific immunity against hepatocellular carcinoma

Objective To investigate the efficacy of microwave ablation combined with intra-tu-moral inoculation of dendritic cells (DCs) in inducing specific immunity against hepatocellular carcino-ma (HCC) in vivo. Methods C57BL/6 bone marrow-derived DCs were grown in vitro for 6 d with use of murine recombinant GM-CSF and IL-4. C57BL/6 mice were inoculated subcutaneously with Hepal-6 cells to establish subcutaneous HCC, and randomized into control group, group receiving in-tratumoral inoculation of DCs (DC group), group receiving microwave ablation of tumor (MWA group), and group receiving microwave ablation of tumor plus intra-tumoral injection of DCs (MWA+ DC group). Immunohistochemical staining was employed to evaluate intra-tumoral infiltration of CD4+ and CD8+ lymphocytes. Cytotoxicity of splenocytes against Hepal-6 was evaluated using MTT method. Tumor growth in each group was compared. Results Immunohistochemical staining illustra-ted that MWA+DCs group had a markedly greater number of CD4+ and CD8+ T lymphocytes inside tumor tissue as compared with the control group, DC group and MWA group (P<0. 05). Splenocytes from MWA+ DCs group had a remarkably higher specific cytotoxicity against Hepal-6 at E/T ratio of 40 and 100 than those from control group, DC group and MWA group (P<0.05). Tumor regression rate was significantly higher in MWA+DCs group as compared with the control group, DC group and MWA group (P<0. 05). Conclusion Intra-tumoral inoculation of DCs following MWA could effec-tively elicit specific anti-tumoral immunity in vivo. It can be used as an effective approach for preven-ting tumor recurrence after MWA. Key words: Carcinoma hepatocellular; Dendritic cell, Immunotherapyl Microwave ablation

In vitro induction,proliferation and function characterization of dendritic cells from rat bone lnRITOW cells

Objective To establish a method of inducing dendritic cells(DC)from rat bone marrow cells in vitro,and identify the phenotype and function characteristics.Methods The rat bone malToW cells were collected and cultured in vitro under the condition of recombinant rat GM-CSF(rrGM-CSF)and recombinant rat IL-4(rrIL-4).After 2 weeks,the morphological character of DCs was observed under light microscope and scanning electron microscope.Expression of MHC-Ⅱ,CD80 and CD86 were detected by flow cytometry.The ability to stimulate allogenic T cells of the cultured DCs was detected by mixed lymphocyte reaction.Results DCs showed typical morphology with elongated dendritic processes under inversion microscope and scanning electron microscope.DCs at day 6 revealed immature phenotype,including MHC-Ⅱ(29.03 ±4.39)%,CD80(21.98±7.08)%and CD86(25.94±6.80)%.DCs at day 12 showed higher expression of MHC-Ⅱ(74.05±5.97)%,CD80(79.85±6.53)%and CD86(81.00±7.47)%,and stimulatory capacity of allogenic T cells,compared with that in DCs at day 6.Conclusion Matured DCs could be generated from rat bone marrow cells and attendance with rrGM-CSF and rrIL-4,which present the feasibility for further research on its application to allograft immunorejection. Key words: Dendritic cells; Bone marrow; Granulocyte-maerophage colony-stimulating factor; Interleukin-4

Two populations of ovine bone marrow-derived dendritic cells can be generated with recombinant GM-CSF and separated on CD11b expression

Whereas studies on dendritic cells in rodents rely largely on bone marrow-derived dendritic cells (BM-DCs), no data are available about BM-DCs in sheep, a species that is largely used for immunology and transplantation studies. We have developed a culture protocol to produce ovine BM-DCs, using 6x(His)-tagged recombinant GM-CSF which was purified from baculovirus-infected insect cells. When ovine bone marrow progenitors were cultured in the presence of recombinant GM-CSF, large numbers of CD11c-positive cells were generated after 6–7 days. The phenotypic appearance of BM-DCs was assessed by flow cytometry and electron microscopy. Two DC subsets were identified that expressed different levels of MHC class II molecules, differed in receptor-mediated endocytosis, and could be separated on CD11b expression. When separated cells were incubated with microbial products, they react differently to those that are considered the TLR2 and TLR4 agonists in other species. Indeed, although CD11b int/hi cells were partially resistant to maturation induced by lipoteichoic acid or lipopolysaccharide, MHC class II upregulation was observed on CD11b dull cells. Moreover, these cells had strong stimulatory capacity for CD4 T cells when assayed in allogeneic reactions. This protocol will help analyzing ovine DC interactions with pathogens, and enables future studies on the development of vaccines.

Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) or tumour necrosis factor-alpha (TNF-alpha) activate human alveolar macrophages to inhibit growth of Mycobacterium avium complex.

We investigated the effects of certain macrophage-active cytokines on the phagocytosis and growth inhibition of Mycobacterium avium complex (MAC) by human alveolar macrophages (AM). We also evaluated the effects of pretreatment with each cytokine on the superoxide anion release (O2-) from AM. The cytokines that we used were recombinant GM-CSF, natural type TNF-alpha, recombinant interferon-gamma (IFN-gamma), and recombinant IL-2. We found that phagocytosis by the various cytokine-stimulated AM was similar to that of unstimulated AM. On the other hand, significant growth inhibition of MAC was observed in the macrophages treated with GM-CSF or TNF-alpha, while no growth inhibition of MAC was observed in the macrophages treated with IFN-gamma or IL-2. Pretreatment with all cytokines tested enhanced the O2- release from AM, but there was no correlation between the enhancement of O2- release and the growth inhibition of MAC. Thus, we concluded that GM-CSF or TNF-alpha could activate AM to inhibit growth of MAC, probably not through the enhanced production of reactive oxygen intermediates.

Chitosan solution enhances the immunoadjuvant properties of GM-CSF

Sustained, local delivery of immunomodulatory cytokines is under investigation for its ability to enhance vaccine and anti-tumor responses both clinically and preclinically. This study evaluates the ability of chitosan, a biocompatible polysaccharide, to (1) control the dissemination of a cytokine, GM-CSF, and (2) enhance the immunoadjuvant properties of GM-CSF. While cytokines have previously been delivered in lipid-based adjuvants and other vehicles, these do not have the clinical safety profile or unique properties of chitosan. We found that chitosan solution maintained a measurable depot of recombinant GM-CSF (rGM-CSF) at a subcutaneous injection site for up to 9 days. In contrast, when delivered in a saline vehicle, rGM-CSF was undetectable in 12–24 h. Furthermore, a single s.c. injection of 20 μg rGM-CSF in chitosan solution (chitosan/rGM-CSF(20 μg)) transiently expanded lymph nodes up to 4.6-fold and increased the number of MHC class II expressing cells and dendritic cells by 7.4-fold and 6.8-fold, respectively. These increases were significantly greater than those measured when rGM-CSF was administered in saline at the standard preclinical dose and schedule, i.e. 4 daily s.c. injections of 20 μg. Furthermore, lymph node cells from mice injected with chitosan/rGM-CSF(20 μg) induced greater allogeneic T cell proliferation, indicating enhanced antigen presenting capability, than lymph node cells from mice injected with rGM-CSF alone. Finally, in vaccination experiments, chitosan/rGM-CSF was superior to either chitosan or rGM-CSF alone in enhancing the induction of antigen-specific CD4 + proliferation, peptide-specific CD8 + pentamer staining and cytotoxic T cell lysis. Altogether, chitosan/rGM-CSF outperformed standard rGM-CSF administrations in dendritic cell recruitment, antigen presentation and vaccine enhancement. We conclude that chitosan solution is a promising delivery platform for the sustained, local delivery of rGM-CSF.

Foreign Body Giant Cell Formation Is Preceded by Lamellipodia Formation and Can Be Attenuated by Inhibition of Rac1 Activation

Macrophages that are recruited to the site of implanted biomaterials undergo fusion to form surface-damaging foreign body giant cells. Exposure of peripheral blood monocytes to interleukin-4 can recapitulate the fusion process in vitro. In this study, we used interleukin-4 to induce multinucleation of murine bone marrow-derived macrophages and observed changes in cell shape, including elongation and lamellipodia formation, before fusion. Because cytoskeletal rearrangements are regulated by small GTPases, we examined the effects of inhibitors of Rho kinase (Y-32885) and Rac activation (NSC23766) on fusion. Y-32885 did not prevent cytoskeletal changes or fusion but limited the extent of multinucleation. NSC23766, on the other hand, inhibited lamellipodia formation and fusion in a dose-dependent manner. In addition, we found that in control cells, these changes were preceded by Rac1 activation. However, NSC23766 did not block the uptake of polystyrene microspheres. Likewise, short interfering RNA knockdown of Rac1 limited fusion without limiting phagocytosis. Thus, phagocytosis and fusion can be partially decoupled based on their susceptibility to NSC23766. Furthermore, poly(ethylene-co-vinyl acetate) scaffolds containing NSC23766 attenuated foreign body giant cell formation in vivo. These observations suggest that targeting Rac1 activation could protect biomaterials without compromising the ability of macrophages to perform beneficial phagocytic functions at implantation sites.

Chemotherapy Enhances CD8+ T Cell-mediated Antitumor Immunity Induced by Vaccination With Vaccinia Virus

The use of immunotherapy or chemotherapy alone is generally ineffective against well-established tumors. To overcome this intrinsic resistance against therapy for tumors, we have attempted to combine immunotherapy with chemotherapy. In this study, we tried to induce a rapid antitumor effect via chemoimmunotherapy using a vaccinia viral vaccine as an immunotherapeutic agent with anticancer agents including epigallocatechin-3-gallate (EGCG) and conventional anticancer drugs. Although a combination of vaccinia-mediated vaccination and chemotherapy led to a strong inhibition of tumor growth, monotherapy alone failed to completely cure tumors. In contrast, intravenous injection of cisplatin (CDDP) or cyclophosphamide (CTX) after vaccinia virus vaccination led to complete regression of the established tumors. Interestingly, anticancer drugs appear to augment the antitumor effect of the vaccinia virus-mediated immunotherapy. This effect is mainly associated with the enhanced tumor-specific CD8(+) T cell immune response induced by vaccinia virus, which was demonstrated by antibody depletion. However, anticancer drugs alone failed to induce a significant enhancement of the tumor-specific CD8(+) T cell immune response. Taken together, these results suggest that combining vaccinia virus-based immunotherapy with anticancer drugs is particularly effective against established tumors by increasing the tumor antigen-specific CD8(+) T cell immune response, which is primed by vaccinia virus-mediated vaccination.

Perioperative sargramostim (recombinant human GM-CSF) induces an increase in the level of soluble VEGFR1 in colon cancer patients undergoing minimally invasive surgery

Introduction Experimentally, laparotomy is associated with increased tumor growth. In humans, abdominal surgery is associated with immunosuppression and elevated plasma VEGF levels that might stimulate tumor growth early after surgery. Avoidance of these surgery-related changes and their consequences may be advantageous. Granulocyte-macrophage colony stimulating factor (GMCSF) is a non-specific immune system up-regulator that has also been associated, experimentally, with increased release of soluble VEGF Receptor 1 (sVEGFR1) which is an endogenous inhibitor of VEGF. This study's purpose was to determine the impact of perioperatively administered recombinant human GMCSF (rhu-GMCSF) on both immune function and plasma sVEGFR1 levels in colorectal cancer patients. Methods This randomized placebo-controlled study included 36 colorectal cancer patients who underwent minimally invasive resection (17 GMCSF, 19 Placebo). Patients received 7 subcutaneous injections of either rhu-GMCSF, 125 μg/m 2, or saline on preoperative days 3, 2 and 1 and on postoperative days (POD) 1, 2, 3 and 4. A number of immune parameters were followed and plasma levels of soluble VEGF Receptor 1 (sVEGFR1) and VEGF were determined. Results The total WBC, neutrophil, eosinophil, and monocyte counts were significantly higher after surgery in the GMCSF group; no differences were noted for the other immune parameters. In the GMCSF group, median plasma sVEGFR1 levels were significantly elevated on POD 1 (188.1 pg/ml), and on POD 5 (142.8 pg/ml) when compared to pre-GMCSF levels (0 pg/ml)( p-value < 0.05 for all comparisons). In the placebo group, the POD5 median sVEGFR1 level (116.3 pg/ml) was elevated and of borderline significance ( p = 0.05) vs the pre-treatment result (0 pg/ml). Of note, both groups had significantly elevated median plasma VEGF levels on POD 5 (Control 435.7 pg/ml; GMCSF 385.3 pg/ml) when compared to their preoperative results (Control 183.3 pg/ml, p = 0.0013; GMCSF 171.5 pg/ml, p = 0.0055). Conclusions Perioperative GMCSF was not associated with an immune function benefit in this study, however, such treatment leads to increased plasma sVEGFR1 levels. Colorectal resection, with or without GMCSF, was also associated with increased VEGF levels postoperatively. Increased plasma levels of sVEGFR1 after surgery might limit the pro-angiogenic tumor stimulatory effects of VEGF. Further study of GMCSF's impact on angiogenesis appears warranted.

Treatment-Enhanced CD4+Foxp3+ Glucocorticoid-Induced TNF Receptor Family RelatedHigh Regulatory Tumor-Infiltrating T Cells Limit the Effectiveness of Cytokine-Based Immunotherapy

Regulatory T cells can suppress activated CD4+ and CD8+ T effector cells and may serve as an impediment to spontaneous or therapeutic type 1 antitumor immunity. In a previous study, we observed minimal therapeutic impact, but significantly enhanced T cell cross-priming and lesional infiltration of tumor-reactive CD8+ T cells into established CMS4 sarcomas after combined treatment of BALB/c mice with rFLt3 ligand (rFL) and recombinant GM-CSF (rGM-CSF). In this study, we show that this cytokine regimen also results in the profound enhancement of CD4+ tumor-infiltrating lymphocytes (TIL) expressing FoxP3, IL-10, and TGF-beta mRNA, with 50 or 90% of CD4+ TIL coexpressing the CD25 and glucocorticoid-induced TNFR family related molecules, respectively. Intracellular staining for Foxp3 protein revealed that combined treatment with rFL plus rGM-CSF results in a significant increase in CD4+Foxp3+ T cells in the spleen of both control and tumor-bearing mice, and that nearly half of CD4+ TIL expressed this marker. In addition, CD4+ TIL cells were of an activated/memory (ICOS(high)CD62L(low)CD45RB(low)) phenotype and were capable of suppressing allospecific T cell proliferation and IFN-gamma production from (in vivo cross-primed) anti-CMS4 CD8+ T cells in vitro, via a mechanism at least partially dependent on IL-10 and TGF-beta. Importantly, in vivo depletion of CD4+ T cells resulted in the ability of previously ineffective, rFL plus rGM-CSF therapy-induced CD8+ T cells to now mediate tumor regression.

Biologically active human GM-CSF produced in the seeds of transgenic rice plants

Rice flour is a well-known and characterized source of pharmaceutical ingredients, which are gluten-free and incorporated in many drug delivery applications such as excipient starch. To further exploit this uniqueness, the synthetic capacity of rice endosperm tissue, the basis of rice flour, was extended by genetic transformation. Recombinant human GM-CSF, a cytokine used in treating neutropenia and with other potential clinical applications, has been expressed in transgenic rice seeds using a rice glutelin promoter. Rice seeds accumulated human GM-CSF to a level of 1.3% of total soluble protein. The rice seed-produced human GM-CSF was found to be biologically active when tested using a human cell line TF-1. Use of rice as a host plant offers not only attractive features of safe production in seeds but also self-containment of foreign genes, as rice is primarily a self-pollinated crop plant.

Ligation of intercellular adhesion molecule 3 induces apoptosis of human blood eosinophils and neutrophils

Intercellular adhesion molecule 3 (ICAM-3) is highly expressed on human granulocytes, including eosinophils and neutrophils, but the functions of ICAM-3 in these cells are not well understood. Our studies test the hypothesis that ICAM-3 regulates granulocyte apoptosis. Intercellular adhesion molecule 3 was activated by a mAb that recognizes an ICAM-3 epitope that binds its ligand, CD11a/CD18. In some experiments with eosinophils, recombinant human IL-5 or GM-CSF was added to mimic in vivo antiapoptotic conditions. Staining with annexin V-fluorescein isothiocyanate and propidium iodide identified apoptotic cells. Binding of ICAM-3 increased apoptosis of both eosinophils (18 and 48 hours) and neutrophils (18 hours). At 18 hours, eosinophil apoptosis increased from 31.4% +/- 3.5 SE (IgG control) to 45.2% +/- 3.8 SE (anti-ICAM-3), and neutrophil apoptosis increased from 48% +/- 4.1 SE (IgG control) to 55.3% +/- 4.5 SE (anti-ICAM-3). At 48 hours, eosinophil apoptosis increased 2-fold under baseline conditions and also in the presence of recombinant human IL-5 or GM-CSF. In both eosinophils and neutrophils, incubation with a blocking antibody against CD18 integrins blunted ICAM-3-induced apoptosis. In eosinophils, blocking peptides for caspases 8 and 9, proteases critical to apoptosis, also decreased ICAM-3-induced apoptosis to control levels. Through its effect on eosinophil and neutrophil apoptosis, ICAM-3 may be an important anti-inflammatory molecule that can oppose the proinflammatory effects of IL-5 and GM-CSF. These findings provide a mechanism for apoptotic clearance of eosinophils and neutrophils involved in allergic inflammation that, unlike necrosis, does not cause nonspecific tissue injury.

In vitro leukemic cell differentiation and WT1 gene expression

Cell differentiation and four WT1 isoforms were assessed in CD34 + cells from patients with acute myelogenous leukemia in presence or absence of recombinant human GM-CSF and G-CSF, on days 0, 10 and 20 of culture. We found that WT1 isoforms expression was consistently higher in AML-derived CD34 + cell-enriched cell fractions, as compared to their normal counterparts, and interestingly, in both cases, cells had differentiation towards the myeloid lineage with WT1 expression different patterns. This data suggest that WT1 expression seems to be modulated by the presence of cytokines, especially on day 20 of culture.

Multigene/Multisubtype HIV-1 Vaccine Induces Potent Cellular and Humoral Immune Responses by Needle-Free Intradermal Delivery

Gene vaccination encounters problems different from those of gene therapy since both a short half-life of the gene and a strong immune response to the gene product are desirable. We have evaluated a DNA vaccine consisting of seven plasmids encoding nine HIV-1 proteins. Using a needle-free delivery device, the Biojector, together with recombinant mouse GM-CSF, this vaccine induced strong gp160 Env- and p24 Gag-specific cellular and humoral immune responses in mice. The rGM-CSF was crucial for inducing both antibodies and antigen-specific CD8+ T cell responses against both gp160 and p24. A GMP-produced lot of this vaccine, intended for human use, was delivered intradermally or intramuscularly into BALB/c mice at a GLP-accredited animal facility. This vaccine induced strong cellular responses independent of the route of immunization; moreover, no signs of toxicity were detected after histopathological examination of various tissues. Overall, the results indicate that the intradermal delivery of multigene/multisubtype HIV DNA in combination with recombinant GM-CSF is a safe and efficacious strategy for inducing high levels of specific CD8+ T cells and unusually high titers of antibodies. This vaccine has been approved by the Swedish Medicinal Products Agency and is currently in a Phase I clinical trial.

Lethal and severe coronary arteritis in DBA/2 mice induced by fungal pathogen, CAWS, Candida albicans water-soluble fraction

CAWS is a microbial pathogen-associated molecular patterns (PAMPs) produced by Candida albicans. CAWS is a mannoprotein–beta-glucan complex and secreted into the culture supernatant. CAWS has various biological effects, causing acute shock and disrupting vascular permeability. Intraperitoneal administration of CAWS induces coronary arteritis in various strains of inbred mice. The CAWS-induced coronary arteritis is strain-dependent and most severe in DBA/2 mice with a significant number of these animals expiring with cardiomegaly during the observation period. In vivo and in vitro, splenocytes of DBA/2 mice produced various cytokines, such as IL-6, TNF-α, and IFN-γ in response to CAWS. GM-CSF was also produced in response to CAWS. The production of cytokines was significantly enhanced in the presence of recombinant GM-CSF. In contrast, anti-GM-CSF significantly reduced the production of TNF-α and IFN-γ. Augmented production of cytokines in response to CAWS would be a key to the severity of coronary arteritis.

Safety and immunological efficacy of a prostate cancer plasmid DNA vaccine encoding prostatic acid phosphatase (PAP)

Prostatic acid phosphatase (PAP) is a prostate tumor antigen currently being investigated as a target antigen in several human vaccine trials, some with evidence of clinical benefit. We have previously demonstrated that plasmid DNA vaccines encoding either human or rat PAP can elicit antigen-specific cellular and humoral immunity in rat models. The current study was performed to determine the safety and potential immunological efficacy in rodents of large and repetitive doses of a GMP-grade plasmid DNA vaccine encoding human PAP, pTVG-HP. Fifty-four male Lewis rats were immunized intradermally at 2-week intervals with 100, 500, or 1500 μg pTVG-HP with 5 μg recombinant rat GM-CSF protein given as a vaccine adjuvant. An additional 12 male Lewis rats served as controls with groups immunized with 1500 μg of a parental DNA vector not encoding human PAP, and a group that received GM-CSF protein only without plasmid DNA. Groups of animals ( n = 3–6) were euthanized after two, four, or six immunizations with collections of tissues and blood for toxicity assessment and immunological analysis. No significant toxicities were observed in terms of animal weights, histopathology, hematological changes, or changes in serum chemistries. Six of fifty-four were found to have subtle evidence of possible renal toxicity, however these findings were not statistically different from control animals. The vaccine was found to be effective in eliciting PAP-specific CD4 and CD8 T cells, predominantly Th1 in type, in all immunized animals at all doses and numbers of immunizations. PAP-specific IgG were detected in a dose-dependent fashion, with titers increasing after multiple immunizations. These studies demonstrate that, in rats, immunization with the pTVG-HP vaccine is safe and effective in eliciting PAP-specific cellular and humoral immune responses. These findings support the further clinical evaluation of pTVG-HP in patients with prostate cancer.

Immune Monitoring in a Phase 1 Trial of a PSA DNA Vaccine in Patients with Hormone-Refractory Prostate Cancer

Prostate cancer remains a leading cause of cancer illness and death among men in Europe. No curative treatment exists when the disease has spread beyond the prostate. Immunotherapy with DNA vaccines has emerged as a potential therapeutic approach for the induction of antitumor specific cytotoxic T lymphocytes. In this study six patients with hormone-refractory prostate cancer were monitored for their ability to mount PSA-specific cellular responses after receiving a pVAX/PSA DNA vaccine (patients 1-3, 100 microg; patients 7-9, 900 microg) with recombinant GM-CSF and IL-2 as adjuvants. IFNgamma ELISPOT showed that naturally processed PSA protein and PSA peptides are recognized by T cells in the blood of some prostate cancer patients after a PSA DNA vaccine. Analysis of other cytokines showed the production of IL-4 and IL-6 but importantly did not show an increase in the number of IL-10-producing cells after vaccination in any of the patients. The authors conclude that a pVAX/PSA DNA vaccine can induce PSA-specific cellular immune responses in patients with hormone-refractory prostate cancer, thus emphasizing the potential for PSA as a target molecule for the immunotherapy of prostate cancer.

Effects of UVB on fascin expression in dendritic cells and Langerhans cells

Fascin is an actin-binding protein that regulates the rearrangement of cytoskeletal elements and their interactions with the cell membrane. Previous studies have indicated that fascin expression is enhanced in DC upon maturation and plays a critical role in T cell activation. Ultraviolet irradiation exerts immunosuppressive effects. We examined the effects of UVB irradiation on the interaction of DC/LC with T cells through fascin. Murine bone marrow-derived DC (BM-DC) were induced by recombinant murine GM-CSF and LPS, and UVB irradiation was applied prior to supplementation with LPS. I-A(+) cells (Langerhans cells (LC)) in the epidermal cell suspensions were exposed to UVB irradiation at the beginning of the 24-h culture. BM-DC and LC were analysed by immunohistochemical staining and flow cytometric analyses. To evaluate the effects of UVB irradiation on DC-T cell binding, we examined the clustering of BM-DC with allogeneic CD4(+) T cells under a confocal microscope. Fascin expression in BM-DC and LC was decreased by UVB irradiation. Furthermore, UVB irradiation reduced the ability of BM-DC to cluster with allogeneic CD4(+) T cells. Polarization of fascin and filamentous actin (F-actin) at the point of contact of BM-DC with T cells was also disturbed by UVB irradiation. These results suggest that the suppression of fascin expression by UVB irradiation down-regulates the rearrangement of the cytoskeleton and, thereby, antigen presentation in DC/LC.

Suppressor of Cytokine Signaling (SOCS) Proteins Indirectly Regulate Toll-like Receptor Signaling in Innate Immune Cells

Suppressor of cytokine signaling (SOCS) proteins constitute a class of negative regulators for Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways. These intracellular proteins are induced by cytokine signaling, but they can also be induced by stimulation of Toll-like receptors (TLR). It has even been suggested that SOCS proteins are important negative regulators of TLR signaling. Here we have elucidated the nature of the regulatory role of SOCS in TLR signaling. Induction of SOCS-3 and cytokine-inducible Src homology 2-containing protein (CIS) by TLR stimulation was strictly dependent on MyD88 but showed differing needs in case of SOCS-1. However, induction of SOCS proteins by TLR ligands was independent of type I interferon. In macrophages overexpressing SOCS, we were not able to observe an inhibitory effect of SOCS-1, SOCS-2, SOCS-3, or CIS on prototypical TLR target genes such as tumor necrosis factor-alpha. However, we found that TLR-2, TLR-3, TLR-4, and TLR-9 stimulation induced interferon-beta (IFN-beta), which is able to exert auto- and paracrine signaling, leading to the activation of secondary genes like IP-10. SOCS-1 and, to a lesser extent, SOCS-3 and CIS were able to inhibit this indirect signaling pathway following TLR stimulation, whereas neither MAP kinase nor NF kappa B signaling were affected. However, STAT-1 tyrosine phosphorylation following TLR triggering was severely impaired by SOCS-1 overexpression. Thus, our data suggest that SOCS proteins induced by TLR stimulation limit the extent of TLR signaling by inhibiting type I IFN signaling but not the main NF kappa B pathway.

Serum neutralizing capacity of GM-CSF reflects disease severity in a patient with pulmonary alveolar proteinosis successfully treated with inhaled GM-CSF

Existence of anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) neutralizing antibody and treatment with recombinant GM-CSF are new topics in idiopathic pulmonary alveolar proteinosis (PAP). We have hypothesized inhaled GM-CSF is effective and neutralizing capacity of GM-CSF, not concentration of anti-GM-CSF antibody in serum reflect disease severity. A 57-year-old female smoker with idiopathic PAP was treated with inhaled GM-CSF. The response to the treatment was evaluated by diffusing capacity for carbon monoxide (DLCO), alveolar-arterial oxygen gradient ([A-a]DO2). Conventional serum markers, including KL-6, surfactant apoprotein (SP)-A, SP-D, carcino-embryonic antigen and cytokeratin fragment 19 (CYFRA), and concentration of anti-GM-CSF antibody were examined. The neutralizing capacity of GM-CSF in serum was evaluated using a GM-CSF dependent cell line, TF-1. Ground glass opacity disappeared at the end of the treatment. Her DLCO, [A-a]DO2 remarkably improved after treatment. The neutralizing capacity of GM-CSF declined in line with disease remission and it correlated significantly with DLCO (P = 0.0137). The concentration of anti-GM-CSF antibody had no significant relation with disease severity and serum markers including neutralizing capacity. Conventional serum markers other than CYFRA showed no significant correlation with Inhaled GM-CSF was effective for idiopathic PAR Serial measurement of neutralizing capacity of GM-CSF was useful to evaluate disease severity and the anti-GM-CSF antibody was proved to be a causative factor for PAR In the future, inhaled GM-CSF may replace whole lung lavage and response to GM-CSF and its optimal amount may be decided by the capacity.

A phase I study of sequential vaccinations with recombinant Fowlpox-PSA (L155)-TRICOM (rF)alone, or in combination with recombinant vaccinia-PSA (L155)-TRICOM (rV), and the role of GM-CSF, in patients (Pts) with prostate cancer

2522 Background: Vaccine strategies targeting PSA represent a novel approach in the treatment of prostate cancer. Since PSA is a “self” antigen, vaccine strategies utilizing this as a target must be developed to enhance its immunogenicity. Previous studies have shown that the induction of T-cell responses directed against a self-antigen is associated with anti-tumor activity but not toxicity. Two novel vaccines have been developed: (a) a recombinant vaccinia virus containing the entire PSA transgene with a modified agonist epitope and three costimulatory molecule transgenes rV and (b) a similar recombinant fowlpox virus rF. Methods: This Phase I study evaluated the safety of a diversified prime/boost vaccine strategy: priming with rV and monthly boosts using rF. All pts had progressive metastatic prostate cancer. This trial also examined the combination of these agents with recombinant GM-CSF (rG) as well as two doses of fowlpox-GM-CSF (rF-G). The first of 5 cohorts utilized a fixed dose of rF alone, the second cohort tested the safety of a priming vaccination with rV followed by monthly rF boosting. Cohorts 3,4, and 5 provided safety data combining cohort 2 with rG or two doses of rF-G respectively. Results: Accrual (n=15) has been achieved. Thirteen pts remain on study. No dose limiting toxicities have been observed. Pt characteristics: median age = 64 years (range 48–80), median PSA = 191.7 ng/ml (range 2.6–2943), median Gleason score = 8 (range 7–9). Seven of 9 evaluable pts treated for at least 3 months have not demonstrated progression on CT or bone scan and remain on study. One pt in cohort 2 has a PSA decline of >35%. Immunologic studies are ongoing to measure changes in PSA specific T cells. Conclusions: Based on the safety data from this clinical trial, we have initiated the randomized Phase II component of this study to compare the immunologic effects of the above vaccine strategy alone, with r-G, or with either of 2 doses of rF-G. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Therion Biologics